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T S Tracy

Publications and source records attributed to T S Tracy.

At least 19 recordsLinked to original sources
PubMed1998

Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites.

Some cytochrome P450 catalyzed reactions show atypical kinetics, and these kinetic processes can be grouped into five categories: activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. A two-site model in which the enzyme can bind two substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 and naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as dapsone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and naproxen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics suggestive of a low Km, low Vmax component for the first substrate molecule and a high Km, high Vmax component for the second substrate molecule. 7, 8-Benzoflavone activation of phenanthrene metabolism by CYP3A4 and dapsone activation of flurbiprofen and naproxen metabolism by CYP2C9 were also observed. Furthermore, partial inhibition of 7, 8-benzoflavone metabolism by phenanthrene was observed. These results demonstrate that various P450 isoforms may exhibit atypical enzyme kinetics depending on the substrate(s) employed and that these results may be explained by a model which includes simultaneous binding of two substrate molecules in the active site.

Aryl Hydrocarbon Hydroxylases↗
PubMed1998

Cytochrome P450: new nomenclature and clinical implications.

Many drug interactions are a result of inhibition or induction of cytochrome P450 enzymes (CYP450). The CYP3A subfamily is involved in many clinically significant drug interactions, including those involving nonsedating antihistamines and cisapride, that may result in cardiac dysrhythmias. CYP3A4 and CYP1A2 enzymes are involved in drug interactions involving theophylline. CYP2D6 is responsible for the metabolism of many psychotherapeutic agents. The protease inhibitors, which are used to treat patients infected with the human immunodeficiency virus, are metabolized by the CYP450 enzymes and consequently interact with a multitude of other medications. By understanding the unique functions and characteristics of these enzymes, physicians may better anticipate and manage drug interactions and may predict or explain an individual's response to a particular therapeutic regimen.

Aged↗
PubMed1998

Effects of gestational and overt diabetes on human placental cytochromes P450 and glutathione S-transferase.

The placenta possesses the ability to metabolize a number of xenobiotics and endogenous compounds by processes similar to those seen in the liver. Animal and in vivo studies have observed that the presence of diabetes alters the expression of hepatic metabolizing enzymes (cytochrome P450 and glutathione S-transferase); however, it is unknown whether similar alterations occur in the human placenta. To evaluate whether diabetes has any effect of placental xenobiotic metabolizing activity, the catalytic activities of 7-ethoxyresorufin O-deethylation (EROD, CYP1A1), chlorzoxazone 6-hydroxylation (CYP2E1), dextromethorphan N-demethylation (CYP3A4), dextromethorphan O-demethylation (CYP2D6), and 1-chloro-2, 4-dinitrobenzene (CDNB) conjugation with glutathione (glutathione S-transferase, GST) from placentas of diet (class A1) and insulin-dependent (class A2) gestational diabetics and overt diabetics were compared with matched controls. EROD activity (CYP1A1) ranged from 0.29 to 2.67 pmol/min/mg protein. However, no differences were observed among overt or gestational diabetics and their respective matched controls. CDNB conjugation (GST) ranged from 0.275 to 1.65 units/min/mg protein. In contrast to that observed with CYP1A1, a small but statistically significant reduction in GST activity was noted in overt diabetics as compared with their matched controls and gestational diabetics. CYP2E1, 2D6, and 3A4 enzymatic activities were not detected in human placental tissue. GST protein was detectable in all tissues studied, but no CYP protein could be detected in any of the tissues. Thus, it seems that pregnant women with overt diabetes have reduced GST activity in the placenta, which could potentially result in the exposure of the fetus to harmful electrophiles. However, the full clinical significance of this finding remains to be elucidated.

Adolescent↗
PubMed1997

Rapid and sensitive high-performance liquid chromatographic assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomes.

A high-performance liquid chromatographic assay was developed for the quantitation of chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone. These compounds along with phenacetin, the internal standard, were extracted from incubation mixtures using ether extraction. The extracts were analyzed on a Brownlee Spheri-5 C8 column with a mobile-phase of acetonitrile-0.5% phosphoric acid (30:70, v/v). The assay utilized UV detection at 287 nm which provided sensitivity and specificity to simultaneously quantify chlorzoxazone and 6-hydroxychlorzoxazone from liver microsomal samples at amounts of 10 ng and greater. The mean correlation coefficient of the standard curves for 6-hydroxychlorzoxazone and chlorzoxazone was 0.998 and 0.993, respectively, over the range of 25-400 ng, and the regression curves were found to be linear at least through 1600 ng. All components eluted within 7 min, resulting in a total analysis time of 8 min. The inter-day and intra-day coefficients of variation were <7 and <3%, respectively. This method provides a rapid, sensitive and cost-effective assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomal incubations.

Animals↗
PubMed1997

Involvement of multiple cytochrome P450 isoforms in naproxen O-demethylation.

OBJECTIVE: A series of studies was undertaken to determine the cytochrome P450 isoform(s) involved in naproxen demethylation and whether this included the same isoforms reported to be involved in the metabolism of other NSAIDs. METHODS: (S)-Naproxen was incubated with human liver microsomes in the presence of a NADPH-generating system and the formation of desmethylnaproxen was measured by high-performance liquid chromatography (HPLC). To further clarify the specific isoforms involved, experiments were conducted with preparations expressing only a single P450 isoform (vaccinia virus-expressed cells and microsomes derived from a lymphoblastoid cell line, each transfected with specific P450 cDNAs) as well as inhibition studies using human liver microsomes and putative specific P450 inhibitors. RESULTS: In human liver microsomes (n = 7), desmethylnaproxen formation was observed with a mean kM of 92 (21) mumol.l-1, Vmax of 538 pmol.min-1.mg-1 protein and Cint2 (reflective of a second binding site) of 0.36 microliter.min-1.mg-1 protein. This Cint2 term was added since Eadie-Scatchard analysis suggested the involvement of more than one enzyme. Studies using putative specific P450 inhibitors demonstrated inhibition of this reaction by sulfaphenazole, (apparent Ki = 1.6 mumol.l-1), warfarin (apparent Ki = 27 mumol.l-1), piroxicam (apparent Ki = 23 mumol.l-1) and tolbutamide (apparent Ki = 128 mumol.l-1). No effect was observed when alpha-naphthoflavone and troleandomycin were employed as inhibitors, but reaction with furafylline produced, on average, a maximum inhibition of 23%. At a naproxen concentration of 150 mumol.l-1, formation of desmethylnaproxen was observed in cells expressing P450 1A2, 2C8, 2C9 and its allelic variant 2C9R144C. To further characterize these reactions, saturation kinetics experiments were conducted for the P450s 1A2, 2C8 and 2C9. The kM and Vmax for P450 1A2 were 189.5 mumol.l-1 and 7.3 pmol.min-1.pmol-1 P450, respectively. Likewise, estimates of kM and Vmax for P450 2C9 were 340.5 mumol.l-1 and 41.4 pmol. min-1.pmol-1 P450, respectively. Reliable estimates of kM and Vmax could not be made for P450 2C8 due to the nonsaturable nature of the process over the concentration range studied. CONCLUSION: Multiple cytochrome P450 isoforms (P450 1A2, 2C8 and 2C9) appear to be involved in naproxen demethylation, although 2C9 appears to be the predominant form.

Chromatography, High Pressure Liquid↗
PubMed1996

Studies of flurbiprofen 4'-hydroxylation. Additional evidence suggesting the sole involvement of cytochrome P450 2C9.

Flurbiprofen, a non-steroidal anti-inflammatory drug (NSAID), is metabolized by both oxidation via the cytochrome P450 system and by glucuronidation. The major oxidative pathway in flurbiprofen metabolism is to a 4'-hydroxy metabolite, and recently we demonstrated that cytochrome P450 2C9 and its R144C variant were involved in this process (Tracy et al., Biochem Pharmacol 49: 1269-1275, 1995). Using complementary DNA (cDNA)-expressed cell systems, it has been demonstrated that at physiological concentrations of flurbiprofen there is a lack of involvement of P450s 1A2, 2C8, 2E1, and 3A4. In evaluating flurbiprofen as a potential probe for cytochrome P450 2C9, it is important to assess the involvement of additional P450s in this process. To this end, further studies were undertaken using specific inhibitors of P450 2C9 and P450 cDNA-expressed microsomes for P450 1A1, 2A6, 2B6, 2C19, and 2D6 to assess their potential involvement. We observed the inhibition of (R)- and (S)-flurbiprofen 4'-hydroxylation by an inhibitor of P450 2C9, sulfaphenazole (Ki = 0.07 and 0.06 microM, respectively), and the NSAID piroxicam (Ki = 10 and 7 microM, respectively). Furthermore, using microsomes from a lymphoblastoid cell line, we found that P450s 1A1, 2A6, 2B6, 2C19, and 2D6 were not involved in flurbiprofen hydroxylation at physiological concentrations of flurbiprofen. This finding is particularly important due to the sequence homology and potential substrate overlap of P450 2C9 and 2C19. These studies then provide additional evidence to suggest that P450 2C9 may be the only isoform involved to any substantial degree in flurbiprofen 4'-hydroxylation, and thus this reaction is useful as an in vitro probe for this particularly cytochrome P450 isoform and may be useful as an in vivo probe.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1996

Regiospecificity of placental metabolism by cytochromes P450 and glutathione S-transferase.

The placenta possesses the ability to metabolize numerous xenobiotics and endogenous steroids. However, it is unknown whether regional differences in these enzymatic reactions exist in the human placenta. To this end, we undertook a study of four regions of the placenta, the chorionic plate, maternal surface, placental margin and whole tissue, to assess the activities of cytochrome P450 1A1 and 19A1 (aromatase) and glutathione S-stransferase in these fractions. No differences in either P450 1A1 or glutathione S-transferase activities were noted among any of the placental fractions. However, with respect to P450 19A1 activity, the placental margin differed significantly from all other fractions (p < 0.05). This study demonstrates that whole tissue samples of the human placenta are adequate for placental cytochrome P450 and glutathione S-transferase metabolism studies.

Adult↗
PubMed1995

Role of cytochrome P450 2C9 and an allelic variant in the 4'-hydroxylation of (R)- and (S)-flurbiprofen.

Flurbiprofen is a chiral non-steroidal anti-inflammatory drug used in the treatment of pain or inflammation. The primary routes of biotransformation for (R)- and (S)-flurbiprofen are oxidation (presumably cytochrome P450) and conjugation. To date, the specific cytochrome P450 (P450) involved in the oxidative metabolism of this compound (specifically 4'-hydroxylation) has not been elucidated. Experiments were conducted to characterize the kinetic parameters (Km and Vmax) for the 4'-hydroxylation of (R)- and (S)-flurbiprofen in human liver microsomes, to determine if enantiomeric interactions occur when both enantiomers are present, and to identify the specific P450 form(s) involved in this reaction. In human liver microsomes, the Km and Vmax (mean +/- SD) for (R)-4'-hydroxy-flurbiprofen formation were 3.1 +/- 0.8 microM and 305 +/- 168 pmol.min-1.mg protein)-1, respectively. In comparison, the Km and Vmax (mean +/- SD) for (S)-4'-hydroxy-flurbiprofen formation were 1.9 +/- 0.4 microM and 343 +/- 196 pmol.min-1.mg protein-1, respectively. Enantiomeric interaction studies revealed a decrease in Km and Vmax for both enantiomers and an apparent loss of stereoselectivity. Racemic-warfarin, tolbutamide, alpha-naphthoflavone and erythromycin were studied as potential inhibitors of this process. The estimated Ki values for the inhibition of (R)- and (S)-4'-hydroxy-flurbiprofen formation by racemic-warfarin were 2.2 and 4.7 microM. This reaction was also inhibited by tolbutamide. In contrast, erythromycin and alpha-naphthoflavone had no appreciable effect on 4'-hydroxy-flurbiprofen formation. cDNA-expression of individual forms was used to determine which P450 was involved in 4'-hydroxy-flurbiprofen formation. P450 2C9 and an allelic variant (R144C) readily catalyzed the formation of 4'-hydroxy-flurbiprofen. P450 1A2 was also active albeit with a turnover rate 1/140th that of P450 2C9R144C (P450s 2C8, 2E1 and 3A4 were not active toward either enantiomer). The results of these studies indicate that the enantiomers of flurbiprofen may exhibit stereoselectivity with respect to enzyme affinity but have roughly equal maximum formation velocities. Additionally, these two enantiomers may compete for the enzyme resulting in lower maximum velocities for both enantiomers. Finally, of those P450 forms examined, only P450 2C9 and an allelic variant catalyzed the 4'-hydroxylation of both (R)- and (S)-flurbiprofen.

Aryl Hydrocarbon Hydroxylases↗
PubMed1995

Stereochemistry in pharmacotherapy: when mirror images are not identical.

OBJECTIVE: To describe how drug stereoisomers may differ in pharmacokinetic and pharmacodynamic properties and how these differences may affect therapeutic outcomes. STUDY SELECTION: Representative studies were chosen from the drug literature demonstrating stereoisomeric differences in drug absorption, protein binding and distribution, metabolism, and elimination. Furthermore, examples of pharmacodynamic differences between drug stereoisomers are presented to demonstrate that these stereoisomers not only may differ in pharmacologic potency, but may possess entirely different pharmacologic actions. DATA SYNTHESIS: Examples are presented demonstrating that when stereoiosomeric differences in pharmacokinetics are linked to pharmacodynamic differences, alterations in therapeutic effect can result. Additionally, drug interactions are discussed in which 1 isomer is affected to a greater extent than the other, potentially causing not only an increase or decrease in effect, but also a change in pharmacologic action. Examples also are presented of the marketing of single isomer entities, with a discussion of the use of these products. Finally, preliminary policies of the Food and Drug Administration are discussed, as well as the potential implications of these policies. CONCLUSIONS: Drugs that are administered as stereoisomers can differ with respect to both pharmacokinetics and pharmacodynamics, and these differences may have profound implications in pharmacotherapy. All future investigations of drugs that exist as stereoisomers must take into account the pharmacokinetics and pharmacodynamics of both isomers to understand fully the observed phenomena.

Drug Therapy↗
PubMed1994

Methotrexate disposition following concomitant administration of ketoprofen, piroxicam and flurbiprofen in patients with rheumatoid arthritis.

The effects of three non-steroidal anti-inflammatory drugs (NSAIDs) on the pharmacokinetics of methotrexate were studied in 10 patients with rheumatoid arthritis. Ketoprofen (3 mg kg-1 day-1), flurbiprofen (3 mg kg-1 day-1), piroxicam (20 mg day-1), or a non-NSAID control (paracetamol/acetaminophen) were administered to patients for at least 6 days (13 days in the case of piroxicam to establish steady state) in a randomized crossover design prior to receiving a weekly oral dose of methotrexate. In the non-NSAID control portion of the study, MTX oral clearance (CLo) was 11.0 +/- 3.9 l h-1, renal clearance (CLR) was 7.9 +/- 2.8 l h-1, percent excreted unchanged was 72 +2- 19% and fraction unbound (fu) was 0.54 +/- 0.11. Values of oral clearance, renal clearance, fraction unbound and percentage excreted unchanged of methotrexate varied no more than 12.2% from non-NSAID control during concomitant administration of ketoprofen, flurbiprofen or piroxicam and were not statistically different from non-NSAID control. In contrast to other NSAIDs such as ibuprofen and salicylates, ketoprofen, flurbiprofen or piroxicam in clinically relevant doses do not appear to affect methotrexate disposition and may be used safely in combination with methotrexate.

Administration, Oral↗
PubMed1992

The effects of a salicylate, ibuprofen, and naproxen on the disposition of methotrexate in patients with rheumatoid arthritis.

We have studied the pharmacokinetics of methotrexate in patients with rheumatoid arthritis concurrently treated with choline magnesium trisalicylate, ibuprofen, naproxen, or a non-NSAID analgesic (control treatment). The apparent systemic clearance of methotrexate was significantly reduced by all three treatments. Trisalicylate and ibuprofen both significantly reduced methotrexate renal clearance, but only the trisalicylate significantly displaced methotrexate from protein, increasing the fraction unbound by 28%. These data show that NSAIDs can affect the disposition of methotrexate, possibly increasing the potential for toxicity and necessitating dosage adjustments. However, large inter-subject variability precludes specific dosage recommendations.

Adult↗
PubMed1992

Ability of nifedipine to prolong parturition in rats.

Rats were randomly assigned to treatments: (i) no surgery control; (ii) saline control; (iii) 0.25, 0.5, 1.0 or 2.0 micrograms nifedipine kg-1 min-1; or (iv) 5.0 micrograms ritodrine kg-1 min-1. All drug treatments increased the interval between pup deliveries compared with the no surgery and saline controls. Apparent complete tocolysis was observed in 20, 60, 80 and 80% of the animals receiving 0.5, 1.0 or 2.0 micrograms nifedipine kg-1 min-1 or 5.0 micrograms ritodrine kg-1 min-1, respectively. A positive pharmacodynamic relationship was observed for the nifedipine doses. Analysis of pup viability showed no statistically significant difference among treatments. Treatment with 2.0 micrograms nifedipine kg-1 min-1 gave a delay in pup delivery comparable to that with ritodrine.

Animals↗
PubMed1991

Determination of the epimeric composition of ibuprofenyl-CoA.

Ibuprofen [racemic2-(4-isobutylphenyl)propionic acid] is a 2-arylpropionic acid nonsteroidal anti-inflammatory drug which undergoes unidirectional, R to S chiral inversion in vivo. It has been proposed that this chiral inversion phenomenon occurs via a coenzyme A (CoA) thioester intermediate. To characterize the formation and metabolism of this metabolic intermediate, ibuprofenyl-CoA, reference standards were needed and thus the CoA derivatives of (R)-, (S)-, and racemic ibuprofen were chemically synthesized. An HPLC assay employing a C18 reverse-phase column was developed to quantitate "total" ibuprofenyl CoA. Samples collected from this assay were then analyzed for ibuprofenyl-CoA epimeric composition by chiral chromatography employing a Chiral-AGP alpha 1-acid glycoprotein column. The applicability of these methods was demonstrated by assessing (R)- and (S)-ibuprofenyl-CoA hydrolysis and epimerization following incubation with rat liver homogenates. Rat liver homogenate catalyzed the complete and rapid epimerization of ibuprofenyl-CoA and the rate constants for (R)- and (S)-ibuprofenyl-CoA hydrolysis were equal. ATP and CoA were found to inhibit rat liver-catalyzed ibuprofenyl-CoA hydrolysis by 70-80% with no effect on epimerization. Additionally, it was demonstrated that traditional indirect ibuprofenyl-CoA assays which employ basic hydrolysis result in erroneous epimeric ratio determinations due to chemical epimerization.

Acyl Coenzyme A↗
PubMed1989

Nitroglycerin delivery through a polyethylene-lined intravenous administration set.

Adsorption and delivery of nitroglycerin through a new polyethylene-lined (PEL) i.v. administration set was compared with adsorption and delivery through an identical set composed of polyvinyl chloride (PVC) rather than PEL tubing. The new delivery system consisted of PEL tubing, a transparent PVC chamber, and a silastic segment for insertion in a peristaltic pump. Nitroglycerin was prepared in concentrations of 50, 125, and 200 micrograms/mL in 0.9% sodium chloride injection and run through both administration sets at flow rates of 12 and 60 mL/hr. Samples were obtained at 0, 0.5, 1, 2,4, and 8 hours from each of three sites: bottle, junction before silastic segment, and distal end of tubing. Nitroglycerin content was assayed using a modified high-performance liquid chromatography technique. A slight but significant average loss of nitroglycerin (2.3 +/- 9.3%) was observed at the distal end with the PEL set, whereas the PVC set showed a significant average nitroglycerin loss of 39.7 +/- 12.7% at the distal end. These differences were independent of infusion rate, nitroglycerin concentration, or time of sampling. Flow rate, concentration, and time had no significant effect on nitroglycerin adsorption with the PEL set, but all three had a significant effect on nitroglycerin adsorption with the PVC set. An unexpected finding was the approximately 14% loss of nitroglycerin from the admixture bottle over time. This phenomenon, which has been observed by other investigators, needs further investigation to determine its cause. It appears that a partially PVC-based administration set should provide consistent delivery of i.v. nitroglycerin to the patient.

Chemistry, Pharmaceutical↗
PubMedDate not supplied

The effects of diabetes on placental aromatase activity.

Diabetes complicates 2-3% of all pregnancies and is associated with an increase in both perinatal morbidity and mortality, though reasons for these adverse outcomes are unknown. Estrogen biosynthesis is a critical factor during pregnancy and is carried out in the placenta via aromatase (cytochrome P450 19A1), which catalyzes the conversion of C-19 androgens to C-18 estrogens. Previous studies have shown that hormones such as insulin-like growth factors and insulin regulate aromatase activity when studied in vitro. Interestingly, levels of these hormones are altered in patients with diabetes. Thus, we hypothesized that the presence of maternal diabetes may alter placental aromatase activity and thus estrogen biosynthesis, possibly serving as one factor in the adverse outcomes of babies born to mothers with diabetes. To this end, we measured the production of 19-hydroxyandrostenedione, 19-oxoadrostenedione and estrone in 30 placental tissues from diabetic patients, using [7-3H]androst-4-ene-3,17-dione as a model substrate for aromatase (P450 19A1). A statistical difference was detected in the percentage of 19-oxoandrostenedione formed between the overt and control groups (P < 0.05). Additionally, NADPH P450-reductase levels were measured in these same tissues to determine whether alterations in this enzyme necessary for aromatase activity could be affected by diabetes. No differences in reductase levels were detected among the patient groups. However, a statistical correlation was found between NADPH P450-reductase activity and the formation velocities of all three estrogen products (P < 0.05). Thus, it appears that the presence of diabetes does not affect placental aromatase activity.

Adult↗
PubMedDate not supplied

Calcium modulators: future agents, future uses.

The calcium modulators have been a significant therapeutic advancement for the treatment of angina. Structural analogs of verapamil and nifedipine have been synthesized, as have structurally unique compounds. As the role of calcium in body processes is further elucidated, the efficacy of the calcium modulators is being evaluated for numerous disorders. It is anticipated that the newly synthesized compounds will have specificity toward particular body processes, thus providing efficacy with minimal side effects.

Asthma↗
PubMedDate not supplied

Metabolic inversion of (R)-ibuprofen. Epimerization and hydrolysis of ibuprofenyl-coenzyme A.

Ibuprofen [(racemic)2-(4-isobutylphenyl)propionic acid] has been proposed but not directly demonstrated to undergo unidirectional inversion from the (R)- to the (S)-configuration via a coenzyme A (CoA) thioester intermediate. Chemically synthesized (R)- and (S)-ibuprofenyl-CoA, and rat and human liver homogenates were used to investigate the relative rates of ibuprofenyl-CoA epimerization and hydrolysis. Rat whole liver homogenate completely epimerized (R)- or (S)-ibuprofenyl-CoA, whereas hydrolysis of this intermediate occurred at a much slower rate. Rat liver mitochondria was the most efficient at both epimerizing and hydrolyzing ibuprofenyl-CoA, whereas rat liver microsomes hydrolyzed ibuprofenyl-CoA at a rate similar to whole liver homogenate but had very little epimerization activity. Rat liver cytosol was the poorest at hydrolyzing ibuprofenyl-CoA but had substantial epimerization capability. Whole liver homogenate from human tissue was less efficient at epimerizing but as efficient at hydrolyzing ibuprofenyl-CoA as rat whole liver homogenate. No stereoselectivity of either epimerization or hydrolysis was noted for any of the enzyme preparations studied. This study demonstrates that the inversion of (R)-ibuprofen occurs, at least in part, via the epimerization of the metabolic intermediate, ibuprofenyl-CoA, in both rat and human liver tissues.

Animals↗