Cell culture metabolic stability

Because clearance at the whole-body level often is determined by metabolism at the cellular level, it is possible to use a variety of human-derived in vitro systems to determine rates of metabolism. These systems include pure human enzymes (such as cytochrome P450 enzymes) (13) and human liver subcellular fractions (S9 and microsomes) (14). However, with enzymes and subcellular fractions, some information is lost because the whole-cell integration of subcellular processes has been disrupted. The use of cultured human hepatocytes retains the whole-cell integration at the expense of greater experimental complexity (15). Each system provides a different window on the metabolic processes, is relatively easy to use, and can be obtained from commercial sources. Rates and pathways of metabolism may be compared with a series of discovery compounds to identify those with the greatest relative metabolic stability or with a benchmark compound of known human PK characteristics to provide a more absolute estimate of hepatic metabolic clearance. 

HBS helices have proven to be remarkably effective tools for the regulation of protein interactions. Significantly, HBS helices outperform their unconstrained counterparts in cell culture studies reflecting their enhanced metabolic stability and cellular uptake properties. The potential of HBS helices has been demonstrated with inhibitors designed to modulate HIV fusion [35], transcription of hypoxia inducible genes [54], p53/HDM2 [33], and Ras/Sos interactions [134]. The selected examples below highlight the potential of HBS helices as inhibitors of PPIs. 

This similar dependence of in vivo potency and mustard reactivity on substituent electronic properties (cf Equations I and 4) has been cited (8) as evidence that the biological activity of these compounds is directly due to the rate at which they alkylate cellular DNA. However, in complex in vivo systems the possible roles of drug transport and metabolism as well as DNA repair have also to be considered. Surprisingly, there is virtually no quantitative data available on the cytotoxicity of substituted aniline mustards in mammalian cell culture systems, where the effects of substituent electronic effects on cytotoxicity and stability can be examined in the... 

MS2D medium (MS salts and vitamins (9) with 30 g/L sucrose, 100 mg/L myo-inositol, 100 mg/L casein hydrolysate, and 2 mg/L 2,4-D at pH 5.8) was utilized in the routine maintenance of the wheat cell culture, as well as for conducting metabolism and media stability testing. 

Due to the high cost of cell culture, Caco-2 assays are usually used as a follow-up to PAMPA in ADME screening [78], and as a result, the sample burden for bioanalysis is not as heavy as for some first-hne assays, such as metabolic stability. There have been a number of reports in the literature that use automated optimization and single LC-MS/MS for sample analysis for Caco-2 assay support [46,79-81]. Nevertheless, Caco-2 samples pose a unique bioanalytical challenge. Unlike plasma or microsomal samples rich in proteins that help solubilize compounds and prevent adsorptive loss, Caco-2 samples are essentially aqueous buffer samples with very little protein. As a result, compounds with low solubility and/ or adsorption problems tend to exhibit poor recoveries in the assay due to precipitation and adsorptive losses [82,83]. An effective solution to this problem is the use of organic solvent to catch compounds immediately after incubation, but prior to analysis, in order to maintain solubility and prevent adsorptive loss to container surfaces. Another approach involves the addition of some protein such as bovine serum albumin (BSA) to the assay buffer system, thus reducing compound loss/ precipitation and improving recoveries [84]. 

However, the isolated cells may develop altered morphology, functions, and have different levels of cell-cell interaction and usually also a dramatically decreased metabolism that could result in an altered response to test chemicals when compared to the in vivo situation [34], Furthermore, primary neuronal cultures consist predominantly of postmitotic neurons which do not proliferate, giving the model stability but also a limited life span. Consequently, the cultures always need to be freshly isolated and may not be fully suitable for high-throughput screening. Neuronal and glial primary cultures from the PNS and CNS can be derived from... 

The demonstration that modificationsmay induce nuclease stability sufficient to enhance activity in cells in tissue culture and in animals has proved to be much more complicated because of the presence of 5 -exonucleases and -endonucleases. In our laboratory, 3 -modifi-cations and internal point modifications have not provided sufficient nuclease stability to demonstrate pharmacological activity in cells (183). In fact, even a 5-nucleotide-long phos-phodiester gap in the middle of a phosphoro-thioate oligonucleotide resulted in sufficient loss of nuclease resistance to cause complete loss of pharmacological activity (164). In mice, neither a 5 -cholesterol nor 5 -C18 amine conjugate altered the metabolic rate of a phospho-rothioate oligodeoxynucleotide in liver, kid-... 

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