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.

Structural design, synthesis, and anti-Trypanosomatidae profile of new Pyridyl-thiazolidinones.

The present work reports the synthesis of a novel series of pyridine-thiazolidinones with anti-Trypanosoma cruzi and leishmanicidal activities (compounds 10-27), derived from 2 or 4-pyridine thiosemicarbazones (1-9). The in vitro assays were performed with Trypanosoma cruzi trypomastigotes and amastigotes, as well as with Leishmania amazonensis promastigotes and amastigotes. The cytotoxicity profile was evaluated using the cell line RAW 264.7. From the 18 pyridine-thiazolidinones, 5 were able to inhibit trypomastigotes. Overall, all compounds inhibited amastigotes, highlighting compounds 15 (0.60 µM), 18 (0.64 µM), 17 (0.81 µM), and 27 (0.89 µM). Compounds 15 and 18 were able to induce parasite cell death through necrosis induction. Analysis by scanning electron microscopy showed that T. cruzi trypomastigotes treated with compounds 15 and 18 induced morphological changes such as shortening, retraction and curvature of the parasite body and leakage of internal content. Regarding the antiparasitic evaluation against Leishmania amazonensis, only compound 27 had a higher selectivity compared to Miltefosine against the amastigote form (IC50 = 5.70 µM). Our results showed that compound 27 presented an antiparasitic activity for both Trypanosoma cruzi and Leishmania amazonensis. After in silico evaluation, it was suggested that the new pyridine-thiazolidinones had an appropriate drug-likeness profile. Our results pointed out a new chemical frame with an anti-Trypanosomatidae profile. The pyridine-thiazolidinones presented here for the first time could be used as a starting point for the development of new antiparasitic agents.

Redução da taxa de filtração glomerular em pacientes críticos em uso de vancomicina: um estudo transversal / Decrease of the glomerular filtration rate in critically ill patients using vancomycin: a cross-sectional study

Objetivo: o presente trabalho teve como objetivo avaliar os fatores clínicos e medicamentosos relacionados com a redução da Taxa de Filtração Glomerular (TFG) em pacientes críticos em uso de vancomicina. Métodos: trata-se de um estudo transversal em que pacientes em uso de vancomicina, maiores de 18 anos, hospitalizados em terapia intensiva, foram selecionados no período de agosto a dezembro de 2019. Foram excluídos os pacientes que tiveram permanência inferior a 48h na unidade, aqueles com doença renal crônica e/ou que tiveram antimicrobiano suspenso nas primeiras 48h. Os dados clínicos e laboratoriais foram coletados do prontuário nas mesmas datas das coletas de amostras sanguíneas. As amostras de sangue foram coletadas no vale a partir do terceiro dia de tratamento. Os níveis de vancomicina foram medidos usando VANC VITROS ®. Os dados foram analisados através do software R. Resultados: 54 pacientes foram incluídos, sendo 68,5% do sexo masculino, 98,1% em ventilação mecânica, com foco respiratório (51,2%) e isolado Acinetobacter baumanni (38,0%). As concentrações de vancomicina variaram entre 5,0 e 50,0µg/mL, média 21,6 (DP: 10,6) µg/mL; 50% dos pacientes apresentaram concentração acima de 20µg/mL e 66,7% piora da TFG após o tratamento. A concentração de vancomicina foi a única variável diretamente relacionada com o desenvolvimento da alteração na função renal (p=0.0037). Não foi possível estabelecer a influência da comedicação na redução da taxa de filtração glomerular (TFG). Conclusão: as doses usuais de vancomicina ajustadas por meio da função renal não atingiram os níveis séricos terapêuticos recomendados de vancomicina, sendo relacionados à nefrotoxicidade.

Objective: we aimed to evaluate clinical and drug factors related to the Glomerular Filtration Rate (GFR) reduction in critically ill patients using vancomycin. Methods: This is a cross-sectional study where critically ill patients using vancomycin, aged over 18 years, were selected from August to December 2019. Patients were excluded when hospitalized for less than 48 hours in the unit, those with chronic kidney disease, and/or who had their antimicrobial suspended in the first 48 hours. Clinical and laboratory data were collected from the medical record on the same days as the blood sample collection. All blood samples were collected at the trough during the third day of vancomycin treatment. Vancomycin levels were measured using VANC VITROS ®. Data analysis was analyzed by R software. Results: 54 patients were included, 68.5% male, 98.1% mechanical ventilation, respiratory focus (51.2%), and isolation of Acinetobacter baumanni (38.0%). Vancomycin concentrations ranged between 5.0 and 50.0µg/mL, mean of 21.6 (SD: 10.6) µg/mL; 50% of patients with concentrations above 20µg/mL and 66.7% worsened GFR after vancomycin treatment. Vancomycin concentration was the only variable directly related to the development of renal malfunction (p=0.0037). It was not possible to establish the influence of co-medication in the reduction of the glomerular filtration rate (GFR). Conclusion: the usual doses of vancomycin adjusted by renal function did not reach the recommended therapeutic serum levels of vancomycin, being related to nephrotoxicity.

Development of the stability-indicating method, structural elucidation of new photodegradation products from terconazole by LC-MS TOF, and in vitro toxicity.

Terconazole (TCZ) was the first triazole antifungal drug launched in the market and has been used in the treatment of vulvovaginal candidiasis. It is also indicated to treat dermatophytosis and fungal ocular infections. However, some of the degradation products from triazole drugs have been reported to be toxic, justifying the need of further investigations about the stability of TCZ. identification of its degradation products and evaluation of their toxicity considering the new possibilities of therapeutic indications. Therefore, in this work a systematic investigation regarding photostability of TCZ was conducted. The active pharmaceutical ingredient (API) and its methanolic solution (100 µg mL-1) were kept into a photostability chamber under a UV light (200 Wh/m2; 1.2 × 106 lux/h) during 5 days and 90 min, respectively. A high-efficiency liquid chromatography method was developed for separation and identification of TCZ and its degradation products. The solid-state API remained stable throughout the test, whereas an extensive degradation was observed when in solution. In this case, four degradation products not yet reported in the literature were identified and characterized by electrospray ionization high-resolution quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). Two degradation products presented m/z of 498 and the other two of 496 and 464, respectively. The results suggest that the degradation follows a first-order kinetic and involves the loss of chlorine atoms from the 2,4-dichlrophenyl moiety. Finally, TCZ submitted to the same stress condition as the API solution, increased significantly the opacity during the bovine corneal opacity and permeability test method (BCOP) indicating a potential to cause ocular toxicity. Further studies using the pure photolysis degradation products of TCZ characterized in this work are still necessary to confirm this find.

Population pharmacokinetic modeling of benznidazole in Brazilian patients with chronic Chagas disease.

The present study aimed to establish a population pharmacokinetic (PopPK) modeling of benznidazole (BZD) in Brazilian patients with chronic Chagas disease. This was part of a Brazilian prospective cohort study with eight patients diagnosed with Chagas disease during the beginning of BZD treatment up to the 60th day. On the 15th day of treatment, a blood sampling was collected and analyzed. A one-compartment PK model was developed using Pmetrics. Patients with an average age of 50.3 (SD: 6.2) years old, 6 female patients and 2 males, 70.2 kg (14.2), receiving a 5 mg/Kg/day dose were included. PK parameters estimated for CL, V and Ka were 6.27 L/h, 38.97 L and 1.66 h-1, respectively. This is the first study to establish a population pharmacokinetic modeling of BZD in Brazilian patients with chronic Chagas disease. Therefore, further studies are needed to obtain the complete characterization of BZD pharmacokinetics.

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.

Population pharmacokinetic modeling of benznidazole in Brazilian patients with chronic Chagas disease

ABSTRACT The present study aimed to establish a population pharmacokinetic (PopPK) modeling of benznidazole (BZD) in Brazilian patients with chronic Chagas disease. This was part of a Brazilian prospective cohort study with eight patients diagnosed with Chagas disease during the beginning of BZD treatment up to the 60th day. On the 15th day of treatment, a blood sampling was collected and analyzed. A one-compartment PK model was developed using Pmetrics. Patients with an average age of 50.3 (SD: 6.2) years old, 6 female patients and 2 males, 70.2 kg (14.2), receiving a 5 mg/Kg/day dose were included. PK parameters estimated for CL, V and Ka were 6.27 L/h, 38.97 L and 1.66 h-1, respectively. This is the first study to establish a population pharmacokinetic modeling of BZD in Brazilian patients with chronic Chagas disease. Therefore, further studies are needed to obtain the complete characterization of BZD pharmacokinetics.

Mupirocin ointments: In vitro x In vivo bioequivalence evaluation

Abstract Bioequivalence (BE) assessment of topical drug products is a long-standing challenge. Agencies such as the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have published several drafts in recent years suggesting different approaches as alternative to evaluate the BE. A proposed Topical Classification System (TCS) has even been discussed. Given the above, the objective of this research was to use in vitro and in vivo BE approaches to evaluate Brazilian marketed mupirocin (MPC) ointments, previously classified as TCS class The in vitro permeation test (IVPT) was performed by applying formulations to pig skin by Franz cells. The in vivo methodology was dermatopharmacokinetic (DPK). These approaches (in vivo tape stripping and IVPT) demonstrated capability of distinguishing among different formulations, thus making them useful methodologies for BE evaluation.

Characterization, in vitro dissolution, and pharmacokinetics of different batches of efavirenz raw materials.

OBJECTIVE: To perform the solid-state characterization and the in vitro-in vivo correlation (IVIVC) of three batches of efavirenz (EFV) active pharmaceutical ingredients. SIGNIFICANCE: EFV is an effective anti-HIV drug. Due to the poor aqueous solubility, the rate and extent of EFV absorption deeply depend on its dissolution characteristics. METHODS: Thermal analyses, x-ray diffraction, and particle size distribution were performed. The saturation solubility and dissolution profiles were assessed in 0.5% (w/v) sodium lauryl sulfate (SLS), fasted-state simulated intestinal fluid (FaSSIF), and fed-state simulated intestinal fluid (FeSSIF) using a flow-through cell. Each batch was orally administered to Wistar rats and the pharmacokinetic parameters were correlated with those obtained from in vitro dissolution. RESULTS: All batches of EFV consisted polymorph I. EFV-A presented the lowest particle size distribution [d(v,0.5) = 197.8 µm; d(v,0.9) = 444.6 µm] followed by EFV-B [d(v,0.5) = 223.9 µm; d(v,0.9) = 481.1 µm], and EFV-C [d(v,0.5) = 240.8 µm; d(v,0.9) = 497.3 µm]. The saturated solubility in FaSSIF was 36% and 40% of that in FeSSIF and SLS, respectively. EFV-A presented the fastest rate and largest extension of dissolution than EFV-B and C (79.15%, 69.93% and 54.22%, respectively, as well as the highest maximum plasma concentration. Levels B, C, and multiple-C of IVIVC models were achieved. CONCLUSION: The FaSSIF medium discriminated the dissolution profiles of EFV APIs. Small differences in particle size distribution had a significant impact on the biopharmaceutical parameters of EFV, suggesting that strict control of such parameter is an important aspect during API development and drug formulation.

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.

Ethephon and fosetyl residues in fruits from São Francisco Valley, Brazil.

This survey describes the occurrence of Ethephon (ETH) and Fosetyl in fruits intended for export produced in the São Francisco Valley, Brazil. The determination of these compounds was carried out by the QuPPe-method (Quick Polar Pesticides Method), which was optimised and successfully validated according to the SANTE/11813/2017. From January 2016 to December 2018 a total of 1048 samples were analysed for ETH residues. In 547 (53%) of the samples, residues below the EU MRL of 1 mg kg-1 were present. In 17 samples (2%) ETH residues were above 1 mg kg-1. Overall, the mean ETH level decreased after 2016. Analyses of fosetyl showed that (18%) of 109 mango samples were positive for this compound. Phosphonic acid was found in concentrations ranging from 0.12 to 3.2 mg kg-1 and 5% of the measurements were above the EU MRL of 2 mg kg-1. The results emphasise the quality control of fruits produced in this region.

Systematic evaluation of the impact of solid-state polymorphism on the bioavailability of thalidomide.

Thalidomide (TLD) is used to treat erythema nodosum leprosum (ENL), multiple myeloma, aphthous ulceration and wasting syndrome in HIV patients. The API can be found in two crystalline habits known as α-TLD and ß-TLD. The saturation solubility (Cs) and the dissolution profiles under non-sink and sink conditions of both polymorphs were assessed. In addition, mini-capsules containing α-TLD or ß-TLD without excipients were orally given (10 mg/kg) to Wistar rats. An intravenous (i.v.) dose was also administrated (5 mg/kg). The Cs values for α-TLD and ß-TLD were not significantly different (α = 56.2 ±â€¯0.5 µg·mL-1; ß = 55.2 ±â€¯0.2 µg·mL-1). However, the dissolution profile of α-TLD presented the fastest rate and the largest extension of drug dissolution than that from ß-TLD (80% in 4 h versus 55% in 4 h). The α-TLD provided a more favorable pharmacokinetic than the ß-TLD (maximum plasma concentration - Cmax: 5.4 ±â€¯0.90 µg·mL-1versus 2.6 ±â€¯0.2 µg·mL-1; area under the curve of the concentration-time profile from time zero to infinity - AUC0-∞: 44.3 ±â€¯8.8 µg·h·mL-1versus 33.9 ±â€¯4.7 µg·h·mL-1; absolute bioavailability - F: 92.2 ±â€¯18.5% versus 70.5 ±â€¯9.9%, respectively). Drug suppliers and pharmaceutical companies should strictly control the technological processes involved in the TLD API synthesis as well as in the production of the pharmaceutical dosage form in order to guarantee the inter-batch homogeneity and therefore, product compliance.

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.

Metronidazole thermogel improves retention and decreases permeation through the skin

ABSTRACT Metronidazole (MTZ) is widely used as the standard antibiotic for the treatment of rosacea and, more recently, is being used off label in Brazilian hospitals for the treatment of wounds. Following oral administration, minimal amounts of active agent reaches the skin and side effects are strongly induced. Consequently, MTZ is currently being applied topically in order to improve the therapeutic efficacy with reduced side effects, with Rozex(r) (RZ) (an MTZ gelled formulation) being the only marketed product. This study examined whether the use of MTZ 0.75% from thermogel formulations could improve drug retention and reduce dermal exposure compared to that by Rozex(r). Following a 21 h permeation study, the highest total amount of MTZ permeated through the rat healthy and disturbed skin was seen with Rozex(r), but similar to all formulations regardless of the skin condition. On the other hand, the amount retained in the epidermis/dermis was larger for thermogel formulations; at least 4 fold that of Rozex(r), when the stratum corneum was present as a barrier. In conclusion, thermogel formulations can be favorable alternatives to Rozex(r) for the topical application of MTZ with improved efficacy and reduced side effects.

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.

Determination of indinavir in human plasma and its use in pharmacokinetic study

We report the development and validation of a new sensitive, accurate and precise HPLC method with ultraviolet detection for the determination of indinavir sulfate (IND) in human plasma and its application to a bioequivalence study of a new generic formulation. The extraction of IND from plasma samples was achieved by using liquid-liquid extraction with a mean recovery of 73.9 percent. The lower limit of quantification was 0.05 µg/mL. Bioequivalence between the products was determined by calculating 90 percent confidence intervals (CI) for the ratio of Cmax, AUC0-t and AUC0-inf values for the test and reference products, within the 0.80-1.25 interval proposed by ANVISA and FDA. Therefore the medications are bioequivalent and inter-exchangeable.

Foi desenvolvido e validado um novo método, sensível, exato e preciso por Cromatografia a Líquido de Alta Eficiência com detecção em UV para a determinação do sulfato de indinavir (IND) em plasma humano. O fármaco foi extraído do plasma através de extração líquido-líquido obtendo recuperação média de 73.9 por cento, com limite inferior de quantificação de 0.05 µg/mL. O método foi aplicado em estudo de farmacocinética para verificar a bioequivalência entre um novo medicamento genérico e seu produto de referência. A bioequivalência entre os produtos foi determinada calculando-se um intervalo de confiança (IC) de 90 por cento para a razão das médias do produto teste e referência compreendidas entre o intervalo de 0.80-1.25 proposto pela ANVISA e O FDA. Os produtos estudados são bioequivalentes e, portanto, intercambiáveis.

Development and validation of a new method for the quantification of norfloxacin by HPLC-UV and its application to a comparative pharmacokinetic study in human volunteers

The development and validation of a simple and accurate method based on HPLC with ultraviolet detection for the quantification of norfloxacin (NFX) in human plasma and its application to a bioequivalence study between two norfloxacin formulations is described. NFX and the internal standard (cyprofloxacin) were extracted from plasma using liquid-liquid extraction. Chromatographic separation of norfloxacin, cyprofloxacin and plasma interferents was achieved with a C-18 column and a mobile phase consisting of 20 mM sodium hydrogen phosphate buffer pH 3.0 and acetonitrile (88:12, v/v) and quantitation was done at 280 nm. The method was linear from 25 to 3000 ng mL-1 (r² > 0.997578), and norfloxacin and cyprofloxacin had an average recovery from plasma of 93.9 percent and 91.2 percent respectively. The RSD of inter-day quality control samples at the lower limit of quantification was less than 15 percent. After a single oral dose (400 mg) of norfloxacin administered to healthy human volunteers using a randomized 2x2 crossover design, pharmacokinetic parameters (AUC0-t, AUC0-00, Cmax, t1/2) were derived from the plasma concentration curves for both formulations. Pharmacokinetic analysis of the data showed that the two formulations were bioequivalent, while no adverse reactions to the drug were observed.

O desenvolvimento e validação de um método simples e preciso por CLAE-UV para quantificação de norfloxacino (NFX) em plasma humano e a sua aplicação a um estudo de bioequivalência entre duas formulações são descritos. NFX e o padrão interno (ciprofloxacino, PI) foram extraídos do plasma através de extração líquido-líquido. A separação cromatográfica do NFX, do PI e dos interferentes do plasma foi realizada com uma coluna C-18 e fase móvel composta de tampão fosfato de sódio 20 mM pH 3,0 e acetonitrila (88/12, v/v) e quantificado em 280 nm. A resposta do detector aos analitos mostrou-se linear entre 25 a 3000 ng mL-1 (r² > 0,997578) e a recuperação média de NFX e PI foi de 93,9 por cento e 91,2 por cento respectivamente. O desvio padrão relativo de amostras analisadas ao nível do limite inferior de quantificação foi menor que 15 por cento. Foi administrada uma dose de NFX (400 mg) por via oral a voluntários humanos em um estudo aberto, aleatório e cruzado 2x2 entre duas formulações. Os parâmetros farmacocinéticos (AUC0-t, AUC0-00, Cmáx, T1/2) foram observados a partir da curva de concentração versus tempo. A análise farmacocinética mostrou que as duas formulações são bioequivalentes entre si. Nenhum efeito adverso foi observado.