Development and Applicability
6.5 EXPERIMENTAL TOOLS USED IN PRECLINICAL DEVELOPMENT
6.5.1
In Vitro Systems for Metabolism and Toxicology Studies The human systemic hierarchical organization of cells gives rise to tissues, organs, and systems. Thus, their use in isolated environments is believed to reflect the functions of the body (Table 6.1). Cells are fundamental functional units or machinery in the living organisms that perform vital functions to sustain life. Cell cultures are finding increasing applicability in replicating the functions of the organs, in vitro systems are comprised of assemblies of various organ-specific cells that exhibit functions close to those in the human body. Cell-based studies could be customized to variousSocial Aspects of Drug Discovery, Development and Commercialization 136
kinetic parameters of substrates or inhibitors. In vitro cell cultures are em- ployed in evaluation of drug efficacy through monitoring of the biological activity of the drug compound on the cell of interest [16].
Cells grown as monolayers (two-dimensional models) are routinely used model systems for identifying the most viable compounds that progress to preclinical animal studies before advancing to human clinical trials. Grow- ing cells as three-dimensional (3D) models tend to depict more closely the natural environment as they are cocultured with other neighboring cells and cellular components in the physiological microenvironment, in order to make them more clinically relevant.
In vitro experimental cell cultures have greatly enhanced the ability to generate parameters to predict transporter-mediated drug distribution and elimination and the associated drug interactions (Table 6.2). In vitro cell cultures increase in complexity from single-gene overexpressing immortalized cell lines to isolated hepatocytes and 3D cultured hepatocyte systems. Cells expressing individual uptake transporters have been widely used to determine potential inhibitory function of a transporter or substrate [17–19]. For compounds that have low passive permeability and/or are
Table 6.2 In vitro screening assays for identifying the corresponding function of an orally ingested drug
Assay Function
Caco-2 permeabililty, PAMPA Intestinal absorption P450 inhibition; induction Drug–drug interaction Hepatocyte liver microsome metabolism Hepatic metabolism Hepatocyte biliary excretion Bile excretion
Hepatocyte cytotoxicity Organ toxicity
Source: Adapted from Ref. [20]
Table 6.1 Toxicity associated with organ-specific cell
Type of toxicity Organ-specific cells
Liver toxicity Hepatocytes
Nephrotoxicity Renal proximal tubule
Cardiotoxicity Cardiomyocytes
Rhabdomyolysis Skeletal myocytes
Vascular toxicity Vascular endothelial cells
Cytotoxicity Organ specific
efflux transporter substrates, apparent intrinsic clearance in hepatocytes is usually lower than that in enzyme systems, due to lower free hepatocyte.
Other types of in vitro data that address drug disposition properties (e.g., membrane penetrability, P450 inhibition, others) are also gathered early us- ing high throughput methods.
In vitro screening assays provide the first line of information about the potential effect of the drug compound on the biological system.
They eliminate the complexities associated with the use of the whole systems by providing options that would have required extensive experi- mental work and yet with the promise of reliable conclusions. The cell cultures are pivotal and only used for pilot and backup data for animal studies. The specialized cells of various organs that retain their specific characteristics are important experimental systems for early toxicity screening [21].
Cytotoxicity is concerned with biochemical or physical malfunctions of the important functional organelles or units of the cells, which include mitochondrial functions, lysosomal functions, and cellular metabolites.
Mechanisms of known functions are important. Cytotoxicity endpoints are mitochondrial functions and lysosomal functions, cellular metabolite con- tent, and membrane integrity. Another advantage of cellular screens is that they employ the high-throughput screening (HTS) technology, which is deployed when molecular targets could not be tested in an isolated form or due to the extent of patent protection. This allows only a whole-cell system to be tested. Such cellular screens permit the evaluation of multiple targets in a pathway and identification of allosteric effectors, which are early indi- cators of toxicity.
6.5.2 Intestinal Absorption Studies Models: Caco-2 Cells
Caco-2 cells have been of great utility in intestinal absorption studies due to their exclusive ability to model human absorption characteristics and Caco-2 is the most commonly used intestinal permeability model. Caco-2 cells and intestinal enterocytes cells have enabled the validation of the appli- cation of Caco-2 cells in drug absorption studies. Caco-2 cells have special features: they develop into small intestinal cells with tight junctions and microvilli, expressing the drug metabolizing enzymes involved in uptake of the agents crucial in drug metabolism [22,23].
The limitations of Cacao-2 cells are that they are unable to be used in screening of formulations that are sensitive to pharmaceutical excipients since dilution is a poor representation of the original formulation potency
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and functionality; they have a low tolerability to organic solvents, limiting their use in this kind of permeability study. they are assayed in microwells, which do not represent the whole system; and they do not accurately pre- dict the side or adverse effects, which is a common experience with drugs in the context of the human body or target organ system. The whole intact system encompassing the natural physiological environment comprising discrete functions that integrate into a network of functions could not be recapitulated in the in vitro systems, which is critical to identifying toxicity.
But since the intestinal absorption in rodents and humans are well corre- lated, their utility is essential [24]
6.5.3 Advances in Cell Culture Systems
Cellular disease models permit a detailed study of disease processes and there- fore this function need to be validated for a better in vivo correlation with Organs on a Chip. The complexity of human biology and the networks often requires viewing each isolated case within the context of another and thus highlights the necessity for a multiparametric approach to certain biologi- cal investigations. Organs-on-chips are intelligent fabrications that represent the various aspects of the physiological environment. This is applicable for disease modeling and drug discovery tailored to various needs, particularly for the study of drug toxicity and metabolism [25,26]. Compartmentalization-based applications termed “semiconductor processing” have been introduced [27].
For example, the Integrated discrete Multiple Organ Culture system has been introduced to coculture organ-specific cells with interconnection of blood circulation. It is used to evaluate the effect of drugs and metabolites [28]. There have been proposals for creating personalized organs-on-chips, which is already under way [29] not only for individualized screening plat- forms but to accelerate drug screening in a way that addresses speed and efficiency.
Some recent progress in four areas has been described that covers in- tegrated microdevices for cell culture; 3D cell patterning and culture;
multilayered microfluidic structures; and perfusion-based microdevices, as explained in Ref. [30] and as follows.
6.5.3.1 Microwell Array Technology: Cell Function/Toxicity Assays The utility of microfabrication techniques technology has been applied to cell culture systems with compartmentalization-integrated microwell arrays and cell culture analogs [31]. These were introduced to address the limitation of lack of interconnectivity and interactions of organs in the cell
culture format. They are known as “small wells within large wells,” wells within a well such that cells form organs that are cultured within the small wells, surrounded by the drug-containing large wells described in [32].
This device permitted the maintenance of the human liver cells’ pheno- typic functions for several weeks. It allowed the evaluation of gene ex- pression profiles, Phase I/II metabolism proteins, secretion of liver-specific products, and susceptibility to hepatotoxins. Application of this technique shows prospects in overcoming aspects of the preclinical failures in drug discovery.
6.5.3.2 Integrated Microcell Culture Systems
In a similar arrangement, the microcell culture analog (mCCA) approach was developed to create a cell culture environment that is close to that of humans, to improve the credibility of the drug PK and PD profiles [33].
The device consists of separated chambers connected with microchannels to link the different chambers of cultured cells of the liver, tumor, and mar- row, to simulate the blood flow. The microenvironment provided by the mCCA was intended to simulate more of the in vivo environment in than a conventional monolayer culture.
6.5.3.3 Three-Dimensional Cell Patterning and Culturing
Cell culture systems created to very closely represent the human physi- ological characteristics are interaction prone, as they produce a fertile envi- ronment for cell–cell and cell–matrix interactions [34,35]. This platform is adaptable to variable cell patterning methods.
6.5.3.4 Membrane-Based Multilayer Microfluidic Devices
This technology reproduces the in vivo microenvironment of kidney tubular cells. It was created in an attempt to resolve the issue of the lack of a tissue- like microarchitecture in traditional two-dimensional systems. Using this device, cell polarization and cytoskeletal rearrangement studies have been made more predictive [36].