In vitro metabolic stability

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... 

Lau, Y.Y. et al. 2002. The use of in vitro metabolic stability for rapid selection of compounds in early discovery based on their expected hepatic extraction ratios. Pharm. Res. 19 1606. 

Plobeck N, Delorme D, Wie Z-Y, Yang H, Zhou F, Schwarz P, Gawell L, Gagnon H, Pelcman B, Schmidt R, Yue SY, Walpole C, Brown W, Zhou E, Labarre M, Payza K, St-Onge S, Kamassah A, Morin P-E, Projean D, Ducharme J, Roberts E (2000) New Diarylmethylpiperazines as Potent and Selective Nonpeptidic %a Opioid Receptor Agonists with Increased In Vitro Metabolic Stability. J Med Chem 43 3878-3894... 

Many types of modeling techniques are available in the discovery phase of drug development, from structure activity relationships (SAR) to physiology based pharmacokinetics (PBPK) and pharmacokinetics-/pharmacodynamics (PK/PD) to help choosing some of the lead compounds. Some tests that are carried out by discovery include techniques related to structure determination, metabolism, and permeability NMR, MS/MS, elemental analysis, PAMPA, CACO-2, and in vitro metabolic stability. Although they are important as a part of physicochemical molecular characterization under the biopharmaceutics umbrella, they will not be discussed here. The reader can find relevant information in numerous monographs [9,10]. 

W.Z. Shou, L. Magis, A.C. Li, W. Naidong, M.S. Bryant, A novel approach to perform metabolite screening during the quantitative LC-AIS-MS analyses of in vitro metabolic stability samples using a Q-LIT-MS, J. Mass Spectrom., 40 (2005) 1347. 

Pobleck N, Delorme D, Wei Z-Y et al (2000) New diarylmethylpiperazines as potent and selective nonpeptidic delta opioid receptor agonists with increased in vitro metabolic stability. J Med Chem 43 3878-3894... 

Rajanikanth, M., Madhusudanan, K.R, and Gupta, R.C., Simultaneous quantitative analysis of three drugs by high-performance liquid chromatography/electrospray ionization mass spectrometry and its application to cassette in vitro metabolic stability studies, Rapid Commun. Mass Spectrom., 17(18), 2063, 2003. 

Baker, T. et al., High-throughput in vitro metabolic stability determinations of pharmaceuticals using automated sample preparation and multiple-compound HPLC/MS/MS, Adv. Mass Spectrom., 15, 657, 2001. 

However, this approach requires significant deconvolution efforts when activity is found in mixtures. To increase the success rate and decrease the number of compounds screened to a manageable size, the search for active metabolites could be limited to those compounds/chemotypes showing a high clearance rates in in vitro metabolic stability or in vivo exposure screens. 

Zhao SX, Forman D, Wallace N, Smith BJ, Meyer D, Kazolias D, Gao F, Soglia J, Cole M, Nettleton D.Simple strategies for reducing sample loads in in vitro metabolic stability high-throughput screening experiments a comparison between traditional, two-time-point and pooled sample analyses. J Pharm Sci 2005 94 38-45. 

Figure 10. New analogues of GB-ll-5 (7) and GB-ll-150 (12). TG-ll-36 (19) displays both potent antimitotic antikinetoplastid activity and increased in vitro metabolic stability compared to GB-ll-150 (12).

Madgula, V.L., B. Avula, R.S. Pawar, et al. 2008. In vitro metabolic stability and intestinal transport of P57AS3 (P57) from Hoodia gordonii and its interaction with drug metabolizing enzymes. PlantaMed. 74(10) 1269-1275. 

Klopf W, Worboys P. Scaling in vivo pharmacokinetics from in vitro metabolic stability data in drug discovery. Comb Chem High Throughput Screen. 2010 13(2) 159—69. 

A standard model for higher-throughput oral screening is shown in Fig. 12.5. As shown in Fig. 12.5, a common scenario for an oral bioavailability screen would be to combine the results of an in vitro absorption screen with the results on an in vitro metabolic stability screen to select compounds to go into an in vivo oral PK screen. The NCEs that still appeared to be promising lead compounds after these various screens could then be selected for full PK studies (oral and intravenous dosing in three to four animals for each dose route). In this way, only those compounds that were likely to exhibit good oral bioavailability would be tested in the full PK studies. 

FIGURE 12.5 Schematic representation of a typical process for oral bioavaUability screening in a drug discovery setting. The results of both an in vitro absorption screen as well as an in vitro metabolic stability screen are combined to select compounds for an in vivo oral PK screen. The compounds that survive these various screens can then be assayed in the standard in vivo full PK study. 

In vitro metabolic stability assays are a valued first-pass assessment of potential metabolic liabilities. However, detailed information about the location of metabolic soft-spots is particularly useful in understanding whether the observed liabilities are specific to a molecule s core or introduced as part of a side chain in the lead optimization process. Whether the metabolic liability is associated with the core or side chain has clear implications for the degree to which the liability can be engineered out of a chemical series. Up until recently, metabolic stability screening and metabolite detection have been decoupled processes, that is, the metabolic stability assays are typically performed at a... 

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