Trypanosoma cruzi killing and immune response boosting by novel phenoxyhydrazine-thiazole against Chagas disease.

Trypanosoma cruzi (T. cruzi) causes Chagas, which is a neglected tropical disease (NTD). WHO estimates that 6 to 7 million people are infected worldwide. Current treatment is done with benznidazole (BZN), which is very toxic and effective only in the acute phase of the disease. In this work, we designed, synthesized, and characterized thirteen new phenoxyhydrazine-thiazole compounds and applied molecular docking and in vitro methods to investigate cell cytotoxicity, trypanocide activity, nitric oxide (NO) production, cell death, and immunomodulation. We observed a higher predicted affinity of the compounds for the squalene synthase and 14-alpha demethylase enzymes of T. cruzi. Moreover, the compounds displayed a higher predicted affinity for human TLR2 and TLR4, were mildly toxic in vitro for most mammalian cell types tested, and LIZ531 (IC50 2.8 µM) was highly toxic for epimastigotes, LIZ311 (IC50 8.6 µM) for trypomastigotes, and LIZ331 (IC50 1.9 µM) for amastigotes. We observed that LIZ311 (IC50 2.5 µM), LIZ431 (IC50 4.1 µM) and LIZ531 (IC50 5 µM) induced 200 µg/mL of NO and JM14 induced NO production in three different concentrations tested. The compound LIZ331 induced the production of TNF and IL-6. LIZ311 induced the secretion of TNF, IFNγ, IL-2, IL-4, IL-10, and IL-17, cell death by apoptosis, decreased acidic compartment formation, and induced changes in the mitochondrial membrane potential. Taken together, LIZ311 is a promising anti-T. cruzi compound is not toxic to mammalian cells and has increased antiparasitic activity and immunomodulatory properties.

Selective LC-MS/MS determination of citalopram enantiomers and application to a pharmacokinetic evaluation of generic and reference formulations.

Two methods using LC-MS/MS were validated to quantify citalopram (CTP) racemate [(R/S)-CTP] and the enantiomers (R)-CTP and (S)-CTP in human plasma, respectively. Paroxetine hydrochloride was used as the internal standard, and samples were extracted by protein precipitation with acetonitrile. The non-enantioselective method was conducted using a C18 column, and the mobile phase consisted of water for solvent A and acetonitrile for solvent B, both with 0.1% formic acid. For the chiral method, an analytical column Lux Cellulose-1 was used. Mobile phase A was composed of water with 0.025% of formic acid and 0.05% of diethylamine, and mobile phase B consisted of acetonitrile:2-propanol (95:5, v/v). No significant matrix effects were observed at the retention times of analytes and internal standard. The mean recovery was 89%, and the assays were linear in the concentration range of 1-50 and 5-30 ng/mL for the non-enantioselective and enantioselective methods, respectively. The intra- and inter-day precisions of both methods were less than 12.30%, and the accuracies were less than 12.13%. The validated methods were successfully applied to a pharmacokinetic study in which 20-mg CTP tablets were administered to healthy volunteers, and their plasma levels were monitored over time in a bioequivalence study. HIGHLIGHTS: Simple and rapid LC-MS/MS method for the quantification of citalopram and its enantiomers in human plasma. Both methods were demonstrated to be selective, reliable, and sensitive. Both methods have sufficient sensitivity to quantify the steady state through concentrations already reported for citalopram and escitalopram. Validated method presented in this study can be suitably applied to pharmacokinetic studies involving citalopram and escitalopram. Bland-Altman analysis suggested that non-enantioselective and enantioselective methods can be applied in pharmacokinetic studies.

Levobupivacaine-Loaded Liposome Associated with Thermogel for Prolonged Analgesia.

Pain is a phenomenon present in the majority of the population, affecting, among others, the elderly, overweight people, and especially recently operated patients, analgesia being necessary. In the specific case of relief of postoperative pain, different kinds of anesthetics are being used, among them bupivacaine, a widely used drug which promotes long-lasting analgesic effects. However, cardiotoxicity and neurotoxicity are related to its repetitive use. To overcome these shortcomings, Novabupi® (a racemic mixture) was developed and is marketed as an injectable solution. This formulation contains an enantiomeric excess of the levogyre isomer, which has reduced toxicity effects. Seeking to rationalize its use by extending the duration of effect and reducing the number of applications, the objectives of this work were to develop and evaluate liposomes containing Novabupi (LBPV), followed by incorporation into thermogel. Liposomes were prepared using the lipid hydration method, followed by size reduction using sonication, and the developed formulations were characterized by hydrodynamic diameter, polydispersity index (PDI), surface zeta potential, and encapsulation efficiency. The selected optimal liposomal formulation was successfully incorporated into a thermogel without loss of thermoresponsive properties, being suitable for administration as a subcutaneous injection. In the ex vivo permeation studies with fresh rodent skin, the thermogel with liposomes loaded with 0.5% LBPV (T-gel formulation 3) showed higher permeation rates compared to the starting formulation, thermogel with 0.5% LBPV (T-Gel 1), which will probably translate into better therapeutic benefits for treatment of postoperative analgesia, especially with regard to the number of doses applied.

Levobupivacaine Thermogel for Long-acting Analgesia.

The adequate management of analgesia, by pharmacological methods or not, is a great challenge. Local anesthetics are used for pain relief, mainly by parenteral, intramuscular, catheter, and other routes of administration. The use of in situ forming systems becomes an alternative for the control of pain. The present research investigates development of thermogels containing poloxamer and levobupivacaine. All formulations were prepared by the cold method; the compatibilities of the excipients were evaluated by DSC, rheology and viscosities, transition temperature, syringeability, release kinetics, and permeation. The compatibility of the tested excipients with the drug was initially observed; all formulations had a viscosity increase at 37°C. Different delivery rates were observed in both the release and permeation studies. The developed systems maintained the in vitro release of the drug for a long period, likely decreasing side effects in vivo and avoiding the need for supplementary analgesia by other routes.

Microemulsion Formulations for the Transdermal Delivery of Lapachol.

This project was carried out to investigate the feasibility of using microemulsions for transdermal delivery of lapachol. From the screening of surfactants and oils, a range of microemulsions were developed using oleic acid, a mixture of Cremophor EL and Tween 20 and water. The solubility of lapachol was determined in these ingredients and in the formulated microemulsions. The microemulsions were characterised using cross-polarising light microscopy, their electrical conductivity, pH, zeta potential and rheology were analysed, and they were also investigated using small-angle X-ray scattering and differential scanning calorimetry. Ex vivo studies were performed using porcine ear skin and Franz diffusion cells to investigate the permeation and retention of lapachol. Systems containing different concentrations of Cremophor EL (8.4-41.6%), Tween 20 (5.4-41.6%) and oleic acid (12-31.9%) are able to form microemulsions. Lapachol was delivered more effectively through the skin from all of the microemulsions tested than by the control (oleic acid). These studies indicated that microemulsions incorporating lapachol were formed successfully and that these enhanced drug delivery and retention in the skin. Microemulsion systems may, therefore, provide promising vehicles for percutaneous delivery of lapachol.

Third-Generation Transdermal Delivery Systems Containing Zidovudine: Effect of the Combination of Different Chemical Enhancers and a Microemulsion System.

This study aimed to examine the influence of the combination of chemical enhancers and a microemulsion on the transdermal permeation of zidovudine (AZT). Ethanol, 1,8-cineole, and geraniol were incorporated in a microemulsion. The droplet size, zeta potential, rheology, and SAXS analysis were performed. The permeation enhancer effect was evaluated using pig ear skin. Snake skin (Boa constrictor) treated with the formulations was also used as a stratum corneum model and studied by attenuated total reflectance-infrared spectroscopy. As a result, it was observed that the incorporation of the chemical enhancers promoted a decrease of the droplet size and some rheological modifications. The 1,8-cineole associated with the microemulsion significantly increased the permeated amount of AZT. Conversely, ethanol significantly increased the quantity of the drug retained in the skin. The probable mechanism for the cineole and ethanol effects was respectively: fluidization and increasing of the diffusion coefficient, and increasing of the partition coefficient. Surprising, geraniol + microemulsion drastically decreased both the permeated and the retained amount of AZT into the skin. Thus, the adequate association of microemulsion and chemical enhancers showed to be a crucial step to enable the topical or transdermal use of drugs.

Dimethoate absorption: A complementary in vitro and in vivo assessment.

This work attempts to evaluate dermal exposure (DE) of farm workers to dimethoate after 4 h of routine application to a lemon plantation. Dimethoate was measured on the workers' clothes as well as in stratum corneum (SC) and in saliva. In vitro permeation tests (IVPT) were performed through rat, pig and human skin and pig buccal, esophageal and sublingual mucosas. The mean of dimethoate DE was 342.19 ± 487.14 mg/d, the percentage of toxic dose per hour was higher than the other pesticides, and the SC penetration factors ranged between 0.5 and 14.81 and 0.05-53.96 % for back of neck and arms respectively. In the supporting IVPT study, dimethoate absorption through human skin was 14.75 % and the default value in the absence of experimental data for this product is 70%. These results show that in family farming the deficiency of correct clothing during the application of pesticides leaves workers more vulnerable.

Quality by design applied to olanzapine and quetiapine LC-MS/MS bioanalysis.

One major challenge in quantifying drugs in biological matrices is to manage interfering compounds. A technique such liquid chromatography coupled to mass spectrometry in tandem (LC-MS/MS) is especially suitable for this application due to its high sensitivity and selectivity in detecting low concentrations of analytes in a complex system. Due to the complexity of LC-MS/MS systems, a number of experimental parameters must be optimized to provide an adequate separation and detection of the analyte. In the present work, a design of experiments approach was developed to optimize an LC-MS/MS-based bioanalytical method to extract olanzapine (OLZ) and quetiapine (QTP) from human plasma. Three steps for the optimization process were conducted: central composite face-centered design to optimize chromatographic parameters (Step 1), ionization in mass spectrometry (Step 2) and a full 32 factorial design to optimize analyte extraction conditions (Step 3). After the optimization process, resolutions and QTP and OLZ retention time (2.3 and 4, respectively) were optimum with pH of 4.7 and 85.5% of acetonitrile for the chromatographic step. Mass spectrometry optimization step provided an increase of (±50%) in the average peak area with high signal-to-noise relationship for the analytes studied. The proposed extraction method was 70% more efficient than the initial method for all drugs analyzed.

Topical bio(in)equivalence of metronidazole formulations in vivo.

The topical bioavailabilities of metronidazole from a commercially available 'reference' product (Rozex®) and two extemporaneous test formulations were compared. With the reference drug product, a full skin pharmacokinetic profile, in vivo in human volunteers (following a 6-h uptake and clearance over the subsequent 22 h), was obtained using an improved stratum corneum (SC) sampling procedure. Then, a two-time point SC sampling method enabled the bio(in)equivalence of the test formulations to Rozex® to be evaluated. One test formulation was shown to be bioequivalent to Rozex®, both for uptake and clearance, whereas the other (more viscous and less spreadable) formulation was not. The delivery of metronidazole into the underlying viable epidermal tissue from Rozex® and from the equivalent test formulation was 2.5 to 3.5-fold higher than that from the inequivalent extemporaneous vehicle. The results highlight that the quantitative composition of a formulation, as well as its physical properties that influence events that take place at the vehicle-skin interface, can have a dramatic impact on the delivery of drug into the SC and subsequently to the viable skin layers below. The reproducible, sensitive and facile in vivo methodology employed may prove of particular value where regulatory approval of generic formulations lacks objective rigour.

Evaluation of Microemulsion and Lamellar Liquid Crystalline Systems for Transdermal Zidovudine Delivery.

This study proposed to investigate and to compare colloidal carrier systems containing Zidovudine (3'-azido-3'-deoxythymidine) (AZT) for transdermal administration and optimization of antiretroviral therapy. Microemulsion (ME) and lamellar phase (LP) liquid crystal were obtained and selected from pseudoternary diagrams previously developed. Small-angle X-ray scattering and rheology analysis confirmed the presence of typical ME and liquid crystalline structures with lamellar arrangement, respectively. Both colloidal carrier systems, ME, and LP remained stable, homogeneous, and isotropic after AZT addition. In vitro permeation study (using pig ear skin) showed that the amount of permeated drug was higher for ME compared to the control and LP, obtaining a permeation enhancing effect on the order of approximately 2-fold (p < 0.05). Microscopic examination after in vivo skin irritation studies using mice suggested few histological changes in the skin of animals treated with the ME compared to the control group (hydrogel). Thus, ME proved to be adequate and have promising effects, being able to promote the drug permeation without causing apparent skin irritation. On the order hand, LP functioned as a drug reservoir reducing AZT partitioning into the skin.

Correlation between microstructure and bioequivalence in anti-HIV drug efavirenz.

Polymorphism and particle size distribution can impact the dissolution behaviour and, as a consequence, bioavailability and bioequivalence of poorly soluble drugs, such as Efavirenz (EFV). Nevertheless, these characteristics do not explain some failures occurring in in vitro assays and in in vivo studies. EFV belongs to Class II and the High Activity Antiretroviral Therapy (HAART) is considered the best choice in the treatment of adults and children. EFV is a drug that needs bioequivalence studies for generic compounds. In this work, six raw materials were analyzed and two of them were utilized with human volunteers (in vivo assays or bioequivalence). All the routine pharmaceutical controls of raw materials were approved; however, the reasons for the failure of the bioequivalence assay could not be explained with current knowledge. The aim of this work was to study microstructure, a solid-state property of current interest in the pharmaceutical area, in order to find an explanation for the dissolution and bioequivalence behaviour. The microstructure of EFV raw materials was studied by Whole Powder Pattern Modelling (WPPM) of X-ray powder diffraction data. Results for different EFV batches showed the biorelevance of the crystalline domain size, and a clear correlation with in vitro (dissolution tests) and in vivo assays (bioequivalence).

Comparative in vitro performance of three small-volume valved holding chambers with beclomethasone/formoterol pressurized metered dose inhaler.

BACKGROUND: The use of valved holding chambers (VHCs) with pressurized metered dose inhalers (pMDIs) is reported to reduce the oral deposition of inhaled drugs and to facilitate the handling of these devices by patients, especially children. Although the number of commercially available VHCs is increasing, the correct choice of VHC in clinical practice is important, because VHCs are not equally effective regarding medication delivery. Hence, we aimed to evaluate the use of three small-volume VHCs-Vortex(®), AeroChamber(®) Plus (ACP), and Able Spacer™ (AS)-along with a commercial pMDI containing a combination of beclomethasone and formoterol (Innovair(®)) frequently used by asthma patients. METHODS: Evaluation of the delivered dose of both drugs and analysis of particle size distribution of aerosols emitted for the inhaler were performed using the Next Generation Impactor with and without the tested VHCs. RESULTS: The VHCs retained significant quantities of both drugs and dramatically reduced the quantity of drugs deposited in the throat of the impactor, indicating that particles with large size were preferably retained in the VHCs. Interestingly, although the delivered dose of both drugs was reduced by the use of VHCs, the use of the Vortex and the ACP resulted in comparable fine particle doses (FPDs) to that obtained when the pMDI was used alone, whereas the AS VHC significantly reduced the FPDs of both drugs. This may be due to the fact that, unlike the AS VHC, the Vortex and the ACP VHCs are made of antistatic materials that minimize the electrostatic interaction with emitted aerosols, enhancing medication delivery. CONCLUSION: The Vortex and the ACP VHCs present interesting advantages over the AS VHC to be used with Innovair pMDI. However, these results are based on an in vitro evaluation and need to be validated in an in vivo study in order to clinically assess the performance of these VHCs.

Relative bioavailability of two formulations of venlafaxine extended-release 75-mg capsules in healthy brazilian male volunteers: A single-dose, randomized-sequence, open-label, two-period crossover study in the fasting and fed states.

BACKGROUND: The oral antidepressant venlafaxine hydrochloride is a selective serotonin-norepinephrine reuptake inhibitor. OBJECTIVE: The aim of this study was to evaluate the bioequivalence of a new generic formulation of venlafaxine extended-release 75-mg capsules (test) and the available branded formulation (reference) to comply with regulatory criteria for marketing of the test product in Brazil. METHODS: This single-dose, randomized-sequence, open-label, 2-period crossover study was conducted in healthy male volunteers and consisted of separate fast- ing and fed phases. A single oral dose of the test or reference formulation was followed by a 7-day washout period, after which subjects received the alternative formulation. There was a 3-month interval between the fasting and fed portions of the study. There was no standardization of race because of the difficulty of achieving standardization in the Brazilian population. Blood samples were collected before dosing and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 24, 36, and 48 hours after dosing. Venlafaxine concentrations were determined using an HPLC-MS/MS method. The formulations were considered bioequivalent if the 90% CIs of the geometric mean ratios (test:reference) for C(max) and AUC(0-t) were within the regulatory range of 80% to 125%. Adverse events were monitored through-out the study based on vital signs, laboratory tests, interviews, and spontaneous patient reports. RESULTS: Forty-eight subjects were enrolled in both phases of the study; all 48 subjects completed the fasting phase, and 1 subject withdrew during the fed phase. The mean (SD) age of participants in the fasting and fed phases was 24.96 (5.5) and 24.90 (4.7) years, respectively; their mean weight was 69.65 (9.6) and 71.00 (10.6) kg and their mean height was 172.0 (6.9) and 173.0 (6.6) cm. Under fasting conditions, the arithmetic mean venlafaxine C(max) was 35.705 (23.946) ng/mL for the test formulation and 34.470 (20.639) ng/mL for the reference formulation, with a geometric mean ratio of 1.04. The arithmetic mean AUC(0-t) for the respective formulations was 562.015 (481.875) and 508.509 (439.456) ng · h/mL, with a geometric mean ratio of 1.11. The arithmetic mean T(max) was 6.188 (1.560) and 5.885 (1.648) hours. Under fed conditions, the arithmetic mean venlafaxine C(max) was 42.892 (24.348) ng/mL for the test formulation and 46.275 (23.011) ng/mL for the reference formulation, with a geometric mean ratio of 0.93. The arithmetic mean AUC(0-t) for the respective formulations was 737.218 (603.998) and 682.124 (524.713) ng · h/mL, with a geometric mean ratio of 1.08. The arithmetic mean T(max) was 6.787 (1.769) and 5.957 (1.661) hours. There were no significant increases in venlafaxine C(max), AUC(0-t), or T(max) for either formulation in the fed phase compared with the fasting phase. In both the fasting and fed portions of the study, the 90% CIs for the ratio (test:reference) of log-transformed C(max) (fasting: 93.24-105.93; fed: 84.67-97.85) and AUC(0-t) (fasting: 102.90-116.71; fed: 98.19-114.41) were within the acceptance range for bioequivalence. The most common adverse events (≥ 5% of subjects) in the fasting phase were nausea (46%), diarrhea (29%), headache (29%), vomiting (15%), and colic (6%); the most common adverse events in the fed phase were nausea (15%), headache (13%), and dizziness (9%). CONCLUSION: In this single-dose study in healthy fasting and fed volunteers, the test formulation of venlafaxine extended-release 75-mg capsules met Brazilian regulatory criteria for bioequivalence to the reference formulation.

Determination of ampicillin in human plasma by solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) and its use in bioequivalence studies.

A simple, fast, sensitive and selective solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method for the quantitative analysis of ampicillin (CAS 69-53-4) in human plasma was developed using amoxicillin as internal standard, and sample extraction by solid-phase extraction (SPE). Extracts were separated by reversed-phase C18 with aqueous mobile phase (acetonitrile, 80:20, v/v) with 0.1% formic acid. The method was validated and successfully applied in a bioequivalence study of capsules 500 mg of ampicillin. Using a short running time of 2.5 min, the lower limit of quantification (LLOQ) for obtained ampicillin was 0.1 microg/ml for a plasma sample of 250 microl and a recovery of 94.38% +/- 4.05. Bioequivalence between the products was determined by calculating 90% confidence intervals (CI) for the ratio of Cmax, AUC0-t and AUC0-inf values for the test and reference products, which were within the 0.80-1.25 interval proposed by FDA and EMEA. It is concluded that the two formulations are bioequivalent in their rate and extent of absorption, and thus, may be used interchangeably.