Indice de daño Paso catorce de cálculo CnL 5 Indice de daño Paso catorce de cálculo

In vivo determination of Vss typically relies on the collection of systemic concentrations (typically in plasma but also possible in blood) of a drug over a period of time after intravenous administration. The value of Vss can be derived from the administered dose and its resulting area under the curve (Fan and de Lannoy, 2014).

Drug distribution is most commonly investigated with in vivo animal models in preclinical stages (Yanni, 2015). However, relying on animals to extrapolate human PK has been demonstrated to be complex as well as unreliable (Tsaioun et al., 2016). Various OATPs,

for example, are found in preclinical species but are absent in humans, which creates possible issues in extrapolating findings from preclinical stages into human trials (Lai, 2013e). Due to the need to improve both throughput capacity and predictive power, as well as the need to reduce animal testing, it has become common to also use in vitro and in silico models to predict distribution during drug discovery. These played a vital role in improving the efficiency in the pharmaceutical industry, as discussed in the first section of the Introduction (Yanni, 2015, Tsaioun et al., 2016).

In a typical drug discovery workflow, plasma protein binding is measured during hit-to-lead screening, followed by lead optimization where tissue distribution is studied in one rodent species. Lastly, prior to administration in humans in Phase I, for advanced lead optimization, a candidate’s distribution is assessed on one non-rodent species (dogs or non-human primates) and on one rodent species (Tsaioun et al., 2016, Zhang et al., 2012).

For plasma protein binding determination, a number of assays are used by the pharmaceutical industry such as equilibrium dialysis and ultrafiltration, which have high throughput (allowing 96-well plate testing). Other used methods include ultracentrifugation and chromatographic separation, and some less used methods such as exclusion chromatography, dynamic dialysis and circular dichroism (Yanni, 2015).

Animal studies for the assessment of the distribution of drug candidates are done through the administration of radiolabelled drug. Such studies include mass balance and quantitative whole-body autoradiography. In these, the labelled drug is administered orally or intravenously to the animal. In mass balance studies blood samples are collected over time, and tissues, urine and faeces are collected at specific times to determine whole-body distribution. The second technique, autoradiography, allows mapping distribution across all tissues and organs with time, however with the caveat of requiring a large amount of animals for each time point, and the inability to distinguish between metabolites and the parent compound (Yanni, 2015).

Radiolabelled studies are also performed in humans, namely positron emission tomography and magnetic resonance imaging allow monitoring the distribution of a drug into different organs. However, both in animals and humans, radiolabelled studies have the disadvantage of presenting possible safety risks associated with radioactivity exposure. Additionally, the cost and labour-intensive work associated with producing radiolabelled compounds makes this a prohibitive approach to investigate a drug’s distribution, especially in humans (Yanni, 2015).

Drug distribution is also assessed by carrying out in vitro transporter studies. The standard parameter used for classifying a compound as a substrate or non-substrate of a given

transporter is the observed efflux/uptake ratio between the basal-to-apical partition and the apical-to-basal partition across a cell culture monolayer. This will typically be used to monitor the uptake by a given transporter of interest. However, it should be noted that if a cell line which expresses a variety of transporters, such as Caco-2, is used it is likely that the observed efflux ratio is a result of several transport routes (Crivori et al., 2006).

Some in vitro assays to carry out transport interaction studies include membrane vesicle assays where cells transfected with ABC transporters (or membrane vesicles obtained from transporter-expressing organs) are incubated with and without ATP to investigate dependencies between ATP content and permeation (Yanni, 2015, Tsaioun and Kates, 2012, Zhang et al., 2012). As a refinement of the vesicle assays, oocyte transport expression systems are used. These have also been reported to allow more precise assessment of transport by a specific transporter than cell lines (Shirasaka et al., 2012), which are another model to carry out transported impact studies.

Cultured cell models typically use Caco-2 or MDCK-transfected cells to study both efflux and uptake. Caco-2 cells have the advantage of differentiating into polarized enterocytes, acquiring tight-junctions and expressing transporters in a way that resembles the human epithelium. These are commonly employed in bi-directional permeability assays. As for transfected cell lines, such as MDCK, CHO, HEK293 or LLC-PK1, they have the advantage of allowing to study isolated (typically over-expressed) transporters (Yanni, 2015, Tsaioun and Kates, 2012, Zhang et al., 2012).

The impact of transporters on distribution into specific tissues can also be assessed by using primary cell lines such as hepatocytes, proximal tubular cells, or co-cultures of glial cells and brain capillary endothelial cells (Yanni, 2015, Tsaioun and Kates, 2012). However primary cell culture is more challenging from the technical point of view. For more specific analyses, primary cells collected from genetically polymorphic human subjects, or from transporter-deficient animals, are used. To address tissues such as the brain, MDCK cells expressing P-gp are commonly used as they form tight junctions also found in the blood- brain barrier (Yanni, 2015, Tsaioun and Kates, 2012).

In situ organ perfusion models are the closest surrogate of in vivo drug transport physiological processes, with the liver perfusion being the most used model. After a drug is perfused through the organ, it is possible to determine the amount of uptaken drug among other outcomes (Zhang et al., 2012).