Enzymes, CYP

The numerous biotransformations catalyzed by cytochrome P450 enzymes include aromatic and aliphatic hydroxylations, epoxidations of olefinic and aromatic structures, oxidations and oxidative dealkylations of heteroatoms and as well as some reductive reactions. Cytochromes P450 of higher animals may be classified into two broad categories depending on whether their substrates are primarily endogenous or xenobiotic substances. Thus, CYP enzymes of families 1-3 catalyze... 

The assessment of clearance is complicated by the numerous mechanisms by which compounds may be cleared from the body. These mechanisms include oxidative metabolism, most commonly by CYP enzymes, but also in some cases by other enzymes including but not limited to monoamine oxidases (MAO), flavin-containing monooxygenases (FMO), and aldehyde oxidase [45, 46], Non-oxidative metabolism such as conjugation or hydrolysis may be effected by enzymes such as glucuronyl transferases (UGT), glutathione transferases (GST), amidases, esterases, or ketone reductases, as well as other enzymes [47, 48], In addition to metabolic pathways, parent compound may be excreted directly via passive or active transport processes, most commonly into the urine or bile. 

Other assays for assessing CYP clearance are also employed, although often less widely or with lower compound throughput. Recombinant CYP enzymes allow the determination of the kinetic parameters for metabolism of individual compounds by individual CYPs. Recombinant CYPs also provide an avenue to assessing and understanding the potential for drug-drug interactions that may occur between two or more compounds. 

Disulfiram inhibits CYP enzymes 1A2, 2C9, and 3A4 many benzodiazepines are metabolized via these pathways lorazepam, temazepam, and oxazepam are NOT metabolized via the CYP4S0 system and are reasonable alternatives. 

Most pharmacokinetic interactions in transplantation occur due to interactions with the CYP enzyme system however, several interactions have been shown to occur via alternative mechanisms. One of the most notable is that seen between tacrolimus and some of the prokinetic agents. Cisapride and metoclopramide have been shown to increase the absorption of tacrolimus by enhancing gastric emptying.41... 

Ethnic differences have been shown to influence response to psychotropic medications. Much of the focus on the explanation for such differences has been on drug-metabolizing (CYP) enzymes of the liver and their sway over pharmacokinetic factors. It is now well recognized that differences in the distribution of polymorphic variants of CYP enzymes exist between different ethnic groups. However, within ethnic groups there are considerable inter-individual variations in drug kinetics, which may not be accounted for solely by genetic variation. Responses to pharmacotherapy are multifaceted and involve the interaction of environmental and... 

Advantages of Using CYP Enzymes for Producing Drug Metabolites... 

An alternative to chemical synthesis is to use human CYP enzymes to generate the desired human drug metabolites. Various means of making human P450s have been used, all with certain drawbacks [81]. The most common source is pooled HLMs, which has been described in detail previously, but these microsomal preparations contain a mixture of many different enzymes, and their cost, batch-to-batch variability in activity and restrictions on availability can limit the usefulness of HLMs for preparative synthetic work. These limitations can become particularly acute when the required amount of a pure metabolite exceeds 5-10 mg. 

Human CYPs are multicomponent enzyme systems, requiring at a minimum the CYP enzyme component and a reductase component to be functional. The reductase requires a reduced nicotinamide cofactor, typically NADPH, and this cofactor must be regenerated to provide a steady supply of reducing equivalents for the reductase. Regeneration is accomplished with a separate substrate and enzyme. Glucose-6-phosphate and glucose-6-phosphate dehydrogenase have been widely used for this purpose. The overall complexity of the reaction mixtures and their cost have been barriers to the widespread use of recombinant human CYPs for metabolite synthesis in the past. 

All the major human liver CYPs are available as HCBs, allowing each human CYP enzyme to be screened in parallel to determine which one is the active enzyme for metabolism of a given drug. Once identified, the most active human CYP can be scaled up and used to produce larger quantities of the desired metabolite. 

A reaction mixture containing 25 mL MicroCyp reaction mix, 1 /am MicroCyp variant 1, and 1 mM diclofenac were placed into 250 mL baffled Erlenmeyer flasks and incubated with agitation at 30 °C. The reaction was complete in less than an hour, reaching 75% conversion (0.23 g L J). The concentration of product achieved in this reaction with the BM3 mutant CYP enzyme represents an improvement of more than an order of magnitude compared with the reaction carried out with the recombinant human CYP2C9 (0.02 gL 1). 

The catalytic activity of CYP enzymes requires functional coupling with its redox partners, cytochrome P450 NADPH oxidoreductase (OR) and cytochrome bs. Measurable levels of these two proteins are natively expressed in most cell lines. Therefore, introduction of only the CYP cDNA is generally needed for detectable catalytic activity. However, the levels of expression of the redox partner proteins may not support maximal CYP catalytic activity, and therefore enhancement of OR levels may be desirable. This approach has been used successfully with an adenovirus expression system in LLC-PKi cells [12],... 

HU, a freely water-soluble molecule, crosses the intestinal wall and other cells by passive diffusion [5, 6], and tissue concentration of HU rapidly matches its blood concentration [7]. The oral bioavailability of HU is nearly complete and hence therapeutically simple to administrate. HU undergoes biotransformation and is converted into urea by a yet-to-be identified hepatic P450 monooxygenase (CYP) enzyme [8, 9], Elimination of HU and its metabolites involves both renal and non-renal mechanisms. 

The majority of CYP enzymes are located in a hydrophobic environment in the endoplasmic reticulum of cells, although cytosolic enzymes also exist, such as CYP101. In order to mimic the physiological environment of CYP enzymes, a number of groups have used phospholipids to construct biosensors such as DDAB, dimeristoyl-L-a-phosphatidylcholine (DMPC), dilauroylphosphatidylethanolamine (DLPE) and distearoylphosphatidylethanolamine (DSPE). Phospholipid layers form stable vesicular dispersions that bear structural relationship with the phospholipid components of biologically important membranes. By this way a membranous environment is created that facilitates electron transfer between the enzyme s redox center and the electrode. 

Besides catalyzing styrene and benzaldehyde, CYP enzymes play an important role in the metabolism of endogenous compounds as well as in pharmacokinetics and toxicokinetics. Joseph [228] developed a biosensor with human CYP3A4 as a novel drugscreening tool. It was constructed by assembling enzyme films on Au electrodes by alternate adsorption of a layer of CYP3A4 on top of a layer of PDDA. The biosensor was applied to detect verapamil, midazolam, quinidine, and progesterone. 

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