Microsomes human liver

Ibrahim RB, Wilson JG, Thorsby ME, et al Effect of buprenorphine on CYP3Aactivity in rat and human liver microsomes. Life Sci 66 1293—1298, 2000 Iguchi MY, Handelsman L, Bickel WK, et al Benzodiazepine and sedative use/abuse by methadone maintenance clients. Drug Alcohol Depend 32 257—266, 1993 Isbell H Manifestations and treatment of addiction to narcotic drugs and barbiturates. Med Clin North Am 34 423 38, 1950... 

Figure 5.52 Accurate-mass cone-voltage spectrum of the ethylhydroxy metabolite of Gly-buride, formed in vitro by human liver microsomes. Reprinted with permission from Zhang, H., Henion, J., Yang, Y. and Spooner, N., Anal. Chem., 72, 3342-3348 (2000). Copyright (2000) American Chemical Society.

In efforts designed to replace the imidazopyridine ring system, indolizi-dines such as (32) [101] (human H3 if = 13 nM), and related heterocycles [102-104] were identified as potent H3 antagonists. Indolizidine (32) suffered from rapid metabolism in human liver microsomes, however, substitution on the indolizidine ring suppresses this liability. 

Ruangyuttikam, W. Skiles, G. L. Yost, G. S. Identification of a cysteinyl adduct of oxidized 3-methylindole from goat lung and human liver microsomal proteins. Chem. Res. Toxicol. 1992, 5, 713-719. 

The figures that follow provide examples of some ways in which in vitro clearance data for two series can be compared and assessed to identify key questions, trends, or hypotheses. While the data presented here are for clearance in a human liver microsomal (HLM) incubation, the analysis could be applied in the same way to other data sets - including other experimental ADME or safety end points, or computationally predicted end points. 

Fig. 3 Human liver microsome apparent intrinsic clearance (Clint,app) vs clogD. Open squares and filled triangles represent two different chemical series (series A and B, respectively)...

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). 

Linnet, K. (2002). Glucuronidation of olanzapine by cDNA-expressed human UDP-glucuronosyltransferases and human liver microsomes. Hum. Psychopharmacol, 17, 233-8. 

Liu, Z. Q., Shu, Y., Huang, S. L. et al. (2001). Effects of CYP2C19 genotype and CYP2C9 on fluoxetine N-demethylation in human liver microsomes. Acta Pharmacologica Sinica, 22 85-90. 

MgCl2 (10 mM) increased the apparent Km (83 to 173 /am) and reduced the Vjnax (3.4 to 2.4 min-1) of triazolam 4-hydroxylation by expressed CYP3A4 [21]. However, both MgCl2 (30 mM) and CaCl2 (30 mM) significantly increased reaction rates of testosterone 6/3-hydroxylation (approximately threefold) and nifedipine oxidation (three- to six-fold) by human liver microsomes (HLMs) or recombinant CYP3A4 (reconstituted with b5 and GSH) [15]. It was suggested that divalent cation stimulation on the activity was related to involvement of b5 in CYP 3A4 reaction. 

Fisher, M.B., Campanale, K., Ackermann, B.L. et al. (2000) In vitro glucuronidation using human liver microsomes and the pore-forming peptide alamethicin. Drug Metabolism and Disposition The Biological Fate of Chemicals, 28, 560-566. 

Chauret, N., Gauthier, A. and Nicoll-Griffth, D.A. (1998) Effect of common solvents on in vitro cytochrome P450 mediated metabolic activities in human liver microsomes. Drug Metabolism and Disposition The Biological Fate of Chemicals, 26, 1-4. 

Crespi, C. L., Greenblatt, D. J., Comparison between cytochrome P450 (CYP) content and relative activity approaches to scaling from cDNA-expressed CYPs to human liver microsomes ratios of accessory proteins as sources of discrepancies between the approaches, Drug Metab. Dispos. 2000, 28, 1493-1504. 

Overall, the human intestine is capable of metabolizing UDP-glucuronyltransferase substrates, although the rates of metabolism are between 5- and 10-fold lower than those observed in human liver microsomes. However, the presence of a metabolic capacity towards UDP-glucuronyltransferase substrates at the level of the enterocyte can exert a significant gut wall first-pass extraction on oral administration. 

The metabolism of several CYP3A4 substrates in microsomes from the upper small intestine has been compared with liver microsomal metabolism. The results are summarized in Table 13.3. Thus, microsomes from the human upper small intestine can metabolize CYP3A4 substrates at rates approaching those found in human liver microsomes. However, the rate of metabolism in intestinal microsomes can be highly variable (8-fold for sirolimus [17] and 18- to 29-fold for midazolam [19]). 

Comparative study of fluorescence CYPs assays of niclosamide with that obtained by conventional HPLC assay using human liver microsomes and recombinant CYPs was developed [72]. 

Bangchang et al. [142] studied a number of antimalarial drugs for their effect on the metabolism of primaquine by human liver microsomes (N = 4) in vitro. The only metabolite generated was identified as carboxyprimaquine by cochromatography with the authentic standard. 

Guengerich FP. Mechanism-based inactivation of human liver microsomal cytochrome P-450 IIIA4 by gestodene. Chem Res Toxicol 1990 3[4] 363 371. 

Fig. 14.7 In vitro glucuronidation of SN-38 in human liver microsomes genotyped for UGT1A1 promoter polymorphism. Each bar represents the mean ( standard error) SN-38G production in livers with 6/6 (n = 19), 6/7 (n = 21), and 7/7 (n = 4) genotype Significantly less than 6/6, p<0.05.

Iyer L, King CD, Whitington PF et al. Genetic predisposition to the metabolism of irinotecan (CPT-11). Role of uridine diphosphate glucuronosyltransferase isoform 1A1 in the glucuronidation of its active metabolite (SN-38) in human liver microsomes. J Clin Invest 1998 101 847-854. 

Yamazaki H et al. Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes. Arch Toxicol 1999 73(2) 65—70. 

Two papers described the optimization of LLE and physicochemical properties in a series of pyrazole HTV nonnucleoside reverse transcriptase inhibitors (NNRTIs) and the selection of lersivirine (6) as a development candidate [15,16]. The early lead (7) was relatively lipophilic (clogP = 4.3), rapidly metabolized in human liver microsomes and had an LLE of only 1.9 [pIC50 (HIV RT) - clogP] [15]. An optimization program targeting increased LLE in less lipophilic compounds of low MW (to... 

FIGURE 6.53 Multiplexing P450 substrate analysis. Four substrates for each CYP450 were incubated in the absence (A) or presence (B) of human liver microsomes at room temperature for 20 min. The reactions were then quenched and samples were analyzed using /rPLC. The principle of this experiment is shown in the top panel. 

Human liver microsomes (HLMs) are the most common in vitro sources of enzymes for inhibition studies, and selective probe substrates are required. Recombinant human P450 enzymes have become commercially available. They are widely used for screening, and less selective probe substrate can be used. Hepatocytes and liver slices48 have also been used for P450 inhibition screening to a lesser extent. 

CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes. Rapid Commun. Mass Spectrom. 14 207. 

Di, L. et al. 2006. Comparison of cytochrome P450 inhibition assays for drug discovery using human liver microsomes with LC-MS, rhCYP450 isozymes with fluorescence, and double cocktail with LC/ S. Int. J. Pharmaceut. http //dx.doi.org/10.1016/j.ijpharm.2006.10.039. 

The species differences in biotransformation pathways, rates of elimination, and intrinsic hepatic clearance of esfenvalerate and deltamethrin using rat and human liver microsomes were examined [33]. Esfenvalerate was eliminated primarily via NADPH-dependent oxidative metabolism in both rat and human liver microsomes. The CLint of esfenvalerate was estimated to be threefold greater in rodents than in humans on a per kg body weight basis. Deltamethrin was also eliminated primarily via NADPH-dependent oxidative metabolism in rat liver microsomes however, in human liver microsomes, deltamethrin was eliminated almost entirely via... 

NADPH-independent hydrolytic metabolism. The CLint for deltamethrin was estimated to be twice as rapid in humans as in rats on a per kg body weight basis. Metabolism by purified rat and human CESs was used to examine further the species differences in hydrolysis of deltamethrin and esfenvalerate. Results of CES metabolism revealed that hCEl was markedly more active toward deltamethrin than the Class I rat CESs, hydrolase A and B, and the Class II human CES, hCE2 however, hydrolase A metabolized esfenvalerate twice as fast as hCEl, whereas hydrolase B and hCEl hydrolyzed esfenvalerate at equal rates. These studies demonstrated a significant species difference in the in vitro pathways of biotransformation of deltamethrin in rat and human liver microsomes, which was due in part to differences in the intrinsic activities of rat and human CESs. 

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