Prediction of acute fish toxicity (AFT) and fish embryo toxicity (FET) tests by cytotoxicity assays using liver and embryo zebrafish cell lines (ZFL and ZEM2S).

Fish cell-based assays represent potential alternative methods to vertebrates' use in ecotoxicology. In this study, we evaluated the cytotoxicity of thirteen chemicals, chosen from OECD guidelines 236 and 249, in two zebrafish cell lines (ZEM2S and ZFL). We aimed to investigate whether the IC50 values obtained by viability assays (alamar blue, MTT, CFDA-AM, and neutral red) can predict the LC50 values of Acute Fish Toxicity (AFT) test and Fish Embryo Toxicity (FET) test. There was no significant difference between the values obtained by the different viability assays. ZFL strongly correlated with AFT and FET tests (R2AFT = 0.73-0.90; R2FET48h = 0.79-0.90; R2FET96h = 0.76-0.87), while ZEM2S correlated better with the FET test (48h) (R2 = 0.70-0.86) and weakly with AFT and FET tests (96h) (R2AFT = 0.68-0.74 and R2FET96h = 0.62-0.64). The predicted LC50 values allowed the correct categorization of the chemicals in 76.9% (AFT test) - 90.9% (FET test) using ZFL and in 30.7% (AFT test) - 63.6% (FET test) using ZEM2S considering the US EPA criterion for classifying acute aquatic toxicity. ZFL is a promising cell line to be used in alternative methods to adult fish and fish embryos in ecotoxicity assessments, and the method performed in 96-well plates is advantageous in promoting high-throughput cytotoxicity assessment.

Use of In silico Methodologies to Predict the Bioavailability of Oral Suspensions: A Regulatory Approach.

BACKGROUND: Oral suspensions are heterogeneous disperse systems, and the particle size distribution, crystalline form of the dispersed solid, and composition of the formulation can be listed as parameters that control the drug dissolution rate and its bioavailability. OBJECTIVE: The aim of this work was to develop a discriminative dissolution test, which, in association with in silico methodologies, can make it possible to safely anticipate bioavailability problems. METHODS: Nimesulide and ibuprofen (BCS class II) and cephalexin (BCS class I) oral suspensions were studied. Previously, solid-state structure and particle size in active pharmaceutical ingredients were characterized and the impact of differences on solubility was evaluated for the choice of discriminative medium. Afterwards, particle size distribution (0.1 to 360 µm), dissolution profile, and in vitro permeability in Caco-2 cell of commercial suspensions, were determined. These parameters were used as input for the establishment of the in vitro-in vivo correlation (IVIVC) for the suspensions using the GastroPlus™ with Wagner-Nelson and Loo- Riegelmann deconvolution approach. RESULTS: The predicted/observed pharmacokinetic model showed good correlation coefficients (r) of 0.960, 0.950, and 0.901, respectively. The IVIVC was established for one nimesulide and two ibuprofen suspensions with r between 0.956 and 0.932, and the percent prediction error (%PE) did not exceed 15%. CONCLUSION: In this work, we have performed a complete study combining in vitro/in silico approaches with the aim of anticipating the safety and efficacy of oral pharmaceutical suspensions in order to provide a regulatory tool for this category of products in a faster and more economical way.

In Vitro-In Vivo Correlation for Desvenlafaxine Succinate Monohydrate Extended Release Tablets.

The objective of this study was to develop a dissolution test in order to establish an in vitro-in vivo correlation (IVIVC) model for desvenlafaxine succinate monohydrate (DVSM) extended release (ER) tablets. The in vitro release characteristics of the drug were determined using USP apparatus 1 at 75 rpm, with volume of HCl pH 1.2, acetate buffer solution (ABS) pH 4.5, or phosphate buffer solution (PBS) pH 6.8. In vivo plasma concentrations and pharmacokinetic parameters in healthy volunteers were obtained from a bioequivalence study. The similarity factors f1 and f2 were used to compare the dissolution data. The IVIVC model was developed using fraction dissolved and fraction absorbed of the reference product. For predictability, the results showed that the percentage prediction error (%PE) value of Cmax was 7.63%. The observed low prediction error for Cmax demonstrated that the IVIVC model was valid for this parameter.

Enantioselective in vitro metabolism and in vitro-in vivo correlation of the herbicide ethofumesate in a human model.

Ethofumesate (ETO) is a chiral herbicide that is marketed as a racemic mixture in the European Union and the United States. The growing consumption of pesticides in the world, along with their presence in water and food, has increased human exposure to these chemicals. Another issue concerning these compounds is that each enantiomer of a chiral pesticide may interact with biomolecules differently. For this reason, this study aimed to investigate the in vitro metabolism of ethofumesate (the racemic mixture as well as the isolated enantiomers) by human liver microsomes (HLM) and to explore the in vitro-in vivo correlation. Before the kinetics was determined, the method was fully validated by evaluating its selectivity, linearity, precision, accuracy, carryover, and stability. All the evaluated parameters agreed with the European Medicines Agency guideline. The enzyme kinetic parameters and the in vitro-in vivo correlation demonstrated that there was no enantioselective difference for the metabolism and bioavailable fraction of each enantiomer. The enzyme kinetics was biphasic; the KM1 values were 15, 5.8, and 5.6 for rac-ETO, (+)-ETO, and (-)-ETO, respectively. The total in vitro intrinsic clearance was 0.10 mg mL min-1 mg-1 for rac-ETO and its enantiomers. The enantiomer (-)-ETO was only metabolized by CYP2C19, while (+)-ETO was metabolized by both CYP2C19 and CYP3A4. CYP2C19 polymorphism and/or inhibition may represent a risk for humans exposed to this pesticide.

Sex-by-formulation interaction assessed through a bioequivalence study of efavirenz tablets.

Although sex-related differences in gastrointestinal physiology have been vastly reported, its impact on drug oral bioavailability and bioequivalence (product discrimination) is often ignored. On this work results from an average bioequivalence study between tablets containing 600mg of the antiretroviral efavirenz (EFV), carried out with 14 healthy subjects (8 female and 6 men) in a randomized 2-period, 2-treatment crossover design, are analyzed from a sex-based approach. Sequences were balanced within each sex group. Considering all subjects, no differences were observed on EFV absorbed amount, as shown by the estimated 90CI of the AUC96 Test/Reference bioequivalence ratio (T/R): 0.950-1.05. However, results were not conclusive due to the 90CI for CMAX T/R was 0.743-1.07. Over this parameter, a significant sex-by-formulation interaction was detected: 90CI CMAX T/R was 0.838-1.36 in women and 0.540-0.920 in men; with a 52% relative difference between point estimates. Formulation differences were therefore evidenced only by male subjects. In vitro dissolution and disintegration tests for both products were carried out in two aqueous media: A) SLS 0.25% and B) HCl/KCl pH1.2. T/R results for dissolution efficiency and tablet disintegration times of formulations in both A and B media were highly correlated with CMAX T/R bioequivalence results observed in women and men respectively, showing that a dissimilar gastrointestinal environment between sexes affected EFV oral absorption. This work shows how sex-by-formulation interaction can affect bioequivalence conclusions. Sex effect on product discrimination should be specially disclosed in bioequivalence studies, mainly for drugs aimed to be given to both sexes.

Determinação das curvas de absorção, perfis de dissolução e estabelecimento de correlação in vitro-in vivo por métodos in silico utilizando o GastroPlusTM e o DDDPlusTM / Determination of absorption curves, dissolution profiles and establishment of in vitro-in vivo correlation by in silico methods using GastroPlusTM and DDDPlusTM

O uso de programas de computador para prever a absorção de fármacos em humanos e simular perfis de dissolução tem se tornado uma ferramenta bastante valiosa na área farmacêutica. O objetivo deste trabalho foi utilizar métodos in silico por meio dos programas de computador GastroPlusTM e DDDPlusTM para simular curvas de absorção de fármacos, perfis de dissolução e estabelecer correlações in vitro-in vivo (CIVIVs). O material aqui apresentado é constituído por cinco capítulos incluindo os fármacos cetoprofeno, pirimetamina, metronidazol, fluconazol, carvedilol e doxazosina. No capítulo 1 são apresentadas curvas plasmáticas simuladas para comprimidos matriciais de cetoprofeno, sendo estabelecida a CIVIV. A utilização de simulações de ensaios de dissolução intrínseca para os fármacos pirimetamina e metronidazol como uma ferramenta para classificação biofarmacêutica é detalhada no capítulo 2. No capítulo 3, a simulação de curvas plasmáticas a partir de cápsulas de fluconazol contendo diferentes perfis de dissolução é demonstrada como uma ferramenta para bioisenção. Estudos de CIVIV foram também realizados para comprimidos de liberação imediata de carvedilol a partir dos perfis de dissolução no capítulo 4. Já o capítulo 5 trata da aplicação de simulações de ensaios de dissolução para o desenvolvimento de formulações de liberação prolongada de doxazosina. As simulações das curvas plasmáticas, assim como a CIVIV, obtidas com o auxílio do programa GastroPlusTM, além dos ensaios de dissolução intrínsica e os perfis de dissolução obtidos por meio do uso do programa DDDPlusTM apresentaram-se como ferramentas de grande aplicação na previsão de características biofarmacêuticas sobre os fármacos e formulações, permitindo redução de tempo e custo com trabalho experimental em laboratório

The use of computer programs to predict drug absorption in humans and to simulate dissolution profiles has become a valuable tool in the pharmaceutical area. The objective of this study was to use in silico methods through software GastroPlusTM and DDDPlusTM to simulate drug absorption curves and dissolution profiles, and to establish in vitro-in vivo correlations (IVIVCs). The work presented herein is divided into five chapters and includes the drugs ketoprofen, pyrimethamine, metronidazole, fluconazole, carvedilol and doxazosin. In Chapter 1, simulated plasma curves for ketoprofen matrix tablets are presented and IVIVC was established. The use of simulated intrinsic dissolution tests for pyrimethamine and metronidazole as a tool for biopharmaceutics classification is detailed in Chapter 2. In Chapter 3, simulation of plasma curves for fluconazole capsules with different dissolution profiles is demonstrated as a tool for biowaiver. IVIVC studies were also conducted for carvedilol immediate-release tablets from dissolution profiles in Chapter 4. Chapter 5 covers the application of simulated dissolution tests for development of doxazosin extended-release formulations. Simulation of plasma curves and IVIVC using the software GastroPlusTM as well as intrinsic dissolution tests and dissolution profiles using the software DDDPlusTM proved to be a tool of wide application in predicting biopharmaceutical characteristics of drugs and formulations, allowing the reduction of time and costs of experimental laboratory work

Gemifloxacin mesylate (GFM): dissolution test based on in vivo data.

Gemifloxacin mesylate (GFM) is a synthetic, broad-spectrum, fluoroquinolone antibacterial agent. It is different from other class members because it achieves adequate plasma concentrations to inhibit both topoisomerase IV and gyrase. The aim of this study was to develop and validate a dissolution test for GFM in coated tablets, using a simulated absorption profile based on in vivo data obtained from the literature. The fraction and percentage of the dose absorbed were calculated using model-dependent Loo-Riegelman approach for two compartments. The best in vitro dissolution profile was obtained using 900 mL of pH 6.0 phosphate buffer as a dissolution medium at 37 °C ± 0.5 °C and paddles at 50 rpm. The in vitro dissolution samples were analyzed using a liquid chromatography method, and the validation was performed according to USP 34 (2011). The method showed specificity, precision, accuracy, robustness and linearity. Under these conditions, a level-A in vitro-in vivo correlation was suggested (r = 0.9926). The prediction errors were calculated to determine the validity and accuracy of the suggested correlation. The dissolution test can be used to evaluate the dissolution profile of GFM-coated tablets and minimize the number of bioavailability studies as part of new formulation development.

Correlação in vitro-in vivo para formas farmacêuticas sólidas de liberação modificada contendo diclofenaco de sódio / In vitro-in vivo correlation for sodium diclofenac modified release tablet formulations

A correlação in vitro-in vivo (CIVIV) refere-se ao estabelecimento de uma relação racional entre as propriedades biológicas, ou parâmetros derivados destas, produzidos por uma forma farmacêutica e suas propriedades ou características físico-químicas. O estabelecimento desse tipo de correlação de dados pode possibilitar a substituição dos estudos in vivo, necessários à demonstração da bioequivalência, pelos estudos in vitro, no caso de alterações no processo de fabricação pós-registro. Os sistemas matriciais apresentam, como principal exemplo de material controlador da liberação, substâncias poliméricas formadoras de matrizes hidrofílicas. Hidroxipropilmetilcelulose (HPMC) é um excipiente de escolha para o preparo de matrizes hidrofílicas, devido à capacidade de formação de gel e controle da liberação. O diclofenaco de sódio (DCL) é um antiinflamatório não esteroidal com ação analgésica e antipirética. Considerando suas características físico-químicas e farmacológicas, é objetivo deste trabalho o estabelecimento de uma CIVIV para DCL incorporado em sistemas matriciais. Os comprimidos de DCL com HPMC foram desenvolvidos e submetidos aos ensaios de dissolução utilizando os aparatos 1, 2, 3 e 4 conforme as especificações farmacopeicas. Foi realizado o estudo de biodisponibilidade, seguindo as normas éticas, com as formulações selecionadas. A partir dos dados de absorção obtidos pela técnica de deconvolução e dos dados de dissolução foi estabelecida a correlação. Os resultados demonstraram que o aumento da concentração de HPMC produziu a redução da velocidade de dissolução e, dependendo da condição de estudo, estas diferenças foram mais ou menos significativas. Os comprimidos com concentração intermediária de HPMC (15 a 20%) foram mais sensíveis às alterações de formulação e das condições do ensaio de dissolução. As formulações contendo 30% de HPMC praticamente não modificaram o perfil de dissolução, mesmo com alterações na formulação e condições de estudo. No...

The term in vitro-in vivo correlation (IVIVC) refers to the establishment of a rational relationship between the biological properties, or a parameter derived from a biological property produced by a dosage form, and a physicochemical characteristic or property of the same dosage form. The establishment of IVIVC enables the substitution of in vivo studies for in vitro studies to evaluate bioequivalence, e.g. in case of post-approval changes. Matrix tablets employ mainly hydrophilic polymers to control drug release. Hydroxypropylmethylcellulose (HPMC) is an excipient of choice for preparation of hydrophilic matrices, due to its gel formation and controlled drug release capacities. Sodium diclofenac (SD) is a non-steroidal anti-inflammatory drug with analgesic and antipyretic effects. Considering its physicochemical and pharmacological characteristics, the objective of this work is to establish an IVIVC for HPMC matrix tablets containing SD. HPMC matrix tablets with SD were formulated and submitted to dissolution testing using apparatus 1, 2, 3 and 4 in accordance with pharmacopoeial specifications. The bioavailability study was carried out under ethical guidelines, using the selected formulations. The correlation was obtained by plotting absorption data, obtained from diclofenac plasmatic curves through a deconvolution technique, against dissolution data. The results showed that the increase of HPMC concentration produces a decrease of the drug dissolution rate and these differences were more or less significant, depending on the study conditions. Tablets with intermediate HPMC concentrations (15 to 20%) were more sensitive to changes in dissolution conditions. Formulations containing 30% HPMC do not present changes in dissolution profiles, even when the formulation or the study conditions change. Formulations F1, F2A, F3 and Voltaren® 50 mg as reference product were used in the bioavailability study to establish IVIVC. The linear correlation between...