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Lactoferrin is a multifunctional iron-binding glycoprotein in milk. Due to its potential for the treatment of various diseases, interest in products containing lactoferrin is increasing. However, as a protein, it is prone to degradation, which critically affects the quality of products. Therefore, the main purpose of our work was to develop a stability-indicating analytical approach for stability evaluation of lactoferrin. We were focused on two complementary methods: reversed-phase and size-exclusion chromatography. The stability-indicating nature of the selected methods was confirmed. They were successfully validated by following the ICH guidelines and applied to preliminary lactoferrin stability studies. Up to three degradation products, as well as aggregates and fragments of lactoferrin, were detected in various samples using complementary reversed-phase and size-exclusion chromatographic methods. The analytical approach was additionally extended with three spectroscopic techniques (absorbance, intrinsic fluorescence, and bicinchoninic acid method), which may provide valuable complementary information in some cases. The presented analytical approach allows the stability evaluation of lactoferrin in various samples, including the ability to detect differences in its degradation mechanisms. Furthermore, it has the potential to be used for the quality control of products containing lactoferrin.
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Vitamin D3 has numerous beneficial effects, such as musculoskeletal, immunomodulatory, and neuroprotective. However, its instability is the main obstacle to formulating quality products. Despite increased attention and growing use, data on vitamin D3 stability is scarce because data from individual studies is inconclusive and mostly qualitative. Therefore, we have systematically investigated the influence of various factors (temperature, light, oxygen, pH, concentration, and metal ions) on its stability in aqueous media using a stability-indicating HPLC-UV method. First-order kinetics fitted its degradation under all tested conditions except light and oxygen. In both cases, the established models in chemical kinetics were inappropriate and upgraded with the Weibull model. Metal ions and acidic conditions had the main destabilizing effect on vitamin D3 in aqueous media, but these solutions were successfully stabilized after the addition of ethylenediaminetetraacetic acid (EDTA), ascorbic acid, and citric acid, individually and in combination. EDTA showed the most significant stabilizing effect. Synergism among antioxidants was not observed. Our findings on vitamin D3 instability in aqueous media also correlated with its instability in commercial products. Vitamin D3 aqueous products require proper stabilization, thereby signifying the importance and contribution of the obtained results to the formulation of stable and quality products.
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The efficiency of coenzyme Q10 (CoQ10) supplements is closely associated with its content and stability in finished products. This study aimed to provide evidence-based information on the quality and stability of CoQ10 in dietary supplements and medicines. Therefore, ubiquinol, ubiquinone, and total CoQ10 contents were determined by a validated HPLC-UV method in 11 commercial products with defined or undefined CoQ10 form. Both forms were detected in almost all tested products, resulting in a total of CoQ10 content between 82% and 166% of the declared. Ubiquinol, ubiquinone, and total CoQ10 stability in these products were evaluated within three months of accelerated stability testing. Ubiquinol, which is recognized as the less stable form, was properly stabilized. Contrarily, ubiquinone degradation and/or reduction were observed during storage in almost all tested products. These reactions were also detected at ambient temperature within the products' shelf-lives and confirmed in ubiquinone standard solutions. Ubiquinol, generated by ubiquinone reduction with vitamin C during soft-shell capsules' storage, may lead to higher bioavailability and health outcomes. However, such conversion and inappropriate content in products, which specify ubiquinone, are unacceptable in terms of regulation. Therefore, proper CoQ10 stabilization through final formulations regardless of the used CoQ10 form is needed.
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BACKGROUND: Retinoids are widely used in different cosmetic products because of general improvement of skin appearance. However, retinoid concentration in cosmetics is restricted, and one particular form-retinoic acid, is banned in cosmetics due to safety reasons. AIMS: Within this study, we aimed to examine the quality of a considerable number of commercial retinoid cosmetic products in terms of their content and labeling, including also screening for the presence of retinoic acid. METHODS: An appropriate analytical methodology, based on HPLC-UV for the simultaneous determination of common retinoids, along with a screening method for retinoic acid, was developed and validated. Structural identity confirmation of the newer retinoid-hydroxypinacolone retinoate, was performed by LC-MS. RESULTS: Retinol and retinyl palmitate were most often found, in concentrations mostly below 0.3%, and up to 1.3% retinol equivalents. Determined contents deviated significantly from the quantitatively declared ones in seven products (0%-130%). In more than half of the tested products, inconsistencies between the contained and labeled retinoid were noticed. These products, as well as 14 additional anti-age cosmetics, were screened for retinoic acid, which was detected in two products. CONCLUSIONS: The obtained results from retinoids assay in commercial cosmetic products confirmed that the proposed method is appropriate for their routine analysis. The presence of retinoic acid in two products and determined retinoid contents above the Scientific Committee on Consumer Safety recommendations in 20% of the tested cosmetics reveal the need for their more strict regulation and quality control to ensure their efficacy and safety.
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Cosméticos , Retinoides , Humanos , Control de Calidad , Tretinoina , Vitamina ARESUMEN
BACKGROUND: Retinoids as dermatological agents are effective against acne, psoriasis, skin aging, and other skin conditions. However, their susceptibility to degradation is a limiting factor for their widespread use. OBJECTIVES: Within this study, we aimed to provide comprehensive and evidence-based information on retinoid stability and degradation kinetics in commercial cosmetics, focusing on different factors affecting their stability. METHODS: A validated HPLC-UV methodology was utilized for determination of the most common retinoids in cosmetics (retinol, retinyl palmitate, ß-carotene) and a newer promising retinoid (hydroxypinacolone retinoate). The stability of 16 retinoid derivatives in 12 commercial cosmetics was evaluated within 6 months of long-term and accelerated stability testing in addition to a one-week photostability study. Retinoid degradation in the tested formulations followed first-order kinetics, which was further applied to shelf-life prediction. RESULTS: Long-term and accelerated stability testing revealed retinoid instabilities in almost all products, resulting in a 0%-80% decline after 6 months at 25°C and a 40%-100% decline at 40°C, which were kinetically evaluated. Light degradation was more pronounced than temperature-induced degradation. Among the studied retinoids, the stability of the newer hydroxypinacolone retinoate was the most prominent. This study also identifies correlations between retinoid concentrations, price, formulation, and their stability in cosmetics. CONCLUSIONS: Retinoid instabilities were formulation-dependent and associated with lower contents than declared in some cosmetics. Retinoid chemical stability and physical stability in topical formulations need to be evaluated by real-time stability studies, instead of the more frequently used accelerated stability studies.
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Cosméticos , Envejecimiento de la Piel , Humanos , Cinética , Retinoides , Vitamina ARESUMEN
Bioequivalence studies are an integral part of clinical pharmacology strategy for drug development. Physiologically based biopharmaceutics modeling (PBBM) can be a helpful tool to assess potential bioequivalence risks and predict the outcome of bioequivalence studies. In this study, GastroPlus™ was used for virtual bioequivalence (VBE) assessment of 6 case studies which includes four BCS 2, and one each of BCS 1 and 3 molecules. The purpose was to investigate if bioequivalence in fed state can be accurately predicted based on model developed on data from bioequivalence study in fasted state and known food effect from clinical studies. Our results show that we were able to successfully predict passing (5 cases) and failed (1 case) bioequivalence studies. Ultimately, if there is confidence in such models, a case can be made to waive fed bioequivalence study, on a case-by-case basis (e.g. for BCS class 1 and 2 molecules with known food effect mechanism, reliable estimate of human pharmacokinetic parameters, and available in vivo data in fasted state for model verification). This has the potential to reduce clinical burden in drug development, increase confidence in pivotal BE studies and support regulatory applications such as justify waiving of BE study for Scale-Up and Post Approval Changes (SUPAC). Hence VBE can significantly reduce time and cost of drug development, as well as minimize drug exposure to healthy volunteers.
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Biofarmacia , Preparaciones Farmacéuticas , Humanos , Modelos Biológicos , Solubilidad , Equivalencia TerapéuticaRESUMEN
Coenzyme Q10 (CoQ10) supplements are widely used because of their antioxidant and anti-inflammatory effects, especially in the management of cardiovascular diseases. The latest pharmaceutical approach to increase CoQ10 bioavailability and efficiency is the formulation of its reduced form. Regardless of the growing number and usage of CoQ10 preparations, their analytics and quality control is inadequate, neglecting interconversion between the two CoQ10 forms. Therefore, this study proposes a HPLC-UV method for the simultaneous quantification of both reduced and oxidized coenzyme Q10, as well as total CoQ10, as a sum of its individual forms. The suitability of the developed method was confirmed by two additional approaches for total CoQ10 determination - its total reduction and oxidation, differing from the proposed procedure only in the final stage of sample preparation. The results for total CoQ10 content were consistent between the three procedures and also with the official USP method for total CoQ10 determination. The proposed method was applied to 13 dietary supplements and medicines in the form of soft- and hard-shell capsules, revealing the co-existence of both CoQ10 forms in 85% of the tested preparations. CoQ10 forms that were undeclared accounted for up to 75% of the CoQ10 content, which is overlooked by current official methods that evaluate only the total CoQ10 content. This validated HPLC-UV method for the unequivocal quantification of reduced and oxidized CoQ10 is therefore appropriate for the routine analysis of CoQ10 preparations in quality control laboratories, as well as for stability studies, and is suggested to be adopted as an official method.
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Antioxidantes , Suplementos Dietéticos , Cápsulas , Cromatografía Líquida de Alta Presión , Suplementos Dietéticos/análisis , Ubiquinona/análogos & derivadosRESUMEN
The significance of thermodynamic solubility in biopharmaceutical compound or drug characterization as well as the importance of having methods that accurately establish it have been extensively addressed. Nonetheless, its precise determination continues to remain a challenging task to accomplish. Even more so when the number of compounds to evaluate is high and the available amount of each compound is low, both of which are inevitable for the compound characterization during the drug development process. Except for the shake-flask method which is still considered as the 'gold standard' in obtaining thermodynamic data, it is currently difficult to say that another satisfactory model which is routinely used to determine thermodynamic solubility is being applied. Therefore, this review summarizes the various experimental approaches which are based on the classical shake flask method but have yet attempted to speed up the experimental process of obtaining such data more conveniently. The most important experimental features of these approaches are provided to the reader. Some advantages and disadvantages associated with each approach are also highlighted, consequently offering a resource to those looking for the most appropriate of the approaches that have already fared well at determining the biopharmaceutically relevant drug solubility.
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Productos Biológicos/química , Química Farmacéutica/métodos , Solubilidad , TermodinámicaRESUMEN
A simple, fast, and cost-effective LC-MS/MS method for quantification of rifampicin in human plasma was developed and fully validated. The plasma samples containing rifampicin and isotopically labelled internal standard rifampicin D8, were cleaned up using a Captiva ND Lipids filtration plate. Chromatographic separation was achieved on an 1290 Infinity liquid chromatograph coupled to 6460 Triple Quadrupole operated in positive mode on a core-shell Kinetex C18 column (50 × 2.1 mm, 2.6 µm) by gradient elution using 0.1% formic acid in water and acetonitrile as a mobile phase. The proposed method is the fastest method published by now, both in terms of sample preparation (approximately one minute per sample) and chromatographic analysis (total run time 2.4 min). Another key benefit is the outstanding sensitivity and wide analytical range (5-40000 µg/L) with good linearity, accuracy, and precision. The method showed almost complete recovery (92%) and absence of any significant matrix effect as demonstrated by uniform responses from QC samples prepared in blood plasma from 6 volunteers (RSD <5%). The proposed method was successfully applied to rifampicin quantification in 340 patients' plasma samples, thus demonstrating its suitability for both therapeutic drug monitoring and pharmacokinetic analysis.
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Pharmaceuticals in wastewater have clearly raised concern and a broad range of analytical methods has been used to assess the risk as accurately as possible. The aim of our study was to measure and compare the concentrations of atorvastatin, bisoprolol, carbamazepine, ciprofloxacin, clofibric acid, diclofenac, fluoxetine, metoprolol, and sertraline in wastewater samples taken from one municipal and one hospital wastewater treatment plant in Slovenia and to predict the potential environmental burden using the risk quotient. In both effluents only clofibric acid and fluoxetine were not detected. The measured concentrations of the remaining seven pharmaceuticals varied between the ng L-1 and the µg L-1 range. Hospital effluent showed higher concentrations, except for diclofenac and carbamazepine. However, high risk quotient was found only for ciprofloxacin and diclofenac in both municipal and hospital effluent. In conclusion, our method can provide a useful tool for systematic monitoring of pharmaceuticals commonly found in wastewater, which will enable a reliable assessment of the risks for the aquatic biota and humans. Knowing the risks will help to plan wastewater treatment and preserve our environment.
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Diclofenaco/análisis , Residuos Sanitarios/análisis , Preparaciones Farmacéuticas/análisis , Aguas Residuales/análisis , Contaminantes Químicos del Agua/análisis , Ciudades , Monitoreo del Ambiente/métodos , Humanos , Medición de RiesgoRESUMEN
Bazedoxifene, a novel selective estrogen receptor modulator, has complex pharmacokinetics with rapid absorption, high metabolic clearance, low oral bioavailability (6.25 %) and a slow elimination phase. Our hypothesis is that drug uptake and efflux transporters may play an important role in its disposition. To adequately cover all aspects of bazedoxifene transport, several approaches were undertaken: PAMPA assay, ATPase assay, membrane inside-out vesicles and Caco-2 and CHO cell lines. The results obtained from PAMPA experiments showed moderate passive permeability of bazedoxifene (P app ≈ 2 × 10(-6)cm/s), suggesting the existence of an active transport during the rapid absorption phase. The Caco-2 transport assay showed large and significant changes in the measured efflux ratios of bazedoxifene when selective transporter inhibitors were applied: verapamil (a Pgp inhibitor), MK571 (an MRP inhibitor), Ko143 (a BCRP inhibitor) and DIDS (an OATP inhibitor). Additionally, membrane preparation experiments demonstrated the interaction of bazedoxifene with P-gp, MRP2 and BCRP. CHO experiments did not show any interactions of bazedoxifene with OATP1B1 or OATP1B3; therefore, bazedoxifene may be a substrate of other OATP isoform(s). The comprehensive in vitro study indicates a strong involvement of Pgp, MRP, BCRP and OATP in bazedoxifene disposition.
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Indoles/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Transporte Biológico Activo/fisiología , Células CHO , Células CACO-2 , Línea Celular , Línea Celular Tumoral , Cricetulus , Dicetopiperazinas/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Humanos , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Permeabilidad , Propionatos/metabolismo , Quinolinas/metabolismo , Verapamilo/metabolismoRESUMEN
A mixed lipid-mixed surfactant self-microemulsifying drug delivery system (SMEDDS) was developed to exploit the health benefits of resveratrol, a Biopharmaceutical Classification System Class 2 natural polyphenol, subject to extensive intestinal presystemic metabolism. SMEDDS with a mixed lipid phase (castor oil/Capmul MCM 1:1) and a mixed surfactant phase (Kolliphor EL/Kolliphor RH 40 1:1) was developed and evaluated for its self-emulsifying properties and in vitro dispersion. The impact of SMEDDS on the permeability properties of resveratrol and its metabolite fluxes through the rat intestine and Caco-2 cells was monitored. The inhibitory effect of selected SMEDDS components on the efflux transporters multidrug resistance-associated protein and P-gp as well as cytotoxicity was assessed on Caco-2 cells. The formulation allowed for high resveratrol loading (122.5 mg/g SMEDDS), excellent self-emulsifying properties, and very rapid release. When formulated in SMEDDS, resveratrol metabolite efflux significantly declined. The formulation (SMEDDS without incorporated resveratrol) and its individual components did not compromise in vitro cell vitality and integrity. Mixed lipid-mixed surfactant SMEDDS is a prospective formulation to improve resveratrol biopharmaceutical, pharmacokinetic, and toxicological properties, leading the way to resveratrol use not only as a supplement but also as a pharmacological drug.
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Caprilatos/química , Aceite de Ricino/química , Portadores de Fármacos , Glicéridos/química , Yeyuno/metabolismo , Estilbenos/metabolismo , Tensoactivos/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Células CACO-2 , Química Farmacéutica , Emulsiones , Humanos , Absorción Intestinal , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Permeabilidad , Ratas , Ratas Sprague-Dawley , Resveratrol , Solubilidad , Estilbenos/administración & dosificación , Estilbenos/química , Estilbenos/toxicidad , Tecnología Farmacéutica/métodos , ViscosidadRESUMEN
Targeting long-term diabetic complications, as well as inflammatory pathologies, aldose reductase inhibitors (ARIs) have been gaining attention over the years. In the present work, in order to address the poor membrane permeation of previously reported ARIs, derivatives of N-phenylpyrrole, bearing groups with putative pKa≥7.4, were synthesized and evaluated for aldose reductase inhibitory activity. The 2-fluorophenol group proved the most promising moiety, and further modifications were explored. The most active compound (31), identified as a submicromolar inhibitor (IC50=0.443µM), was also selective against the homologous enzyme aldehyde reductase. Cross-docking revealed that 31 displays a peculiar interaction network that may be responsible for high affinity. Physicochemical profiling of 31 showed a pKa of 7.64, rendering it less than 50% ionized in the physiological pH range, with potentially favorable membrane permeation. The latter was supported from the successful inhibition of sorbitol formation in rat lenses and the ability to permeate rat jejunum.
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Aldehído Reductasa/antagonistas & inhibidores , Permeabilidad de la Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Yeyuno/efectos de los fármacos , Fenoles/farmacología , Pirroles/farmacología , Aldehído Reductasa/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Yeyuno/citología , Yeyuno/metabolismo , Lentes , Masculino , Modelos Moleculares , Estructura Molecular , Fenoles/síntesis química , Fenoles/química , Pirroles/síntesis química , Pirroles/química , Ratas , Ratas Wistar , Sorbitol/antagonistas & inhibidores , Sorbitol/metabolismo , Relación Estructura-ActividadRESUMEN
Local delivery to the affected area represents the optimal means by which advantageous pharmacological properties of curcumin may be fully exploited as currently, due to the biopharmaceutical limitations associated with this polyphenol, its full beneficial effects remain limited. Curcumin-containing liposomes coated with bioadhesive polymers of natural and synthetic origin (chitosan and Carbopol) were evaluated in vitro. For these purposes, an in vitro model of vaginal mucus was developed allowing the monitoring of curcumin permeability in the conditions mimicking vaginal environment. The model was optimized by varying the amounts of glycoproteins, as compared to the permeabilities determined through isolated bovine mucus. The strength of bioadhesion was evaluated using the isolated bovine mucosa. Both curcumin solution and non-coated curcumin liposomes served as controls. Bioadhesive polymers enabled significantly higher (p<0.05) curcumin permeability through the artificial and isolated bovine mucus compared to the controls. Polymer coating of liposomes resulted in an increase in their bioadhesiveness. Mucoadhesive liposomes can be considered as potential novel drug delivery systems intended for vaginal administration of curcumin.
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Resinas Acrílicas/química , Quitosano/química , Curcumina/administración & dosificación , Portadores de Fármacos/química , Membrana Mucosa/efectos de los fármacos , Vagina/efectos de los fármacos , Resinas Acrílicas/farmacocinética , Adhesividad , Administración Intravaginal , Animales , Bovinos , Quitosano/farmacocinética , Curcumina/química , Curcumina/farmacocinética , Portadores de Fármacos/farmacocinética , Femenino , Técnicas In Vitro , Liposomas , Mucinas/química , Membrana Mucosa/química , Membrana Mucosa/metabolismo , Permeabilidad , Porcinos , Vagina/metabolismoRESUMEN
Imatinib is a potent selective inhibitor of tyrosine kinases and is used primarily in the treatment of chronic myeloid leukemia and the gastrointestinal stromal tumour. Although, it is well established that imatinib is a substrate of several transport proteins which are also active in the intestinal mucosa, the mechanisms of imatinib intestinal absorption and elimination were not systematically investigated yet. To do that, we used a Sweetana-Grass type of diffusion chambers with segments of rat intestine as a model of the intestinal mucosa, measured the permeability coefficients of imatinib and its major metabolite (N-desmethyl imatinib) in both directions with and without specific and general inhibition of active transport, and calculated the efflux ratios. The results show that the good bioavailability of imatinib is highly likely achieved by its active absorption from the intestine and that its active elimination through the intestinal mucosa is mediated by a synergistic activity of organic cation transporter 1 in the basolateral membrane and the added activity of two efflux proteins (P-glycoprotein and breast cancer resistant protein) in the apical membrane of enterocytes of the rat ileum. Interestingly, it was found that N-desmethyl imatinib is only transported by P-glycoprotein.
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Antineoplásicos/farmacocinética , Benzamidas/farmacocinética , Yeyuno/metabolismo , Piperazinas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacocinética , Pirimidinas/farmacocinética , Animales , Transporte Biológico , Mesilato de Imatinib , Técnicas In Vitro , Absorción Intestinal , Permeabilidad , Ratas , Ratas Sprague-DawleyRESUMEN
To study the release of liposome-associated drugs into hydrogels, we designed and synthesized two pH-sensitive rhodamine derivatives to use as model compounds of different lipophilicities. The dyes were fluorescent when in the free form released from liposomes into the chitosan hydrogel, but not when incorporated within liposomes. The effect of liposomal composition, surface charge and vesicle size on the release of those incorporated dyes was evaluated. The lipophilicity of the rhodamine derivatives affected both the amount and rate of release. While liposome size had only a minor effect on the release of dyes into the hydrogel, the surface charge affected the release to a greater extent. By optimizing the characteristics of liposomes we could develop a liposomes-in-hydrogel system for application in wound therapy. We further characterized liposomes-in-hydrogel for their rheological properties, textures and moisture handling, as well as their potential to achieve a controlled release of the dye. The polymer-dependent changes in the hydrogel properties were observed upon addition of liposomes. The charged liposomes exhibited stronger effects on the textures of the chitosan hydrogels than the neutral ones. In respect to the ability of the system to handle wound exudates, chitosan-based hydrogels were found to be superior to Carbopol-based hydrogels.
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Vendas Hidrocoloidales , Colorantes Fluorescentes/química , Rodaminas/química , Resinas Acrílicas , Quitosano/química , Hidrogeles , Lípidos/química , Liposomas , Polivinilos/químicaRESUMEN
The success of imatinib therapy in chronic myeloid leukemia is highly influenced by its active transport into target cells. However, the methodology for analytical evaluation of intracellular drug concentration is rare and usually reliant upon the use of radioactively labeled drugs. More specifically, there is no published method available in the literature for the determination of imatinib concentration in granulocytes. To gain further insight into the intracellular drug uptake a very reliable two-stage sample concentration procedure was devised and coupled with a sensitive ultra-high performance liquid chromatography tandem mass spectrometry. The reliability of this sample preparation and sensitivity of the analysis was confirmed by a successful validation of all necessary method parameters to an impressive lower limit of quantification of 0.5 ng imatinib per 10(6) cells still at the signal to noise ratio of 670. The usefulness of the method is further improved with only 6 mL of blood being necessary for patient analysis. The method has been applied to blood samples of 13 CML patients treated with imatinib and all the measured intracellular drug concentrations were within the validated range. These and further measurements will enable the research of factors which may, besides blood plasma concentration, influence the individual's response to imatinib therapy. Furthermore, individualisation of dosing based on the directly measured targeted drug delivery could be possible.
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Antineoplásicos/farmacocinética , Benzamidas/farmacocinética , Monitoreo de Drogas/métodos , Granulocitos/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Piperazinas/farmacocinética , Pirimidinas/farmacocinética , Antineoplásicos/administración & dosificación , Antineoplásicos/sangre , Antineoplásicos/uso terapéutico , Benzamidas/administración & dosificación , Benzamidas/sangre , Benzamidas/uso terapéutico , Calibración , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Monitoreo de Drogas/instrumentación , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Límite de Detección , Piperazinas/administración & dosificación , Piperazinas/sangre , Piperazinas/uso terapéutico , Pirimidinas/administración & dosificación , Pirimidinas/sangre , Pirimidinas/uso terapéutico , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masas en Tándem/instrumentación , Espectrometría de Masas en Tándem/métodosRESUMEN
Garlic phytochemicals and garlic supplements influence the pharmacokinetic and pharmacodynamic behavior of concomitantly ingested drugs. In this paper we have summarized the mechanisms responsible for first-pass intestinal pharmacokinetic interactions by investigating the intestinal permeability of some cardiovascular, antiviral drugs, their transport with hepatic transporters and CYP3A4 metabolism. Transporter-enzyme interplay was studied with several in vitro models of varying complexity: rat small intestine and Caco-2 cell monolayers were used in studies of intestinal processes, and hepatic pharmacokinetics was monitored in HepG2 cells, isolated rat hepatocytes and rat liver slices. Garlic phytochemicals from aged garlic extract modified the activities of secretory and absorptive transporters in both intestine and liver and competitively inhibited CYP3A4 enzyme. The increased activities of the most important intestinal efflux (P-glycoprotein - Pgp, Multidrug Resistance Associated Protein 2 - MRP-2, Breast Cancer Resistance Protein - BCRP) and uptake (MonoCarboxylate Transporter 1 - MCT1, Organic Anion Transporting Polypeptide - OATP, Peptide transporter 1 - PepT1) transporters were caused by changes in electrophysiological membrane properties and by allosteric modifications. Because clinical studies investigating interactions between garlic and human immunodeficiency virus protease inhibitors saquinavir and ritonavir have already been performed, we used these in vivo data to evaluate the in vitro results and the reliability of the models employed as screening tools for forecasting the potential of first-pass intestinal metabolism changes. We also assessed the probability of pharmacokinetic interactions with garlic of the novel drug darunavir and other cardiovascular drugs. Finally, selected garlic phytochemicals were tested for their ability to influence P-glycoprotein and CYP3A4 activities.
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Ajo/química , Interacciones de Hierba-Droga , Extractos Vegetales/farmacología , Animales , Antivirales/farmacocinética , Transporte Biológico , Células CACO-2 , Fármacos Cardiovasculares/farmacocinética , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A , Darunavir , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Masculino , Proteínas de Transporte de Membrana/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Sulfonamidas/farmacocinéticaRESUMEN
BCS based biowaivers are recognized by major regulatory agencies. An application for a biowaiver can be supported by or even based on "in vitro" measurements of drug permeability. However, guidelines limit the application of biowaivers to drug substances that are transported only by passive mechanisms. Regarding published permeability data as well as measurements obtained in our institution, one can rarely observe drug substances that conform to this very strict criterion. Therefore, we measured the apparent permeability coefficients of 13 drugs recommended by FDA's Guidance to be used as standards for "in vitro" permeability classification. The asymmetry of permeability data determined for both directions (mucosal-to-serosal and serosalto- mucosal) through the rat small intestine revealed significant active transport for four out of the nine high-permeability standards and for all four low-permeability standard drugs. As could be expected, this asymmetry was abolished at 4°C on rat intestine. The permeability of all nine high-permeability, but none of the low permeability standards, was also much lower when measured with intestinal tissue, Caco-2 cell monolayers or artificial membranes at 4°C compared to standard conditions (37°C). Additionally, concurrent testing of several standard drugs revealed that membrane transport can be affected by the use of internal permeability standards. The implications of the results are discussed regarding the regulatory aspects of biopharmaceutical classification, good practice in drug permeability evaluation and regarding the general relevance of transport proteins with broad specificity in drug absorption.
Asunto(s)
Guías como Asunto , Absorción Intestinal , Preparaciones Farmacéuticas/clasificación , Animales , Transporte Biológico , Biofarmacia/normas , Células CACO-2 , Humanos , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Masculino , Permeabilidad , Preparaciones Farmacéuticas/metabolismo , Ratas , Ratas Sprague-Dawley , Temperatura , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Imatinib, dasatinib and nilotinib are three tyrosine kinase inhibitors currently used to treat Bcr-Abl1 positive chronic myelogenous leukaemia (CML). However, achieving maximum benefit with these drugs may require optimal dosing and adherence to therapy. In those cases, therapeutic drug monitoring (TDM) can be a useful tool in managing patients with CML. The paper presents simple and high throughput method for simultaneous determination of all three TKIs in dried blood spot (DBS) samples from CML patients. DBS samples were prepared by applying 10 µL of spiked whole blood onto an Agilent DBS cards. Whole blood spot was punched out of the card, transferred to a well in a 96-well Captiva ND Lipids filter plate. After the addition of isotopically labelled internal standard, the drug was extracted with 0.1% formic acid in methanol. The collected extract (1 µL) was injected onto a Phenomenex Kinetex 50 mm × 2.1 mm C18 column and eluted with acetonitrile gradient into a triple quadrupole ESI-MS/MS Agilent 6460 operated in positive mode. The total run time was only 2.6 min. The method was validated in terms of linearity, selectivity, specificity, accuracy, precision, absolute and relative matrix effect and stability. The effect of haematocrit (Hct) on the accurate concentration determination was also examined. The method was linear in the range of 50-5000 µg/L for imatinib and nilotinib and in the range of 2.5-250 µg/L for dasatinib, with correlation coefficient values higher than 0.997. Lower limits of quantification (LLOQ) were 50 µg/L for imatinib and nilotinib and 2.5 µg/L for dasatinib. The method proved to be accurate (% bias < 13.2) and precise (CV < 10.3%) on intra- as well as on inter-day basis. Sample matrix (% ME=94.5-106.7) and different Hct values had no significant effect on the accuracy of measured concentrations. Samples proved to be stable whilst stored on DBS cards at room temperature or in the refrigerator; however, at 40 °C the stability of dasatinib was compromised. The method presented was successfully applied to clinical samples.