Human liver microsomal stability

IkB kinase-p is a key regulatory enzyme in the NF-kB pathway, and inhibition of this enzyme has the potential for yielding treatments for inflammatory and autoimmune diseases. Morwick et al. [53] report on the optimization of a pM IKKp inhibitor with low aqueous solubility, moderate human liver microsome stability, and inhibition of several CYPs (3A4, 2C9, 1A2) with pM potencies. Modulation of the thiophene core (other thiophene isomer, pyrimidine and oxazole) produces compounds of similar potency to the hit. Fusing the 5-phenyl moiety to the thiophene to form a thieno[2,3-b]pyridine core increases aqueous solubility of the series as well as reduces the CYP liability. While the optimized compound still shows pM IKK(S potency, the aqueous solubility, HLM stability and CYP profiles are much improved. A pharmacophore model was generated that enabled scaffold hopping to yield this new chemotype (Scheme 7). 

Human liver microsome stability testing Broad-spectrum selectivity screen then, rat receptor affinity assay... 

Sakiyama, Y., Yuki, H., Moriya, T., Hattori, K., Suzuki, M., Shimada, K., Honma, T. Predicting human liver microsomal stability with machine learning techniques. J. Mol. Graph. Model. 2008,... 

Fig. 10.10. (a) Experimental LogD (eLogD) vs. human liver microsome stability (FiLM %Rem 1p,M). A threshold value of eLogD < 2.7 is required for >70% stability in FILM, (b) Experimental vs. calculated LogD. eLogD < 2.7 translates to eLogD < 2.0 at pH 7.4. 

Fig. 10.12. (a) Experimental human liver microsome stability (FILM %Rem 1 . ) vs. calculated Log of solubility (c LogS). (b) Experimental human liver microsome stability expressed as apparent intrinsic clearance (FILM CL(int) xL/min/mg) vs. calculated Log of solubility (c LogS). A threshold value of cLogS > -3 and -4.0 is required for stability in FILM based on %R and intrinsic clearance, respectively. 

Brown, M.F. et al. (2007) Piperazinyl CCRl antagonists-optimization of human liver microsome stability. Bioorganic S, Medicinal Chemistry Letters, 17, 3109-3112. 

Hit to lead Physicochemical properties (solubility, Log P) PAMPA Plasma protein binding Efficacy species and human liver microsomal stability, CAR and PXR transactivation assay... 

The pyrazole NH moiety present in 9 represented a synthetic handle which enabled further derivitization to improve potency and modulate ADME properties. Introduction of a methyl group at this position was tolerated (compound 11) and many other substituents were subsequently incorporated in this location. In particular, introduction of a 4-piperidine moiety afforded compound 12 which displayed the most potent biochemical and cellular ICsoS among aU analogs synthesized. The active enantiomer of 12, compound 1 (crizotinib), was later synthesized using a chiral benzyl alcohol obtained from the biotransformation method mentioned previously. Crizotinib displayed improved biochemical LipE compared with compound 8 (6.4 vs. 2.2) and exhibited enhanced human liver microsomal stability relative to the pyrrolindine-containing compound 4 (HLM% remaining at 1 pM 44% vs. 5.7%). 

Several kinetic parameters can be measured on different experimental systems to account for the interaction of a compound with CYPs. For example when studying the metabolic stability of a compound, it could be measured in a recombinant CYP system, in human liver microsomes, in hepatocytes and so on. Each system increases in biological complexity. Although in the recombinant CYP system only the cytochrome under consideration is studied, in the case of the human liver microsomes, there is a pool of enzyme present that includes several CYPs, and finally in the hepatocyte cell system, metabolizing enzymes play an important role in the metabolic compound stability. In addition, transport systems are also present that could involve recirculation or other transport phenomena. The more complex the experimental system, the more difficult it is to extract information on the protein/ligand interaction, albeit it is closer to the in vivo real situation and therefore to the mechanism that is actually working in the body. 

In addition, compound 15 also had good metabolic stability in human liver microsome in vitro assay (hLM ti/2 = 39min) and in rat in vivo pharmacokinetic studies (ty2 = 3.3 h, po), with a rat oral bioavailability of 15%, showing a significant improvement in these PK parameters over the lead compound 1. The observed improvement in PK during the optimization was another validation of the strategy discussed above. This part of the optimization process is summarized in Scheme 19.2. 

Kantharaj, E., Ehmer, P. B., Tuytelaars, A., Van Vlaslaer, A., Mackie, C., and Gilissen, R. A. (2005b). Simultaneous measurement of metabolic stability and metabolite identification of 7-methoxymethylthiazolo[3,2-a]pyrimidin-5-one derivatives in human liver microsomes using liquid chromatography/ion-trap mass spectrometry. Rapid Commun. Mass Spectrom. 19 1069-1074. 

When chemical inhibition experiments are conducted with a relatively metabolically stable drug candidate (one that must be incubated with relatively high concentrations of human liver microsomes for a relatively long time in order to generate quantifiable levels of metabolite), it is important to take into account the metabolic stability of the inhibitors themselves. Lack of metabolic stability makes some compounds poor choices as chemical inhibitors despite their selectivity. For example, coumarin is a selective substrate of CYP2A6 (Km 0.25 to 0.5 pM) (111) and it would be a good selective competitive inhibitor of CYP2A6 if it were not metabolized so rapidly by human liver microsomes. 

Lee SH, Slattery JT (1997) Cytochrome P450 isozymes involved in lisofylline metabolism to pentoxifylline in human liver microsomes. Drug Metab Dispos 25 1354-1358 Li AP (1999) Cryopreserved human hepatocytes characterization of DME activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability and drug-drug interaction potential. Chem Biol Interact 121 17-35... 

Busby WF, Ackermann JM, Crespi CL (1999) Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450. Drug Metab Dispos 27 246-249 Clohs L, Wong J (2002) Validation of a capillary electrophoresis assay for assessing the metabolic stability of verapamil in human liver microsomes. J Cap Elec Microchip Tech 7 113 Coughtrie MWH, Fisher MB (2003) The role of sulfotransferase and UDP-glucuronosyltransferases. In Lee JS, Obach RS, Fisher MB (eds) Drug Metabolizing Enzymes. Marcel Dekker, New York pp 541-575... 

Figure 11-13. Time-course human liver microsomal incubation profiles for a number of positive and negative controls, as well as project compounds. Metabolic stability profiles are represented both in tabular format and graphically above.

Several TRK-850 derivatives were synthesized based on the general strategies mentioned above, and their metabolic stabilities were evaluated in the presence of human liver microsomes. Metabolic rates were determined by incubating the compounds with human liver microsomal enzymes and then quantifying the remaining parent peak by HPLC analysis. The data were expressed as the elimination rate constant (Kel) and the relative metabolic rate, or the /C , [ of the compound compared with the /C, [ of TRK-850 (ATF, ratio). 

Another approach to human bioavailability estimation is based on in vitro data using Caco-2 as a measure of permeability and human liver microsomes for metabolism estimates. These data are combined in a graphical method to get a rough estimate of human oral bioavailability [22]. In principle, but not yet proven, this method could also be applied by using calculated permeability and metabolic stability. 

The use of fast gradient elution LC-MS techniques for metabolic screening was first described by Ackermann and coworkers in 1998 [91] and Korfmacher and coworkers in 1999 [69], In the method developed by Ackermann et al., a HPLC column-switching apparatus is used to desalt and analyze lead candidates incubated with human liver microsomes. The resulting data can be quickly resolved into specific categories of metabolic stability high (>60%) moderate (>30%-59%) low (>10%-29%) and very low (<10%). 

Pfizer has recently evaluated open source descriptors and model building algorithms using a training set of approximately 50 000 molecules and a test set of approximately 25 000 molecules with human liver microsomal metabolic stability data. A C5.0 decision tree model demonstrated that the Chemistry Development Kit descriptors together with a set of SMARTS keys had good statistics (Kappa = 0.43, sensitivity = 0.57, specificity 0.91, positive predicted value (PPV) = 0.64) equivalent to models built with commercial MOE2D and the same set of SMARTS keys (Kappa = 0.43, sensitivity = 0.58, specificity... 

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