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In this chapter, we have provided a step-by-step protocol of an accelerated differentiation 5 day culturing Caco-2 (HTB-37) model in a 24-well plate format. The 5-day accelerated Caco-2 assay is based on previous literature protocols where inserts are coated with collagen protein and the cult ...

This chapter will focus on the techniques used in the evaluation of red blood cell partitioning/binding (RCB) of drugs in discovery and development. Certain therapeutic compounds have a high degree of affinity for the red blood cell fraction of whole blood and have large RBC-to-plasma concen ...

The mouse lymphoma assay (MLA) quantifies genetic alterations affecting expression of the thymidine kinase (Tk) gene which is located on chromosome 11. This assay is widely used for evaluating the genotoxic potential of various agents. It can also be used for photo-mutagenicity. The Tk-de ...

The Salmonella/microsome bacterial mutagenicity test (Ames test) is used worldwide as a simple and rapid mutagenicity testing system. Several modified versions of the Ames test have been developed, subsequent to the original “plate incorporation assay” using the Salmonella bact ...

It is of extreme importance to determine the genotoxicity of potential pharmaceutical products as it can drastically affect the potential use of those compounds. Described in this chapter is a system based in eukaryotic yeast cells that utilizes an endogenous DNA damage-responsive gene ...

The Comet assay, also known as the single cell gel electrophoresis (SCGE) assay, is widely applied as one of the standard methods to assess DNA damage caused by a range of DNA damaging agents. The unique aspect of this method is its ability to detect DNA damage in individual cells. During the last 2 decades, the C ...

Methodology is described to evaluate the reactivity of acyl glucuronides of carboxylic acid-containing compounds. In this chapter, zomepirac is presented as an example where the reactivity is determined in two complimentary ways: (1) Acyl glucuronides are prepared with UDPGA-for ...

Determination of plasma protein binding is important in drug discovery and development for developing PK-PD relationships, and projecting clinical doses because the free drug concentration at the site of action is responsible for the pharmacological activity. This chapter will d ...

Quantitation of reactive intermediates from bioactivation of drug via covalent protein binding using radiolabeled drug is the gold standard for quantitation of reactive metabolite formation in the absence of synthetic standard. However, radiolabeling many compounds during ...

Metabolism catalyzed by the cytochrome P450 enzymes (CYPs) represents the most important clearance pathways for most drugs in humans. However, CYP-mediated metabolism can also lead to drug bioactivation resulting to formation of reactive metabolites that can potentially induce ...

We provide here step-by-step protocols for the quantification of drugs/endogenous metabolites on dried blood spots (DBS) cards. DBS is a micro-volume blood collection technique in which aliquots of whole blood are deposited on specially manufactured filter paper, dried at ambient te ...

Early knowledge on the structures of metabolites from in vitro and in vivo metabolism studies is very useful for improving the biopharmaceutical, efficacy and safety properties of lead candidates in drug discovery. The recognition of the value in what metabolite identification brings ...

In this chapter, we will discuss the choice of proper chromatographic and mass spectrometric conditions that are critical for establishing sensitive and robust in vitro and in vivo assays. Liquid chromatography combined with mass spectrometry (LC/MS) has been a primary tool for quantit ...

The human transporters organic anion transporter 1 (OAT1), organic anion transporter 3 (OAT3) and organic cation transporter 2 (OCT2) are membrane proteins involved in the renal clearance of substances from the body. While the original purpose of these clearance pathways was likely the r ...

The hepatic transporters OATP1B1 and OATP1B3 contribute (to varying degrees, depending on the drug) to the uptake of many anionic drugs, including several of the widely used statins. Because statins are prescribed for many patients and the consequences of pharmacokinetic interactio ...

The transporter field has grown extensively over the past decade. Analogous to drug metabolizing enzymes such as the cytochrome P450s, transporters play a major role in defining pharmacokinetic, safety and efficacy profiles of drugs. Multidrug resistance-associated protein 2 (M ...

The gel retardation assay (GRA), also referred as the electromobility shift assay (EMSA), is commonly used technique to examine DNA binding of transcription factors including activated nuclear receptors to their specific DNA recognition sites. GRA of the aryl hydrocarbon receptor ( ...

Analysis of pregnane X receptor (PXR, NR1I2) activation to determine induction of drug metabolizing enzymes and transporters and predict drug-drug interactions (DDIs) is a wildly used technique among in vitro assays. Direct assessment of PXR activation is a cell-based assay that requi ...

As the closest relative of the aforementioned PXR, the constitutive androstane/activator receptor (CAR, NR1I3) also governs the transcription of numerous hepatic drug-metabolizing enzymes and transporters in response to various xenobiotic exposures. Unlike most prototy ...

The Ah receptor (AhR) is a ligand-dependent transcription factor that mediates a wide range of biological and toxicological effects from exposure to structurally diverse synthetic and naturally occurring chemicals. The role of the AhR and its signaling pathway in endogenous physio ...