DDI assays

Many factors may confound the assessment of the D DI potential of early discovery compounds [93], Limited or no solubility data exist to understand the likelihood that the compound will precipitate out of an in vitro incubation. The compounds have generally not been analyzed from a spectroscopic perspective their characteristics may interfere with a fluorogenic DDI assay. Metabolism data are typically not available. The binding of a compound to plasma proteins or microsomal incubation constituents is not well understood, which may lead to underprediction of its inhibitory potential. The compounds are typically delivered in DMSO, which may cause solvent-related inhibition of the enzymatic assay. Also, since little is known about in vivo concentrations or projected dose, framing the consequences of an early DDI in vitro experiment may be difficult. With these factors in mind, general experimental paradigms have been developed to help minimize their potential impact. 

A competitive inhibition screen is often the first step in understanding the DDI potential of a NCE. The definitive assessment of inhibition is the inhibition constant (K ), which provides not only the inhibition potency but also information on the mechanism of inhibition (competitive, non-competitive). However in the hit to lead profiling environment this approach is over-complex for the question being asked, and generates far too many samples to enable rapid screening of compound series. DDI assays based upon the IC50 principle are therefore favored. The relationship between K and IC50 for a competitive inhibitor is ... 

Robotic Liquid-Handling Systems In Vitro DDI Assays... 

DDI CYP inhibition (rCYP, HLM) (see Section 6.3.2.3) CYP induction (PXR-TA, Fa2N-4, hepatocytes) (see Section 6.3.2.3) CYP reaction phenotyping Assessing in vitro CYP inhibition and induction potentials Predicting in vivo DDIs Assay (HLM CYP inhibition) ° Human hve microsomes ° 0.5-100 rM probe concentration depending on each isozyme Analysis ° PPT followed by LC-MS/MS or onhne SPE-MS/MS... 

Bioanalytical support for DDI assays with EC-MS is distinctively different from support for most other ADME screens in that only the probe metabolite (instead of all test compounds) is monitored in all samples. As a result, no MS/MS method optimization (other than that for the probe metabolite) is needed. On the other hand, the EC-MS/MS sample burden for CYP inhibition assays is probably the heaviest among all in vitro ADME screening assays, as CYP assays are typically conducted in 384-well plates and involve full IC50 (half maximal inhibitory concentration) curves, and many times in a time-dependent fashion to measure inhibition potentials for both the test compound and its metabohte(s). As a result, bioanalytical research for CYP inhibition support has mostly focused on improving sample analysis speed and throughput. (Data handling and report generation are now mostly automated and do not represent a bottle neck.) Several assay and bioanalytical approaches used either individually or in combination have been reported for CYP inhibition sample analysis. One approach is to incubate multiple substrates for multiple isozymes in the same well, and use an SRM method with multiple MS/MS transitions to analyze all the metabolites and assess the inhibition... 

In vitro techniques for studying DDI potential are based on the metabolism of known marker substrates. Two assay types are typically used to study DDIs the turnover of drug-like probes monitored by LC-MS/MS methods or the use of spectrophotometer (plate reader) based methods. As each technique has unique advantages and shortcomings, assay use has not been standardized across the industry. Although techniques based on the turnover of radiolabeled substrates have also been developed [94—97], these methods are used infrequently and will not be discussed further. 

The DDI fluorescent inhibition screen is a high-throughput screen, primarily as a result of reduced analysis time, and can range from a few hundred compounds per week to a thousand per week. The assay uses recombinant microsomes prepared from insect cells infected with recombinant baculovirus containing a human CYP enzyme and individual fluorogenic substrates. In this instance the substrates are not specific and hence cannot be used in a mixed enzyme system such as HLM. In addition, this assay is more prone than the conventional inhibition screen (Section 8.3.2) to NCE interferences within the assay (i.e., inherently fluorescent NCEs, fluorescent quenching by the NCE). 

The pharmacokinetic results obtained with HPLC/ UV and HPLC/MS/MS are shown in Figure 12.10. Dotted lines indicate the LOQ for F-ddA by both assay methods. Data for F-ddI obtained by either method show good agreemeiiL being well above the LOQ for both techniques. On the other hand, all of the F-ddA data points are below the LOQ by HPLC/UV. Nonetheless, measurements reported below LOQs can be useful in that they help define what assay sensitivity must be achieved for pharmacokinetic data analysis. 

The CYP inhibition assay utilizes the 96-well plate format with a robotic system, where both incubation and analysis are performed in the same plates. The setup of the sample plates is shown in Figure 4.1. For each compound, both direct inhibition and metabolism/mechanism-based inhibition, which is caused by a metabolite of the NCE that is either a more potent direct reversible inhibitor (metabolism-based) or a time-dependent irreversible inhibitor (mechanism-based), are evaluated. Both direct and mechanism-based inhibitors could result in inhibitory DDIs [51,52],... 

How can in vitro transactivation assays be used to predict the clinical significance of inductive DDIs What are some of the drawbacks or limits to this strategy ... 

The in vivo assay for evaluation of antitumor activity of other tannins and related polyphenols was thus routinely performed by administration of tannins with a single intraperitoneal injection (10 mg/kg) to female ddY mice 4 days before inoculation of sarcoma 180 cells (1 x 10 ). Sixty days after inoculation of the tumor cells, survivors were killed and autopsied. The antitumor activity of each tannin was estimated by the number of regressors and the % increases in the life span (% ILS) [107]. Among approximately 100 polyphenols of different types tested, most of the oligomeric hydrolyzable tannins exhibited a potent activity in contrast to the negligible activity of the monomeric hydrolyzable tannins, proanthocyanidins and their related low-molecular weight polyphenols such as gallic acid, catechins and caffeoyl derivatives. 

The use of different in vitro and in vivo models depends largely on the objective of the studies. Multiple methodologies are often needed for better understanding of a transporter-mediated efflux or uptake of drug molecules. To address the potential DDI in humans, currently FDA suggests the use of the bidirectional transport assays in human transporter expressing cells in their draft guidance. 

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