CYP proteins

Ho vever this approach does not address inter-individual variability in CYP expression nor the apparent substrate specificity of RAFs. This may be overcome through the use of intersystem extrapolation factors (ISEFs) vhich compare the intrinsic activities of rCYP versus liver microsomes and provide CYP abundance scaling by mathematical means. This employs the RAF approach and adjusts for the actual amount of liver microsomes CYP present (measured by immunochemistry) rather than a theoretical amount (Equation 8.4). Such corrections can be made using nominal specific contents of individual CYP proteins in liver microsomes or more appropriately employ modeling and simulation software (e.g., SIMCYP www.simcyp.com) which takes into account population-based variability in CYP content. 

A recent paper by Chauret et al. described the discovery of a novel fluorescent probe that is selectively metabolized by CYP3A in human liver microsomes (32). This probe, DFB [3-[(3,4-difhiorobenzyl)oxy]-5,5-dimethyl-4-[4-(methylsulfonyl) phenyl] furan-2(5F/)-one], is metabolized to DFH [3-hydroxy-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5//)-one], which has fluorescent characteristics (Fig. 7). In vitro CYP reaction phenotyping studies (cDNA-expressed CYP proteins and immunoinhibition experiments with highly selective anti-CYP3A4 antibodies) demonstrated that DFB was metabolized primarily by CYP3A4 (Fig. 8). Furthermore, metabolism studies performed with human liver microsomes obtained from different donors indicated that DFB dealkylation and testosterone 6P-hydroxylation correlated well (Fig. 9). 

The growth in available CYP protein structures from X-ray crystallography, presented in Figure 3, has inspired several investigators to detailed... 

Mechanism Direct chemical effect on enzyme Indirect effect through enhanced production of CYP protein... 

A Accounts for about 30% of CYP proteins in the liver also present in the... 

Seven isoforms account for the total CYP protein mass foimd in human liver [12]. These are summarised in Fignre 5.1. 

Figure 5.1 Major CYP enzymes expressed in human liver. Amounts are expressed as a percentage of total CYP protein.

The accessibility component, represents the recognition between the specific CYP-protein and the ligand when the ligand is positioned in the CYP-protein and exposes the atom i to the heme. It depends on the ligand 3D structure, conformation, chirality, and on the 3D structure and sidechain flexibility of the CYP-enzyme. Thus the score is proportional to the exposure of the ligand atom i to the heme group of a specific CYP-enzyme. 

In Vivo 3-132,-141,-149 4-149 Sprague-Dawley rats (male) Thyroid hormone reduction (T3, T4) in blood Increased thyroid gland weight hepatic CYP protein levels [183]... 

Irreversible or quasi-irreversible metabolic inhibition occurs when either the parent compound or a metabolic intermediate binds to the reduced ferrous heme portion of the P450 enzyme/ thereby inactivating it (51). In irreversible inhibition, or "suicide inhibition/" the intermediate forms a covalent bond with the CYP protein or its heme component/ causing permanent... 

IV. Cultured Hepatocytes Effects of Endogenous and Exogenous NO on Total CYP Heme, Total Microsomal Heme, and CYP Protein — -----------------------------... 

The results of a representative experiment are shown in Table IV (Kim et al., 1995). As expected, dialysis versus heme has little effect on the CYP heme content in microsomes from control hepatocytes (no NO exposure) or in hepatocytes treated with CME in the presence of NMMA. However, this dialysis restored most of the total CYP heme that was lost due to treatment with either endogenous (CME) or exogenous (SNAP) -NO. It is important to note that this restoration results in reappearance of virtually only the peak at 450 nm [no increase at 420 nm (Kim et al., 1995)]. This result demonstrates that the -NO exposure results in liberation of heme from CYP proteins, and that the apoproteins remain relatively intact after heme removal and available for heme reconstitution to the native heme coordination. 

Figure 2 depicts a working hypothesis for these results. Activation of NO synthesis results in nitrogen oxide-induced loss of protein-bound heme from CYP proteins, which remain relatively intact. This heme liberation results in a decrease in heme synthesis (decreased ALAS) and an increase in heme degradation (increased HO). In addition, -NO synthesis results in direct inhibition of ferrochelatase, which further contributes to inhibition of heme synthesis. There also appears to be a mechanism to repair or resynthesize CYP after -NO synthesis is inhibited. Finally, a result of this effect may be protection against cellular injury, since increased HO is an important response against cellular injury from a variety of insults. 

Figure 9.17 Left Percentage of total CYP protein content (pmol/mg) in one sample of pooled HLMs. Numbers don t add up to 100, since not all CYPs are shown. Right Contribution of individual CYPs to the metabolism of403 marketed drugs. Note how a disproportionately large amount of this pie belongs to only two enzymes, CYP3A4 and CYP2D6.

Figure 10.3 Formation and possible fates of a reactive metabolite. Once formed at the active site of a CYP, it might dissociate and go on to form a covalent adduct with glutathione (GSH) or nucleophilic groups in cellular components. Alternately, reaction at the active site of the CYP can lead to quasi-irreversible inhibition by complexation with the iron atom of heme or to irreversible CYP inhibition by covalent bond formation either with the heme unit or with a nucleophilic component of the CYP protein.

He XJ, Ejiri N, Nakayama H, Doi K (2005) Changes in cytochrome P450 isozymes (CYPs) protein levels during lactation in rat liver. Exp Mol Pathol 79 224-228... 

Plasma protein binding should be accounted for in the development of tissue blood partition coefficients and microsomal and CYP protein binding in the development of metabolic rate constants. Plasma protein binding was accounted for in the development of tissue blood partition coefficients in Appendix D (Berezhkovskly 2004a Poulin and Thiele 2000). values may need to be adjusted for any nonspecific microsomal or CYP binding that occurs during kinetic measurements (Austin et al. 2002). 

Kni by the substrate (decimal amount) that is unbound to microsomal or CYP protein. Mirfazaelian et al. (2006) used flow- and diffusion-limited compartments in their deltamethrin model. The addition of permeability areas (PA) having diffusion limits, such as the fat and slowly perfused compartments, enabled the investigators to bring model predictions in line with in vivo data. 

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