Differences in the absorption,metabolism and biliary excretion of a diastereomeric pair of α v β 3 - antagonists in rat: limited role of P-glycoprotein

1. The study investigated mechanisms underlying the pharmacokinetic differences of two zwitterionic diastereomers ((3 S) -3-{ (3 R or 3 S) -2-oxo-3-\[3-(5,6,7,8-tetrahydro-1,8- naphthyridin-2-yl)propyl]pyrrolidin-1-yl}-3-quinolin-3-ylpropanoic acid) with different lipophilicities using a combination of in vivo and in vitro approaches. 2. In rat, both isomers possessed comparable plasma clearances (CL). However, the more lipophilic diastereomer I exhibited a higher metabolic clearance (>2-fold higher than II), whereas the hydrophilic zwitterion II exhibited a higher biliary clearance (~5- fold higher than I) . Following oral administration, the bioavailability (F) of I (17%) was much higher than that of II (1%). 3. Consistent with these in vivo observations and the expectation based on their lipophilicity differences, the metabolism in rat liver microsomes was faster and the permeability in Caco-2 and LLC-PK1 cells and in situ rat intestinal loop was better for I than for II. 4.Only the absorption of the more lipophilic diastereomer I was subjected to an efflux system in the Caco-2 and in situ rat intestinal loop models. I was a good substrate for Pglycoprotein (P-gp) in both the human MDR1 and mouse mdr1a transfected cell lines, and in the wild-type mdr1a (+/+) mouse when compared with the P-gp-deficient mdr1a (-/-) mouse. Concomitant administration of I with verapamil in rat caused significant increases in oral AUC, F and C max of I without affecting its CL, further supporting the effect of P-gp in limiting the intestinal absorption of I in vivo in this animal model. 5. Since the findings that the lipophilic diastereomer I, but not II, was a good P-gp substrate were not in line with the observations that I was excreted to bile much slower than II and that I was absorbed better than II, the results suggested that P-gp played a minor role to the observed differences in the biliary excretion and intestinal absorption of the diastereomers I and II in rat.     

Renal elimination of a novel and potent αvβ3 integrin antagonist in animals

1. Compound A (3-{2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]napthyridin-2-yl)propyl]-imidazolidin-1-yl}-3(S)-(6-methoxy-pyridin-3-yl)propionic acid), a hydrophilic zwitterion, is a potent and selective α_vβ₃ integrin antagonist currently under clinical development for the treatment of osteoporosis. The mechanism of renal excretion of compound A was investigated using a combination of in vivo and in vitro approaches.

2. In rats, renal excretion of compound A involved tubular secretion; ratios between renal clearance, corrected for unbound fraction in plasma (CL_r,u) and glomerular filtration rate (GFR) were greater than unity (2–5). The tubular secretion of compound A was saturable at high plasma levels (>?26?μM), and was inhibited significantly, although modestly (about twofold) by relatively high plasma concentrations of the organic anion PAH (160?µM) and the cation cimetidine (about 400?μM), but not by the P-gp inhibitor quinidine (about 50?μM). However, compound A (about 100?μM) had a minimal effect on CL_r/GFRs for cimetidine and PAH.

3. In rhesus monkeys, renal elimination of compound A also involved tubular secretion, with a CL_r,u/GFR ratio of about 30. The renal secretion of compound A was not affected by either cimetidine (about 120?μM) or PAH (about 80?μM). Similarly, compound A (about 40?μM) had a minimal effect on the renal tubular secretion of both cimetidine and PAH.

4. At the doses studied, neither rat nor monkey plasma protein binding of compound A, cimetidine or PAH was affected in the presence of each other.

5. In vitro transport studies showed that compound A was not a substrate for P-gp in the Caco-2, human MDR1 and mouse mdr1a transfected LLC-PK1 cell lines. In an uptake study using rOAT1 and rOAT3 transfected HEK cell lines, compound A was shown to be a substrate for rat OAT3 (K_m?=?15?μM), but not rat OAT1.

6. The results suggest that the tubular secretion of compound A is not mediated by P-gp, but rather is mediated, at least in part, via the organic anion transporter OAT3, the renal transporter shown to be capable of transporting both the organic anion PAH and the organic cation cimetidine. Although there is a possibility for pharmacokinetic interactions between compound A and substrates or inhibitors of OAT3, at the renal excretion level, the magnitude of interaction would likely be modest in humans at clinically relevant doses.

     

αvβ3 Integrin antagonists as inhibitors of bone resorption

The _vβ₃ integrin is a non-covalent, heterodimeric, cell-surface protein that is expressed with varying density on numerous cell types, including osteoclasts, vascular smooth muscle cells, endothelial cells and a variety of tumour cells. Functionally, _vβ₃ mediates a diverse range of biological events including the adhesion of osteoclasts to bone matrix, smooth muscle cell migration and angiogenesis. Specifically, there has been significant attention focused on the preparation of inhibitors of _vβ₃ for use as inhibitors of bone resorption, in recognition of the medical need for improved prevention and treatment of osteoporosis. Herein, we summarise the pertinent chemistry and biological advances in the medicinal design and biological evaluation of peptide and small molecule _vβ₃ antagonists as inhibitors of bone resorption.     

Stereoselective metabolism of metoprolol in isolated rat liver

目的：研究美托洛尔光学异构体在离体大鼠肝灌流上的药物动力学的立体选择性差异．方法：美托洛尔光学异构体在离体大鼠肝灌流中作循环式灌流，用高效液相色谱法进行定量分析．结果：０１６，０３２和０６４ｍｇ三种不同剂量的美托洛尔光学异构体在离体大鼠肝脏中消除过程，经叠加原则认为不存在饱和机制，符合单室模型一级动力学过程，在三种剂量下，两种异构体间的动力学参数Ｔ１／２，Ｋ和Ｃｌ均存在着显著性差异（Ｐ＜００１），其Ｓ（－）Ｍｅｔ／Ｒ（＋）Ｍｅｔ的Ｃｌ比值为０１４－０１７．结论：离体大鼠肝脏对美托洛尔两种光学异构体的代谢具有立体选择性，其肝消除符合线性动力学过程．     

Selective α1B- and α1D-adrenoceptor antagonists suppress noradrenaline-induced activation,proliferation and ECM secretion of rat hepatic stellate cells in vitro

Aim:

To explore the effects of noradrenaline (NA) on hepatic stellate cells (HSCs) in vitro and to determine the adrenoceptor (AR) subtypes and underlying mechanisms.

Methods:

The distribution and expressions of α_1A-, α_1B-, and α_1D-ARs in HSC-T6 cells were analyzed using immunocytochemistry and RT-PCR. Cell proliferation was evaluated with MTT assay. The expression of HSC activation factors [transforming factor-β₁ (TGF-β₁) and α-smooth muscle actin (α-SMA)], extracellular matrix (ECM) secretion factors [tissue inhibitor of metalloproteinase-1 (TIMP-1) and collagen-Ι (ColΙ)] and PKC-PI3K-AKT signaling components (PKC, PI3K, and AKT) in the cells were detected by Western blotting and RT-PCR.

Results:

Both α_1B- and α_1D-AR were expressed in the membrane of HSC-T6 cells, whereas α_1A-AR was not detected. Treatment of the cells with NA concentration-dependently increased cell proliferation (EC₅₀=277 nmol/L), which was suppressed by the α_1B-AR antagonist CEC or by the α_1D-AR antagonist BMY7378. Furthermore, NA (0.001, 0.1, and 10 μmol/L) concentration-dependently increased the expression of TGF-β₁, α-SMA, TIMP-1 and ColΙ, PKC and PI3K, and phosphorylation of AKT in HSC-T6 cells, which were suppressed by CEC or BMY7378, or by pertussis toxin (PT), RO-32-0432 (PKC antagonist), {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (PI3K antagonist) or GSK690693 (AKT antagonist).

Conclusion:

NA promotes HSC-T6 cell activation, proliferation and secretion of ECM in vitro via activation of G_α-coupled α_1B-AR and α_1D-AR and the PKC-PI3K-AKT signaling pathway.     

The utility of endogenous glycochenodeoxycholate-3-sulfate and 4β-hydroxycholesterol to evaluate the hepatic disposition of atorvastatin in rats

The liver is an important organ for drugs disposition, and thus how to accurately evaluate hepatic clearance is essential for proper drug dosing. However, there are many limitations in drug dosage adjustment based on liver function and pharmacogenomic testing. In this study, we evaluated the ability of endogenous glycochenodeoxycholate-3-sulfate (GCDCA-S) and 4β-hydroxycholesterol (4β-HC) plasma levels to evaluate organic anion-transporting polypeptide (Oatps)-mediated hepatic uptake and Cyp3a-meidated metabolism of atorvastatin (ATV) in rats. The concentration of ATV and its metabolites, 2-OH ATV and 4-OH ATV, was markedly increased after a single injection of rifampicin (RIF), an inhibitor of Oatps. Concurrently, plasma GCDCA-S levels were also elevated. After a single injection of the Cyp3a inhibitor ketoconazole (KTZ), plasma ATV concentrations were significantly increased and 2-OH ATV concentrations were decreased, consistent with the metabolism of ATV by Cyp3a. However, plasma 4β-HC was not affected by KTZ treatment despite it being a Cyp3a metabolite of cholesterol. After repeated oral administration of RIF, plasma concentrations of ATV, 2-OH ATV and 4-OH ATV were markedly increased and the hepatic uptake ratio of ATV and GCDCA-S was decreased. KTZ did not affect plasma concentrations of ATV, 2-OH ATV and 4-OH ATV, but significantly decreased the metabolic ratio of total and 4-OH ATV. However, the plasma level and hepatic metabolism of 4β-HC were not changed by KTZ. The inhibition of hepatic uptake of GCDCA-S by RIF was fully reversed after a 7-d washout of RIF. Plasma concentration and hepatic uptake ratio of GCDCA-S were correlated with the plasma level and hepatic uptake of ATV in rats with ANIT-induced liver injury, respectively. These results demonstrate that plasma GCDCA-S is a sensitive probe for the assessment of Oatps-mediated hepatic uptake of ATV. However, Cyp3a-mediated metabolism of ATV was not predicted by plasma 4β-HC levels in rats.     

Specific α2-adrenoreceptor antagonists induce behavioural activation in the rat

The behavioural effects of the specific and selective α(2)-adrenoreceptor antagonists, idazoxan, efaroxan and RX811059, have been investigated in the rat. All three drugs induced periods of behavioural activation characterized by increased locomotion and exploration (rearing and hole dipping). However, these effects were only apparent in animals which were fully habituated to their environments and thus displayed low baseline activity. The behaviour observed lay within the normal range of activity and was not apparent under conditions when exploration was stimulated such as in a novel environment. α( 2)-Adrenoreceptor antagonist- induced activation was a weak response when compared with the intense and prolonged hyperactivity, in both novel and non-novel environments, induced by the amine releaser D- amphetamine. Possible mechanisms involving a direct action of noradrenaline at postsynaptic α( 1)-adrenoreceptors (subsequent to enhanced presynaptic α(2)-receptor feedback blockade) or an indirect action of α(2)-antagonists on dopamine function in mesolimbic pathways are discussed.     

Bioavailability and disposition of ‘H-solanine in rat and hamster

1. The toxicokinetics of [³H]-α-solanine after oral (p.o.) and intravenous (i.v.) administration in rat and hamster were studied, in order to decide which is the most appropriate model in risk assessment studies. The i.v. Dose was 54 βg/kg; the oral dose was 170 βg/kg.

2. After i.v. Administration, the toxicokinetics of total radioactivity in blood were comparable in rat and hamster. However, the clearance of total radioactivity from plasma was more effective in rat than in hamster. The half-lives of distribution and of the terminal phase of unchanged α-solanine were not different between rat and hamster, whereas the systemic and metabolic clearance were, respectively, about 1.6 and 2.7 times higher in rat than in hamster. The clearance of unchanged α-solanine is more effective than of total radioactivity.

3. After p.o. Administration in rat and hamster, the mean bioavailability of total radioactivity is about 29 and 57%, respectively. The bioavailability of unchanged α-solanine is only 1.6 and 3.2%, respectively, when compared with i.v. administration.

4. T_1/2el of α-solanine after p.o. Administration was in rats a factor of four and in hamsters a factor of two shorter than after i.v. Administration. A strong retention of radioactivity was seen in the hamsters after p.o. Administration; only 40% of the dose was excreted within 7 days versus 90% in rat.

5. Based on these and toxicological data from literature, it was decided that the hamster is a more appropriate model in (sub) chronic toxicity studies with α-solanine than the rat.     

Metabolism of α-thujone in human hepatic preparations in vitro

This study aims to characterize the metabolism of α-thujone in human liver preparations in vitro and to identify the role of cytochrome P450 (CYP) and possibly other enzymes catalyzing α-thujone biotransformations. With a liquid chromatography-mass spectrometry (LC-MS) method developed for measuring α-thujone and four potential metabolites, it was demonstrated that human liver microsomes produced two major (7- and 4-hydroxy-thujone) and two minor (2-hydroxy-thujone and carvacrol) metabolites. Glutathione and cysteine conjugates were detected in human liver homogenates, but not quantified. No glucuronide or sulphate conjugates were detected. Major hydroxylations accounted for more than 90% of the primary microsomal metabolism of α-thujone. Screening of α-thujone metabolism with CYP recombinant enzymes indicated that CYP2A6 was principally responsible for the major 7- and 4-hydroxylation reactions, although CYP3A4 and CYP2B6 participated to a lesser extent and CYP3A4 and CYP2B6 catalyzed minor 2-hydroxylation. Based on the intrinsic efficiencies of different recombinant CYP enzymes and average abundances of these enzymes in human liver microsomes, CYP2A6 was calculated to be the most active enzyme in human liver microsomes, responsible for 70-80% of the metabolism on average. Inhibition screening indicated that α-thujone inhibited both CYP2A6 and CYP2B6, with 50% inhibitory concentration values of 15.4 and 17.5 μM, respectively.     

Metabolism of α-thujone in human hepatic preparations in vitro

1. This study aims to characterize the metabolism of α-thujone in human liver preparations in vitro and to identify the role of cytochrome P450 (CYP) and possibly other enzymes catalyzing α-thujone biotransformations.

2. With a liquid chromatography–mass spectrometry (LC-MS) method developed for measuring α-thujone and four potential metabolites, it was demonstrated that human liver microsomes produced two major (7- and 4-hydroxy-thujone) and two minor (2-hydroxy-thujone and carvacrol) metabolites. Glutathione and cysteine conjugates were detected in human liver homogenates, but not quantified. No glucuronide or sulphate conjugates were detected. Major hydroxylations accounted for more than 90% of the primary microsomal metabolism of α-thujone.

3. Screening of α-thujone metabolism with CYP recombinant enzymes indicated that CYP2A6 was principally responsible for the major 7- and 4-hydroxylation reactions, although CYP3A4 and CYP2B6 participated to a lesser extent and CYP3A4 and CYP2B6 catalyzed minor 2-hydroxylation. Based on the intrinsic efficiencies of different recombinant CYP enzymes and average abundances of these enzymes in human liver microsomes, CYP2A6 was calculated to be the most active enzyme in human liver microsomes, responsible for 70–80% of the metabolism on average.

4. Inhibition screening indicated that α-thujone inhibited both CYP2A6 and CYP2B6, with 50% inhibitory concentration values of 15.4 and 17.5 µM, respectively.

     

Ligands to the integrin receptor αvβ3

The vitronectin receptor α_vβ₃ is a member of a family of membrane bound adhesion receptors that mediate cell–cell and cell–matrix interactions. This integrin recognises extracellular matrix proteins that express the tripeptide Arg-Gly-Asp (RGD). A number of patents and patent applications have recently appeared describing peptidic and non-peptidic α_vβ₃ antagonists as potential therapeutic agents for the treatment of osteoporosis, cancer, diabetic retinopathy and rheumatoid arthritis. This review covers those issued patents and published patent applications that disclose novel ligands to α_vβ₃ that have appeared since 1999.     

Ligands to the integrin receptor αvβ3

The vitronectin receptor _vβ₃ is a member of the integrin superfamily of membrane bound glycoprotein receptors that is responsible for cell-cell and cell-matrix interactions and shares the same β subunit as the fibrinogen receptor _IIbβ₃ (also known as GPIIb/IIIa). Both these integrins recognise extracellular proteins that express the peptide sequence arginine-glycine-aspartic acid (RGD). Non-peptide RGD mimetics that bind with high affinity and selectivity to _IIbβ₃ were previously prepared as anti-thrombotic agents. More recently, medicinal chemistry groups have modified these non-peptide fibrinogen receptor antagonist lead structures to impart potency and integrin selectivity for _vβ₃. Numerous patent applications and issued patents have appeared throughout the last decade claiming structurally novel _vβ₃ antagonists as agents for the prevention and/or treatment of osteoporosis, cancer, diabetic retinopathy and rheumatoid arthritis. This review encompasses those issued patents and published patent applications that disclose ligands to _vβ₃.     

Selective inhibitors of the integrin αvβ6

The patent describes the production, purification, identification and characterisation of antibodies to the integrin αvβ6. The integrin αvβ6 has been implicated in several pathological conditions. Inhibition of the integrin is therefore of therapeutic value. Since the integrins are ubiquitous and involved in normal functioning of cells, selectivity is very important. The authors have shown that the antibody is selective for αvβ6. Use of this antibody in animal models is not shown.     

Increase in urinary excretion of 6β-hydroxycortisol in common marmosets as a marker of hepatic CYP3A induction

The ratio of urinary 6β-hydroxycortisol (6β-OHF) to free cortisol (F), i.e., the 6β-OHF/F ratio, has been reported to be a specific marker for human CYP3A induction by in vivo studies of human subjects. In the development of drugs, it is quite beneficial to predict human CYP3A induction in preclinical safety studies using urine samples from experimental animals. We examined the 6β-OHF/F ratio in urine of common marmosets administered with rifampicin, a potent inducer of CYP3A, to evaluate the usefulness of common marmosets for the prediction of CYP3A induction. Rifampicin was orally administered to three groups of four male common marmosets at doses of 0, 10, and 20 mg/kg per day for 4 days. Amounts of 6β-OHF and F in urine samples were determined by means of high-performance liquid chromatography (HPLC) during the experimental period. One day after the 4th dosing, animals were killed, and P450 contents and P450-catalyzed, 7-alkoxycoumarin O-dealkylase (ACD) activities in the liver were measured. Western blot analysis of liver microsomes was also performed using anti-rat P450 (CYP1A1, 2B1/2, 3A, and 4A) antibodies. The results indicated elevations in the 6β-OHF/F ratios that were dependent on both the dosing period and dose levels adopted. The ratios on day 4 reached 4.7- and 5.3-fold the pre-administration values in the 10 and 20 mg/kg per day groups, respectively. P450 contents and ACD activities were also elevated in all of the groups. Western blot analysis showed specific increases in the protein which cross-reacts with anti-rat CYP3A antibody in all of the groups. Furthermore, the 6β-OHF/F ratio was well correlated with the CYP3A contents in liver (r = 0.906). These results indicated that increase in urinary excretion of 6β-OHF is a specific marker of the induction of hepatic CYP3A in common marmosets just as in humans. Consequently, the present study suggested that human CYP3A induction elicited by chemical agents can be predicted in common marmosets by measuring the urinary excretion of 6β-OHF. Received: 7 December 1998 / Accepted: 9 March 1999     

Antagonists of integrin αv in brain cancer

Angiogenesis is required for full growth and development of primary neoplasms and enhances the establishment and growth of secondary metastases. The observation that angiogenesis is dependent on the interaction of endothelial cell surface integrin adhesion molecules with the surrounding extracellular matrix (ECM) environment has encouraged many pharmaceutical and biotechnology companies to develop α_v integrin antagonists. This article reviews a recent patent application which claims methods for inhibiting tumour growth in the brain using antagonists of α_v integrins including α_vβ₃ and α_vβ₅.     

Synthesis and blocking activities of a new class of α₁-adrenoceptor antagonists

Finding effective chemotherapeutic agents for clinical use is a long-lasting goal in medicinal chemistry. In this study, we report a new class of α₁-adrenoceptor (α₁-AR) antagonists. Specifically, we describe the synthesis and the blocking activities toward α₁-AR of 7-(2-hydroxypropoxy)-3,4-dihydroisoquinolin-1(2H)-one 1 and its structurally perturbed analogs 2 – 11 that were designed according to the principle of bioisosterism. Their structures were identified with IR, ¹H NMR, MS, HRMS and elemental analysis. The blocking activities of compounds 1 – 11 were evaluated on isolated rat anococcygeus muscles. The results indicated that these compounds showed moderate to good activities. Among them, compound 1 exhibited the highest activity that was comparable to those of known α₁-AR antagonists tamsulosin and DDPH (1-(2,6-dimethylphenoxy)-2-(3,4-di- methoxyphenylethylamino)propane hydrochloride) and thus may be exploitable as a lead compound for the discovery of promising α₁-AR antagonists.     

The use of α-adrenoceptor antagonists in lower urinary tract disease

α-adrenoceptor antagonists have traditionally been used in the treatment of hypertension but in recent years they have become increasingly common in the treatment of benign prostatic enlargement (BPE), where they reduce the ‘dynamic’ component of bladder outlet obstruction and appear to have additional actions to reduce irritative symptoms of the disease. Prazosin (Hypovase^®, Alza), doxazosin (Cardura^®, Pfizer), indoramin (Doralese^®, Wyeth-Ayerst Pharmaceuticals Inc.) and terazosin (Hytrin^®, Abbott Laboratories) are currently available in the UK for BPE but these agents have cardiovascular actions in a significant number of patients, inducing effects which must be considered adverse unless the patient also requires treatment for mild-to-moderate hypertension. The uroselective α-adrenoceptor antagonists tamsulosin (Flomax^®, Yamanouchi Pharmaceutical Co. Ltd.) and alfuzosin (Xatral^®, Sanofi-Synthelabo) have recently been introduced. These agents exert their selectivity via different mechanisms; selective tissue distribution for alfuzosin and α-adrenoceptor subtype selectivity for tamsulosin. The incidence of cardiovascular side effects for both drugs is similar to placebo. Several lines of evidence suggest that the α-adrenoceptor antagonists may relieve lower urinary tract (LUT) symptoms by other mechanisms additional to those which account for the reduction in bladder outlet obstruction. If correct, these agents may be of use in the treatment of other bladder conditions.     

Determination of α1-adrenoceptor antagonists in plasma by radioreceptor assay

A simple, rapid and sensitive radioreceptor assay (RRA) for the quantification of α₁-adrenoceptor antagonists such as prazosin in plasma is described. The method involves the use of an RRA based on [³H]prazosin displacement in rat cerebral cortical membranes. The method is reliable, with intra-assay and inter-assay RSDs ranging from 5.9 to 9.2%. The limit of detection is 0.2 (prazosin hydrochloride), 0.05 (tamsulosin hydrochloride) and 0.3 (bunazosin hydrochloride) pmol per assay. Using this method the plasma levels of prazosin hydrochloride were determined in beagle dogs administered orally 2.39 μmol kg⁻¹ of this drug. The plasma levels of prazosin in beagle dogs are in good agreement with those obtained using a high-performance liquid chromatography (HPLC). This RRA proved to be applicable to the monitoring of plasma prazosin levels in patients with essential hypertension and/or benign prostatic hypertrophy receiving therapy with this drug with the therapeutic dosage schedule. Thus, the concentrations of α₁-adrenoceptor antagonists in plasma can be adequately monitored by RRA as well as by HPLC.