Solubility evaluation of didanosine: a comparison between the equilibrium method and intrinsic dissolution for biopharmaceutics classification purposes

ABSTRACT BCS (Biopharmaceutics Classification System) and BDDCS (Biopharmaceutics Drug Disposition Classification System) were proposed as tools for classifying drugs into four categories. Both systems consider the solubility as an important characteristic for the classification of compounds in drug development and in vivo disposition prediction. Although some results of drug solubility can be found in the literature, the aforementioned characteristic is not entirely clear when considering didanosine (ddI). Based on that, the solubility of ddI was evaluated using equilibrium and intrinsic dissolution methods. For the equilibrium method, excess amount of ddI was added to each media until obtaining a supersaturated solution and the mixture was submitted to agitation at 37 °C. For the intrinsic dissolution method, the drug was compressed into the Wood's apparatus matrix and subjected to dissolution in each media with agitation at 37 °C. The results obtained from the equilibrium method indicated that it was necessary 139.37 mL of pH 1.2 media, 87.72 mL of pH 4.5 media, 12.54 mL of pH 6.8 media, 5.03 mL of pH 7.5 media and 7.65 mL of purified water for drug solubilization. Furthermore, a very fast intrinsic dissolution rate (IDR) was obtained for each media: 0.1 mg/min/cm² (pH 1.2), 0.2 mg/min/cm² (pH 4.5), 0.2 mg/min/cm² (pH 6.8), 0.1 mg/min/cm² (pH 7.5) and 0.1 mg/min/cm² (purified water). Based on these results, ddI can be considered as a highly soluble drug for both equilibrium and intrinsic dissolution methods.

Avaliação dos mecanismos envolvidos na permeabilidade de fármacos antirretrovirais por meio do modelo de perfusão in situ em ratos / Evaluation of mechanisms involved in the permeability of antiretroviral drugs using the intestinal in situ perfusion model in rats

Para medicamentos administrados oralmente, as etapas de liberação do fármaco a partir da forma farmacêutica e sua subsequente absorção constituem importantes processos para que a adequada biodisponibilidade oral ocorra. Deste modo, as características de solubilidade e de permeabilidade são de extrema importância para que mecanismos relacionados à absorção sejam compreendidos no âmbito das propriedades ADME (absorção, distribuição, metabolismo e excreção). Com base nisso, o Sistema de Classificação Biofarmacêutica (SCB) foi proposto como uma ferramenta que permite a classificação de fármacos em quatro classes distintas de acordo com a solubilidade e permeabilidade. De maneira complementar, o Sistema de Classificação Biofarmacêutica de Distribuição de Fármacos (SCBDF) foi proposto levando em consideração a solubilidade e o metabolismo das substâncias, além de considerar o impacto de transportadores presentes nos tecidos biológicas, inclusive no trato gastrintestinal (TGI). Assim, o presente trabalho teve como objetivo avaliar os mecanismos envolvidos na permeabilidade de fármacos antirretrovirais (estavudina, lamivudina e zidovudina) por meio do modelo de perfusão in situ com coleta de sangue mesentérico em ratos, considerando os aspectos relacionados ao efluxo e ao metabolismo pré-sistêmico que ocorrem nos enterócitos. Além disso, estudos in vitro em culturas celulares MDCK e MDCK-MDR1 foram realizados a fim de auxiliar na elucidação dos mecanismos de transporte dos referidos fármacos. Para a realização dos estudos de perfusão in situ, ratos Wistar foram anestesiados e a porção do jejuno foi canulada para permitir a entrada do fármaco solubilizado no interior do intestino, bem como a coleta das amostras de perfusato em intervalos regulares de tempo. A veia mesentérica também foi canulada para viabilizar a obtenção das amostras de sangue durante os experimentos. Para os estudos de efluxo, o verapamil foi adicionado à solução de perfusão como inibidor de Pgp (glicoproteína-P), enquanto que o cetoconazol foi empregado como inibidor de enzimas CYP3A. Em modelo in vitro MDCK e MDCK-MDR1, os experimentos foram conduzidos bidirecionalmente com o uso de GG918 como inibidor de P-gp. Em todos os experimentos realizados, o metoprolol e a ranitidina foram empregados como marcadores de alta e de baixa permeabilidade, respectivamente. Os resultados de permeabilidade mostraram que a estavudina e a zidovudina apresentam características de alta permeabilidade, enquanto a lamivudina apresentou o menor resultado dentre os três fármacos. Os ensaios bidirecionais em MDCK, no entanto, mostraram que os três antirretrovirais apresentam baixos valores de permeabilidade, uma vez que seus resultados foram significativamente menores que o valor encontrado para o metoprolol. Com relação à avaliação do mecanismo de efluxo, tanto a lamivudina quanto a zidovudina apresentaram interações significativas com a P-gp nos dois métodos empregados (perfusão in situ e MDCK-MDR1), uma vez que o aumento nos valores de permeabilidade foi constatado quando o inibidor de P-gp foi empregado. Os estudos de metabolismo intestinal realizados por meio do modelo de perfusão in situ mostraram que nenhum dos fármacos antirretrovirais apresentou interação significativa com as enzimas CYP3A quando o cetoconazol foi empregado como inibidor, uma vez que não foram constatadas mudanças significativas nos valores de permeabilidade. A comparação entre os resultados de permeabilidade efetiva (Pef) e de permeabilidade aparente (Pap), obtidos por meio da quantificação das amostras de perfusato e de plasma, respectivamente, permitiu verificar que as diferenças estatísticas entre estes dois parâmetros podem indicar variados mecanismos de transporte, uma vez que a Pap constata a quantidade da substância que realmente foi capaz de superar as barreiras físicas e bioquímicas presentes na parede do TGI. Logo, a Pap é considerada um parâmetro mais próximo das condições in vivo. Esta diferença foi constatada apenas para a zidovudina nos ensaios de transporte de efluxo, uma vez que o valor médio de Pef não representou a mesma conclusão fornecida pelo valor médio de Pap. Os resultados obtidos permitiram concluir que os fármacos antirretrovirais apresentam permeabilidade de moderada a alta em função do possível envolvimento de carreadores de influxo. Além disso, os três fármacos antirretrovirais interagiram de alguma forma com a P-gp, sendo os resultados referentes à lamivudina e à zidovudina mais significativos. Embora tenha sido constatada o envolvimento da P-gp na permeabilidade da zidovudina, sua elevada fração absorvida indica que a absorção desta substância não é limitada por este mecanismo e que a ação do carreador de efluxo não é clinicamente relevante neste caso. Com relação aos estudos de metabolismo, a presença de enzimas CYP3A nos enterócitos também não representou uma condição desfavorável para a absorção dos antirretrovirais. Assim, a avaliação dos mecanismos no presente trabalho contribuiu para a caracterização biofarmacêutica da estavudina, lamivudina e zidovudina e as metodologias descritas podem ser empregadas nas etapas iniciais de desenvolvimento farmacêutico com o objetivo de assegurar a segurança e a eficácia de medicamentos.

For orally administered pharmaceutical products, the drug release from the dosage form and its absorption are considered important processes for adequate oral bioavailability. Thus, the solubility and permeability characteristics are extremely important for understanding of mechanisms related to the absorption in the scope of the ADME properties (absorption, distribution, metabolism and excretion). Based on that, the Biopharmaceutics Classification System (BCS) was proposed as a tool for classifying drugs into four classes considering their solubility and permeability characteristics. In an additional way, the Biopharmaceutics Drug Disposition Classification System (BDDCS) was proposed considering the solubility and metabolism of compounds. Besides, the BDDCS also considers the impact of transporters in biological tissues, such as in the gastrointestinal tract (GIT). Thus, this study aimed to evaluate the mechanisms involved in the permeability of antiretroviral drugs (stavudine, lamivudine and zidovudine) using the intestinal in situ perfusion model with mesenteric blood sampling in rats, considering efflux and intestinal pre-systemic metabolism that occur in the enterocytes. Furthermore, in vitro studies in cell cultures MDCK and MDCK-MDR1 were performed in order to elucidate the transport mechanisms of the drugs. For intestinal in situ perfusion studies, Wistar rats were anesthetized and a portion of jejunum was cannulated to allow the drug entry into the intestine, as well as the perfusate sampling at regular time intervals. The mesenteric vein was also cannulated to allow blood sampling during the experiments. For efflux studies, verapamil was used as P-gp (P-glycoprotein) inhibitor while ketoconazole was used as CYP3A inhibitor. In in vitro model MDCK and MDCK-MDR1, the experiments were performed bidirectionally using GG918 as P-gp inhibitor. For all experiments, metoprolol and ranitidine were used as markers of high and low permeability, respectively. Permeability results showed that stavudine and zidovudine present high permeability characteristics while lamivudine showed the lowest value. However, bidirectional studies in MDCK showed that the antiretroviral drugs present low permeability since their results are far from metoprolol's results. Regarding efflux studies, both lamivudine and zidovudine presented relevant interaction with P-gp in in situ perfusion and MDCK-MDR1 models, since the increase in permeability values was observed when P-gp inhibitor was added. Metabolism studies performed through intestinal in situ perfusion showed that none of antiretroviral drugs interact significantly with CYP3A enzymes, since that no variation in permeability results were noticed. Comparison between effective permeability (Peff) and apparent permeability (Papp), obtained from prefusate and plasma, respectively, allowed to check that statistical differences between these two parameters can indicate different transport mechanisms, since Papp is related to the drug amount that really overcome the physical and biochemical barriers in the gut. Thus, Papp is considered the closest parameter to in vivo condition. This difference was observed for zidovudine in efflux studies, since Peff values does not match with the conclusion provided by the Papp. The results obtained allowed to conclude that the antiretroviral drugs presents moderate to high permeability due to the involvement of influx carriers. Furthermore, the antiretroviral drugs interacted with the P-gp, but lamivudine and zidovudine showed significant results. Although the involvement between zidovudine and P-gp is observed, its high fraction absorbed indicates that the absorption is not limited by efflux transporter, which is not relevant clinically. Regarding 32 metabolism studies, CYP3A enzymes is not considered as a limiting condition for absorption of the antiretroviral drugs. Thus, the evaluation of the mechanisms contributes for biopharmaceutical characterization of stavudine, lamivudine and zidovudine and the methodologies used in this study can be applied in early drug development to ensure adequate safety and efficacy of pharmaceutical products.

Classification of natural products as sources of drugs according to the biopharmaceutics drug disposition classification system (BDDCS) / 中国天然药物

Natural products (NPs) are compounds that are derived from natural sources such as plants, animals, and micro-organisms. Therapeutics has benefited from numerous drug classes derived from natural product sources. The Biopharmaceutics Drug Disposition Classification System (BDDCS) was proposed to serve as a basis for predicting the importance of transporters and enzymes in determining drug bioavailability and disposition. It categorizes drugs into one of four biopharmaceutical classes according to their water solubility and extent of metabolism. The present paper reviews 109 drugs from natural product sources: 29% belong to class 1 (high solubility, extensive metabolism), 22% to class 2 (low solubility, extensive metabolism), 40% to class 3 (high solubility, poor metabolism), and 9% to class 4 (low solubility, poor metabolism). Herein we evaluated the characteristics of NPs in terms of BDDCS class for all 109 drugs as wells as for subsets of NPs drugs derived from plant sources as antibiotics. In the 109 NPs drugs, we compiled 32 drugs from plants, 50% (16) of total in class 1, 22% (7) in class 2 and 28% (9) in class 3, none found in class 4; Meantime, the antibiotics were found 5 (16%) in class 2, 22 (71%) in class 3, and 4 (13%) in class 4; no drug was found in class 1. Based on this classification, we anticipate BDDCS to serve as a useful adjunct in evaluating the potential characteristics of new natural products.

Pharmacokinetic aspects and in vitro-in vivo correlation potential for lipid-based formulations

Lipid-based formulations have been an attractive choice among novel drug delivery systems for enhancing the solubility and bioavailability of poorly soluble drugs due to their ability to keep the drug in solubilized state in the gastrointestinal tract. These formulations offer multiple advantages such as reduction in food effect and inter-individual variability, ease of preparation, and the possibility of manufacturing using common excipients available in the market. Despite these advantages, very few products are available in the present market, perhaps due to limited knowledge in the in vitro tests (for prediction of in vivo fate) and lack of understanding of the mechanisms behind pharmacokinetic and biopharmaceutical aspects of lipid formulations after oral administration. The current review aims to provide a detailed understanding of the in vivo processing steps involved after oral administration of lipid formulations, their pharmacokinetic aspects and in vitro in vivo correlation (IVIVC) perspectives. Various pharmacokinetic and biopharmaceutical aspects such as formulation dispersion and lipid digestion, bioavailability enhancement mechanisms, impact of excipients on efflux transporters, and lymphatic transport are discussed with examples. In addition, various IVIVC approaches towards predicting in vivo data from in vitro dispersion/precipitation, in vitro lipolysis and ex vivo permeation studies are also discussed in detail with help of case studies.