CYP inhibition

Reactive Metabolite Formation, Mechanism-Based CYP Inhibition,... 

A lead is variously defined in the pharmaceutical industry as a compound derived from a hit with some degree of in vitro optimization (potency in primary assay, activity in functional and/or cellular assay), optimization of physical properties (solubility, permeability), and optimization of in vitro ADME properties (microsomal stability, CYP inhibition). Moreover, a lead must have established SAR/SPR around these parameters such that continued optimization appears possible. A lead may also have preliminary PK and in vivo animal model data. However, it is the task of the lead optimization chemist to improve PK and in vivo activity to the levels needed for identification of a clinical candidate. 

Gentest (now BD Biosciences) was the first to develop spectrophotometric assays to study CYP inhibition [98]. These assays are based on the turnover of mildly fluorescent substrate probes to moderately fluorescent metabolites, where metabolite formation is monitored by an increase in fluorescence using a plate reader [99,100]. Problems with these methods include background interference due to low signal-to-noise ratio, chemotype-specific interference and fluorescence quenching. Aurora Biosciences (now Vertex) has designed probes that exhibit larger fluorescence... 

Moody, G.C., Griffin, S.J., Mather, A.N., McGinnity, D.F. and Riley, R.J. (1999) Fully automated analysis of activities catalysed by the major human liver cytochrome P450 (CYP) enzymes assessment of human CYP inhibition potential. Xenobiotica, 29 (1), 53-75. 

H. and Pelkonen, O. (2006) Cytochrome P450 (CYP) inhibition screening comparison of three tests. European... 

The are several clearance and toxicological aspects that have to be considered in the drug discovery process such as metabolic stability, enzyme selectivity, CYP inhibition and type of inhibition. Among these factors, the prediction of the site of metabolism has become one of the most successful parameters for prediction. The knowledge of the site of metabolism enhances the opportunity to chemically modify the molecule to improve the metabolic stability. There are several approaches based on database mining, chemical reactivity, protein interaction or both that have been developed for the prediction of this property, with different degree of success and applicability. 

To develop safer drugs, it is important to identify irreversible CYP inhibitors at an early stage in the drug discovery process. Certain substructures, reviewed in this chapter, are frequently responsible for irreversible CYP inhibition and either their use in medicinal chemistry programs should be limited or their potential for irreversible binding to CYPs should be carefully monitored. 

Irreversible CYP inhibition can arise from different chemical mechanisms. However, a common initial step is the metabolic activation of a substrate into a reactive metabolite that is trapped within the active site of the CYP to form a tightly bound complex causing a long-lasting inactivation of enzyme activity. Enzymatic activity can be restored only through the new synthesis of the enzyme. For this reason, irreversible CYP inhibition is often referred to as mechanism-based inhibition , metabolite-based inhibition or suicide inhibition . 

Mechanism-based CYP inhibition is time dependent, as it is a consequence of metabolic activation. For the same reason, it also depends on substrate concentration. It also, usually, depends on the presence of NAD PH in the incubation system (or an NADPH regeneration system), as most of the CYP-catalyzed reactions require NADPH to occur [5],... 

A rare but serious event that can result from irreversible CYP inhibition is the development of a hypersensitivity reaction. The bioactivation of a drug and the formation of a covalent adduct between the activated substrate and the enzyme can lead to hapten formation and eventually to an idiosyncratic autoimmune response (usually in the form of autoimmune hepatitis) [14]. The hapten formation is the first key step toward the autoimmune response. The CYP macromolecule is made immunogenic ( foreign ) by the covalent binding of the electrophilic metabolites, and the immune reaction follows with the production of autoantibodies against the target molecule (not necessarily alkylated). 

Structural Features Often Responsible for Mechanism-Based CYP Inhibition 271... 

Irreversible CYP inhibition, especially when considered as a cause of chemically reactive metabolites, raises a number of issues that should be considered early in the drug discovery process. Careful evaluation of the structural features of the lead series as well as screening for MBIs early in the drug development process should guide series selection and optimization with the goal of avoiding the introduction of potentially risky moieties into new chemical entities. 

Different food components (e.g., fruit extracts) and commonly used pharmaceutical excipients (e.g., Tween 80) can interfere with secretory transport systems, leading to drug-food interactions [31, 43, 46, 167], As for CYPs, inhibition and induction of efflux transporters have been reported as possible... 

LC/MS/MS with selected reaction monitoring (SRM) offers a fast and simple means to analyze biological matrices, which is a key factor in high-throughput CYP inhibition screens using liver microsomes. Potentially, the LC/MS/MS technique is suitable for analyses of cocktail substrates in other in vitro drug metabolism evaluations such as CYP induction/activation assays, rapid analysis of pooled liver microsomes, rapid reaction phenotyping of tissue (hepatic and extrahepatic) samples, as well as evaluation of hepatocytes/tissue slice CYP activity. ° ... 

Human Biologies International offers various in vitro data, screens (Hepato-Screen ), kits, and analysis software (HepatoSoft ) for CYP inhibition, reactions, and stability (323). 

Ricerca offers in vivo and in vitro ADMET services. Specifically, it provides in vitro assays for CYP inhibition, metabolism, ADME, pharmacokinetic, and metabolism profiling (330). 

The key descriptors used to predict the likelihood of a CYP inhibition are incredibly vast. Descriptors can, for example, be defined as spatial, electronic or conformational. Some of the key structural properties include lipophilicity, pKa and PSA and are relatively easy to interpret, while others are more complex. For further information on descriptors used in DDI modeling, please refer to De Groot et al. [6]. 

Table 8.2 Comparison of pharmacological target and CYP inhibition potencies for selected oral drugs, in order of DDI magnitude. Data compiled from [104-106],...

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