Structural basis of the thrombin selectivity of a ligand that contains the constrained arginine mimic (2S)-2-amino-(3S)-3-(1-carbamimidoyl- piperidin-3-yl)-propanoic acid at P1

We have studied the thrombin and trypsin complexed structures of a pair of peptidomimetic thrombin inhibitors, containing different P1 fragments. The first has arginine as its P1 fragment, and the second contains the constrained arginine mimic (2S)-2-amino-(3S)-3-(1-carbamimidoyl-piperidin-3-yl)-propano ic acid (SAPA), a fragment known to enhance thrombin/trypsin selectivity of inhibitors. On the basis of an analysis of the nonbonded interactions present in the structures of the trypsin and thrombin complexes of the two inhibitors, the calculated accessible surfaces of the enzymes and inhibitors in the four complexes, data on known structures of trypsin complexes of inhibitors, and factor Xa inhibitory potency of these compounds, we conclude that the ability of this arginine mimic to increase thrombin selectivity of an inhibitor is mediated by its differential interaction with the residue at position 192 (chymotrypsinogen numbering). Thrombin has a glutamic acid at residue 192, and trypsin has a glutamine. The analysis also suggests that this constrained arginine mimic, when present in an inhibitor, might enhance selectivity against other trypsin-like enzymes that have a glutamine at residue position 192.     

The effects of enzyme induction and enzyme inhibition on labetalol pharmacokinetics.

The oral and intravenous pharmacokinetics of labetalol were determined in five subjects before and after a 3 week course of glutethimide 500 mg/day. After glutethimide there was a significant reduction in the AUC after the oral dose of labetalol, from 40,596 +/- 11,534 (mean +/- s.e. mean) to 22,057 +/- 6,276 ng ml-1 min (2P less than 0.05), and systemic bioavailability was reduced from 30.3 +/- 2.8 to 17.0 +/- 3.5% (2P less than 0.001). There was no significant change in labetalol plasma concentration-time curve (AUC) following an intravenous dose, half-life, volume of distribution, and plasma clearance. The oral and intravenous pharmacokinetics of labetalol were determined in six subjects before and after a 3 day course of cimetidine 1.6 g/day. After cimetidine there was a significant reduction in the volume of distribution of labetalol, from 520 +/- 51 to 445 +/- 24 1 (2P less than 0.05). The AUC of labetalol after the oral dose increased by 66%, from 51,029 +/- 7,950 to 84,772 +/- 19,444 ng ml-1 min (2P = 0.06). The systemic bioavailability of labetalol increased from 25.1 +/- 2.4 to 39.0 +/- 7.6% (2P = 0.06). There was no significant change in labetalol AUC after the intravenous dose, half life, and plasma clearance. There were no significant changes in resting heart rate and supine systolic and diastolic blood pressure following labetalol plus glutethimide, or labetalol plus cimetidine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacious and orally bioavailable thrombin inhibitors based on a 2,5-thienylamidine at the P1 position: discovery of N-carboxymethyl-d-diphenylalanyl-l-prolyl[(5-amidino-2-thienyl)methyl]amide

Thrombin, a crucial enzyme in the blood coagulation, has been a target for antithrombotic therapy. Orally active thrombin inhibitors would provide effective and safe prophylaxis for venous and arterial thrombosis. We conducted optimization of a highly efficacious benzamidine-based thrombin inhibitor LB30812 (3, K(i) = 3 pM) to improve oral bioavailability. Of a variety of arylamidines investigated at the P1 position, 2,5-thienylamidine effectively replaced the benzamidine without compromising the thrombin inhibitory potency and oral absorption. The sulfamide and sulfonamide derivatization at the N-terminal position in general afforded highly potent thrombin inhibitors but with moderate oral absorption, while the well-absorbable N-carbamate derivatives exhibited limited metabolic stability in S9 fractions. The present work culminated in the discovery of the N-carboxymethyl- and 2,5-thienylamidine-containing compound 22 that exhibits the most favorable profiles of anticoagulant and antithrombotic activities as well as oral bioavilability (K(i) = 15 pM; F = 43%, 42%, and 15% in rats, dogs, and monkeys, respectively). This compound on a gravimetric basis was shown to be more effective than a low molecular weight heparin, enoxaparin, in the venous thrombosis models of rat and rabbit. Compound 22 (LB30870) was therefore selected for further preclinical and clinical development.     

Dietary pretreatment with green tea polyphenol, (−)-epigallocatechin-3-gallate reduces the bioavailability and hepatotoxicity of subsequent oral bolus doses of (−)-epigallocatechin-3-gallate

Human case-studies have reported an association between green tea-based dietary supplements and hepatotoxicity. Studies have demonstrated the hepatotoxicity of high-dose oral bolus dosing with the tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) in mice and dogs. We examined the effect of pretreatment with dietary EGCG on the hepatotoxicity and bioavailability of acute oral bolus dosing with EGCG in CF-1 mice. EGCG (750 mg/kg, i.g., once daily for 3 days) increased plasma alanine aminotransferase by 80-fold, decreased both reduced (by 59%) and total (by 33%) hepatic glutathione, and increased hepatic levels of phosphorylated histone 2AX. Pretreatment with dietary EGCG (3.2 mg/g diet) for 2 weeks mitigated hepatotoxicity. Acute oral EGCG also decreased mRNA expression of glutathione reductase. Dietary pretreatment prevented these decreased and increased glutathione peroxidase (Gpx)2, Gpx3, Gpx5, and Gpx7 expression. We found that dietary EGCG reduced the plasma (57% reduction) and hepatic (71% reduction) EGCG exposure following oral bolus dosing compared to mice that were not pre-treated. Overall, it appears that EGCG can modulate its own bioavailability and that dietary treatment may reduce the toxic potential of acute high oral bolus doses of EGCG. These data may partly explain the observed variation in hepatotoxic response to green tea-containing dietary supplements.     

The transport of a hepatoprotective agent, isopropryl 2-(l-3-dithiethane-2-ylidene)-2[N(4-methyl-thiazole-2-yl) carbamoyl] acetate (YH439), across Caco-2 cell monolayers

Isopropryl 2-(1-3-dithiethane-2-ylidene)-2[N-(4-methyl-thiazole-2-yl) carbamoyl] acetate (YH439) is currently under phase II clinical trials by the Yuhan Research Center for use as a hepatoprotective agent. Unfortunately, the oral bioavailbility of YH439, which is sparingly soluble in water (i.e., 0.3 microg/ml or 0.91 microM at room temperature), reportedly, is negligible regardless of the dose administered to rats in the 10-300 mg/kg range. The bioavailability of the compound increased up to 24%, when administered in the form of a micellar solution (700 microg/ml or 2.1 mM for YH439) at a dose of 10 mg/kg, suggesting that its limited solubility is associated with its negligible bioavailability. In order to obtain additional information concerning the bioavailability of YH439, the mechanism(s) involved in gastrointestinal (GI) absorption were investigated in the present study. For this purpose, the transport of YH430 across a Caco-2 cell monolayer was measured in a Transwell. A permeability of 4.07 x 10(-5) cm/s was obtained for the absorptive (i.e., apical to basolateral direction) transport of 0.42 pM YH439, implicating that the in vivo GI absorption is nearly complete. The absorptive transport exhibited a slight concentration-dependency with an intrinsic clearance (CLi) of 0.38 microLL/cm2/sec, which accounted for 28.1% of the total intrinsic clearance (i.e., CLi plus the intrinsic clearance for the linear component) of the transport. Thus, saturation of the absorption process appears to be a minor factor in limiting the bioavailability of the compound. The apparent permeability of YH439 from the basolateral to the apical direction (i.e., efflux, 6.67 x 10(-5) cm/s) was comparable to that for absorptive transport, but, interestingly, a more distinct concentration-dependency was observed for this transport. However, the efflux does not appear to influence the bioavailability of the compound, as evidenced by the sufficiently high permeability in the absorption direction. Rather, a reportedly extensive first-pass hepatic metabolism appears to be a principal factor in limiting the bioavailability. In this respect, reducing the first-pass metabolism by some means would lead to a higher bioavailability of the compound. Thus, elevation of the absorption rate of YH439 becomes a necessity. From a practical point of view, increasing the concentration of YH439 in the GI fluid appears to be a feasible way to increase the absorption rate, because the compound is primarily absorbed via a linear mechanism. In summary, the solubilization of YH439, as previously demonstrated for a micellar solution of the compound, appears to be a practical way to increase the oral bioavailability of YH439.     

Effects of citronellal, a monoterpenoid in Zanthoxyli Fructus, on the intestinal absorption of digoxin in vitro and in vivo

Complementary and alternative medicines can be applied concomitantly with conventional medicines; however, little drug information is available on these interactions. Previously, we reported on the inhibitory effects of an extract and monoterpenoids (e.g., (R)-(+)-citronellal) contained in citrus herbs on P-glycoprotein (P-gp) using P-gp-overexpressed LLC-PK1 cells. The objective of the present study was to investigate the effects of (R)-(+)-citronellal on P-gp-mediated transport in the intestinal absorption process in vitro and in vivo. Transcellular transport of [(3)H]digoxin across Caco-2 cell monolayers was measured in the presence or absence of (R)-(+)-citronellal. (R)-(+)-citronellal reduced the basolateral-to-apical transport and efflux ratio for [(3)H]digoxin significantly. Serum concentration-time profiles and pharmacokinetic parameters of digoxin after intravenous and oral administration were analyzed in rats pretreated with oral (R)-(+)-citronellal. The bioavailability of digoxin after oral administration decreased significantly to 75.8% of that after intravenous administration at the same dose. (R)-(+)-citronellal increased the bioavailability of oral digoxin to 99.9% but had no effects on total body clearance, volume of distribution, or elimination rate. These findings suggest that (R)-(+)-citronellal can increase the bioavailability of oral digoxin based on the blockade of P-gp-mediated efflux of digoxin from intestinal epithelia to the lumen in the absorption process.     

Pharmacokinetics of S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro- 3-trifluoromethyl-phenyl)-propionamide in rats, a non-steroidal selective androgen receptor modulator

1: S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide (also known as S-4) is a non-steroidal selective androgen receptor modulator demonstrating tissue-selective androgenic and anabolic effects. The purpose of the present study was to examine the systemic pharmacokinetics, elimination and oral bioavailability of S-4 in rats. 2: Thirty-five male Sprague-Dawley rats weighing approximately 250 g were randomly assigned to one of seven treatment groups. Intravenous doses of 0.5, 1, 10, and 30 mg kg(-1) were given via a jugular catheter. Oral doses of 1, 10 and 30 mg kg(-1) were administered via gavage. Plasma concentrations were determined using a validated high-performance liquid chromatography or by a high-performance liquid chromatography/mass spectrometry method. 3: Clearances ranged between 1.0 and 2.1 ml min(-1) kg(-1) and varied with dose. The volume of distribution was approximately 0.448 l kg(-1) in all treatment groups. Oral bioavailability was also dose dependent, with the lower doses showing complete oral bioavailability. The half-life of S-4 over the dose range tested was between 2.6 and 5.3 h. 4: It was demonstrated that S-4 is rapidly absorbed, slowly cleared, and has a moderate volume of distribution in rats. The pharmacokinetics and oral bioavailability of S-4 indicate that it is an excellent candidate for clinical development.     

Nonpeptide factor Xa inhibitors: DPC423, a highly potent and orally bioavailable pyrazole antithrombotic agent

DPC423, 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide, is a synthetic, orally bioavailable, competitive, and selective inhibitor of human coagulation factor Xa (K(i) [nM]: factor Xa, 0.15; trypsin, 60; thrombin, 6000; plasma kallikrein, 61; activated protein C, 1800; factor IXa, 2200; factor VIIa, >15,000; chymotrypsin, >17,000; urokinase, >19,000; plasmin, >35,000; tissue plasminogen activator, >45,000; complement factor I, 44,000 [IC(50)]). In vitro, DPC423 produced anticoagulant effects in human plasma in which it doubled prothrombin time, activated partial thromboplastin time, and Heptest clotting time at 3.1 +/- 0.4, 3.1 +/- 0.4, and 1.1 +/- 0.5 microM, respectively. In dogs, DPC423 had a good pharmacokinetic profile with an oral bioavailability of 57%, a plasma clearance of 0.24 L/kg/h, and a plasma half-life of 7.5 h. In rabbit and rat models of arteriovenous shunt thrombosis, DPC423 was an effective antithrombotic agent with an IC(50) of 150 and 470 nM, respectively. The antithrombotic effect of DPC423 is likely to be related to the inhibition of factor Xa but not to the inhibition of thrombin or due to direct inhibition of platelet aggregation. Therefore, based on potency, selectivity, efficacy, and oral bioavailability, DPC423 was selected for clinical development as an oral anticoagulant for the potential treatment of thrombotic disorders. Preliminary human data suggest that DPC423 is orally bioavailable in humans and has a long plasma half-life.     

肠道CYP3A和P-gp:口服药物的吸收屏障

细胞色素P4 5 0 3A (CYP3A)亚族是人类药物代谢最重要的I相酶。由Mdr1基因编码的外向转运载体蛋白P糖蛋白 (P gp)为药物外排泵。这两种蛋白质在口服药物吸收的主要部位胃肠道均有高表达 ,同时二者的底物具有显著的重叠性。近来 ,大量研究表明 ,决定口服药物生物利用度的主要因素是肠道细胞CYP3A对已吸收药物的生物转化作用和肠道细胞中P gp对已吸收药物的主动外排作用。如果药物为CYP3A和 (或 )P gp的底物 ,当其与CYP3A和P gp的抑制剂同时服用后 ,药物的口服生物利用度将可能升高     

Design, synthesis, and biological evaluation of novel potent and selective alphavbeta3/alphavbeta5 integrin dual inhibitors with improved bioavailability. Selection of the molecular core

A novel series of potent and selective alpha(v)beta(3)/alpha(v)beta(5) dual( )()inhibitors was designed, synthesized, and evaluated against several integrins. These compounds were synthesized through a Mitsunobu reaction between the guanidinium mimetics and the corresponding central templates. Guanidinium mimetics with enhaced rigidity (i.e., (2-pyridylamino)propoxy versus the 2-(6-methylamino-2-pyridyl)ethoxy) led to improved activity toward alpha(v)beta(3). Exemplary oral bioavailability in mice was achieved using the indole central scaffold. Although, oral bioavailability was maintained when the indole molecular core was replace with the bioisosteric benzofuran or benzothiophene ring systems, it was found to not significantly impact the integrin activity or selectivity. However, the indole series displayed the best in vivo pharmacokinetic properties. Thus, the indole series was selected for further structure-activity relationships to obtain more potent alpha(v)beta(3)/alpha(v)beta(5) dual antagonist with improved oral bioavailability.     

The pharmacokinetics of a thiazole benzenesulfonamide beta 3-adrenergic receptor agonist and its analogs in rats, dogs, and monkeys: improving oral bioavailability.

The pharmacokinetics and oral bioavailability of (R)-N-[4-[2-[[2-hydroxy-2-(pyridin-3-yl)ethyl]amino]ethyl]phenyl]-4-[4-[4-(trifluoromethylphenyl]thiazol-2-yl]benzenesulfonamide (1), a 3-pyridyl thiazole benzenesulfonamide beta3-adrenergic receptor agonist, were investigated in rats, dogs, and monkeys. Systemic clearance was higher in rats (approximately 30 ml/min/kg) than in dogs and monkeys (both approximately 10 ml/min/kg), and oral bioavailability was 17, 27, and 4%, respectively. Since systemic clearance was 25 to 40% of hepatic blood flow in these species, hepatic extraction was expected to be low, and it was likely that oral bioavailability was limited either by absorption or a large first-pass effect in the gut. The absorption and excretion of 3H-labeled 1 were investigated in rats, and only 28% of the administered radioactivity was orally absorbed. Subsequently, the hepatic extraction of 1 was evaluated in rats (30%) and monkeys (47%). The low oral bioavailability in rats could be explained completely by poor oral absorption and hepatic first-pass metabolism; in monkeys, oral absorption was either less than in rats or first-pass extraction in the gut was greater. In an attempt to increase oral exposure, the pharmacokinetics and oral bioavailability of two potential prodrugs of 1, an N-ethyl [(R)-N-[4-[2-[ethyl[2-hydroxy-2-(3-pyridinyl)ethyl]amino]ethyl]phenyl]-4-[4-[4-(trifluoromethyl)phenyl]thiazol-2-yl]benzenesulfonamide; 2] and a morpholine derivative [(R)-N-[4-[2-[2-(3-pyridinyl)morpholin-4-yl]ethyl]phenyl]-4-[4-[4-(trifluoromethyl)- phenyl]thiazol-2-yl]benzenesulfonamide; 3], were evaluated in monkeys. Conversion to 1 was low (<3%) with both derivatives, and neither entity was an effective prodrug, but the oral bioavailability of 3 (56%) compared with 1 (4%) was significantly improved. The hypothesis that the increased oral bioavailability of 3 was due to a reduction in hydrogen bonding sites in the molecule led to the design of (R)-N-[4-[2-[[2-hydroxy-2-(pyridin-2-yl)ethyl]amino]ethyl]phenyl]-4-[4-(4-trifluoromethylphenyl)thiazol-2-yl]benzenesulfonamide (4), a 2-pyridyl beta3-adrenergic receptor agonist with improved oral bioavailability in rats and monkeys.     

Biarylaniline phenethanolamines as potent and selective beta3 adrenergic receptor agonists

The synthesis of a series of phenethanolamine aniline agonists that contain an aniline ring on the right-hand side of the molecule substituted at the meta position with a benzoic acid or a pyridyl carboxylate is described. Several of the analogues (e.g., 34, 36-38, 40, and 44) have high beta(3) adrenergic receptor (AR) potency and selectivity against beta(1) and beta(2) ARs in Chinese hamster ovary (CHO) cells expressing beta ARs. The dog pharmacokinetic profile of some of these analogues showed >25% oral bioavailability and po half-lives of at least 1.5 h. Among the compounds described herein, the 3,3'-biarylaniline carboxylate derivatives 36, 38 and the phenylpyridyl derivative 44 demonstrated outstanding in vitro properties and reasonable dog pharmacokinetic profiles. These three analogues also showed dose dependent beta(3) AR mediated responses in mice. The ease of synthesis and superior dog pharmacokinetics of compound 38 relative to that of 44 in combination with its in vitro profile led us to choose this compound as a development candidate for the treatment of type 2 diabetes.     

Characterization of three diaminopyrimidines as potent and selective antagonists of P2X3 and P2X2/3 receptors with in vivo efficacy in a pain model

BACKGROUND AND PURPOSE

P2X3 and P2X2/3 receptors are highly localized on the peripheral and central pathways of nociceptive signal transmission. The discovery of A-317491 allowed their validation as chronic inflammatory and neuropathic pain targets, but this molecule has a very limited oral bioavailability and CNS penetration. Recently, potent P2X3 and P2X2/3 blockers with a diaminopyrimidine core group and better bioavailability were synthesized and represent a new opportunity for the validation of P2X3-containing receptors as targets for pain. Here we present a characterization of three representative diaminopyrimidines.

EXPERIMENTAL APPROACH

The activity of compounds was evaluated in intracellular calcium flux and electrophysiological recordings from P2X receptors expressed in mammalian cells and in a in vivo model of inflammatory pain (complete Freund''s adjuvant (CFA) in rat paws).

KEY RESULTS

Compound A potently blocked P2X3 (pIC₅₀ = 7.39) and P2X2/3 (pIC₅₀ = 6.68) and showed no detectable activity at P2X1, P2X2, P2X4 and P2X7 receptors (pIC₅₀ < 4.7). Whole-cell voltage clamp electrophysiology confirmed these results. Compounds showed good selectivities when tested against a panel of different classes of target. In the CFA model, compound B showed significant anti-nociceptive effects (57% reversal at 3 mg·kg⁻¹).

CONCLUSIONS AND IMPLICATIONS

The diaminopyrimidines were potent and selective P2X3 and P2X2/3 receptor antagonists, showing efficacy in vivo and represent useful tools to validate these receptors as targets for inflammatory and neuropathic pain and provide promising progress in the identification of therapeutic tools for the treatment of pain-related disorders.     

Pharmacokinetics of the novel, high-affinity and selective dopamine D3 receptor antagonist SB-277011 in rat, dog and monkey: in vitro/in vivo correlation and the role of aldehyde oxidase

1. In vitro studies with the selective dopamine D3 receptor antagonist SB-277011 were conducted in liver microsomes and homogenates from rat, dog, cynomolgus monkey and human to correlate the rate of metabolism with the in vivo pharmacokinetics of the compound in rat, dog and cynomolgus monkey. 2. In the presence of NADPH, SB-277011 was relatively stable in the presence of liver microsomes from rat, dog, cynomolgus monkey and human with an intrinsic clearance (CLi) of < 2 ml min(-1) g(-1) liver for all species. In total liver homogenates, SB-277011 was metabolized at a similar rate in rat and dog (CLi < 2 ml min(-1) g(-1) liver) to that in liver microsomes but in cynomolgus monkey and human (CLi = 9.9 and 45 ml min(-1) g(-1) liver, respectively) the intrinsic clearance was approximately 6- and 35-fold higher, respectively, than that in liver microsomes. 3. In the absence of NADPH, SR-277011 was rapidly cleared in liver homogenates from cynomolgus monkey and human (CLi = 7.4 and 27 ml min(-1) g(-1) liver, respectively) demonstrating that a significant pathway of metabolism of this compound was via an NADPH-independent non-microsomal oxidative route. This pathway was sensitive to inhibition with isovanillin suggesting that the enzyme responsible was aldehyde oxidase. 4. The in vivo pharmacokinetics showed that the plasma clearance of SB-277011 was low in rat (20 ml min(-1) kg(-1)), moderate in dog (14 ml min(-1) kg(-1)) and high in cynomolgus monkey (58 ml min(-1)kg(-1)), which is consistent with the in vitro findings and demonstrated a greater capacity for the monkey to metabolize this compound. The oral bioavailability of SB-277011 in rat, dog and cynomolgus monkey was 35, 43 and 2%, respectively. Given the high clearance of this compound in cynomolgus monkey, the low oral bioavailability is probably as a result of high first-pass elimination, specifically by aldehyde oxidase, rather than poor absorption. 5. The high in vitro clearance of SB-277011 in human liver homogenates and the involvement of aldehyde oxidase in the metabolism of SB-277011 indicates that the bioavailability of the compound is likely to be low in human.     

Discovery and evaluation of potent P1 aryl heterocycle-based thrombin inhibitors

In an effort to discover potent, clinically useful thrombin inhibitors, a rapid analogue synthetic approach was used to explore the P(1) region. Various benzylamines were coupled to a pyridine/pyrazinone P(2)-P(3) template. One compound with an o-thiadiazole benzylic substitution was found to have a thrombin K(i) of 0.84 nM. A study of ortho-substituted five-membered-ring heterocycles was undertaken and subsequently demonstrated that the o-triazole and tetrazole rings were optimal. Combination of these potent P(1) aryl heterocycles with a variety of P(2)-P(3) groups produced a compound with an extraordinary thrombin inhibitory activity of 1.4 pM. It is hoped that this potency enhancement in P(1) will allow for more diversification in the P(2)-P(3) region to ultimately address additional pharmacological concerns.     

Effects of pravastatin on the pharmacokinetic parameters of nimodipine after oral and intravenous administration in rats: Possible role of CYP3A4 inhibition by pravastatin

Objective:

The aim of this study was to investigate the effects of pravastatin on the pharmacokinetics of nimodipine in rats.

Materials and Methods:

The effect of pravastatin on P-glycoprotein (P-gp) and cytochrome P450 (CYP) 3A4 activity was evaluated. Nimodipine was administered to rats intravenously (3 mg/kg) and orally (12 mg/kg) with pravastatin (0.3 and 1 mg/kg).

Results:

Pravastatin inhibited CYP3A4 enzyme activity in a concentration-dependent manner with a 50% inhibition concentration (IC₅₀) of 14 µM. Compared with the oral control group, the area under the plasma concentration-time curve (AUC_0-∞) of nimodipine was increased significantly. Consequently, the absolute bioavailability (AB) of nimodipine with pravastatin (1 mg/kg) was 31.1%, which was significantly enhanced compared with the oral control group. Moreover, the relative bioavailability (RB) of nimodipine was 1.12- to 1.31-fold greater than that of the control group.

Conclusions:

The enhanced oral bioavailability of nimodipine might be mainly due to inhibition of the CYP3A-mediated metabolism of nimodipine in the small intestine and/or in the liver and due to reduction of the total body clearance rather than both to inhibition of the P-gp efflux transporter in the small intestine and reduction of renal elimination of nimodipine by pravastatin. The increase in the oral bioavailability of nimodipine with pravastatin should be taken into consideration of potential drug interactions between nimodipine and pravastatin.KEY WORDS: Bioavailability, CYP3A4, nimodipine, P-gp, pharmacokinetics, pravastatin     

The gut as a barrier to drug absorption: combined role of cytochrome P450 3A and P-glycoprotein

Intestinal phase I metabolism and active extrusion of absorbed drug have recently been recognised as major determinants of oral bioavailability. Cytochrome P450 (CYP) 3A, the major phase I drug metabolising enzyme in humans, and the multidrug efflux pump, P-glycoprotein, are present at high levels in the villus tip of enterocytes in the gastrointestinal tract, the primary site of absorption for orally administered drugs. The importance of CYP3A and P-glycoprotein in limiting oral drug delivery is suggested to us by their joint presence in small intestinal enterocytes, by the significant overlap in their substrate specificities, and by the poor oral bioavailability of joint substrates for these 2 proteins. These proteins are induced or inhibited by many of the same compounds. A growing number of preclinical and clinical studies have demonstrated that the oral bioavailability of many CYP3A and/or P-glycoprotein substrate drugs can be increased by concomitant administration of CYP3A inhibitors and/or P-glycoprotein inhibitors. We believe that further understanding the physiology and biochemistry of the interactive nature of intestinal CYP3A and P-glycoprotein will be important in defining, controlling, and improving oral bioavailability of CYP3A/P-glycoprotein substrates.     

Alpha-hydroxylation of tamoxifen and toremifene by human and rat cytochrome P450 3A subfamily enzymes

An increased risk of developing endometrial cancer is observed in breast cancer patients treated with tamoxifen (TAM) and in healthy women undergoing TAM chemoprevention therapy. TAM-DNA adducts were detected in the endometrium of women taking TAM (Shibutani, S., et al. (2000) Carcinogenesis 21, 1461-1467) and are formed primarily through O-sulfonation of alpha-hydroxytamoxifen (alpha-OHTAM). To explore the genotoxicic mechanisms of TAM, TAM was incubated with one of multiple human cytochrome P450 enzymes, i.e., P450 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, 4A11, 4F2, 4F3A, or 4F3B, in a NADPH regenerating system, and the metabolites were identified using HPLC/UV analysis with authentic standards. Among the 18 human P450 enzymes, P450 3A4 generated a significant amount of alpha-OHTAM. When some rat P450 enzymes were examined, P450 3A2 also catalyzed alpha-hydroxylation of TAM. Similarly, human P450 3A4 and rat P450 3A1 and 3A2 converted toremifene (TOR, a chlorinated TAM analogue) to alpha-hydroxytoremifene (alpha-OHTOR). The formation of alpha-OHTAM and alpha-OHTOR by these P450 enzymes was confirmed by tandem mass spectroscopy. Only the P450 3A subfamily enzymes are able to alpha-hydroxylate TAM and TOR. Although the formation of alpha-OHTOR by these enzymes was much higher than that of alpha-OHTAM, TOR is known to be much less genotoxic than TAM. The results support our proposed mechanism that the lower genotoxicity of TOR is due to limited O-sulfonation of alpha-OHTOR by hydroxysteroid sulfotransferases, resulting in the poor formation of DNA adducts (Shibutani, S., et al. (2001) Cancer Res. 61, 3925-3931).     

Strategies and progress towards the ideal orally active thrombin inhibitor

Thrombin plays a key role in the control of thrombus formation, for which reason its inhibition has become a target for new antithrombotics. Important issues in the profile of the ideal thrombin inhibitor are: potency, selectivity, oral bioavailability, half-life in the circulatory system and safety. Although many potent direct inhibitors of thrombin have been discovered, most of these inhibitors lack sufficient oral bioavailability. This is often associated with the presence of highly basic functionalities such as guanidine or amidine. These basic functionalities in the P1 moiety are preferred by thrombin and are present in the first generation of thrombin inhibitors. Recently, several orally active direct thrombin inhibitors have been disclosed. Most of these inhibitors originate from leads of the first generation. Two major optimization strategies could be identified to further improve these leads: A: maintain the highly basic P1 moiety and compensate its negative effects, and B: reduce the basicity of the P1 moiety and compensate for the decrease in inhibitory activity. The progress made using these strategies is evaluated. In addition, screening large sets of compounds yielded new structures that provide useful starting points for optimization. The optimization strategy used to convert leads from screening into potent orally active thrombin inhibitors is also be evaluated.     

Single-dose pharmacokinetics of nizatidine (Axid) in children

The pharmacokinetics of nizatidine following a single 5.0 mg/kg oral dose given as an extemporaneous liquid formulation in apple juice was examined in 12 healthy children (8.0 +/- 2.4 years, 30.7 +/- 8.4 kg). Nizatidine and N-desmethylnizatidine were quantitated by HPLC/MS from five post dose blood samples taken over a 12-hour period. The apparent terminal elimination rate constant for nizatidine in the pediatric subjects (0.58 +/- 0.8h(-1)) was virtually identical to that (0.54 +/- 0.13 h(-1)) previously reported from adult studies. When corrected for an estimated 30% reduction in nizatidine oral bioavailability observed in adults upon coingestion of the drug with other fruit/vegetable juices, nizatidine pharmacokinetic parameter estimates (e.g., Cmax, CL/F, Vss/F) in our pediatric subjects were similar to those previously reported in adults who were administered dimensionally similar (e.g., approximately 4 mg/kg) solid oral doses of the drug. Examination of the mean area under the curve (i.e., AUC0-infinity for nizatidine and N-desmethylnizatidine suggested an approximate 15% metabolic conversion of the parent drug. Finally, nizatidine plasma concentrations in pediatric patients following a single 5.0 mg/kg oral dose exceeded the EC50 value of the drug for gastric acid suppression determined from adult studies for approximately 6 hours.