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Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada
(Received September 15, 2004; accepted January 14, 2005)
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Abstract |
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(TNF). On day 6, rats were administered single i.v. (2 mg/kg) or oral (20 mg/kg) doses of racemic verapamil, and S- and R-verapamil concentrations were determined by high-performance liquid chromatography. Hepatic cytochrome P450 (P450) content and verapamil protein binding were also measured. All experiments were carried out in both pre-AA and control rats. Serum nitrite, CRP, and TNF levels were significantly elevated in pre-AA rats while signs of pain and arthritis were absent. Pre-AA also significantly elevated plasma concentrations of S- and R-verapamil after both i.v. and oral doses, due, likely, to decreased drug clearance. This was accompanied by a significant reduction in hepatic cytochrome P450, CYP3A, and CYP1A content as well as significantly reduced verapamil free fraction in pre-AA. The early phase of AA is marked by increased proinflammatory mediators and reduced verapamil clearance, as well as decreased hepatic P450 enzymes. Hence, pre-AA is a suitable model of inflammation for pharmacokinetic studies that avoids unnecessary exposure of animals to the pain and distress of fully developed adjuvant arthritis.
; Philippe et al., 1997). Although this model of inflammation has been used extensively to study pharmacokinetics of drugs (Walker et al., 1986; Belpaire et al., 1989; Pollock et al., 1989; Piquette-Miller and Jamali, 1993; Emami et al., 1998), it subjects animals to significant pain (Nagakura et al., 2003). AA causes increased expression of proinflammatory mediators, which is associated with suppression of hepatic metabolic processes, and, hence, reduces clearance of efficiently cleared drugs (Piquette-Miller and Jamali, 1993; Morgan, 1997; Kulmatycki and Jamali, 2001). The elevated levels of proinflammatory mediators, however, appear to occur a few days after injection of adjuvants when animals are still asymptomatic and relatively free of the pain and distress associated with the disease progression (Philippe et al., 1997; Szekanecz et al., 2000; Coulthard et al., 2002; Nagakura et al., 2003). Since increased exposure to proinflammatory mediators may be responsible for inflammation-induced inhibition of drug clearance (Kulmatycki and Jamali, 2001), relevant pharmacokinetic data may be obtained during the early phase of AA (pre-AA) and in the absence of the pain and stress associated with the experimental disease. We, therefore, evaluated the effect of pre-AA on some proinflammatory mediator concentrations, hepatic P450 content, and p.o. and i.v. pharmacokinetics of verapamil, a drug with well known efficient hepatic metabolism (Laethem et al., 1994; Mayo et al., 2000; Sattari et al., 2003). Since decreased clearance of drugs during inflammatory conditions may also be due to increased binding to plasma proteins secondary to an elevation of acute-phase protein 1-acid glycoprotein (AAG) (Belpaire et al., 1982), we also studied the effect of pre-AA on the protein binding of verapamil. |
Materials and Methods |
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Ultrasensitive ELISA kit was purchased from BioSource International (Camarillo, CA). Animals. Experiments were performed on male Sprague-Dawley rats (250–300 g) and were approved by the Health Sciences Animal Policy and Welfare Committee of the University of Alberta. Animals had free access to water, but food was withheld for 12 h before pharmacokinetic experiments. They were housed under a 12-h light/dark cycle.
Protocol. The day of adjuvant injection (0.2 ml of 50 mg/ml M. butyricum suspended in squalene into the tail base) was marked as day 0. Control animals received 0.2 ml of saline into the tail base. Animals were weighed and assessed for symptoms of arthritis, and blood samples were taken from the tail vein for the determination of serum nitrite, C-reactive protein and plasma TNF concentrations on days 0, 1, 3, 5, and 6.
On day 5, the right jugular vein was cannulated for serial blood collection while animals were under halothane/O2 anesthesia. Briefly, a polyethylene (PE-50; Clay Adams, Parsippany, NJ) cannula tipped with 2 cm of Silastic (Dow Corning, Midland, MI) tubing was inserted into the right jugular vein and exteriorized by subcutaneous tunneling to an incision made in the inter-scapular area.
On day 6, pre-AA and control rats received single doses of racemic verapamil either intravenously (2 mg/kg in saline) (n = 6/group) or orally via gastric gavage (20 mg/kg suspended in polyethylene glycol 400) (n = 4–6/group). Blood samples (
0.2 ml) were collected at 0, 0.08, 0.17, 0.25, 0.5, 1, 2, 4, and 8 h after intravenous doses, and at 0, 0.25, 0.5, 0.75, 1, 2, and 4 h after oral doses. Plasma was separated and kept at -80°C for verapamil assay.
Microsomal Experiments. A separate set of control and pre-AA animals (n = 4/group) were subjected to the protocol described above. The animals were sacrificed on day 6, and their livers removed and microsomes prepared (Barakat et al., 2001). Briefly, livers were rinsed in 0.15 M KCl solution and homogenized in 0.25 M sucrose solution. The homogenates were centrifuged at 12,000g at 4°C for 10 min. The S9 fraction was collected and microsomes were precipitated by addition of 1 M CaCl2. The suspension was centrifuged at 27,000g at 4°C for 15 min; then, the pellet was resuspended in 0.15 M KCl solution and centrifuged at 27,000g at 4°C for 15 min. The pellet was again resuspended in 0.25 M sucrose solution and stored at -80°C.
The microsomal protein concentration was determined by the method of Lowry et al. (1951). Briefly, microsomes were incubated with 1% CuSO4/2% sodium potassium tartarate/10% Na2CO3 anhydrous in 0.5 M NaOH (1:1:20 v/v) at room temperature for 10 min and then with 10% Folin-phenol reagent at 50°C for 10 min. The sample was analyzed by spectrophotometry at 570 nm.
Total cytochrome P450 content was determined according to the method of Omura and Sato (1964). Briefly, microsomes were suspended in 0.05 M phosphate buffer, pH 7.4, at a protein concentration of 1 mg/ml. A few milligrams of solid sodium dithionite were added to the 1 ml of suspension, and a baseline was determined using the recording spectrophotometer by scanning from 500 to 400 nm. Carbon monoxide was bubbled gently into the sample cuvette for 20 s. The spectrum was again recorded from 500 to 400 nm. The quantity of cytochrome P450 was calculated from the optical density difference (450–480 nm) and the molar extinction coefficient of 91 mM-1 cm-1.
Western Blot Analysis. CYP3A and CYP1A protein content was assessed by Western blot analysis. Briefly, microsomal protein (30 µg) was denatured at 100°C for 5 min and then separated by SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide gels and blotted onto a pure nitrocellulose membrane (0.45-µm, Trans-Blot Transfer Medium; Bio-Rad). Nonspecific binding sites were blocked overnight in a solution of 5% skim milk/2% bovine serum albumin/0.05% Tween 20 in Tris-buffered saline buffer. Membranes were incubated with primary antibody [1:1000 dilution; polyclonal rabbit anti-rat CYP3A2 or polyclonal goat anti-rat CYP1A1 (Daiichi Pure Chemicals, Tokyo, Japan)] for 2 h, and then with secondary antibody [1:5000 dilution; horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody or HRP-rabbit anti-goat IgG antibody (Jackson Immunoresearch Laboratories Inc., West Grove, PA)] for 1 h. Immunoreactive proteins were visualized by chemiluminescence (ECL Western Blotting Detection Reagents; Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK), and band density was measured by densitometry (TBX Densitometer; Tobias Associates Inc., Ivyland, PA).
Verapamil Protein Binding. Protein binding was measured in serum obtained from the set of rats used for microsomal experiments. One milliliter of serum from pre-AA (n = 4) and control (n = 4) rats was adjusted to pH 7.4 with 0.1 N HCl. The serum was spiked with 2000 ng/ml racemic verapamil to approximate the serum concentrations of verapamil in rats after i.v. dosing. The serum was incubated at 37°C for 1 h and then transferred to Millipore micropartition chambers (Millipore Corporation, Bedford, MA) for ultrafiltration at 2000g for 1 h. Filtrate and nonfiltrate verapamil concentrations were measured by HPLC. The fraction unbound, fu, was determined as fu = Cu/Ct, where Cu is the unbound concentration and Ct is the total concentration.
Stereospecific Verapamil Assay. A stereospecific HPLC method (Shibukawa and Wainer, 1992) was used to determine plasma concentrations of R- and S-verapamil. Briefly, 75 µl of (+)-glaucine (400 ng/ml) as internal standard was added to 100 µl of plasma, followed by 100 µl of 2 M NaOH and 0.4 ml of sodium phosphate buffer (pH 7.0, ionic strength 0.1). Verapamil was extracted with 6 ml of heptane/heptafluorobutanol (99:1) and vortex mixed, followed by centrifugation. The organic layer was evaporated to dryness, and the residue was reconstituted in mobile phase (hexane/isopropanol/ethanol/triethylamine, 92:4:4:0.1 v/v) and injected into an isocratic HPLC system at a flow rate of 0.7 ml/min. The HPLC apparatus consisted of a Waters WISP 712 autoinjector (Millipore-Waters, Mississauga, ON, Canada), an achiral column (5 cm x 4.6 mm i.d. Supelcosil LCSi; Supelco, Bellefonte, PA) and a chiral column (250 mm x 4.6 mm i.d., 5-µm Chiralpak AD-H column; Daicel Chemical Inc., Tokyo, Japan) maintained at 31°C, a 474 fluorescence detector (Waters, Mississauga, ON, Canada) set at excitation of 272 nm and emission at 317 nm with a bandwidth at 18 nm, and a Hewlett Packard 3390A integrator (Hewlett Packard, Palo Alto, CA). Standard curves were linear over the concentration range of 10 to 1000 ng/ml (r2 
0.99, CV 
10%). The minimum quantifiable concentration was 5 ng/ml for S- and R-verapamil.
Serum Nitrite Analysis. Serum nitrite (NO2-), a stable breakdown product of nitric oxide (NO), was measured using a previously described method (Grisham et al., 1996). Briefly, nitrate (NO3-) was reduced to nitrite (NO2-) by incubating 100 µl of serum with 10 µl of Aspergillus nitrate reductase (10 U/ml) in the presence of 25 µl of 1 M HEPES (pH 7.4), 25 µl of 0.1 mM FAD, and 50 µl of 1 mM NADPH for 30 min at 37°C. Then, 5 µl of lactate dehydrogenase (1500 U/ml) and 50 µl of 100 mM pyruvic acid were added and incubated for an additional 10 min at 37°C. NO2- was determined by addition of 1.0 ml of Griess reagent, and absorbance was measured at 543 nm. Standard curves were linear over the concentration range of 3 to 200 µM (r2 0.99, CV 10%). The minimum quantifiable concentration was 3 µM.
Serum C-Reactive Protein Analysis. A commercially available rat CRP ELISA kit (Helica Biosystems, Inc.) was used. This assay required 100 µl of serum (1:10,000 dilution) to be added to a 96-well plate coated with antibodies to rat CRP. After incubation for 30 min, the plate was washed and 100 µl of conjugate (HRP-labeled rabbit anti-rat CRP-IgG) was added and incubated for 30 min. The plate was again washed, and 100 µl of 3,3',5,5-tetramethylbenzidine substrate solution was added and incubated for 10 min. Stop solution (100 µl) was added to stop the reaction, and absorbance was read at 450 nm. Standard curves were linear over the concentration range of 17.5 to 133 µg/ml (r2 0.99, CV 10%). The limit of detection of the assay was 2.5 µg/ml.
Plasma TNF Analysis. Plasma TNF concentrations were measured using a commercially available rat TNF ultrasensitive ELISA kit (BioSource International). Briefly, 100 µl of plasma was added to a 96-well plate coated with anti-rat-TNF capture antibody. After incubation for 3 h, the plate was washed and 100 µl of biotin conjugate was added and incubated for 45 min. The plate was again washed, and 100 µl of streptavidin-HRP was added and incubated for 45 min. After washing, 100 µl of Chromogen was added and incubated for 20 min followed by addition of 100 µl of stop solution, and the absorbance was read at 450 nm. Standard curves were linear over the concentration range of 2.3 to 150 pg/ml (r2 0.99, CV 10%). The limit of detection of the assay was 1.9 pg/ml.
Data Analysis and Statistics. Pharmacokinetic indices for S- and R-verapamil after i.v. administration were estimated using WinNonlin 4.1 (Phar-sight, Mountain View, CA). The open two-compartment model best described the unweighted data based on the Akaike information criteria. Pharmacokinetic indices for S- and R-verapamil after oral administration were determined by noncompartmental analysis. Elimination rate constants (ß) were calculated using log-linear regression of at least three points in the log-linear terminal phase of the plasma concentration-time curve. The area under the plasma concentration-time curve (AUC) was calculated using the log-linear trapezoidal rule from 0 h to the time of the last measured plasma concentration (Clast). Extrapolation to infinity (AUCt-
) was determined by Clast/ß, and AUC0- was determined as the sum of AUC0-t and AUCt-. The ratios of oral to i.v. AUC were estimated by comparing the partial (0–4 h) AUCs after oral and i.v. doses.
Data are presented as mean ± standard deviation. Significance of difference in pharmacokinetic parameters between pre-AA and control groups was determined by the two-tailed Student's t test at = 0.05. Comparison of animal weight, serum nitrite, and CRP concentrations between the groups and between the experimental days were done by analysis of variance with Tukey's adjustment for multiple comparisons.
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Results |
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Serum nitrite concentrations were significantly elevated in pre-AA rats by day 3 compared with control rats and remained elevated through day 6 (Fig. 1). C-reactive protein concentrations were also significantly elevated in pre-AA rats as early as 1 day postinduction, compared with control rats, and remained elevated through day 6 (Fig. 1). TNF concentrations were elevated slightly but significantly only on day 6 (14.4 ± 4.7 pg/ml versus 9.2 ± 1.8 pg/ml, p = 0.015) in pre-AA rats.
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Pre-AA significantly increased plasma concentrations of verapamil and altered pharmacokinetic indices following both i.v. and oral administration (Figs. 2 and 3). Following i.v. administration, AUC0- increased by 165% for S-verapamil and 45% for R-verapamil (Table 1). A corresponding decrease in clearance was observed (58% S-verapamil; 29% R-verapamil). The apparent volume of distribution at steady state (Vss) and volume of the central compartment (Vc) were unchanged by pre-AA. The unbound fraction of drug was also significantly reduced by 80% for S-verapamil and 67% for R-verapamil.
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Verapamil administered orally to pre-AA rats resulted in significantly elevated plasma concentrations of both enantiomers, as compared with control rats, and to a greater extent than after i.v. doses. Cmax and AUC0–4 h for S-verapamil in pre-AA rats were 6.7- and 9.3-fold higher than in control rats (Table 1). For R-verapamil, Cmax and AUC0–4 h in pre-AA rats were 3.6- and 9.5-fold higher than in control rats. The last measurement was made at 4 h, at which time the elimination phase had not been reached in pre-AA rats. Therefore, estimates of oral clearance and volume of distribution could not be determined. Absolute oral bioavailability also could not be obtained; however, the relative ratio of AUCs after oral dosing for the 4 h postdose was estimated (Table 1). The AUCp.o./AUCi.v. ratio increased 3.7-fold for S-verapamil and 6.5-fold for R-verapamil pre-AA rats.
Total microsomal cytochrome P450 content in pre-AA rats was significantly reduced (49%) compared with control rats (p = 0.001) (Fig. 4). The reduction in total P450 content was significantly correlated with increases in serum pro-inflammatory mediator concentrations (Fig. 5).
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Microsomal content of CYP3A1/2 and CYP1A1/2 isoforms was also significantly down-regulated in pre-AA rats (Fig. 4), and the reduction was correlated with the reduction in total P450 (Fig. 5). The suppression of both CYP3A1/2 and CYP1A1/2 protein expression was also significantly correlated with changes in serum nitrite and TNF concentrations, but not CRP levels (Fig. 6).
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Discussion |
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; Szekanecz et al., 2000) and development of swelling, pain, and deformity of joints 12 to 14 days postadjuvant (Whitehouse, 1988; Philippe et al., 1997). AA-induced inflammation also results in increased levels of acute-phase proteins, impaired drug-metabolizing enzymes, and, consequently, reduced clearance of a variety of drugs (Walker et al., 1986; Belpaire et al., 1989; Pollock et al., 1989; Piquette-Miller and Jamali, 1993; Emami et al., 1998). Although a widely used model, AA is associated with excessive pain and discomfort. Pre-AA can be an alternative approach to AA. However, for the model to be useful for pharmacokinetic studies, at least two characteristics are essential. First, the concentration of proinflammatory mediators should increase during the early phase of AA development and before full development of the experimental disease. Second, the time lapse between the rise in the mediator concentration and their expected effect on pharmacokinetic indices cannot be greater than the time between the adjuvant injection and the full development of the disease. The pre-AA model appears to meet both these criteria: it causes a substantial rise in CRP and nitrite in a few days (Fig. 1) and results in reduction in total cytochrome P450, CYP3A, and CYP1A content 6 days after the adjuvant injection (Figs. 4 and 6). Consequently, a significant decrease in clearance of verapamil is observed on day 6 (Figs. 2 and 3). Our observation confirms earlier reports that indicate an acute inflammatory response and rises in proinflammatory mediators within the first few days of the adjuvant injection (Hirschelmann et al., 1990; Philippe et al., 1997; Szekanecz et al., 2000). In addition, we have shown that this early rise in proinflammatory mediators coincides with an increased extent of protein binding for verapamil (Table 1) and correlates with a reduction in the total cytochrome P450, CYP3A, and CYP1A protein content (Figs. 4 and 6) in as early as 6 days.
Verapamil is a basic drug that is highly bound to the acute-phase protein AAG and is efficiently cleared in the liver. In inflammatory conditions, AAG is increased (Belpaire et al., 1982), resulting in a greater extent of binding and, hence, reduced free fractions of the drug (Laethem et al., 1994). For highly protein-bound drugs, even small changes in protein binding can result in large relative increases in unbound fraction. In our study, we observed significantly decreased unbound fractions for both enantiomers of verapamil in pre-AA rats as compared with control rats. Other models of inflammation have shown varying effect on protein binding of verapamil. Endotoxin-induced inflammation reduces unbound fraction of verapamil (Laethem et al., 1994), whereas interferon -2a-induced inflammation does not significantly affect protein binding (Sattari et al., 2003). This suggests that altered protein levels may be dependent on the type of inflammation.
Following i.v. administration, clearance of highly extracted drugs such as verapamil is dependent primarily on hepatic blood flow (Q) (Gibaldi and Perrier, 1982). Since Q is unaffected by inflammation (Walker et al., 1986), the significant reduction in clearance of verapamil observed in our study indicates that pre-AA causes substantial reductions in intrinsic metabolic activity consistent with the observed reduction in P450 protein content. Suppression of metabolic activity and, hence, intrinsic clearance may reduce drug clearance to such an extent that it becomes dependent on drug free fraction and intrinsic metabolic activity in addition to Q, as with intermediately extracted drugs (Gibaldi and Perrier, 1982). Pre-AA decreased intrinsic clearance of S-verapamil by 70% and R-verapamil by 62%. The apparent extraction ratio (E) determined by the relationship, E = CL/Q, demonstrates that E for R-verapamil decreased from 0.96 to 0.76, and for S-verapamil from 0.96 to 0.49. E of S-verapamil, therefore, is consistent with values associated with intermediately cleared drugs.
Interestingly, the clearance of propranolol, another highly cleared drug with high affinity for AAG, although reduced by adjuvant arthritis after oral doses, is not affected after i.v. dosing (Piquette-Miller and Jamali, 1993). Metabolism of propranolol, which occurs through various enzyme pathways including glucuronidation, may be affected less in inflammation than the metabolism of verapamil, which is metabolized primarily by the CYP 3A and 1A isoenzymes (Kroemer et al., 1993), and, therefore, remained highly cleared even in the presence of inflammation.
Verapamil is subject to extensive first pass metabolism after oral administration, and pharmacokinetic indices are thus influenced by extent of protein binding and alterations in hepatic metabolism. Pre-AA significantly elevated Cmax and AUC0–4 h for both enantiomers of verapamil (Table 1). Similar effects have been observed in acute inflammation (Laethem et al., 1994; Sattari et al., 2003) and confirm that pre-AA is a suitable alternative model of inflammation for pharmacokinetic studies. It is of value to mention that, following oral administration to inflamed rats, a greater extent of variability was observed in the plasma verapamil concentrations (Fig. 3). This is consistent with data reported for verapamil in humans (Mayo et al., 2000) and propranolol in the rat (Piquette-Miller and Jamali, 1995). The inflammation-induced reduced clearance of these highly cleared drugs depends on the severity of the disease. Hence, the variability in concentrations observed in our inflamed rats is likely a reflection of the inherent intersubject variability of the severity of the inflammation.
Reduced cytochrome P450 has been reported in various models of inflammation. There are few direct or indirect data on liver metabolic activity in the early phase of AA. During the acute phase of AA-induced inflammation, prolonged sleep time has been reported for pentobarbital (Dipasquale et al., 1974) and hexobarbital (Baumgartner et al., 1974), possibly due to decreased drug metabolism. On the other hand, cytochrome P450 content appears to be unaltered at day 5 after adjuvant injection (Morton and Chatfield, 1970), and disposition of cyclosporine after 5 days of AA remains unaffected (Pollock et al., 1989). Our study showed a 48% reduction in total cytochrome P450 content on day 6 and corresponding decreases in verapamil intrinsic clearance. Importantly, the expression of the specific isoenzymes involved in verapamil metabolism, CYP3A1/2 and CYP1A1/2, were also down-regulated, by 55% and 44%, respectively, in pre-AA. Furthermore, suppression of total cytochrome P450 was significantly correlated to decreases in both CYP3A and CYP1A isoenzymes. The early rise in proinflammatory mediators in pre-AA may have been responsible for the observed reductions in P450 content and verapamil clearance. Serum CRP concentrations were significantly correlated with the decrease in total P450 content, but not with either of the P450 isoforms, suggesting that this acute phase protein is reflective of systemic inflammatory processes but is not directly involved in P450 down-regulation. TNF levels were only slightly elevated on day 6 but were positively correlated with the down-regulation of both P450 isoforms as well as total P450 content. This suggests that the proinflammatory cytokine TNF has a role in the suppression of P450 enzymes in pre-AA, either directly or indirectly, although the mechanism cannot be inferred by this study. Of the proinflammatory mediators measured in this study, serum nitrite levels had the strongest correlation with the down-regulation of CYP3A and CYP1A isoforms, as well as total cytochrome P450 content. Proinflammatory cytokines have been shown to suppress cytochrome P450 enzyme activity and content (Morgan, 1997), perhaps through cytokine-mediated increase in production of NO, known to inactivate P450 enzymes (Khatsenko et al., 1993). Serum nitrite, a surrogate marker of NO, has been positively correlated with arthritic disease severity and inflammation-induced reductions in drug clearance (Mayo et al., 2000).
In summary, the early phase of AA is marked by increased proinflammatory mediators and reduced verapamil clearance, as well as decreased hepatic P450 protein content. Hence, pre-AA is a suitable model of inflammation for pharmacokinetic studies that avoids unnecessary exposure of animals to the pain and distress of fully developed adjuvant arthritis.
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Footnotes |
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This work was presented in part as an abstract at the 7th Annual Symposium of the Canadian Society for Pharmaceutical Sciences, June 9–12, 2004, Vancouver, BC, Canada.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: AA, adjuvant arthritis; pre-AA, early phase adjuvant arthritis; CRP, C-reactive protein; HPLC, high-performance liquid chromatography; AAG, 1-acid glycoprotein; HRP, horseradish peroxidase; NO, nitric oxide; AUC, area under the plasma concentration-time curve.
Address correspondence to: Dr. Fakhreddin Jamali, 3118 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, Alberta, Canada, T6G 2N8. E-mail: fjamali{at}pharmacy.ualberta.ca
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1-acid glycoprotein and to human serum. Eur J Clin Pharmacol 22: 253-256.[CrossRef][Medline]
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