Lactonization is the critical first step in the disposition of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor atorvastatin.

In an in vitro study, we compared the cytochrome P450 (CYP)-dependent metabolism and drug interactions of the acid and lactone forms of the 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor atorvastatin. Metabolism of atorvastatin acid and lactone by human liver microsomes resulted in para-hydroxy and ortho-hydroxy metabolites. Both substrates were metabolized mainly by CYP3A4 and CYP3A5. Atorvastatin lactone had a significantly higher affinity to CYP3A4 than the acid (K(m): para-hydroxy atorvastatin, 25.6 +/- 5.0 microM; para-hydroxy atorvastatin lactone, 1.4 +/- 0.2 microM; ortho-hydroxy atorvastatin, 29.7 +/- 9.4 microM; and ortho-hydroxy atorvastatin lactone, 3.9 +/- 0.2 microM). Compared with atorvastatin acid, CYP-dependent metabolism of atorvastatin lactone to its para-hydroxy metabolite was 83-fold higher [formation CL(int) (V(max)/K(m)): lactone 2949 +/- 3511 versus acid 35.5 +/- 48.1 microl. min(-1). mg(-1)] and to its ortho-hydroxy metabolite was 20-fold higher (CL(int): lactone 923 +/- 965 versus acid 45.8 +/- 59. 1 microl. min(-1). mg(-1)). Atorvastatin lactone inhibited the metabolism of atorvastatin acid by human liver microsomes with an inhibition constant (K(i)) of 0.9 microM while the K(i) for inhibition of atorvastatin by atorvastatin lactone was 90 microM. Binding free energy calculations of atorvastatin acid and atorvastatin lactone complexed with CYP3A4 revealed that the smaller desolvation energy of the neutral lactone compared with the anionic acid is the dominant contribution to the higher binding affinity of the lactone rather than an entropy advantage. Because atorvastatin lactone has a significantly higher metabolic clearance and the lactone is a strong inhibitor of atorvastatin acid metabolism, it can be expected that metabolism of the lactone is the relevant pathway for atorvastatin elimination and drug interactions. We hypothesize that most of the open acid metabolites present in human plasma are generated by interconversion of lactone metabolites.

Active transport of the angiotensin-II antagonist losartan and its main metabolite EXP 3174 across MDCK-MDR1 and caco-2 cell monolayers.

1. We studied the functional interaction between transport and metabolism by comparing the transport of losartan and its active metabolite EXP 3174 (EXP) across cell monolayers. 2. Epithelial layers of Caco-2 cells as well as MDR1, MRP-1 and MRP-2 overexpressing cells, in comparison to the respective wildtypes, were used to characterize the transcellular transport of losartan and EXP. 3. Losartan transport in MDCK-MDR1 and Caco-2 cells was saturable and energy-dependent with a significantly greater basolateral-to-apical (B/A) than apical-to-basolateral (A/B) flux (ratio=31+/-1 in MDCK-MDR1 and ratio 4+/-1 in Caco-2 cells). The B/A flux of losartan was inhibited by cyclosporine and vinblastine, inhibitors of P-glycoprotein and MRP. In contrast, no active losartan transport was observed in MRP-1 or MRP-2 overexpressing cells. 4. The metabolite was only transported in Caco-2 cells with a B/A-to-A/B ratio of 5+/-1, while lacking active transport in the MDR1, MRP-1 or MRP-2 overexpressing cells. The B/A flux of EXP was significantly inhibited by cyclosporine and vinblastine. 5. In conclusion, losartan is transported by P-glycoprotein and other intestinal transporters, that do not include MRP-1 and MRP-2. In contrast, the carboxylic acid metabolite is not a P-glycoprotein substrate, but displays considerably higher affinity for other transporters than losartan, that again most probably do not include MRP-1 and MRP-2.

Conserved extracellular cysteine pair in the M3 muscarinic acetylcholine receptor is essential for proper receptor cell surface localization but not for G protein coupling.

Most G protein-coupled receptors contain a conserved pair of extracellular cysteine residues that are predicted to form a disulfide bond linking the first and second extracellular loops. Previous studies have shown that this disulfide bond may be critical for ligand binding, receptor activation, and/or proper receptor folding. However, the potential importance of the two conserved cysteine residues for proper receptor cell surface localization has not been investigated systematically. To address this issue, we used the rat M3 muscarinic receptor as a model system. Most studies were carried out with a modified version of this receptor subtype (lacking potential N-glycosylation sites and the central portion of the third intracellular loop) that could be readily detected via western blot analysis. Cys-->Ala mutant receptors were generated, transiently expressed in COS-7 cells, and then examined for their subcellular distribution and functional properties. ELISA and immunofluorescence studies showed that the presence of both conserved cysteine residues (corresponding to C140 and C220 in the rat M3 muscarinic receptor sequence) is required for efficient expression of the M3 muscarinic receptor on the cell surface. On the other hand, these residues were found not to be essential for protein stability (determined via immunoblotting) and receptor-mediated G protein activation (studied in second messenger assays). These results shed new light on the functional role of the two extracellular cysteine residues present in most G protein-coupled receptors.

Grapefruit juice activates P-glycoprotein-mediated drug transport.

PURPOSE: Grapefruit juice (GJ) is known to increase the oral bioavailability of many CYP3A-substrates by inhibiting intestinal phase-I metabolism. However, the magnitude of AUC increase is often insignificant and highly variable. Since we earlier suggested that CYP3A and P-glycoprotein (P-gp) form a concerted barrier to drug absorption, we investigated the role of P-gp in GJ-drug interactions. METHODS: The transcellular bidirectional flux of drugs that are (i) CYP3A-and/or P-gp substrates (Vinblastine, Cyclosporine, Digoxin, Fexofenadine, Losartan) or that are (ii) primary CYP3A-substrates (Felodipine, Nifedipine) was evaluated across MDCK-MDR1 cell monolayers with or without GJ, verifying monolayer integrity at all times. RESULTS: While both apical-to-basal (A-B) and basal-to-apical (B-A) fluxes of all CYP3A/P-gp substrates tested were increased in the presence of GJ, the resulting net efflux (B-A/A-B) was in all cases significantly greater with GJ than control (Vin, 28.0 vs. 5.1; CsA, 9.9 vs. 2.8; Dig, 22. 9 vs. 14.7, Fex, 22.3 vs. 11.1, Los, 39.6 vs. 26). In contrast, no such GJ flux effect was observed with Fel and Nif, substrates of CYP3A only (2 vs. 1.7 and 1.2 vs. 1.3). CONCLUSIONS: GJ significantly activates P-gp-mediated efflux of drugs that are substrates of P-gp, potentially partially counteracting the CYP3A-inhibitory effects of GJ.

Use of a disulfide cross-linking strategy to study muscarinic receptor structure and mechanisms of activation.

To gain insight into the molecular architecture of the cytoplasmic surface of G protein-coupled receptors, we have developed a disulfide cross-linking strategy using the m3 muscarinic receptor as a model system. To facilitate the interpretation of disulfide cross-linking data, we initially generated a mutant m3 muscarinic receptor (referred to as m3'(3C)-Xa) in which most native Cys residues had been deleted or substituted with Ala or Ser (remaining Cys residues Cys-140, Cys-220, and Cys-532) and in which the central portion of the third intracellular loop had been replaced with a factor Xa cleavage site. Radioligand binding and second messenger assays showed that the m3'(3C)-Xa mutant receptor was fully functional. In the next step, pairs of Cys residues were reintroduced into the m3'(3C)-Xa construct, thus generating 10 double Cys mutant receptors. All 10 mutant receptors contained a Cys residue at position 169 at the beginning of the second intracellular loop and a second Cys within the C-terminal portion of the third intracellular loop, at positions 484-493. Radioligand binding studies and phosphatidylinositol assays indicated that all double Cys mutant receptors were properly folded. Membrane lysates prepared from COS-7 cells transfected with the different mutant receptor constructs were incubated with factor Xa protease and the oxidizing agent Cu(II)-(1,10-phenanthroline)3, and the formation of intramolecular disulfide bonds between juxtaposed Cys residues was monitored by using a combined immunoprecipitation/immunoblotting strategy. To our surprise, efficient disulfide cross-linking was observed with 8 of the 10 double Cys mutant receptors studied (Cys-169/Cys-484 to Cys-491), suggesting that the intracellular m3 receptor surface is characterized by pronounced backbone fluctuations. Moreover, [35S]guanosine 5'-3-O-(thio)triphosphate binding assays indicated that the formation of intramolecular disulfide cross-links prevented or strongly inhibited receptor-mediated G protein activation, suggesting that the highly dynamic character of the cytoplasmic receptor surface is a prerequisite for efficient receptor-G protein interactions. This is the first study using a disulfide mapping strategy to examine the three-dimensional structure of a hormone-activated G protein-coupled receptor.

A radioreceptor assay for the analysis of AT1-receptor antagonists. Correlation with complementary LC data reveals a potential contribution of active metabolites.

A reliable and sensitive radioreceptor assay based on rat lung homogenate as receptor preparation was developed to determine the angiotensin-II antagonistic profile of losartan and its main active metabolite EXP 3174 as well as its congeners exemplified by UP 269-6 and SL 91.0102-90 DL. This method proved to be precise with an intra- and interday variability of less than 10% and a limit of quantification < or = 1 ng ml-1. The analysis of the Ki values in protein-free Hepes-buffer versus blank human or rat plasma revealed the distinct high plasma-protein binding of EXP 3174 which consequently caused a dramatic drop of potency from 10-15-fold in the buffer to only about 2-fold in control plasma, when compared to the parent compound losartan and the two congeners investigated. Upon evaluation of clinical samples by both the reported radioreceptor assay (RRA) and the established high-performance liquid chromatography (HPLC), the correlation of the normalized data pairs (concentration equivalents) suggested the contribution of active metabolites to the angiotensin-II antagonistic effect of SL 91.0102-90 DL, but not to the effect of UP 269-6. In the context of an extended preclinical study in rats, the correlation of RRA with the respective HPLC concentration equivalents of losartan and its main active metabolite EXP 3174 confirmed previous findings that only losartan and EXP 3174 exert the angiotensin-II-AT1 receptor blockade without the contribution of other metabolites (P.C. Wong, W.A. Price, A.T. Chiu et al., J. Pharmacol. Exp. Ther. 255 (1990) 211-217).

[Endoluminal stent prosthesis implantation in thoracic aneurysm of the descending aorta--a case report]. / Endoluminale Stentprothesenimplantation bei thorakalem Aneurysma der Aorta descendens--ein Fallbericht.

We report on a 72-year old male with a large aneurysm of descending thoracic aorta which was treated by implantation of a Dacron-covered self-expanding Nitinol stent graft (Talent, HOSMED). The patient was unfit for surgical aneurysm resection because of generalised atherosclerosis and cardiomyopathy. We think that stent implantation for descending thoracic aortic aneurysms is an attractive alternative to surgical aneurysm resection, especially in patients with enhanced operative risk. However, further experience, and especially long term-results, are required before more widespread application of intra-luminal stent implantation in the management of thoracic aortic aneurysms can be recommended.

HPLC assays to simultaneously determine the angiotensin-AT1 antagonist losartan as well as its main and active metabolite EXP 3174 in biological material of humans and rats.

Novel rapid and sensitive HPLC assays were developed to simultaneously determine losartan and its main active metabolite EXP 3174 in biological material of humans and rats following solid-phase or liquid-liquid extraction. The analytes were separated on a 3 microm particle-sized ULTREMEX CN column, which was preceded by a 5 microm particle-sized guard column, using UV-detection at 245 nm. The assays provided high sensitivity with limits of quantification (LoQ) of 5 ng ml(-1) for both compounds in human and rat plasma and 10 ng ml(-1) in human and rat urine, respectively. In rat blood, bile and various tissues, limits of quantifications were achieved that ranged 10-15 ng per ml and per 100 mg tissue, respectively, for both analytes.

Pharmacokinetics of methylprednisolone and prednisolone after single and multiple oral administration.

The pharmacokinetics of methylprednisolone and prednisolone were evaluated in 24 healthy men after oral administration of single and multiple doses for 3 days. For each drug, 6 different administration regimens with doses ranging from 1 to 80-mg of methylprednisolone and 1.25 to 100-mg of prednisolone, and administration intervals ranging from 3 to 24 hours for both were investigated. Plasma was assayed using a normal phase high-performance liquid chromatography (HPLC) method. Methylprednisolone showed linear pharmacokinetics with no apparent dose or time dependency. Prednisolone showed marked dose dependency with higher clearance and volume of distribution for higher doses. This can be explained by its saturable protein binding of plasma, because unbound clearance and unbound volume of distribution were not dose-dependent. After multiple administration, prednisolone showed significant time-dependent pharmacokinetics with increased unbound clearance and increased unbound volume of distribution. Due to the complicated pharmacokinetic properties of prednisolone, it is extremely difficult to determine the dose needed to obtain a desired target concentration. The pharmacokinetics of methylprednisolone are more predictable because methylprednisolone concentrations are proportional to dose, and no determination of plasma protein binding is needed.

The renin-angiotensin-aldosterone system: focus on its distinct role in arterial hypertension and its various inhibitors as a therapeutic strategy to effectively lower blood pressure.

Chronically elevated blood pressure results from pathological alterations in control systems. Current approaches to elucidate the underlying etiology strongly emphasize the (patho)physiological significance of the Renin-Angiotensin-Aldosterone System (RAAS) which interestingly interacts with the sympathetic, the cholinergic and purinergic systems. While the angiotensin-II-receptor subtype 1 (AT1), which mediates the blood-pressure-related effects of angiotensin II (All), has so far been extensively investigated, the physiological relevance of the other angiotensin-II-receptor subtypes-in particular of the AT2-receptor subtype-is about to be evolved by analysis of the various signal transduction mechanisms and by evaluation of transgenic animals, e.g. the knock-out mice, following disruption of the single A-II-receptor subtypes. Based on the clinical success of ACE inhibitors, the blockade of the Renin-Angiotensin-Aldosterone System in many different ways has been recognized as a successful strategy to effectively lower blood pressure.