Evaluation of human intestinal absorption data and subsequent derivation of a quantitative structure-activity relationship (QSAR) with the Abraham descriptors.

The human intestinal absorption of 241 drugs was evaluated. Three main methods were used to determine the human intestinal absorption: bioavailability, percentage of urinary excretion of drug-related material following oral administration, and the ratio of cumulative urinary excretion of drug-related material following oral and intravenous administration. The general solvation equation developed by Abraham's group was used to model the human intestinal absorption data of 169 drugs we considered to have reliable data. The model contains five Abraham descriptors calculated by the ABSOLV program. The results show that Abraham descriptors can successfully predict human intestinal absorption if the human absorption data is carefully classified based on solubility and administration dose to humans.

Molecular modelling of human CYP2E1 by homology with the CYP102 haemoprotein domain: investigation of the interactions of substrates and inhibitors within the putative active site of the human CYP2E1 isoform.

1. The construction of a three-dimensional model of human CYP2E1 is reported. It is based on homology with the haemoprotein domain of the unusual bacterial P450, CYP102, which is of known crystal structure. 2. Interactive docking of a number of human CYP2E1 substrates is consistent with their known positions of CYP2E1-mediated metabolism, where specific interactions with key active site amino acid side-chains appear to rationalize the binding and orientation of substrate molecules. 3. Amino acid residues within the putative active site of human CYP2E1, including those associated with the binding of substrates and inhibitors, are shown to correspond with those identified by site-directed mutagenesis experiments conducted on CYP2 family isoforms, and they are known to affect substrate metabolism regioselectivity. 4. Consequently, it was found that the CYP2E1 active site exhibits complementarity with the structural characteristics of known substrates and inhibitors of this enzyme, including their relatively low molecular weights and disposition of hydrogen bond-forming groups.

Molecular modelling of CYP1 family enzymes CYP1A1, CYP1A2, CYP1A6 and CYP1B1 based on sequence homology with CYP102.

Molecular modelling of a number of CYP1 family enzymes from rat, plaice and human is described based on amino acid sequence homology with the haemoprotein domain of CYP102, a unique bacterial P450 of known structure. The interaction of various substrates and inhibitors within the putative active sites of rat CYP1A1, human CYP1A2, a fish CYP1 enzyme CYP1A6 (from plaice) and human CYP1B1, is shown to be consistent with P450-mediated oxidation in each example or, in the case of inhibitors, mechanism of inhibition. It is reported that relatively small changes between the enzymes' active site regions assist in the rationalization of CYP1 enzyme preferences for particular substrate types, and a template of superimposed CYP1A2 substrates is shown to fit the putative active site of the human CYP1A2 enzyme.

Molecular modelling of the human cytochrome P450 isoform CYP2A6 and investigations of CYP2A substrate selectivity.

(1) The generation of a homology model of CYP2A6, the major catalyst of human hepatic coumarin 7-hydroxylase activity, involves the use of the recently published substrate-bound CYP102 crystal structure as a template. (2) A substantial number of structurally diverse CYP2A6 substrates are found to dock satisfactorily within the putative active site of the enzyme, leading to the formulation of a structural template (or pharmacophore) for CYP2A6 specificity/selectivity. (3) The CYP2A6 model is consistent with available evidence from site-directed mutagenesis studies carried out on CYP2A subfamily isoforms, and enables some explanation of species differences in CYP2A-mediated metabolism of certain substrates. (4) Quantitative structure-activity relationship (QSAR) analysis of CYP2A5 (the mouse orthologue) mutants yields statistically significant correlations between various properties of amino acid residues and coumarin 7-hydroxylase activity.

First principles investigation of singly reduced cytochrome P450.

1. The application of novel ab initio quantum mechanical methods to the states in the catalytic cycle of cytochrome P450 following the first reduction step is described. 2. A good correlation was found between the calculated energy of reduction and the experimentally determined redox potential for a range of substrate- and substrate analogue-bound systems. 3. On reduction of the haem system, the ground state of Fe remains Fe3+. On binding of a CO molecule, Fe adopts a low-spin Fe2+ state, in agreement with experiment. However, on binding of an O2 molecule, calculations indicate that the system adopts a ferric superoxide ground state, in which the Fe is in a low-spin Fe3+ state.

Molecular modelling of CYP2B6, the human CYP2B isoform, by homology with the substrate-bound CYP102 crystal structure: evaluation of CYP2B6 substrate characteristics, the cytochrome b5 binding site and comparisons with CYP2B1 and CYP2B4.

1. Molecular modelling studies of CYP2B isoforms from rat (CYP2B1), rabbit (CYP2B4) and man (CYP2B6) are reported, with particular emphasis on substrate interactions with the human CYP2B isoform, CYP2B6. 2. The findings represent an advance on our previous study that focused primarily on the rat CYP2B isoform, CYP2B1, and involved homology modelling with substrate-free CYP102. 3. The current work utilizes the recently published substrate-bound CYP102 crystal structure as a template for construction of the CYP2B subfamily isoforms and shows, in particular, that known CYP2B6 substrate specificity and regioselectivity can be rationalized by putative active site interactions.

Cytochrome P450 substrate specificities, substrate structural templates and enzyme active site geometries.

The structural characteristics of human cytochrome P450 substrates are outlined in the light of extensive studies on P450 substrate specificity. Templates of superimposed substrates for individual P450 isozymes are shown to fit the corresponding enzyme active sites, where contacts with specific amino acid residues appear to be involved in the interaction with each structural template. Procedures leading to the evaluation of likely P450 specificity, binding affinity and rate of metabolism are described in the context of key examples in which molecular modelling appears to rationalize experimentally observed findings.

Structural determinants of cytochrome P450 substrate specificity, binding affinity and catalytic rate.

The structural characteristics of cytochrome P450 substrates are summarised, showing that molecular descriptors can discriminate between chemicals of differing P450 isozyme specificity. Procedures for the estimation of P450 substrate binding interaction energies and rates of metabolism are described, providing specific examples in both individual compounds binding to P450s, including those of known crystal structure, and within series of structurally related chemicals. It is demonstrated that binding energy components are primarily hydrophobic/desolvation and electrostatic/hydrogen-bonded in nature, whereas electronic factors are of importance in determining variations in reaction rates. It is thus shown that the prediction of P450 substrate binding affinities and catalytic rates may be feasible, provided that sufficient structural information is available for the relevant enzyme-substrate complex.

Molecular modelling of human CYP2C subfamily enzymes CYP2C9 and CYP2C19: rationalization of substrate specificity and site-directed mutagenesis experiments in the CYP2C subfamily.

1. The results of molecular modelling of human CYP2C isozymes, CYP2C9 and CYP2C19, are reported based on an alignment with a bacterial form of the enzyme, CYP102. 2. The three-dimensional structures of the CYP2C enzymes are consistent with known experimental evidence from site-directed mutagenesis, antibody recognition and regiospecificity of substrate metabolism. 3. The variations in substrate specificity between CYP2C9 and CYP2C19 can be rationalized in terms of single amino acid residue changes within the putative active site region, of which I99H appears to be the most significant.

Molecular modelling of cytochrome P4502D6 (CYP2D6) based on an alignment with CYP102: structural studies on specific CYP2D6 substrate metabolism.

1. A molecular model of CYP2D6 has been constructed from the bacterial form CYP102 via a homology alignment between the CYP2D subfamily and CYP102 protein sequences. 2. A number of typical CYP2D6 substrates are shown to fit the putative active site of the enzyme, as can the specific inhibitor quinidine. 3. Some of the allelic variants in CYP2D6, which give rise to genetic polymorphisms in 2D6-mediated metabolism, can be rationalized in terms of their position within the active site region. 4. The results of site-directed mutagenesis experiments are consistent with the CYP2D6 model generated from the CYP102 crystal structure. 5. The possibility of an alternative orientation within the active site may explain the CYP2D6-mediated metabolism of relatively large-sized substrates.

Molecular modelling of CYP3A4 from an alignment with CYP102: identification of key interactions between putative active site residues and CYP3A-specific chemicals.

1. A structural model of CYP3A4 is reported on the basis of a novel amino acid sequence alignment between the CYP3 family and CYP102, a bacterial P450 of known crystal structure. 2. Construction of the CYP3A4 model from CYP102 is facilitated by the relatively high sequence homology between the two protein (52% homology; 27% identity) with many conservative amino acid changes, yielding a structure of low internal energy. 3. A considerable number of specific substrates, and some specific inhibitors, are shown to occupy the putative CYP3A4 active site via interactions with the same amino acid residues in almost all cases investigated. 4. The CYP3A4 model rationalizes the known positions of metabolism for many substrates of this major human P450 such that the route of metabolism in novel development compounds can be predicted.

Absorption and disposition of ranitidine hydrochloride in rat and dog.

1. The pharmacokinetics of ranitidine were studied in the male beagle dog at a dose level of 50 mg (intravenous) or 5 mg/kg (oral). 2. After intravenous administration, Clp was moderate (10.4 ml/min/kg) with Clr accounting for approximately 30% of total clearance. Vdarea was 3.5 l/kg, resulting in a t1/2 of approximately 4 h. 3. After oral administration, F was good (73%) with peak plasma concentrations of ranitidine (2 micrograms/ml) achieved within 0.5-1 h hour after dosing. t1/2 (4.1 h) was similar to that observed after intravenous administration. 4. The absorption, metabolism and excretion of [14C]-ranitidine were studied in rat and dog after oral administration at a dose level of 50 mg/kg. 5. Urinary excretion was the major elimination pathway for radioactive drug-related material in both species (62-75% of the dose). Unchanged ranitidine was the major radioactive component in both rat and dog urine (0-24 h), accounting for approximately 40% of the dose in each case. 6. In dog, ranitidine undergoes N-oxidation (approximately 30% of dose) whereas in rat, N-oxidation, S-oxidation, N-demethylation and oxidative deamination are all evident, with each metabolite accounting for < 6% of the dose. 7. Two previously unreported metabolites of ranitidine were identified in rat urine using newly developed hplc and lc/ms methods. These metabolites result from single and di-N-demethylation of ranitidine and accounted for 4 and 1% of the dose respectively.

The aliphatic oxidation of salmeterol to alpha-hydroxysalmeterol in human liver microsomes is catalyzed by CYP3A.

Salmeterol xinafoate (Serevent) is a long-acting beta2-adrenoceptor agonist, used in the treatment of asthma, that has bronchodilator and anti-inflammatory action. Salmeterol is extensively metabolized by aliphatic oxidation in humans, with the major metabolite being alpha-hydroxysalmeterol. The aim of this investigation was to identify the specific cytochrome P450 (P450) isoform or isoforms involved in the formation of alpha-hydroxysalmeterol in human liver microsomes. [14C]Salmeterol was incubated with a pooled sample (N = 19) of human liver microsomes in the absence or presence of selective chemical inhibitors of the major human P450 isoforms. One microM ketoconazole, a selective inhibitor of CYP3A, substantially inhibited the metabolism of salmeterol to alpha-hydroxysalmeterol. Disulfiram caused a small but consistent decrease in the amount of alpha-hydroxysalmeterol formed, possibly reflecting less than total selectivity for CYP2E1 under the conditions used. Other selective inhibitors had no significant effect on the metabolism of salmeterol. The rates of formation of alpha-hydroxysalmeterol in 10 individual liver microsomal samples showed an approximately 10-fold variation and were found to be highly correlated (r2 = 0.94; p < 0.001) with rates of metabolism of midazolam to 1'-hydroxymidazolam, a marker of CYP3A activity, in the same microsomal samples. No significant correlation was evident for the metabolism of salmeterol with levels of total P450 or other markers of human P450 activities in the same microsomal samples, thus indicating that the formation of alpha-hydroxysalmeterol is catalyzed predominantly by CYP3A. Insect cell microsomes that coexpressed human CYP3A and NADPH-P450 reductase were able to metabolize [14C]salmeterol to alpha-hydroxysalmeterol, thus confirming the role of CYP3A in catalyzing this reaction. The therapeutic dose of salmeterol is very low, so it is unlikely that any clinically relevant interactions will be observed as a consequence of the coadministration of salmeterol and other pharmaceutical agents that are metabolized by CYP3A.

Microbial biotransformation of the angiotensin II antagonist GR117289 by Streptomyces rimosus to identify a mammalian metabolite.

Screening a range of microorganisms incubated with the angiotensin II antagonist GR117289 resulted in the use of Streptomyces rimosus to generate five related biotransformation products. These comprised three compounds hydroxylated on the aliphatic side chain, one further oxidized to a ketone, and one hydroxylated on the phenyl ring. These microbial metabolites were used as standards to identify a human metabolite detected in plasma and urine, but present in insufficient quantities for full structural characterisation. This further demonstrates how the use of microbial biotransformation systems at an early stage of drug metabolism studies can act as a valuable tool in facilitating identification of minor human metabolites.

Characterization of glucuronic acid conjugates of a novel angiotensin receptor antagonist.

Nanogram quantities of glucuronic acid conjugates of GR117289 in rat and dog bile have been analysed by semi-microbore high-performance liquid chromatography (HPLC)/ionspray mass spectrometry with on-line UV diode array detection. The determination of drug metabolites in bile has often proved problematical due to the large number of endogenous components in this biological matrix, in particular the bile acids. Semi-microbore HPLC is useful for concentrating small quantities of material and, in combination with an on-line diode array detector, for distinguishing between drug related and endogenous components. A novel angiotensin II receptor antagonist, GR117289, had proved difficult to analyse by thermospray mass spectrometry because of its thermal lability. The use of the less thermally dependent technique of ionspray mass spectrometry allowed the characterization of nanogram quantities of glucuronic acid metabolites of GR117289 in bile.