Stability metabolic

In vitro methods for the determination of metabolic stability provide a cost-effective solution to analyze the wealth of samples encountered in a drug discovery environment. Detail is sacrificed so that approximate and/or relative results can be obtained with significant savings in resources (i.e., instrumentation, animals) and time. 

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%). 

The LC-MS methods introduced by Ackermann et al. and Korfmacher et al. paved the way for more powerful platforms for high-throughput metabolic stability screening that featured the use of LC-MS/MS [80,92], These powerful methods relied on robotic sample preparation systems that were integrated with an automated LC-MS/MS system. 

Together with the site of metabolism, the rate of metabolism is a fundamental factor since this will contribute to the rate of elimination of the compound from the body. The rate of metabolism can be measured using different in vitro systems (cytochrome recombinant, human liver microsome, human hepatocites, etc). Normally, the data are produced using different compounds and the rate values are extracted from complex systems like liver microsomes, or human hepatocites. 

Often the lack of homogenity of the data source and experimental methods makes derivation of predictive computational models too complex. 

Predictive models can be better produced when recombinant cytochrome data are available, an experimental technique which may increase the probability of obtaining consistent and predictive models, since one protein is involved in the metabolic reaction. The most accurate data to describe the rate and affinity of the ligand towards an enzyme are the kinetic parameters Vmax and Km. Nevertheless, the calculation of these parameters is time consuming. A less precise parameter is the determination of the compound percentage remaining in a cytochrome incubation after a certain period of time. These metabolic data are less accurate and can only be used to classify the compounds in a metabolic system as stable or unstable. This type of data was the basis for a predictive model of metabolic stability towards CYP3A4 [35]. 

Compound AP40 IC50 (nM) Selectivity vs. notch inhibition In vitro T1/2 Tg2576 mice (min) In vitro T1/2 human (min) % A 40 reduction 4 h (po) % Ap42 reduction 4 h (po) 

Incorporation of fluorine at a site adjacent to a metabolic soft spot has also been used as a strategy to increase duration of action. Linopir-dine (24) was among the first clinical compounds that enhanced potassium-evoked release of acetylcholine in preclinical models of AD [22]. Linopirdine showed no clinical efficacy and its human pharmacokinetic profile was suggested as the reason for this lack of clinical efficacy. Specifically noted was the molecule s poor brain exposure and short half-life due to formation of the N-oxides 25 and 26 (Table 3) [23,24]. Optimization of 24 resulted in replacement of the indolone core by the anthracenone 27, which had improved in vitro activity, but still exhibited a short duration of action. To improve the metabolic stability, fluorine 

An alternative set of non-specific enzymatic reactions can reveal masked polar functional groups which might be present in a drug. For example, there are enzymes which can demethylate a methyl ether to reveal a more polar hydroxyl group. Once again, the more polar product (metabolite) is excreted more efficiently. 

These reactions are classed as phase I reactions in the overall process of drug metabolism. They generally involve oxidation, reduction, and hydrolysis (Fig. 8.1). 

The structures most prone to oxidation are AT-methyl groups, aromatic rings, the terminal positions of alkyl chains, and the least hindered positions of alicyclic rings. 

Nitro and carbonyl groups are prone to reduction by reductases, whilst amides and esters are prone to hydrolysis by esterases. 

There is also a series of metabolic reactions classed as phase II reactions (Fig. 8.2). These are conjugation reactions whereby a polar molecule is attached to a suitable polar handle which is either already present on the drug or has been placed there by a phase I reaction. The resulting conjugate has increased polarity, thus increasing its excretion rate in urine or bile even further. 

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An ideal" drug must be safe and effective. The mavimum daily dose should not exceed 200-300 mg. Drugs should be well absorbed orally and bioavailablc. Metabolic stability ensures a reasonable long half-life. Further on, a drug should be non-... 

Shen M, Xiao Y, Golbraikh A, Gombar VK, Tropsha A. Development and validation of k-nearest-neighbor QSPR models of metabolic stability of drug candidates. J Med Chem 2003 46 3013-20. 

Bnrsi R, de Gooyer ME, Grootenhuis A, Jacobs PL, van der Louw J, Leysen D. (Q)SAR stndy on the metabolic stability of steroidal androgens. J Mol Graph Modelling 2001 19 552-6. 

When assayed in HEK293 cells transfected with the cloned human, rat and guinea pig TRPVl, (23a) showed similar potencies. Not unexpeetedly, (23a) showed poor metabolic stability and a structure-activity study to optimize potency and drug-like properties was initiated. Modification on the left-handed A -aryl section showed that ... 

Drug candidates that are intended for oral dosing need to have good ADME properties so that they can be dosed once or twice daily. The drug should be well absorbed, survive first pass metabolism, and have sufficiently low clearance. At the lead identification stage, the primary in vitro ADME assays employed are those that assess permeability and metabolic stability. There are a variety of assays available for both parameters, as described in the previous chapter. 

Abadji Y, Lin SY, Taha G, Griffin G, Stevenson LA, Pertwee RG, Makri-yannis A. (R)-Methanandamide a chiral novel anandamide possessing higher potency and metabolic stability. J Med Chem 1994 37 1889-1893. 

Goutopoulos A, Fan P, Khanolkar AD, Xie XQ, Lin SY, Makriyannis A. Stereochemical selectivity of methanandamides for the CB1 and CB2 cannabinoid receptors and their metabolic stability. Bioorg Med Chem 2001 9 1673-1684. 

Biological activity is not the only criterion required for drug development, as anyone who has been involved in this area is aware. Potency, toxicity, bioavailability, metabolic stability, and plasma half-life are only a few of the critical issues that must be addressed. Although satisfactory potency and spectrum activity had been achieved with WIN54954, which has been clinically evaluated, this compound lacked metabolic stability and consequently displayed a short half-life. 

The question at this point was whether modifications could be made to the oxadiazole molecule to enhance metabolic stability and achieve comparable activity. This approach required knowledge of the site of metabolism and the nature of the metabolic products. This information was obtained from ion mass spectrometry. The identity of these products was determined by comparing the fragmentation pattern of metabolites A and B with the parent compound and the corresponding daughter ions (Fig. 25). 

Kauffman, S. (1969) Metabolic stability and epigenesis in randomly constructed genetic nets. /. Theoret. Biol., 22 437-467, Elsevier, Amsterdam. 

The development of synthetic methods for the selective introduction of short-chain perfluoroalkyl groups into organic molecules is of interest in drug development [464]. Fluoromodifications often confer unique properties on a molecule, for example in terms of increased metabolic stability and lipophilicity and, as a consequence, the pharmacokinetic profiles are often improved [465]. Burger and coworkers developed a domino process consisting of a SN reaction combined with a Claisen and a Cope rearrangement which allows the transformation of simple fluorinated compounds into more complex molecules with fluoro atoms [466]. Treatment of furan 2-917 with 2-hydroxymethyl thiophene (2-918) in the presence... 

Kubiak, T.M., Maule, A.G., Marks, N.J., Martin, R.A. and Weist, J.R. (1996) Importance of the proline residue to the functional activity and metabolic stability of the nematode FMRFamide-related peptide, KPNFIRFamide (PF4). Peptides 17, 1267-1277. 

Mandagere, A. K., T. N. Thompson, and K. K. Hwang. Graphical model for estimating oral bioavailability of drugs in humans and other species from their Caco-2 permeability and in vitro liver enzyme metabolic stability rates, J. Med. Chem. 2002, 45, 304-311... 

In the following section, the calculation of the VolSurf parameters from GRID interaction energies will be explained and the physico-chemical relevance of these novel descriptors demonstrated by correlation with measured absorption/ distribution/metabolism/elimination (ADME) properties. The applications will be shown by correlating 3D molecular structures with Caco-2 cell permeabilities, thermodynamic solubilities and metabolic stabilities. Special emphasis will be placed on interpretation of the models by multivariate statistics, because a rational design to improve molecular properties is critically dependent on an understanding of how molecular features influence physico-chemical and ADME properties. 

Metabolic stability in human liver preparations may represent a suitable experimental tool to obtain relevant pharmacokinetic information in an early phase of drug discovery. Compounds which are metabolized principally by the liver can be identified and studied in more detail to determine the principal sites of biotransformation. 

The aim of the present example was to investigate whether the assessment of an in silico model of metabolic stability from a training set of several hundred drugs or drug-like compounds in human CYP3A4 cDNA-expressed microsomal preparations, would offer a suitable approach to predict the metabolic stability of external compounds. 

A simple protocol was used to build the compounds compounds were modeled with the corresponding net charges, after which 2D-3D structure conversion was carried out using the program Concord [21]. The 3D dataset obtained was submitted to the VolSurf program, and principal component analysis (PCA) was applied for chemometric interpretation. No metabolic stability information was applied to the model. 

Two significant principal components were extracted. The score plot of the two principal components is shown in Fig. 17.5, where the compounds are color-coded according to their metabolic stability (filled points represent unstable compounds,... 

Fig. 17.5. PCA score plot for the example on metabolic stability. Filled points represent compounds with metabolic stability <20% open points refer to compounds with metabolic stability >90%. (See text for explanation.)...

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