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1.
Life Sci ; 77(16): 2018-29, 2005 Sep 02.
Article in English | MEDLINE | ID: mdl-15919096

ABSTRACT

Echinacea is a widely used herbal remedy for the treatment of colds and other infections. However, almost nothing is known about the disposition and pharmacokinetics of any of its components, particularly the alkamides and caffeic acid conjugates which are thought to be the active phytochemicals. In this investigation, we have examined serial plasma samples from 9 healthy volunteers who ingested echinacea tablets manufactured from ethanolic liquid extracts of Echinacea angustifolia and Echinacea purpurea immediately after a standard high fat breakfast. Caffeic acid conjugates could not be identified in any plasma sample at any time after tablet ingestion. Alkamides were rapidly absorbed and were measurable in plasma 20 min after tablet ingestion and remained detectable for up to 12 h. Concentration-time curves for 2,4-diene and 2-ene alkamides were determined. The maximal concentrations for the sum of alkamides in human plasma were reached within 2.3 h post ingestion and averaged 336+/-131 ng eq/mL plasma. No obvious differences were observed in the pharmacokinetics of individual or total alkamides in 2 additional fasted subjects who took the same dose of the echinacea preparation. This single dose study provides evidence that alkamides are orally available and that their pharmacokinetics are in agreement with the one dose three times daily regimen already recommended for echinacea.


Subject(s)
Amides/pharmacokinetics , Caffeic Acids/pharmacokinetics , Echinacea/chemistry , Adult , Amides/blood , Amides/chemistry , Caffeic Acids/blood , Chromatography, High Pressure Liquid , Female , Humans , Male , Mass Spectrometry , Plant Extracts/administration & dosage , Plant Extracts/blood , Plant Extracts/pharmacokinetics , Plants, Medicinal , Tablets/administration & dosage , Time Factors
2.
Drug Metab Dispos ; 30(2): 161-6, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11792685

ABSTRACT

The nonsteroidal anti-inflammatory drug naproxen is primarily metabolized in humans by acyl glucuronidation to form naproxen acyl glucuronide and by O-dealkylation to form 6-O-desmethylnaproxen (DMN). DMN contains both carboxy and phenolic groups and has been shown to form acyl glucuronide and sulfate conjugates. This project aimed to investigate whether DMN formed a phenolic glucuronide and diglucuronide(s) (with both the carboxy and phenolic groups glucuronidated). Male Sprague-Dawley rats (300-350 g) with exteriorized bile flow were dosed i.v. with DMN at 50 mg/kg. Four major DMN-related peaks were detected in bile by high-performance liquid chromatography (HPLC) analysis at 225 nm, including the known acyl glucuronide and sulfate conjugates. Selective hydrolyses using acidic and alkaline conditions and digestion with beta-glucuronidase allowed tentative identification of the two unknown peaks as the phenolic glucuronide of DMN and a novel acyl glucuronide-sulfate diconjugate of DMN (i.e., formed by sulfonation of the phenolic group and glucuronidation of the carboxy group). The identities were confirmed by liquid chromatography-tandem mass spectrometry analysis of individual HPLC fractions. Total recovery of the DMN dose was approximately 80%, with the sulfate conjugate (50%) and unchanged DMN (10%) being excreted predominantly in urine and the acyl glucuronide (10%), phenolic glucuronide (6%), and acyl glucuronide-sulfate diconjugate (4%) being excreted predominantly or exclusively in bile. No evidence for a diglucuronide metabolite of DMN was found in either bile or urine of the DMN-dosed rats.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/metabolism , Bile/metabolism , Glucuronides/metabolism , Naproxen/analogs & derivatives , Naproxen/metabolism , Sulfates/metabolism , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Male , Naproxen/chemistry , Rats , Rats, Sprague-Dawley
3.
Xenobiotica ; 31(6): 309-19, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11513245

ABSTRACT

1. An isolated perfused rat liver (IPRL) preparation was used to investigate separately the disposition of the non-steroidal anti-inflammatory drug (NSAID) naproxen (NAP), its reactive acyl glucuronide metabolite (NAG) and a mixture of NAG rearrangement isomers (isoNAG), each at 30 microg NAP equivalents ml perfusate (n = 4 each group). 2. Following administration to the IPRL, NAP was eliminated slowly in a log-linear manner with an apparent elimination half-life (t 1/2) of 13.4 +/- 4.4h. No metabolites were detected in perfusate, while NAG was the only metablolite present in bile in measurable amounts (3.9 +/- 0.8% of the dose). Following their administration to the IPRL, both NAG and isoNAG were rapidly hydrolysed (t 1/2 in perfusate = 57 +/- 3 and 75 +/- 14 min respectively). NAG also rearranged to isoNAG in the perfusate. Both NAG and isoNAG were excreted intact in bile (24.6 and 14.8% of the NAG and isoNAG doses, respectively). 3. Covalent NAP-protein adducts in the liver increased as the dose changed from NAP to NAG to isoNAG (0.20 to 0.34 to 0.48% of the doses, respectively). Similarly, formation of covalent NAP-protein adducts in perfusate were greater in isoNAG-dosed perfusions. The comparative results suggest that isoNAG is a better substrate for adduct formation with liver proteins than NAG.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/metabolism , Glucuronides/metabolism , Liver/metabolism , Naproxen/metabolism , Acylation , Animals , Bile/metabolism , Half-Life , Hydrolysis , Isomerism , Kinetics , Male , Rats , Rats, Sprague-Dawley
4.
Life Sci ; 68(7): 785-97, 2001 Jan 05.
Article in English | MEDLINE | ID: mdl-11205870

ABSTRACT

The nonsteroidal anti-inflammatory drug zomepirac (ZP) is metabolised to a chemically reactive acyl glucuronide conjugate (ZAG) which can form covalent adducts with proteins. In vivo, such adducts could initiate immune or toxic responses. In rats given ZP, the major band detected in liver homogenates by immunoblotting with a polyclonal ZP antiserum was at 110 kDa. This adduct was identified as ZP-modified dipeptidyl peptidase IV (DPP IV) by immunoblotting using the polyclonal ZP antiserum and monoclonal DPP IV antibodies OX-61 and 236.3. In vitro, ZAG, but not ZP itself, covalently modified recombinant human and rat DPP IV. Both monoclonal antibodies recognized DPP IV in livers from ZP- and vehicle-dosed rats. Confirmation that the 110 kDa bands which were immunoreactive with the ZP and DPP IV antibodies represented the same molecule was obtained from a rat liver extract reciprocally immunodepleted of antigens reactive with these two antibodies. Furthermore, immunoprecipitations with OX-61 antibody followed by immunolotting with ZP antiserum, and the reciprocal experiment, showed that both these antibodies recognised the same 110 kDa molecule in extracts of ZP-dosed rat liver. The results verify that DPP IV is one of the protein targets for covalent modification during hepatic transport and biliary excretion of ZAG in rats.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , DNA Adducts/metabolism , Dipeptidyl Peptidase 4/metabolism , Glucuronides/metabolism , Hydrogen-Ion Concentration , Tolmetin/analogs & derivatives , Tolmetin/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Blotting, Western , CHO Cells , Cricetinae , Humans , Liver/enzymology , Male , Precipitin Tests , Rats , Rats, Sprague-Dawley , Recombinant Proteins/chemistry , Tolmetin/metabolism
5.
Life Sci ; 70(1): 25-36, 2001 Nov 21.
Article in English | MEDLINE | ID: mdl-11764004

ABSTRACT

Many non-steroidal anti-inflammatory drugs (NSAIDs) which form acyl glucuronide conjugates as major metabolites have shown an antiproliferative effect on colorectal tumors. This study assesses the extent to which rearrangement of an acyl glucuronide metabolite of a model NSAID into beta-glucuronidase-resistant isomers facilitates its passage through the small intestine to reach the colon. Rats were dosed orally with diflunisal (DF), its acyl glucuronide (DAG) and a mixture of rearrangement isomers (iso-DAG) at 10 mg DF equivalents/kg. The parent drug DF appeared in plasma after all doses, with maximum concentrations of 20.5+/-2.5, 28.8+/-8.3 and 11.0+/-1.6 microg DF/ml respectively, obtained at 3.8+/-0.3, 3.6+/-1.8 and 7.5+/-0.9 hr after the DF, DAG and iso-DAG doses respectively. At 48 hr, 16.2+/-3.3, 19.8+/-0.8 and 42.9+/-10.1% of the doses respectively were recovered in feces, with < or = 1% remaining in the intestine. About half of each dose was recovered as DF and metabolites in 48 hr urine: for DF and DAG doses, the majority was in the first 24 hr urine, whereas for iso-DAG doses, recoveries in the first and second 24 hr periods were similar. The results show that hydrolysis of both DAG and iso-DAG, and absorption of liberated DF, occur during passage through the gut, but that these processes occur more slowly and to a lesser degree for iso-DAG. The intrinsic hydrolytic capacities of various intestinal segments (including contents) towards DAG and iso-DAG were obtained by incubating homogenates under saturating concentrations of DAG/iso-DAG at 37 degrees C. Upper small intestine, lower small intestine, caecum and colon released 2400, 3200, 9200 and 22800 microg DF/hr/g tissue plus contents respectively from DAG substrate, and 18, 10, 140 and 120 microg DF/hr/g tissue plus contents respectively from iso-DAG substrate. The much greater resistance of iso-DAG to hydrolysis appears attributable to its resistance to beta-glucuronidases. The data suggest that in rats dosed with DF, DAG excreted in bile would be substantially hydrolysed in the small intestine and liberated DF reabsorbed, but that portion which rearranges to iso-DAG would likely reach the colon.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Colon/metabolism , Diflunisal/analogs & derivatives , Diflunisal/pharmacokinetics , Glucuronidase/metabolism , Intestine, Small/metabolism , Animals , Animals, Outbred Strains , Area Under Curve , Biological Transport , Glucuronidase/chemistry , Intestinal Absorption , Male , Models, Animal , Rats , Rats, Sprague-Dawley , Stereoisomerism
6.
Life Sci ; 70(1): 37-48, 2001 Nov 21.
Article in English | MEDLINE | ID: mdl-11764005

ABSTRACT

Many nonsteroidal anti-inflammatory drugs (NSAIDs) which have antiproliferative activity in colon cancer cells are carboxylate compounds forming acyl glucuronide metabolites. Acyl glucuronides are potentially reactive, able to hydrolyse, rearrange into isomers, and covalently modify proteins under physiological conditions. This study investigated whether the acyl glucuronides (and isomers) of the carboxylate NSAIDs diflunisal, zomepirac and diclofenac had antiproliferative activity on human adenocarcinoma HT-29 cells in culture. Included as controls were the carboxylate NSAIDs themselves, the non-carboxylate NSAID piroxicam, and the carboxylate non-NSAID valproate, as well as its acyl glucuronide and isomers. The compounds were incubated at 1-3000 microM with HT-29 cells for 24 hr, with [3H]-thymidine added for an additional 2 hr incubation. IC50 values were calculated from the concentration-inhibition response curves for thymidine uptake. The four NSAIDs inhibited thymidine uptake, with IC50 values about 200-500 microM. All of the NSAID acyl glucuronides (and isomers, tested in the case of diflunisal) showed antiproliferative activity broadly comparable to the parent drugs. This activity may stem from direct uptake of intact glucuronide/isomers followed by covalent modification of proteins critical in the cell replication process. However, hydrolysis during incubation and cellular uptake of liberated parent NSAID will play a role. In HT-29 cells incubated with zomepirac, covalently modified proteins in cytosol were detected by immunoblotting with a zomepirac antibody, suggesting that HT-29 cells do have the capacity to glucuronidate zomepirac. The anti-epileptic drug valproate had no effect on inhibition of thymidine uptake, though, surprisingly, its acyl glucuronide and isomers were active. The reasons for this are unclear at present.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Diclofenac/pharmacology , Diflunisal/pharmacology , Glucuronides/pharmacology , HT29 Cells/drug effects , Tolmetin/analogs & derivatives , Tolmetin/pharmacology , Acylation , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Cell Division/drug effects , Diclofenac/metabolism , Diflunisal/metabolism , Dose-Response Relationship, Drug , Glucuronides/metabolism , HT29 Cells/metabolism , HT29 Cells/pathology , Humans , Piroxicam/pharmacology , Stereoisomerism , Thymidine/metabolism , Tolmetin/metabolism , Valproic Acid/metabolism , Valproic Acid/pharmacology
7.
Drug Metabol Drug Interact ; 16(3): 159-71, 2000.
Article in English | MEDLINE | ID: mdl-11116751

ABSTRACT

A major metabolic pathway for triamterene (a potassium sparing diuretic) is aromatic hydroxylation followed by sulphate conjugation. Diflunisal (a salicylate anti-inflammatory agent) also undergoes sulphate conjugation of its phenolic group as a major pathway. We investigated the possible effect of diflunisal on the elimination of triamterene (competition for phenolic sulphonation) in six healthy volunteers by studying the disposition of single doses of triamterene (100 mg) taken alone and in the presence of steady-state levels of diflunisal. Diflunisal coadministration (500 mg b.i.d.) had no effect on the pharmacokinetics of triamterene itself. However, plasma AUC of p-hydroxytriamterene sulphate was greater (4.6 times), and its renal clearance lower (0.24 times), in the presence of diflunisal. There was no change in the formation clearance or protein binding of p-hydroxytriamterene sulphate in the presence of diflunisal. The data point to competition for renal excretory pathways rather than sulphonation capacity. This interaction could have clinical relevance since p-hydroxytriamterene sulphate is pharmacologically active.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Diflunisal/pharmacology , Diuretics/pharmacokinetics , Triamterene/pharmacokinetics , Adult , Chromatography, High Pressure Liquid/methods , Diuretics/blood , Diuretics/urine , Drug Interactions , Female , Humans , Male , Middle Aged , Triamterene/blood , Triamterene/urine
8.
Epilepsy Res ; 41(3): 259-68, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10962217

ABSTRACT

The plasma profiles of valproate (VPA), its beta-oxidation metabolites E-2-en-VPA and 3-oxo-VPA and its terminal desaturation metabolite 4-en-VPA, have been measured in a patient receiving NaVPA 1000 mg twice per day from early in the course of serious hepatotoxicity and for 2 weeks after the drug was stopped. Concurrent profiles of liver, renal and haematological function parameters were available. Relative to concurrent plasma VPA concentrations, E-2-en-VPA concentrations were not different to those of the VPA-treated epileptic population at any stage of the illness, whereas 3-oxo-VPA concentrations relative to concurrent VPA concentrations were abnormally high early in the toxicity, abnormally low at its peak (3-5 days later), and comfortably within normal limits for the treated epileptic population late in the recovery phase (9-13 days from the onset). When measurable, plasma 4-en-VPA concentrations were not elevated. The elimination half-life of VPA during the recovery phase was 100 h, which is some 6-12 times greater than values reported for this parameter in normal patients. These data clearly define, in this patient, a link between idiosyncratic VPA-associated hepatotoxicity at its onset and peak and the later stages of VPA beta-oxidation. Whether the beta-oxidation abnormalities are causative or a consequence of an as yet undefined defect is unknown. In this patient, 4-en-VPA was unlikely to have been involved in the pathogenesis of the toxicity.


Subject(s)
Anticonvulsants/adverse effects , Anticonvulsants/pharmacokinetics , Chemical and Drug Induced Liver Injury/metabolism , Valproic Acid/adverse effects , Valproic Acid/pharmacokinetics , Adult , Biotransformation , Electroencephalography , Epilepsies, Partial/complications , Epilepsies, Partial/drug therapy , Humans , Liver Function Tests , Male , Tomography, X-Ray Computed
9.
Drug Metab Dispos ; 28(8): 945-50, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10901705

ABSTRACT

The anticonvulsant phenytoin (5,5-diphenylhydantoin) provokes a skin rash in 5 to 10% of patients, which heralds the start of an idiosyncratic reaction that may result from covalent modification of normal self proteins by reactive drug metabolites. Phenytoin is metabolized by cytochrome P450 (P450) enzymes primarily to 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH), which may be further metabolized to a catechol that spontaneously oxidizes to semiquinone and quinone species that covalently modify proteins. The aim of this study was to determine which P450s catalyze HPPH metabolism to the catechol, proposed to be the final enzymatic step in phenytoin bioactivation. Recombinant human P450s were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Novel bicistronic expression vectors were constructed for P450 2C19 and the three major variants of P450 2C9, i.e., 2C9*1, 2C9*2, and 2C9*3. HPPH metabolism and covalent adduct formation were assessed in parallel. P450 2C19 was the most effective catalyst of HPPH oxidation to the catechol metabolite and was also associated with the highest levels of covalent adduct formation. P450 3A4, 3A5, 3A7, 2C9*1, and 2C9*2 also catalyzed bioactivation of HPPH, but to a lesser extent. Fluorographic analysis showed that the major targets of adduct formation in bacterial membranes were the catalytic P450 forms, as suggested from experiments with human liver microsomes. These results suggest that P450 2C19 and other forms from the 2C and 3A subfamilies may be targets as well as catalysts of drug-protein adduct formation from phenytoin.


Subject(s)
Anticonvulsants/metabolism , Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/metabolism , Oxidoreductases, N-Demethylating/metabolism , Phenytoin/metabolism , Autoimmunity , Catechols/analysis , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/immunology , Epitopes/immunology , Humans , Oxidoreductases, N-Demethylating/immunology , Recombinant Proteins/metabolism
10.
Br J Clin Pharmacol ; 49(5): 409-15, 2000 May.
Article in English | MEDLINE | ID: mdl-10792197

ABSTRACT

AIMS: The study aimed to show whether autoinduction of valproate (VPA) along its beta-oxidation pathway occurred upon chronic dosing in humans. METHODS: Twelve young volunteers without active illness took sodium valproate (NaVPA) 200 mg orally 12 hourly for 3 weeks. On days 7 and 21, serial blood samples and all urine passed over an interdosing interval from 08.00 to 20.00 h were collected for analysis of VPA and certain metabolites. RESULTS: Plasma AUC(0,12 h) of VPA was significantly lower on day 21 than on day 7 (2.40 vs 2.84 micromol ml-1 h, 95% CI for the difference 0.13-0.81 micromol ml-1 h). Significant differences in plasma AUC(0,12 h) of the beta-oxidation metabolites E-2-en-VPA and 3-oxo-VPA were not found. However, formation clearances of plasma VPA to urinary E-2-en-VPA and 3-oxo-VPA were significantly increased from day 7 to day 21 (0. 010 vs 0.024 and 2.57 vs 3.60 ml kg-1 h-1, respectively, 95% CI for the differences -0.025 to -0.004 and -1.72 to -0.34 ml kg-1 h-1, respectively). Formation clearances to VPA-glucuronide (0.534 vs 0. 505 ml kg-1 h-1) and 4-OH-VPA (0.112 vs 0.110 ml kg-1 h-1) were not significantly different. CONCLUSIONS: Regular low dose VPA intake in humans over a period of 3 weeks appears to be associated with a small induction of its metabolism by the beta-oxidation pathway, but not by glucuronidation or 4-hydroxylation.


Subject(s)
Anticonvulsants/pharmacokinetics , Valproic Acid/pharmacokinetics , Adolescent , Adult , Female , Humans , Male , Metabolic Clearance Rate , Oxidation-Reduction
11.
Life Sci ; 68(5): 525-37, 2000 Dec 22.
Article in English | MEDLINE | ID: mdl-11197750

ABSTRACT

Acyl glucuronides are reactive electrophilic metabolites of carboxylate drugs, capable of undergoing hydrolysis, rearrangement and covalent binding reactions with proteins in vivo. Such covalent drug-protein adducts may be prerequisites for certain idiosyncratic immune and toxic responses in susceptible individuals. The present study examined the effect of experimental cholestasis on the extent and pattern of formation of protein adducts in plasma and liver of rats given the non-steroidal antiinflammatory drug (NSAID) zomepirac (ZP). Groups of intact, bile-exteriorized and bile duct-ligated rats given a 50 mg/kg i.v. dose of ZP were studied for 24 hr. In intact rats, only 1.4% of the dose was recovered as the sum of ZP, ZP acyl glucuronide (ZAG) and its rearrangement isomers (iso-ZAG) in urine in 24 hr. In bile-exteriorized animals, 0.5% of the dose was recovered in urine in 24 hr, with 31.6% of the dose being recovered in bile (2.7% as ZP, 20.0% as ZAG and 8.9% as iso-ZAG). In the bile duct-ligated group, recovery of dose in 24 hr urine totalled 17.5% (1.7% as ZP, 6.7% as ZAG and 9.1% as iso-ZAG). ZAG and iso-ZAG were measurable in plasma only in the bile duct-ligated group, and covalent binding of ZP to plasma proteins was much higher (5-6 fold) than in intact or bile-exteriorized rats. Total adduct concentrations in liver were not significantly different among the three groups. Immunoblotting using a polyclonal ZP antiserum confirmed that serum albumin was a major target protein in plasma. The major ZP-modified bands in the livers of intact and bile-exteriorized rats were at about 110, 140 and 200 kDa. However, the bands at 110 and 140 kDa were much lower in the livers of bile duct-ligated rats. The results show that about 30% of ZP doses are normally excreted as ZAG and its isomers in bile, with only minor excretion in urine. Bile duct ligation shunts the glucuronide into blood (and urine), strongly promoting adduct formation with plasma proteins, and alters the pattern but not the total quantity of drug-modified proteins formed in the liver.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Bile Ducts/physiology , Liver/metabolism , Tolmetin/analogs & derivatives , Tolmetin/pharmacokinetics , Animals , Anti-Inflammatory Agents, Non-Steroidal/blood , Anti-Inflammatory Agents, Non-Steroidal/urine , Area Under Curve , Blood Proteins/metabolism , Gallbladder/metabolism , Glucuronides/metabolism , Isomerism , Metabolic Clearance Rate , Protein Binding , Rats , Tolmetin/blood , Tolmetin/urine
12.
Eur J Clin Pharmacol ; 56(9-10): 715-21, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11214782

ABSTRACT

OBJECTIVE: The effects of coadministration of the non-steroidal anti-inflammatory drug diflunisal (DF) on glucuronidation and beta-oxidation of the antiepileptic agent valproic acid (VPA), and of VPA on DF glucuronidation, were studied in human volunteers. METHODS: Seven healthy male volunteers received sodium valproate (NaVPA, 200 mg) orally twice daily for 7 days, after which all drug intake ceased for 1 month. The volunteers then took DF (250 mg) orally twice daily for 7 days. Both drugs were then taken (at the same doses as previously) twice daily for 7 days. On day 7 of each dosing phase, serial blood samples and all urine passed over the 12-h inter-dosing interval were collected. VPA, DF and selected metabolites were analysed using validated methods. Statistical comparisons of pharmacokinetic parameters were made using paired Student's t-tests. RESULTS: Mean plasma concentrations of total VPA were lower and apparent plasma clearances significantly higher during DF coadministration. This was associated with a significant 20% increase in the unbound fraction of VPA (from 6.6+/-1.3% to 7.9+/-1.8%). The apparent clearance of unbound VPA was not different. There was no evidence of any significant effect of DF coadministration on VPA metabolism: urinary recoveries of and formation clearances to urinary VPA-glucuronide, E-2-en-VPA, 3-oxo-VPA and 4-en-VPA were not significantly altered. However, there was a highly significant 35% increase in the area under the plasma concentration-time curve from 0-12 h (AUC0-12h) of 3-oxo-VPA and its renal clearance was lower, though not significantly so. VPA coadministration had no effect on DF pharmacokinetics or formation clearances of DF to its acyl glucuronide (DAG), phenolic glucuronide (DPG) or sulfate (DS) conjugates. However, plasma AUC0-12h values of the glucuronides were significantly lower and their renal clearances higher (though significantly so only in the case of DPG) during VPA coadministration. CONCLUSIONS: Steady-state coadministration of VPA and DF leads to a significant displacement of VPA from plasma protein binding sites. There was no evidence of competition for glucuronidation capacity or other metabolic interactions. Rather, the interactions detected appeared to be renal in nature, with renal clearance of 3-oxo-VPA being reduced by DF coadministration, and renal clearance of DPG and perhaps DAG being increased by VPA coadministration.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Anticonvulsants/pharmacokinetics , Diflunisal/pharmacokinetics , Valproic Acid/pharmacokinetics , Adult , Area Under Curve , Biotransformation , Drug Interactions , Glucuronides/metabolism , Half-Life , Humans , Male , Oxidation-Reduction
13.
Biopharm Drug Dispos ; 21(6): 235-42, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11304722

ABSTRACT

The effects of co-administration of the antiepileptic agent valproic acid (VPA) and the non-steroidal anti-inflammatory drug naproxen (NAP) on their relative dispositions (particularly with respect to glucuronidation) were investigated in human volunteers. Seven healthy males received each drug alone and then in combination (orally twice daily for seven days, 500 mg sodium VPA, 500 mg NAP). On day 7 of each dosing phase, serial plasma and 24 h urine samples were collected for analysis. Co-administration of NAP resulted in significant increases (about 20%, p<0.05) in the apparent plasma clearance of total VPA and in the unbound fraction of VPA in plasma, with the apparent plasma clearance of unbound VPA being unchanged. There were associated increases in the formation clearances to urinary VPA-glucuronide and 3-oxo-VPA, though these were relatively greater for the glucuronidation pathway (and remained significant when formation clearances were calculated using the unbound fraction of drug in plasma). The data thus point to a shift towards glucuronidation as a result of the NAP-induced increase in the unbound fraction of VPA in plasma. By contrast, VPA co-administration caused a decrease (of about 10%, p<0.05) in the apparent plasma clearance of total NAP. Taken in hand with in vitro results showing a VPA-induced displacement (of about 40%) of NAP from plasma protein binding sites, the data strongly support a role for diminished glucuronidation of NAP and its desmethyl metabolite in the presence of co-administered VPA.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Anticonvulsants/pharmacology , Anticonvulsants/pharmacokinetics , Naproxen/pharmacology , Naproxen/pharmacokinetics , Valproic Acid/pharmacology , Valproic Acid/pharmacokinetics , Adult , Area Under Curve , Biotransformation , Drug Interactions , Gas Chromatography-Mass Spectrometry , Glucuronides/metabolism , Humans , Male , Middle Aged , Oxidation-Reduction , Spectrophotometry, Ultraviolet
14.
J Pharmacol Toxicol Methods ; 41(1): 27-32, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10507755

ABSTRACT

The covalent binding of drugs or their metabolites to proteins is of increasing interest in the investigation of the toxicity of these compounds. Recent attention on biological consequences of protein adduct formation with carboxylate drugs, derived via their reactive acyl glucuronide metabolites, has focussed on liver tissue. Although the intact animal represents undisturbed hepatic physiology, other hepatic models can offer advantages, e.g., multiple experiments from a single liver. In this study we set out to compare the patterns of covalent binding of zomepirac (ZP) to proteins in the livers of intact rats, isolated rat hepatocytes (in culture or suspension), and in rat liver homogenates. Rats were dosed i.v. with 25 mg ZP/kg, and their livers were removed 3 h later. Isolated hepatocytes or liver homogenates were exposed to ZP at 100 microg/mL for 3 h at 37 degrees C. Liver homogenates were exposed to ZP and also zomepirac acyl glucuronide (ZAG) at 100 microg ZP equivalents/mL for 3 h at 37 degrees C. Covalent binding of ZP species was examined by SDS-PAGE and Western blotting with a polyclonal ZP antiserum. In livers from dosed animals, the strongest staining appeared at about 110120, 140, and 200 kDa. Few similarities existed with the results from isolated hepatocytes and, not surprisingly, liver homogenates. Only the 200-kDa band was common to all treatments. Many proteins seemed to be modified, at least to some extent. The differences in major bands are most likely caused by the loss of liver and hepatocyte architecture. The variability across different model systems in respect to covalent binding to hepatic proteins emphasizes the need for care in interpretation of results.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/metabolism , Glucuronates/metabolism , Liver/metabolism , Tolmetin/analogs & derivatives , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Binding, Competitive , Blotting, Western , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Glycosylation , Liver/cytology , Liver/drug effects , Male , Protein Binding , Rats , Rats, Wistar , Tolmetin/metabolism , Tolmetin/pharmacology
15.
Chem Biol Interact ; 115(2): 153-66, 1998 Sep 04.
Article in English | MEDLINE | ID: mdl-9826947

ABSTRACT

Drugs possessing a carboxylate functional group usually form acyl glucuronides as major metabolites. These electrophilic metabolites can undergo several spontaneous reactions, including covalent adduct formation with proteins. The present study examined whether covalent adducts were formed with microtubular protein (MTP, 85%, alpha/beta-tubulin) and whether this influenced its ability to assemble into microtubules. Bovine brain microtubular protein (MTP) was purified by assembly-disassembly cycles and incubated with the nonsteroidal anti-inflammatory drug (NSAID) zomepirac (ZP), its acyl glucuronide (ZAG) and rearrangement isomers (iso-ZAG) at various concentrations for 2 h at room temperature and pH 7.5. Assembly was monitored by change in turbidity (increase in absorbance at 340 nm). Both ZAG and iso-ZAG caused dose-dependent inhibition of assembly (50% inhibition at about 1 mM), while ZP caused modest inhibition (< 50% inhibition at 4 mM). In a slightly different system, incubation of performed microtubules with 4 mM ZAG caused about 35% inhibition of reassembly ability, while modification of MTP under similar conditions resulted in about 85% reduction of assembly ability. Immunoblotting with a ZP antiserum showed that ZAG and iso-ZAG covalently modified MTP in a dose-dependent manner, while ZP itself caused no modification. Tubulin and many minor proteins comprising MTP were modified. ZP-modified tubulin was shown to be present in the cytosol of livers from rats dosed twice daily for 3 days with ZP at 50 mg/kg, using a sandwich ELISA with ZP and tubulin antisera. Whether any perturbation of microtubule assembly occurs in vivo as a result of this in vivo modification is currently under investigation.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Microtubules/drug effects , Tolmetin/analogs & derivatives , Tubulin/chemistry , Animals , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Cattle , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Liver/drug effects , Liver/metabolism , Microtubules/metabolism , Rats , Stereoisomerism , Structure-Activity Relationship , Tolmetin/metabolism , Tolmetin/pharmacology , Tubulin/metabolism
16.
Life Sci ; 62(8): 751-62, 1998.
Article in English | MEDLINE | ID: mdl-9489511

ABSTRACT

Acyl glucuronides are reactive electrophilic metabolites of carboxylate drugs which can form covalent adducts with endogenous macromolecules such as serum albumin and hepatic proteins. Such adducts have been suggested as initiating factors in certain immune and toxic responses to acidic drugs. In the present study, pretreatment of rats with high daily doses (50 mg/kg orally) of the non-steroidal anti-inflammatory drug (NSAID) diflunisal (DF) for 35 days, followed by perfusion of the isolated liver with 3 mg DF for 3 hr, resulted in appreciable concentrations of covalent adducts of DF with hepatic tissue (3.68 microg DF/g liver). Immunoblotting using a rabbit polyclonal DF antiserum showed the major DF-modified bands at about 110, 140 and 200 kDa. A vehicle-pretreated control group achieved adduct concentrations of only 0.37 microg DF/g liver, with the 200 kDa band not detectable in immunoblots. Elimination of DF from perfusate of the isolated perfused rat liver (IPRL) preparation was the same (t1/2 about 3.4 hr) in both DF- and vehicle-pretreated groups. Appearance of the sulfate (DS) conjugate, the major metabolite in perfusate, was also similar. However, higher concentrations of the acyl glucuronide (DAG) and phenolic glucuronide (DPG) conjugates were found in perfusate at later times, though a statistically significant difference in area under the concentration-time curve was found only in the case of DAG. At 3 hr, recoveries of dose as DAG and DPG were significantly higher in perfusate, but not in bile. No significant differences in uptake and biliary excretion of taurocholate were found between the two groups. The finding of higher perfusate concentrations of DAG and DPG could signal a minor compromise to biliary excretion processes for the glucuronides, though whether such a result is simply coincident with or attributable to DAG-derived covalent DF-protein adducts in liver remains indeterminate.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/metabolism , Bile/metabolism , Diflunisal/metabolism , Glucuronates/metabolism , Liver/metabolism , Taurocholic Acid/metabolism , Animals , Biological Transport , Male , Perfusion , Rabbits , Rats , Rats, Sprague-Dawley
17.
Drug Metab Dispos ; 26(2): 98-104, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9456294

ABSTRACT

Acyl glucuronides are intrinsically reactive metabolites of carboxylate drugs, capable of undergoing hydrolysis, intramolecular rearrangement (isomerization via acyl migration), and intermolecular transacylation reactions. Transacylation with nucleophilic groups located on protein molecules leads to covalent drug-protein adducts. Protein adducts can also form from the rearrangement isomers via a glycation mechanism. In this study, the isolated perfused rat liver preparation was used to separately trace the dispositions of the nonsteroidal anti-inflammatory drug diflunisal (DF), its reactive acyl glucuronide metabolite (DAG), and a mixture of DAG rearrangement isomers (iso-DAG), each administered at 30-microg DF equivalents/ml perfusate (four recirculating perfusions each group). After administration of DF, the drug was eliminated in a log linear manner over 3 hr, with apparent elimination half-life (t1/2) of 2.6 +/- 0.4 hr. The sulfate conjugate (DS), excreted almost exclusively into perfusate, accounted for 14.2% of the dose, with the phenolic glucuronide (DPG) and DAG (11.1 and 7.9% of dose, respectively) excreted primarily in bile. Only a small portion (2.3%) of the dose was recovered as novel "diglucuronides" (D-2G, arising from phenolic glucuronidation of iso-DAG), excreted exclusively in bile. Covalent DF-protein adducts were found in both perfusate (0.98%) and liver (0. 14%). After administration of DAG, rapid hydrolysis occurred (initial DAG t1/2 17.3 +/- 4.2 min). At 3 hr, recoveries (in comparison to DF-dosed perfusions) were similar for DF (51.7%) and DAG (8.3%), significantly decreased for DS (10.6%) and DPG (6.4%), and significantly increased for iso-DAG (0.8%), D-2G (9.1%), and covalent adducts in perfusate (1.49%) and liver (0.30%). After administration of iso-DAG, elimination from perfusate was slower (t1/2 55 +/- 15 min), and hydrolysis to DF was modest by comparison with DAG-dosed perfusions. Recoveries as iso-DAG and D-2G in bile were greatly enhanced (8.2 and 36.4%, respectively). Adduct formation was higher in liver (0.76% of dose) but not in perfusate (1.03%). Immunoblots of liver homogenates revealed drug-modified proteins at ca. 110 and 120 kDa. The results show that (a) DAG undergoes avid systemic deconjugation-conjugation cycling and isomerization to iso-DAG; (b) iso-DAG is more resistant to hydrolysis, is readily taken up by hepatocytes and undergoes novel metabolism (phenolic glucuronidation); and (c) the glycation pathway (i.e. using iso-DAG as substrate) plays a major role in formation of covalent DF-protein adducts in liver.


Subject(s)
Diflunisal/analogs & derivatives , Diflunisal/metabolism , Inactivation, Metabolic/physiology , Liver/metabolism , Animals , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Diflunisal/pharmacokinetics , Glucuronates/metabolism , Glucuronates/pharmacokinetics , Male , Molecular Structure , Perfusion , Proteins/analysis , Proteins/metabolism , Rats , Sulfates/metabolism , Sulfates/pharmacokinetics
18.
Chem Res Toxicol ; 10(9): 1049-58, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9305589

ABSTRACT

The cytochrome P450-dependent covalent binding of radiolabel derived from phenytoin (DPH) and its phenol and catechol metabolites, 5-(4'-hydroxyphenyl)-5-phenylhydantoin (HPPH) and 5-(3',4'-dihydroxyphenyl)-5-phenylhydantoin (CAT), was examined in liver microsomes. Radiolabeled HPPH and CAT and unlabeled CAT were obtained from microsomal incubations and isolated by preparative HPLC. NADPH-dependent covalent binding was demonstrated in incubations of human liver microsomes with HPPH. When CAT was used as substrate, covalent adduct formation was independent of NADPH, was enhanced in the presence of systems generating reactive oxygen species, and was diminished under anaerobic conditions or in the presence of cytoprotective reducing agents. Fluorographic analysis showed that radiolabel derived from DPH and HPPH was selectively associated with proteins migrating with approximate relative molecular weights of 57-59 kDa and at the dye front (molecular weights < 23 kDa) on denaturing gels. Lower levels of radiolabel were distributed throughout the molecular weight range. In contrast, little selectivity was seen in covalent adducts formed from CAT. HPPH was shown to be a mechanism-based inactivator of P450, supporting the contention that a cytochrome P450 is one target of covalent binding. These results suggest that covalent binding of radiolabel derived from DPH in rat and human liver microsomes occurs via initial P450-dependent catechol formation followed by spontaneous oxidation to quinone and semiquinone derivatives that ultimately react with microsomal protein. Targets for covalent binding may include P450s, though the catechol appears to be sufficiently stable to migrate out of the P450 active site to form adducts with other proteins. In conclusion, we have demonstrated that DPH can be bioactivated in human liver to metabolites capable of covalently binding to proteins. The relationship of adduct formation to DPH-induced hypersensitivity reactions remains to be clarified.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Phenytoin/metabolism , Animals , Biotransformation/physiology , Blotting, Northern , Cats , Chlorophyll/analogs & derivatives , Chlorophyll/metabolism , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Female , Humans , In Vitro Techniques , Male , Microsomes, Liver/metabolism , Molecular Weight , Photosensitizing Agents/metabolism , Protein Binding , Rats , Rats, Wistar , Reactive Oxygen Species , Spectrometry, Fluorescence
19.
Br J Clin Pharmacol ; 44(1): 21-7, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9241092

ABSTRACT

AIMS: The study investigated the mechanism of the interaction between valproate and carbamazepine which causes raised plasma carbamazepine-10,11-epoxide concentrations with unchanged plasma carbamazepine concentrations. This interaction has usually been attributed to valproate inhibiting epoxide hydrolase, the enzyme that catalyses the biotransformation of carbamazepine-10,11-epoxide to carbamazepine-10,11-trans-diol. METHODS: Clearances of plasma carbamazepine, carbamazepine-epoxide and carbamazepine-diol to relevant carbamazepine metabolites present in urine were measured under steady-state conditions in 17 adults receiving carbamazepine as anticonvulsant monotherapy, and in 10 adults taking the drug together with valproate. RESULTS: Plasma carbamazepine-epoxide concentrations were higher, relative to carbamazepine dose, in the co-medicated patients. Plasma apparent clearances of carbamazepine, relative to drug dose, were similar whether or not valproate was taken. Formation clearances of carbamazepine-10,11-trans-diol conjugate, and probably of carbamazepine-10,11-trans-diol, were lower in subjects co-medicated with valproate, and a higher proportion of the carbamazepine dose was excreted in urine as carbamazepine-10,11-epoxide. CONCLUSIONS: Valproate appears to inhibit the glucuronidation of carbamazepine-10,11-trans-diol, and probably also inhibits the conversion of carbamazepine-10,11-epoxide to this trans-diol derivative, rather than simply inhibiting the latter reaction only.


Subject(s)
Anticonvulsants/pharmacology , Anticonvulsants/pharmacokinetics , Carbamazepine/pharmacokinetics , Epilepsy/metabolism , Valproic Acid/pharmacology , Adult , Anticonvulsants/administration & dosage , Anticonvulsants/therapeutic use , Carbamazepine/administration & dosage , Carbamazepine/analogs & derivatives , Carbamazepine/blood , Carbamazepine/therapeutic use , Carbamazepine/urine , Drug Therapy, Combination , Epilepsy/blood , Epilepsy/drug therapy , Epilepsy/urine , Female , Humans , Male , Middle Aged , Valproic Acid/administration & dosage , Valproic Acid/therapeutic use
20.
Drugs Aging ; 10(4): 278-89, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9108988

ABSTRACT

Alterations in drug disposition that occur with aging are now becoming widely recognised, and there is an increasing number of drugs for which the approach to therapy in elderly patients can be based on pharmacokinetic data. Both healthy aging and comorbid disease can alter the responsiveness of the body to drugs and to their absorption, distribution and elimination. Altered absorption in the elderly has not been documented after oral ingestion of any anticonvulsant drug. Increased adipose tissue in the elderly may raise the apparent volume of distribution (Vd) of lipid-soluble drugs. An increased Vd in the elderly has been shown for diazepam and clobazam, but not midazolam. The data are inconclusive for phenytoin and valproic acid (sodium valproate). The decreased plasma protein binding that often occurs in the elderly has few clinical consequences. The reduced liver function that to occur with aging seems to affect the elimination of drugs that are mainly cleared by oxidative metabolism [e.g. carbamazepine, phenytoin and phenobarbital (phenobarbitone)]. Reduced clearances for methylphenobarbital (methylphenobarbitone), diazepam, midazolam and clobazam occur in elderly men, but not in women. The reduced renal function that is seen in old age affects the disposition of drugs that are eliminated mainly by direct renal excretion. Thus. the clearances of vigabatrin and gabapentin correlate with creatinine clearance. Such considerations may help guide anticonvulsant dosage in the elderly.


Subject(s)
Aging/metabolism , Anticonvulsants/pharmacokinetics , Absorption , Adult , Aged , Aged, 80 and over , Aging/pathology , Aging/physiology , Anticonvulsants/administration & dosage , Anticonvulsants/pharmacology , Biological Availability , Clinical Trials as Topic , Dose-Response Relationship, Drug , Female , Humans , Kidney/drug effects , Liver/drug effects , Male , Middle Aged , Sex Factors , Structure-Activity Relationship , Tissue Distribution
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