In vitro metabolism of a nitroderivative of acetylsalicylic acid (NCX4016) by rat liver: LC and LC–MS studies

The metabolism of a nitroderivative of acetylsalicylic acid, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX4016), the lead compound of a new class of NO-releasing non steroidal-antiinflammatory drugs has been studied in vitro in rat liver subcellular fractions (S 9000×g, microsomes, cytosol). Samples were extracted with CH₃CN (2 vol.) containing 1% H₃PO₄ (2 M), vortexed for 3 min and then centrifuged for 5 min at 5000 rpm. Supernatants were diluted with 0.02 M phosphoric acid and analysed by reverse-phase LC. Linearity of calibration for NCX4016 and metabolites was observed over the range 0.25–50 μg/ml with coefficients of determination greater than 0.9996. Extraction efficiency from spiked liver samples ranged from 85 to 95% for all the analytes. In the S 9000×g fraction, NCX4016 undergoes rapid metabolization, with the formation of salicylic acid (SA) and [3-(nitrooxymethyl)phenol] (HBN). HBN is then rapidly metabolised to 3-hydroxybenzylalcohol (HBA), and mainly to a new metabolic species, whose formation takes place specifically in the liver cell cytosol. LC–MS analysis (electrospray ionisation) of the cytosol extract in negative and positive-ion modes furnished deprotonated [M−H]⁻ and protonated [M+H]⁺ molecular ions at m/z 412 and 414, respectively, accompanied by the typical clusters with sodium. MS/MS analysis in negative-ion mode, by selection and collision of the ion at m/z 412, gave a fragmentation pattern characterized by the ions at m/z 272 and 254, which allowed to assign the structure of 1-(glutathion-S-yl)methylene-3-hydroxy-benzene, a conjugated product between GSH and the benzyl carbon atom of HBN. In rat liver cytosol HBN is completely metabolised to this thioether adduct within 30 min incubation; the process is enzymatically mediated by GSH transferase and strictly dependent on GSH availability. The relevance of this new metabolic pathway in NCX4016 detoxification by rat liver is discussed.     

Stereoselective conjugation of a uricosuric diuretic with glutathione by glutathione transferase 3-3.

1. The activity of glutathione (GSH) transferases in rat liver cytosol was inhibited by the (-) enantiomer of a uricosuric diuretic (6,7-dichloro-5[N,N-dimethylsulphamoyl]-2,3-dihydrobenzofuran-2-ca rboxylic acid, DBCA) in a concentration-dependent manner. Although the DBCA (+) enantiomer inhibited the activity of liver cytosol GSH transferases, it was less effective. 2. Among four purified GSH transferase isozymes obtained from rat liver cytosol, isozyme 3-3 showed stereoselective interactions with the enantiomers of DBCA. This isozyme most actively and preferentially catalysed the transfer of GSH to DBCA (-) enantiomer.     

7-Glycidoxycoumarin (GOC): a fluorophotometric epoxide substrate for the assay of glutathione S-transferase activity

7- Glycidoxycoumarin ( GOC ), a new fluorophotometric epoxide substrate for glutathione S-transferase (GSH TFase ), was conjugated regiospecifically with GSH at pH 6.5 in rat liver cytosol to yield S-(2-hydroxy-3-(7'- coumaroxy )-1-propyl)glutathione which was isolated by HPLC and identified with an authentic specimen by 13C NMR spectroscopy. The conjugation product formed in the incubation media consisting of GOC , GSH, and 9000 g supernatant fractions from various tissues of the rat, was directly determined by photometry of fluorescence emission at 388 nm at an excitation wavelength of 328 nm after removal of the unreacted substrate and its enzymic hydrolysis product, 7-(1',2'-dihydroxy-3'-propoxy)coumarin, by simple extraction with isobutyl alcohol in the presence of a saturating amount of sodium chloride. Stability of GOC at pH 6.5 markedly retarded its autoconjugation with GSH and made the fluorophotometric method sensitive enough to assay small GSH TFase activities in gel column chromatographic fractions as well as in various tissues of the animals. Apparent Km and Vmax for GOC in rat liver cytosol were 55 microM and 7.41 nmole/mg protein/min, respectively. GSH conjugation of GOC was catalyzed by at least two isozymes, E and AA, of hepatic GSH TFases .     

NCX 4016, a nitric oxide-releasing aspirin,modulates adrenergic vasoconstriction in the perfused rat tail artery

1. The ability of the nitric oxide (NO)-releasing aspirin, NCX 4016, to control vasoconstrictor responses induced by electrical field stimulation (TNS) or by exogenous norepinephrine (NE) was investigated in perfused rat tail artery with intact endothelium. 2. NCX 4016 (25, 50 and 100 microM) dose-dependently antagonized the vasoconstriction caused by TNS (from 0.5 to 64 Hz) and by NE (from 0.01 to 10 microM). The vasorelaxant activity of NCX 4016 (100 microM) in NE-precontracted arteries was concomitant with a marked increase of tissue cyclic GMP (4.9 fold, P<0.001) and was significantly antagonized by the inhibitors of soluble guanylate cyclase, methylene blue and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one. 3. The effect of NCX 4016 was endothelium NO-independent since, in preparations perfused with N(G)-monomethyl-L-arginine (10 microM), this compound prevented the rise in basal perfusion pressure and reversed the accentuation of vasoconstrictor responses caused by NO synthase inhibition. 4. Aspirin-moiety released by NCX 4016 inhibited the 6-keto-PGF(1alpha) formation without interfering with the vasorelaxant activity of NCX 4016, while aspirin (100 microM) was devoid of any activity against vasoconstriction induced by both TNS and NE in perfused rat tail artery. 5. NCX 4016 moderated adrenergic vasoconstriction in perfused rat tail arteries by a direct donation of NO without involving the relaxant factors such as PGI(2) and NO from endothelial cells. 6. The results obtained with NCX 4016 in perfused rat tail artery bears some therapeutical potential in conditions associated with vascular smooth muscle hyperreactivity to adrenergic stimulation.     

Stereoselective conjugation of a uricosuric diuretic with glutathione by glutathione transferase 3-3

1. The activity of glutathione (GSH) transferases in rat liver cytosol was inhibited by the (-) enantiomer of a uricosuric diuretic (6,7-dichloro-5[N,N-dimethylsulphamoyl]-2,3-dihydrobenzofuran-2-carboxylic acid, DBCA) in a concentration-dependent manner. Although the DBCA (+) enantiomer inhibited the activity of liver cytosol GSH transferases, it was less effective.

2. Among four purified GSH transferase isozymes obtained from rat liver cytosol, isozyme 3–3 showed stereoselective interactions with the enantiomers of DBCA. This isozyme most actively and preferentially catalysed the transfer of GSH to DBCA (-) enantiomer.     

Prevention of pulmonary thromboembolism by NCX 4016, a nitric oxide-releasing aspirin

We studied the antithrombotic activity of 2-acetoxybenzoate 2-[1-nitroxy-methyl]-phenyl ester (NCX 4016), a novel nitric oxide (NO)-releasing aspirin derivative, in vivo in different animal models of platelet-dependent and independent pulmonary thromboembolism and compared it with that of aspirin. NCX 4016 protected mice from death induced by the intravenous (i.v.) injection of collagen plus epinephrine, of 9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F(2alpha) (U46619) and of thrombin while aspirin was only active against collagen plus epinephrine. The drop in platelet count and number of lung emboli were reduced by NCX 4016 more effectively than aspirin. NCX 4016 protected mice also from mechanical pulmonary embolism (i.v. injection of hardened rat red blood cells) while aspirin was ineffective. In rabbits, NCX 4016 significantly reduced the accumulation of [111In]oxine-labeled platelets in the pulmonary vasculature induced by collagen and by thrombin while aspirin produced reductions which were significant only versus collagen. In conclusion, NCX 4016 exerts a more pronounced antithrombotic activity than aspirin in vivo in two different animal species, largely due to a deeper inhibitory effect on platelets. NCX 4016 may represent a better antithrombotic agent than aspirin.     

NCX4016 (NO-Aspirin) has multiple inhibitory effects in LPS-stimulated human monocytes

NCX4016 (2 acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester, NicOx S.A., France) is an anti-thrombotic agent, chemically related to acetylsalicylic acid (ASA) and able to release NO. We tested the effects of NCX4016 and ASA on the release of the thromboxane (TX) A(2) metabolite TXB(2), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), expression and activity of tissue factor (TF) in stimulated, adherent human monocytes. Both ASA and NCX4016 1 - 1000 micromol l(-1) dose-dependently reduced TXB(2) concentration, measured by RIA in the supernatant of 10 microg ml(-1) LPS-stimulated cells. NCX4016 activity was comparable to that of equimolar ASA when incubation lasted 6 h (NCX4016 30 micromol l(-1): -86.0+/-10.1%, NCX4016 300 micromol l(-1): -92.2+/-9.0%, ASA 30 micromol l(-1): -92.3+/-7.5%, ASA 300 micromol l(-1): -97.3+/-1.0%, n=6, M+/-s.d.). Most of the activity of NCX4016 up to 100 micromol l(-1) was prevented by 10 micromol l(-1) ODQ, inhibitor of cyclic GMP. NCX4016 100 - 300 micromol l(-1) reduced TNF-alpha (NCX4016 300 micromol l(-1)=-77.2+/-19.9%, n=6) and IL-6 (NCX4016 300 micromol l(-1): -61.9+/-15.2%, n=6) in LPS stimulated monocytes while ASA had no significant effects. TF activity (NCX4016 300 micromol l(-1): 53.7+/-39.9%, n=4) and immunoreactive TF (NCX4016 300 micromol l(-1): -93.9+/-7.9%, n=7), measured in the supernatant of stimulated cells, were also dose-dependently inhibited by NCX4016 but not by ASA. The present results indicate that NCX4016 inhibits TXA(2) generation as well as cytokine release and TF in human monocytes partly via NO-dependent mechanisms. NCX4016 may have a favourable profile of activities in the clinical setting of athero-thrombosis.     

Pharmacologic profile and therapeutic potential of NCX 4016, a nitric oxide-releasing aspirin, for cardiovascular disorders

NCX 4016, 2-(acetyloxy)benzoic acid 3-[(nitrooxy)methyl]phenyl ester, is a new molecule in which a nitric oxide (NO)-releasing moiety is covalently linked to aspirin. After enzymatic metabolism, NCX 4016 releases both components. In vitro and in some animal models, these components exert their pharmacologic effects simultaneously. Nitric oxide (NO) is a small gaseous molecule that exerts several activities which may prevent atherothrombotic disorders. Moreover, it displays a protective activity on the gastric mucosa. NCX 4016 has been shown to inhibit platelet activation in vitro more effectively than aspirin, to inhibit smooth muscle cell proliferation, to exert an endothelial cell protective activity and to suppress the function of several inflammatory cells potentially involved in atherothrombosis. In animal models, NCX 4016 protected from platelet thromboembolism, prevented restenosis in atherosclerosis-prone animals, protected the heart from ischemia/reperfusion injury, and induced neoangiogenesis in critically ischemic limbs. Moreover, it displayed little or no gastric toxicity and appeared to protect stomach from noxious stimuli, including aspirin. NCX 4016 has been evaluated in healthy volunteers and found to inhibit platelet cyclo-oxygenase-1 (COX-1) similarly to or slightly less than aspirin, to raise the circulating levels of NO-degradation products, and to have little or no gastric toxicity in short term studies. In particular, in phase II studies, NCX 4016 had favorable effects on effort-induced endothelial dysfunction in intermittent claudication and on platelet-activation parameters elicited by short-term hyperglycemia in type II diabetics. In patients with type II diabetes the effects of NCX 4016 on microalbuminuria and on some hemodynamic parameters were promising. The pharmacokinetics of in vivo aspirin- and NO- released by NCX 4016, as well as the bioavailability of the two molecules, were not yet adequately studied. Also, the long-term tolerability of NCX 4016, as well as its possible effectiveness in preventing ischemic cardiovascular events and progression of atherosclerosis, should be explored.     

Effects of a new class of no-releasing NSAIDs on platelets and isolated arteries

A new class of nitroderivatives of non-steroidal anti-inflammatory drugs has recently been synthesized (Nicox Ltd., London, UK). In order to improve gastric tolerance of the parent compound, a side-chain, able to release nitric oxide, has been added to the core structure of the molecule. We studied in vitro the effects of nitrofenac and two NO-aspirins (NCX 4215 and NCX 4016) on platelets and isolated arteries to identify any possible effect due to the release of nitric oxide or to the inhibition of cyclo-oxygenase activity. Nitrofenac induced a dose-dependent relaxation both with intact (46% with 1×10⁻³ mol/L) and endothelium-denuded (75% with 1×10⁻³ mol/L) rings of rat aorta precontracted with epinephrine, while diclofenac did not affect this contraction (0% relaxation in intact and 22% in rubbed arteries). Pretreatment with diclofenac 1×10⁻³ mol/L significantly increased the vasorelaxant effects of nitrofenac at each drug concentration, both in intact (86% with 1×10⁻³ mol/L) and rubbed preparations (89%). NO-aspirins, unlike acetylsalicylic acid, were able to relax both intact and endothelium-denuded rings of rat aorta (100% relaxation). Methylene blue and oxyhaemoglobin completely reversed the relaxation induced by nitrofenac and NO-aspirins, both in rubbed and intact aortic rings. Both NO-aspirins exhibited antiaggregating properties in archididonic acid-stimulated human platelets, measured using a turbidimetric method (NCX 4215, 1×10⁻³ mol/L: 70% inhibition; NCX 4016 1×10⁻⁴ mol/L: 100%), NCX 4016 proving as effective as acetylsalicylic acid 1×10⁻⁵ mol/L. Thrombin-induced platelet aggregation was inhibited in acetylsalicylic acid-treated platelets (NCX 4215, 1×10⁻³ mol/L: 50%, NCX 4016, 1×10⁻⁴ mol/L: 92%). NCX 4016 was also able to provent thrombin-induced intracellular free calcium increase, effect not observed with acetylsalicylic acid. In vitro thromboxane A₂ production in human platelets, assayed RIA as thromboxane B₂ serum concentration, was reduced by NCX 4215, 1×10⁻³ mol/L (76%) and virtually abolished by NCX 4016 5×10⁻⁵ mol/L (95% inhibition). These results demonstrate in vitro the antiaggregating activity of NO-aspirins, NCX 4016 being more active than NCX 4215, and the vasorelaxant effects of all the tested molecules. The mechanism involved in two-fold: release of nitric oxide and inhibition of cyclo-oxygenase.     

Nitric oxide-releasing aspirin inhibits vasoconstriction in perfused tail artery of normotensive and spontaneously hypertensive rats

The aim of this study was to investigate the capacity of the 2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester (NCX 4016), a nitric oxide (NO)-releaser derivative of aspirin, to decrease blood pressure in spontaneously hypertensive rats (SHR) and to counteract the adrenergic vasoconstriction in perfused tail artery of these animals. Oral treatment for 10 consecutive days with NCX 4016 (100 micromol/kg) in SHR and their genetic controls Wistar Kyoto (WKY) rats resulted in a reduction of blood pressure in SHR but not in WKY rats. In SHR, the NCX 4016 treatment increased the serum nitrite/nitrate and diminished the serum thromboxane B2, whereas aspirin did not change blood pressure but abolished the serum thromboxane B2. Perfused tail arteries excised from vehicle-treated SHR exhibited a significant impairment of endothelium-dependent vasorelaxant function. These vessels, prepared from SHR or WKY rats treated orally with NCX 4016 (10, 30 and 100 micromol/kg for 7 consecutive days), revealed a dose-dependent decrease in vasoconstriction in response to transmural nerve stimulation and norepinephrine, whereas aspirin was ineffective. Furthermore, in tail arteries of both SHR and WKY rats treated orally with NCX 4016 (100 micromol/kg for 7 consecutive days), the cGMP increased significantly. In conclusion, NCX 4016, by releasing NO and increasing cGMP in vascular tissue, reduces sympathetic-mediated vasoconstriction in resistance vessels and lowers blood pressure in SHR.     

NCX4016 (NO-aspirin) reduces infarct size and suppresses arrhythmias following myocardial ischaemia/reperfusion in pigs

1. The effect of the nitro-derivative of aspirin, NCX4016, was assessed on ischaemic ventricular arrhythmias and myocardial infarct size in anaesthetized pigs in comparison to native aspirin. 2. Pigs were given aspirin (10 mg kg(-1); n=6), low dose NCX4016 (18.4 mg kg(-1); n=6) or high dose NCX4016 (60 mg kg(-1); n=7) orally for 5 days prior to coronary occlusion and reperfusion. None of the interventions had any effect on baseline haemodynamics prior to coronary occlusion in comparison to control pigs (n=9). Aspirin and high dose NCX4016 both prevented the generation of thromboxane A(2) from platelets activated ex vivo with A23187 (30 microM), whereas all three interventions markedly attenuated platelet aggregation in response to collagen in whole blood in comparison to controls. 3. None of the drug interventions had any effect on the incidence of ventricular fibrillation (VF) during myocardial ischaemia (100% in all groups). However, 60 mg kg(-1) NCX4016 significantly attenuated the total number of premature ventricular beats (PVB's) (62+/-16 vs 273+/-40 in control pigs; P<0.05) during the first 30 min of occlusion. The higher dose of NCX4016 also significantly reduced myocardial infarct size (22.6+/-3.7% of area at risk vs 53.0+/-2.8% of area at risk in control pigs; P<0.05). 4. These results suggest that the nitro-derivative of aspirin, NCX4016, is an effective antiplatelet agent, which unlike aspirin also reduces the extent of myocardial injury following ischaemia and reperfusion.     

Detoxification of molinate sulfoxide: comparison of spontaneous and enzmatic glutathione conjugation using human and rat liver cytosol

Previous lab studies implicated the sulfoxidation pathway of molinate metabolism to induce testicular toxicity. Once molinate is metabolized to molinate sulfoxide, it undergoes further phase II metabolism either spontaneously, enzyme catalyzed, or both to form glutathione-conjugated molinate. This study compared the metabolic capability of rat and human liver cytosol to form a glutathione (GSH)-conjugated metabolite of molinate. The GSH conjugation of molinate sulfoxide in rat cytosol was described by the constants Km of 305 microM and Vmax of 4.21 nmol/min/mg cytosol whereas the human values were 91 microM and 0.32 nmol/min/mg protein for Km and Vmax, respectively. At the same 1 mM GSH concentration, the in vitro bimolecular nonenzymatic rate constant of 3.02 x 10(-6) microM(-1) min(-1) was calculated for GSH conjugation of molinate sulfoxide. Specific activity for rat and human glutathione transferase was calculated to equal 1.202 +/- 0.25 and 0.809 +/- 0.45 micromol/min/mg protein, respectively by 1-chloro-2,4-dinitrobenzene (CDNB) assay. Compared to a conventional GSH depletion model (BSO + DEM combination), molinate alone was nearly as effective in reducing GSH levels by approximately 90 and 25% in liver and testes, respectively. The impact of molinate sulfoxide's ability to adduct glutathione transferase and inhibit the production of the glutathione conjugated metabolite was examined and found to be negligible.     

Bioactivation of morphine in human liver: isolation and identification of morphinone, a toxic metabolite

Morphinone, identified in the bile of guinea pigs and rats given morphine, is a reactive electrophile and has the ability to bind to glutathione (GSH) and tissue macromolecules, leading to GSH depletion and cell damage. We previously demonstrated that the livers of various animal species are capable of forming morphinone from morphine. In this study, we examined whether the human liver can produce morphinone from morphine. HPLC analysis revealed that the incubation of morphine with the 9000xg supernatant of human liver in the presence of NAD(P) and 2-mercaptoethanol (ME) gave a peak corresponding to the synthetic morphinone-ME adduct (MO-ME), which is readily formed by a nonenzymatic reaction of morphinone with ME. The reaction product was isolated and was unambiguously identified as MO-ME using FAB-MS and NMR analyses in comparison with synthetic MO-ME. The conversion of morphine to morphinone required NAD(P), and NAD was a preferred cofactor over NADP. All the 9000xg supernatants from six human livers could produce morphinone at different rates, ranging from 30 to 120 nmol/g liver/30 min with NAD at pH 7.4. The enzyme activity responsible for the formation of morphinone from morphine was mainly localized in the microsomes. The microsomal enzyme activity was inhibited by steroids, lithocholic acid and indomethacin. Among these compounds, steroids with a 17beta-hydroxyl group almost completely depressed morphinone formation. In conclusion, the metabolic pathway of morphine to morphinone, a toxic metabolite, in human was shown for the first time in in vitro experiments.     

Glutathione conjugation of 4-hydroxy-trans-2,3-nonenal in the rat in vivo, the isolated perfused liver and erythrocytes.

The formation of glutathione (GSH) conjugates of racemic 4-hydroxy-trans-2,3-nonenal (4-HNE) in the rat in vivo in the perfused rat liver and rat erythrocytes has been studied. An HPLC system was developed for the assay of 4-HNE-glutathione conjugates (HNE-SG). The very sensitive electrochemical detection method (detection limit 5 pmol) can also be used to study endogenously formed HNE-SG. Three diastereomeric HNE-SG conjugates could be separated by this system. Rat liver cytosol catalyzed the formation of 2 of the 3 conjugates. When 17 micromol/kg [(3)H] 4-HNE was injected intravenously in the rat, 21% of the radioactivity was excreted within 90 min in bile and 37% in urine. Most of the 4-HNE in bile was present as 2 of the HNE-SG conjugates (molecular mass 463). In addition, 25% was excreted as a third GSH conjugate (molecular mass of 461), which was identified as the lactone of the 4-hydroxynonenoic acid glutathione conjugate. Erythrocytes in vitro eliminated 4-HNE very rapidly, in part by GSH conjugation, suggesting that they may also play an important role in vivo. To study the role of the liver selectively, we used the recirculating perfused rat liver without erythrocytes in the perfusion medium; the same conjugates were found, but the third conjugate was a minor component. These results present direct evidence for the in vivo formation of 4-HNE glutathione conjugates in which the liver may play an important role.     

Differences in the effect of phenobarbital treatment on the in vitro metabolism of aflatoxin and aniline by duck and rat livers

Groups of ducks and rats were treated with phenobarbital sodium for 14 days (1 mg/ml in the drinking water) and the effects of this treatment on in vitro rates of microsomal aniline and aflatoxin metabolism were observed.

Aniline hydroxylase activity was enhanced in microsomes of both species. Normal levels of activity were comparable in duck and rat livers.

Aflatoxin metabolism was stimulated in crude microsomal preparations (9000 g supernatant fraction) from rat but not from duck liver. Normal mean rates of total aflatoxin metabolism were 0.31 nmole/g/min in rat liver and 47·0 nmoles/g/min in duck liver.

Phenobarbital treatment had the effect of stimulating total aflatoxin metabolism, hydroxylation and demethylation in isolated rat liver microsomes.

The failure of this treatment to stimulate aflatoxin metabolism in the duck liver suggested that microsomal metabolism was not rate limiting. Aflatoxin was actually metabolised 90 times more rapidly by soluble enzymes (105,000g supernatant) from duck liver than from rat liver.     

Estrogen receptor binding and estrogenic/antiestrogenic effects of two new metabolites of nitromiphene, 2-[p-[2-nitro-1-(4-methoxyphenyl)-2-phenylvinyl]phenoxy]-N-ethylpyrrolidine

Reduction of the triarylethylene antiestrogen 2-[p-[2-nitro-1-(4-methoxphenyl)-2-phenylvinyl]phenoxy]-N-ethylpyrrolidine (1) with sodium borohydride-stannous chloride afforded 2-(p-methoxyphenyl)-p'-(2-pyrrolidin-1-ylethoxy)deoxybenzoin (2). Incubation of 1 with rat cecal content suspension under aerobic or anaerobic conditions also resulted in the generation of 2. The lactam analogue of 1 (6) was prepared by condensation of 4-(2-bromoethoxy)-4'-methoxybenzophenone (3) with benzylmagnesium chloride, followed by dehydration, amidation with 2-pyrrolidinone, and nitration. A metabolite with chromatographic and spectral properties identical with those of 6 was found in extracts from incubation mixtures of 1 with phenobarbital-induced rat liver 9000g supernatant. Compound 2 did not exhibit appreciable binding to the rat uterine cytosol estrogen receptor at concentrations of up to 1 X 10(-6) M and had no estrogenic or antiestrogenic activity in the 3-day rat uterotropic assay. By contrast, 6 had estrogen receptor affinity somewhat greater than that of 1 and slightly greater estrogenic activity accompanied by reduced antiestrogenic activity in comparison with those of 1.     

Determination of the plasma pharmacokinetic and tissue distributions of swertiamarin in rats by liquid chromatography with tandem mass spectrometry

An LC-MS/MS method was developed for the quantification of swertiamarin (CAS 17388-39-5) in rat plasma and tissues using gentiopicroside as the internal standard (IS). Swertiamarin and an IS were extracted from plasma and tissues by a simple solid-phase extraction (SPE) procedure. Separation was achieved on a Phenomenex kinetex-C18 column (100 mm×2.1 mm, 2.6 μm) with an isocratic mobile phase consisting of methanol and water (22:78, v/v) with 0.1% acetic acid at a flow rate of 0.2 mL/min. The analyte and IS were detected by negative ion electrospray ionisation in multiple-reaction monitoring mode while monitoring the transitions of m/z 433 [M + CH3COO] - →179 and m/z 415 [M + CH3COO] - →179 for swertiamarin and the IS, respectively. The method was validated with respect to selectivity, matrix effect, linearity, accuracy, precision, recovery and stability. The method was successfully applied in a pharmacokinetic study of swertiamarin after intravenous and oral administration to rats. The pharmacokinetics of swertiamarin showed rapid absorption and elimination, and its absolute bioavailability was low at 10.3%. After oral administration to rats, swertiamarin was rapidly and widely distributed in its tissues. High concentrations were found in the liver and kidney, indicating that swertiamarin was possibly absorbed in the liver and eliminated by the kidney.     

Kinetic study of the reaction of cisplatin with thiols.

The reactions of cisplatin [cis-diamminedichloroplatinum(II), CDDP] with glutathione (GSH) and drug thiols were investigated at 37 degrees C in 100 mM Tris-NO(3), pH approximately 7.4, using a clinically relevant concentration of CDDP (33 micro M), a large excess of GSH (16.5 mM), and [NaCl] of 4.62 mM. The conditions were designed to mimic passage of CDDP through the cytosol. The reactions were studied by UV-absorption spectroscopy, high-pressure liquid chromatography (HPLC), and atomic absorption spectroscopy. The initial rates, detected by UV absorbance, confirmed that the reactions are first order in [CDDP]. The HPLC peak corresponding to CDDP was analyzed for platinum content by atomic absorption spectroscopy, which decreased exponentially with time, confirming that the reactions are first order in [CDDP] and allowing determination of the pseudo first order rate constants (k(1)). For reaction of the dichloro form of CDDP with GSH, the k(1) value was approximately 2.2 x 10(-4) s(-1) (t(1/2) of approximately 53 min), giving the second order rate constant value (k(2)) of approximately 1.3 x 10(-2) M(-)1 s(-1). Reaction of a mixture of the aquated forms of CDDP with GSH gave a lower k(1) value ( approximately 0.9 x 10(-4) s(-1)). Reaction of CDDP with sodium 2-mercaptoethanesulfonate (mesna) gave a k(1) value of approximately 1.8 x 10(-4) s(-1) (t(1/2) of approximately 65 min and k(2) of approximately 1.1 x 10(-2) M(-1) s(-1)). Reaction of CDDP with S-2-(3-aminopropylamino)ethanethiol (WR-1065) gave a k(1) value of approximately 12.0 x 10(-4) s(-1) (t(1/2) of approximately 10 min and k(2) of approximately 7.3 x 10(-2) M(-)1 s(-1)). The relatively slow reaction rate of CDDP with GSH is consistent with the efficient DNA platination by CDDP in the presence of millimolar concentration of GSH in the cytosol.     

In vitro biotransformation and genotoxicity of the drinking water disinfection byproduct bromodichloromethane: DNA binding mediated by glutathione transferase theta 1-1

The drinking water disinfection byproduct bromodichloromethane (CHBrCl(2)) was previously shown to be mutagenic in Salmonella typhimurium that overexpress rat glutathione transferase theta 1-1 (GSTT1-1). Several experimental approaches were undertaken in this study to investigate the DNA covalent binding potential of reactive intermediates generated by GSTT1-1-mediated metabolism of CHBrCl(2). First, rodent hepatic cytosol incubations containing [(14)C]CHBrCl(2), supplemented glutathione (GSH), and calf thymus DNA resulted in approximately 3-fold (rat liver cytosol) and 7-fold (mouse liver cytosol) greater amounts of total radioactivity (RAD) associated with the purified DNA as compared to a control (absence of rodent cytosol) following liquid scintillation counting (LSC) of isolated DNA. The relative increase in DNA labeling is consistent with the conjugation activity of these rodent cytosols toward CHBrCl(2). Second, exposure of GSTT1-1-expressing S. typhimurium to [(14)C]CHBrCl(2) resulted in a concentration-dependent increase of bacterial DNA-associated total radioactivity. Characterization of DNA-associated radioactivity could not be assigned to a specific deoxynucleoside adduct(s) following enzymatic hydrolysis of DNA and subsequent HPLC analysis. A possible explanation for this observation was formation of a 'transient' adduct that was unstable in the DNA isolation and hydrolysis procedures employed. To circumvent problems of adduct instability, reactions of [(14)C]CHBrCl(2) with GSH catalyzed by recombinant rat GSTT1-1 were performed in the presence of calf thymus DNA or, alternatively, the model nucleophile deoxyguanosine. Hydroxyapatite chromatography of [(14)C]-labeled DNA or HPLC chromatography of [(14)C]-labeled deoxyguanosine derivatives demonstrated the covalent binding of [(14)C]CHBrCl(2)-derived metabolites to DNA and deoxyguanosine in low yield (approximately 0.02% of [(14)C]CHBrCl(2) biotransformed by GSTT1-1 resulted in DNA adducts). Cytochrome P450 (CYP)- and GST-catalyzed biotransformation of CHBrCl(2) in rat tissues (kidney and large intestine) that develop tumors following chronic CHBrCl(2) exposure were compared with rat liver (a nontarget tissue). Rat liver had a significant capacity to detoxify CHBrCl(2) (to carbon dioxide) compared with kidney and large intestine as a result of CYP-catalyzed oxidation, liver was approximately 16-fold more efficient than kidney and large intestine when intrinsic clearance values (V(max)/K(m)) were compared. In contrast, the efficiency of GST-mediated GSH conjugation of CHBrCl(2) in kidney and large intestine was only slightly lower than liver (approximately 2- to 4-fold lower), thus, the relative amounts of reactive intermediates that are produced with the capacity to covalently modify DNA may be enhanced in these extrahepatic tissues. The significance of these findings is that conjugation of CHBrCl(2) with GSH can result in the covalent modification of DNA and that cancer target tissues in rats have a much reduced detoxification capacity, but only a modest decrease in bioactivation capacity, as compared to the liver (a nontarget tissue in rats).