Assay stability

Several higher throughput in vitro assays may be used to assess various DMPK properties of NCEs. One common parameter is that HPLC/MS/MS is the method of choice for the analytical step.11 17 26 These higher throughput assays include the Caco-2 assay, p450 enzyme inhibition assay, and in vitro stability assay. Each assay has different requirements and solutions and they will be described individually. 

An important DMPK property of a NCE is oral bioavailability (F) of the compound in various pre-clinical species.3 The oral bioavailability of a compound is dependent on several factors including intestinal permeability (estimated by the Caco-2 assay) and hepatic clearance (estimated with an in vitro metabolic stability assay).3 30 The metabolic stability assay is typically performed by incubating test compounds in liver microsomes or hepatocytes. The results can provide estimates of in vivo stability in terms of metabolic liabilities.3 8 59 62 Several authors described this assay as an important tool for the rapid assessment of the DMPK properties of NCEs.3 6 8111819 26 44 59 62-65... 

One issue related to supporting a metabolic stability assay with HPLC/MS/MS is the need to set up an MS/MS method for each compound. While it may only take 10 min to infuse a compound solution and find the corresponding precursor and product ions (along with minimal optimization of the collision energy), the processes of MS/MS development would require 4 hr per day if one wanted to assay 25 compounds per day. MS vendors have responded to this need by providing software tools that can perform the MS/MS method development step in an automated fashion. Chovan et al.68 described the use of the Automaton software package supplied by PE Sciex (Toronto, Canada) as a tool for the automated MS/MS method development for a series of compounds. The Automaton software was able to select the correct precursor and product ions for the various compounds and optimize the collision energy used for the MS/MS assays of each compound. They found that the Automaton software provided similar sensitivity to methods that would have been developed by manual MS/MS procedures. Chovan et al. also reported that the MS/MS method development for 25 compounds could be performed in about an hour with the Automaton software and required minimal human intervention. 

Some authors searched for common oxidative metabolites as part of metabolic stability assays. Tong et al.75 described a highly automated microsomal metabolic stability assay that achieved a throughput of 50 compounds per day with each compound tested in rats, dogs, monkeys, and humans. In addition to assaying the test compound, they monitored M+16 metabolites by using the... 

Chovan, L.E. et al. 2004. Automatic mass spectrometry method development for drug discovery Application in metabolic stability assays. Rapid Commun. Mass Spectrom. 18 3105. 

Approximately 300 drugs were tested in aqueous solubility, log D, apparent permeability and metabolic stability assays. Compounds having low values for solubility, apparent permeability, or metabolic stability, or extreme log D values were flagged. The frequency of compounds with flags in each human bioavailability (%) bin is shown. 

Saavedra et ah used CE as orthogonal technique to HPLC to confirm the identification of a degradation product in alprazolam tablets during their stability assay. 

Alkaline phosphatase [H,NEt][N03] Enzyme activity and stability assayed by hydrolysis of 29... 

Forewarming (preheating). Heating an unconcentrated milk, especially at 90°C x 10 min, before a heat stability assay, reduces its heat stability,... 

By phase II, stability assays should be validated and a good faith effort made to validate all in-process tests. Release assays should also be validated. Removal of product and process-related impurities should be demonstrated. Stability of cells during growth should be validated. 

O Connor, D., Mortishire-Smith, R., Morrison, D., Davies, A., and Dominguez, M. (2006). Ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry for robust, high-throughput quantitative analysis of an automated metabolic stability assay, with simultaneous determination of metabolic data. Rapid Commun. Mass Spectrom. 20 851-857. 

In Vivo Peptide Stability Assay/Pharmacokinetics in Mice... 

In a general serum stability assay, the peptide is subjected to human serum (see Note 3) at realistic temperature conditions and incubated for various time intervals, comparable to traditional protease assays. The reactions are stopped by TCA or ethanol, precipitating larger serum proteins while leaving peptides... 

Until very recently, metabolic stability screening and metabolite identification (metabolite ID) have been sequential processes that is, the metabolic stability assays are typically performed hrst to identify rapidly metabolized compounds and a follow-up metabolite ID study is performed next, typically at a 10-fold higher substrate concentration to ensure generation of sufficiently high-quality MS/MS information to support structure elucidation. 

The irradiation of a solution of ketoconazole will illustrate that situation The monitoring of the absorption at the spectral maximum of ketoconazole at 242 nm only gives a decrease of 9% after an irradiation period of 100 minutes (Fig. 4A). A comparison of the complete UV absorption spectrum before and after 100 minutes irradiation shows more significant changes in the range around 300 nm (Fig. 4B). But the real extent of decomposition only became apparent with the results of the HPLC stability assay. The HPLC analysis proved that already after 5 minutes 50% of the drug was decomposed by photolysis. 

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