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Clinical Chemistry 45: 1465-1476, 1999;
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Right arrow Drug Monitoring and Toxicology
(Clinical Chemistry. 1999;45:1465-1476.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Drug Monitoring of Antiretroviral Therapy for HIV-1 Infection: Method Validation and Results of a Pilot Study

Thomas P. Moyer1,a, Zelalem Temesgen2, Robert Enger1, Lynn Estes3, Joel Charlson1, Lawrence Oliver1 and Alan Wright2

Departments of
1 Laboratory Medicine and Pathology,
2 Infectious Diseases and Internal Medicine, and
3 Hospital Pharmacy Services, Mayo Clinic, Rochester, MN 55905.
a Author for correspondence.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Antiretroviral therapy for HIV-1 infection has become increasingly complex. The availability of new and potent drugs and progress in understanding the pathogenesis of HIV-1 infection have led to the establishment of new treatment paradigms. The varying dosing regimens, associated toxicities, and the potential for drug-drug and food-drug interactions further complicate treatment. This complexity contributes to patient nonadherence. Because clinicians have no tools to monitor adherence or drug-drug interactions and because response requires that therapy exceed the known inhibiting concentration, serum monitoring of antiretroviral therapy may play a role in improving treatment of HIV-1 infection. We report methods to quantify serum concentrations of antiretroviral drugs used to treat HIV-1 infection, precision and interference studies of these methods, and results observed in a pilot evaluation of blood serum concentrations from 12 human subjects.

Methods: HPLC offers adequate sensitivity to measure peak or trough serum concentrations of delavirdine, lamivudine, nevirapine, indinavir, nelfinavir, ritonavir, and saquinavir and peak serum concentrations of stavudine, zidovudine, and didanosine with reasonable precision.

Results: Peak indinavir serum concentrations in most patients were in the range of 1–10 mg/L, and trough concentrations were in the range of 0.1–0.5 mg/L. Peak stavudine concentrations were in the range of 0.3–1.3 mg/L, and trough concentrations were in the range of 0.1–0.5 mg/L. Peak zidovudine concentrations were in the range of 0.1–1.1 mg/L.

Conclusions: Because this was a blood serum concentration-seeking pilot study to evaluate analytic performance, we do not report on the correlation of drug response to blood concentration. However, the concentrations observed in patients are generally consistent with blood concentrations reported from studies of monotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recent advances in the treatment of HIV-1 infection involving coadministration of reverse transcriptase and protease inhibitors to achieve near-complete suppression of HIV-RNA concentrations have led to considerable improvements in life expectancy of infected individuals (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Highly active antiretroviral therapy (HAART), i.e., the use of aggressive combination antiretroviral regimens consisting of reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs), has become the standard of care (12)(13). Response to therapy is monitored by quantifying HIV-RNA copies (viral load) and CD-4+ T-lymphocyte count. The objective of therapy is to reduce viral load to undetectable by the assay used and to increase CD-4+ T-cell count (6)(14)(15)(16); both are important indicators of therapeutic success. Treatment success in clinical trials reportedly is in the range of 55–70% (8)(9)(11). Continued improvements are anticipated as drug-drug and food-drug interactions and viral resistance patterns become better understood.

Although the benefit of HAART has been clearly demonstrated, it presents several problems for the patient. The large number of pills (n >20) to be taken per day, the associated toxicities, the varying dosage regimens, and drug-drug and drug-food interactions may lead to confusion and nonadherence on the part of the patient. Nonadherence to antiretroviral therapy is particularly critical because it allows continued viral replication and the development of resistance to drugs (17)(18)(19). Several studies have now shown that nonadherence is an independent risk factor for treatment failure or detectable HIV-1 viremia (20)(21)(22)(23)(24)(25). Other possible causes of antiretroviral therapy failure are drug-drug and drug-food interactions and individual patient variability in metabolism and clearance. These may produce suboptimal drug concentrations in some patients with subsequent incomplete viral suppression and the development of resistance. On the other hand, increased blood concentrations may be related to toxicity.

There are no good and reliable methods to assess adherence with medication regimens. Methods that have been used include pill counts, patient interviews, prescription refill histories, and electronic monitoring devices. Each of these methods has limitations and drawbacks. The best results may be obtained by combining several methods. Although it also has its limitations, drug concentration monitoring may provide clinicians an additional tool to evaluate patients' adherence to prescribed medication regimens. It may also enable individualized and optimized dosing regimens based on knowledge of drug-drug and drug-food interactions and individual patient pharmacokinetic parameters (26)(27).

Dosing of antiretroviral agents is very complex, requiring administration of three or more drugs in replicate doses at different times throughout the day. Because of this complexity, specimen collection to simultaneously monitor peak and trough concentrations of all antiretroviral drugs is difficult to accomplish. Peak concentrations of RTIs and PIs occur at different times after dose; therefore, a single sample does not reflect optimum timing for peak concentration for all drugs. For example, in a regimen including lamivudine, indinavir, nevirapine, and zidovudine, a specimen collected just before the morning dose reflects trough concentrations for all drugs (but zidovudine should be undetectable), and a specimen collected 2 h after dose would reflect peak concentrations except for nevirapine; nevirapine reaches peak concentration >4 h after dose. If didanosine replaced lamivudine in the regimen, the dosing schedule would require substantial adjustment because didanosine must be administered without food—the peak concentration for zidovudine would occur 2 h earlier than the peak for didanosine. If nelfinavir replaced indinavir in the regimen, adjustment of dose relative to food intake must be made because food increases absorption of nelfinavir.

The known pharmacokinetic information on the antiretroviral drugs approved for clinical use at the time of this study (May 1998) is outlined in Table 1 . There are several key pharmacokinetic nuances that must be considered for the antiretroviral drugs. Nucleoside analog RTIs (e.g., zidovudine) have a short half-life (1–2 h) but are administered only twice per day. Peak serum concentrations are achieved within 1 h of dose administration, and serum concentrations become undetectable 6–8 h after therapy. Nucleoside analog RTIs become biologically activated after intracellular conversion to phosphorylated metabolites; these phosphorylated metabolites are not usually detected in serum. Protease inhibitors and non-nucleoside RTI drugs are metabolized by cytochrome P450 (28)(29)(30)(31). These drugs exhibit considerable drug-drug interactions.


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  		Table 1. Known pharmacokinetic parameters of antiretroviral drugs.

Because of these complexities, it seems evident that some form of pharmacokinetic monitoring of therapy could substantially improve treatment protocol design. Flexner (8) recently noted that high PI plasma concentration regimens may be more effective in preventing emergence of resistant virus, and Fletcher et al. (10) have shown that a specific concentration range of zidovudine is directly related to improved response. Morse et al. (26), Brundage et al. (27), and others (32)(33)(34) have suggested that a pharmacokinetic approach is likely to provide treatment benefit.

Methods for analysis of the approved anti-HIV-1 agents in blood serum have been developed to support the evaluation of clinical efficacy required by the US Food and Drug Administration. These methods are the basis for the pharmacokinetic information available for each drug (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45). Unfortunately, many of these procedures are either proprietary or have not been published in the peer-reviewed clinical literature. Those that were published before 1996 may not have been evaluated for interference or influence by new PI drugs. We felt that a merging of methods and a current evaluation of interferences from new antiretroviral agents was required to validate these methods for their use in therapeutic drug monitoring. The procedures for individual drugs presented by Morris and Selinger (46), Burger et al. (47), Jarugula and Boudinot (48), Hedaya and Sawchuk (49), Staton et al. (50), and Woolf et al. (51) are the basis for the methods presented here.

At the time this pilot study was completed (May 1998), the approved antiretroviral agents were those listed in Table 1Up . New products are anticipated in each of these drug groups over the next several years. This report represents the findings from the pilot phase of a large adherence-controlled drug concentration monitoring study. Preliminary reports of this work have been presented (52)(53).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
reagents
All inorganic chemicals and organic solvents used were ACS grade obtained from Curtin Matheson Scientific. Solid-phase extraction cartridges were obtained from United Chemical Technologies. All nucleoside analogs and tegafur were purchased from Sigma Chemical Company. Encainide was provided by Bristol-Myers, Princeton, NJ. Non-nucleoside RTIs and PIs were acquired from the Mayo Clinic Pharmacy except for zalcitabine, which was kindly provided by Roche Pharmaceuticals, Nutley, NJ. Samples of pure PI and RTI drug were requested from each respective pharmaceutical supplier, but all declined to provide pure drug to be used for calibrating the assays. Drug extracted from pulverized pill samples was used to calibrate these assays. Purity was assessed by demonstrating a single peak by HPLC, and the concentration was validated by comparing ultraviolet absorbance to the known absorptivity.

patient samples
Serum samples were obtained from patients undergoing routine clinical care for their HIV infections. Patients were on HAART consisting of a PI and at least two RTIs. For the purpose of this study, those with HIV-1 RNA below 400 copies/mL were defined as "responders", those with HIV-1 RNA values above this limit were termed "nonresponders". Adherence was estimated through collaboration between the HIV nurse and the HIV pharmacist who separately interviewed the patients about medications at each visit and reviewed pharmacy refill requests to verify use. Concurrent medications at the time of specimen collection are noted in Table 2 .Drug therapy was individualized for each patient based on life-style, likelihood of adherence, tolerance to side effects, and response to therapy. Blood specimens were drawn just before dose (trough) or 2 h after (approximate peak) the PI dose; the HIV nurse observed the patient take the dose and coordinated blood collection. Viral load and CD4+ T lymphocytes were quantified by PCR (6)(14) and flow cytometry (16)(54), respectively.


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  		Table 2. Summary of HIV-related laboratory and pharmaceutical information for 12 pilot subjects.

antiretroviral measurements
This report presents the blood serum concentrations observed in a pilot study of 12 human subjects in advance of a larger (60 subjects) adherence study currently being performed. Specimens from six subjects not enrolled in the adherence study (Table 2Up , subjects 1–4, 11, and 12) were used to validate the methods; quantification was validated by the method of standard additions in these specimens. Subjects 5–10 (Table 2Up ) were patients enrolled in the adherence study; adherence status was verified in these patients. Results of the larger adherence study will be reported elsewhere.

Antiretroviral drug concentrations in blood serum were quantified by HPLC (46)(47)(48)(49)(50)(51). Three different assays were developed.

Didanosine, lamivudine, and stavudine.
Unbuffered serum (1 mL) mixed with 0.05 mL of 20 mg/L tegafur (internal standard) was adsorbed to a 3-mL solid phase C18 cartridge. Drug was eluted in 1 mL of methanol, and the solvent was evaporated, leaving a dry residue. The residue was reconstituted in 300 µL of mobile phase and separated by HPLC on a 25-cm C18 column (Supelcosil LC18-DB, no. 5-8355; Supelco) with a flow rate of 1.5 mL/min; the effluent was monitored at 248 nm. In a mobile phase of 40 mL/L acetonitrile in 10 mmol/L phosphate, pH 6.9, the elution order was as follows: lamivudine (13.5 min), didanosine (19.9 min), stavudine (22.2 min), and internal standard (24.7 min). A seven-point calibration was performed using human serum to which 0.02, 0.10, 0.50, 1.0, 2.5, and 20 mg/L of each drug had been added. Calibration curves were linear throughout that range, with a simple weighted linear regression constant >0.99.

Nevirapine and zidovudine.
Unbuffered serum (0.5 mL) mixed with 0.05 mL of 10 mg/L 3-isobutyl-1-methylxanthine (internal standard) was extracted with 6 mL of 950 mL/L chloroform-50 mL/L isopropyl alcohol. The organic phase was transferred to a clean tube and evaporated. The residue was reconstituted in 300 µL of mobile phase and separated by HPLC on a 15-cm C8 column (Supelcosil LC8-DB, no. 5-8347; Supelco) with a flow rate of 1.5 mL/min; the effluent was monitored at 266 nm. In a mobile phase of 170 mL/L acetonitrile in 15 mmol/L phosphate (pH 7.5), the elution order was as follows: zidovudine (4.4 min), internal standard (8.0 min), and nevirapine (11.2 min). A seven-point calibration was performed using human serum to which 0.10, 0.25, 1.0, 5, and 10 mg/L nevirapine and 0.01, 0.025, 0.1, 1.0, and 2.0 mg/L zidovudine had been added. Calibration curves were linear throughout that range, with a simple weighted linear regression constant >0.99.

Delavirdine, indinavir, nelfinavir, ritonavir, and saquinavir.
Serum (1 mL) mixed with 0.05 mL of 3 mg/L encainide (internal standard) and 1 mL of 0.19 mol/L borate (pH 9.5) were extracted with 6 mL of 500 mL/L ethyl acetate-500 mL/L hexane. The organic phase was transferred to a clean tube and evaporated. The resulting residue was reconstituted in 300 µL of mobile phase and separated by HPLC on a 15-cm C8 column (Supelcosil LC8-DB, no. 5-8347; Supelco) with a gradient flow rate of 0.8 to 1.5 mL/min through the run; the effluent was monitored at 254 nm. In a mobile phase of 450 mL/L acetonitrile, 50 mL/L methanol in 15 mmol/L phosphate (pH 7.5), the elution order was as follows: indinavir (5.1 min), delavirdine (5.8 min), internal standard (6.6 min), ritonavir (10.4 min), saquinavir (15.0 min), and nelfinavir (22.9 min). A seven-point calibration was performed using human serum to which 0.10, 0.30, 1.0, 5, and 15 mg/L delavirdine, indinavir, nelfinavir, and ritonavir and 0.02, 0.05, 0.3, 1.0, and 3.0 mg/L saquinavir had been added. Calibration curves were linear throughout that range, with a simple weighted linear regression constant >0.99.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
sample preparation
Serum samples for didanosine, lamivudine, and stavudine analysis were prepared for HPLC quantification by adsorption onto a solid-phase cartridge. Serum samples for delavirdine, nevirapine, indinavir, nelfinavir, ritonavir, saquinavir, and zidovudine quantification were prepared for HPLC quantification by organic extraction. The recovery of drug added into normal human serum at low and high concentrations typically achieved during therapy was evaluated by comparing triplicate assayed results to the amount of drug added into the serum. Table 3 shows the recoveries achieved with these assays.


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  		Table 3. Drug recovery during sample preparation for HPLC analysis.

chromatography
HPLC quantification of the serum extract was carried out using three different chromatographic systems. The elution pattern developed from a serum extract of didanosine, lamivudine, and stavudine added into serum at concentrations near cmax is shown in Fig. 1 A. The elution pattern developed from a serum extract to which nevirapine and zidovudine had been added at concentrations near cmax is shown in Fig. 1B . The elution pattern developed from an extract of serum to which delavirdine, indinavir, nelfinavir, ritonavir, and saquinavir had been added at concentrations near cmax is shown in Fig. 1C .



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  		Figure 1. High performance liquid chromatograms of antiretrovirals at concentrations near cmax.

(A), didanosine (ddI), lamivudine (3TC), and stavudine (d4T); (B), neveripine and zidovudine (AZT); (C), indinavir, delavirdine, ritonavir, saquinavir, and nelfinavir. ISTD, internal standard.

analyte stability
Analyte stability was assessed by analyzing native human serum to which the drug had been added at a concentration near cmax (Table 1Up ). The concentration of the drug was constant when the sample was maintained at 56 °C for longer than 1 h but not longer than 2 h. All drug concentrations were constant in serum when stored at room temperature in sealed tubes for 7 days. Serum concentrations were constant in specimens stored at -20 and -65 °C for up to 90 days. Drug concentrations were constant through four freeze-thaw cycles. Extracts prepared for HPLC analysis were reassayed at 8 h and 24 h; no extract degradation was noted over these time intervals.

assay linearity and limit of detection
The assay linearity and limit of detection were evaluated by creating seven serum-based samples to which drug had been added in concentrations ranging from the limit of detection to the upper limit of linearity outlined in Table 4 . The limit of detection was defined as the lowest concentration of drug that could be analyzed with a between-run CV <20%. The upper limit of linearity was identified as the highest concentration that could be measured using a seven-point calibration with weighted (1/x2) linear regression analysis to achieve a linear regression constant >0.99.


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  		Table 4. Assay linearity and limit of detection.

precision
Three human serum-based pools with drug added at three different concentrations were created. Drugs were added to the low control pool at concentrations two times the limit of detection. Drugs at the highest known cmax for each drug were added to serum to create the high control pool. Drug concentrations midway between those in the low and high control pools were added to serum to create a mid-range control pool. A single aliquot from each of these pools was analyzed in three separate analyses performed on 3 different days. Table 5 shows the between-day CVs for each analyte at these three concentrations.


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  		Table 5. Assay precision at three concentrations.

interference studies
A large group of drugs commonly found in plasma were evaluated for possible interference. Pure samples of each drug were chromatographed in the three HPLC systems described. Those that eluted near one of the antiretrovirals were added to human serum at physiologic concentrations and extracted. Those drugs that were extracted and eluted near an antiretroviral were identified as sources of possible interference.4

patient results
Serum concentrations of antiretroviral drugs were determined in 12 patients. Six of those 12 patients provided dual specimens in the controlled arm of the study collected at the optimal time for trough and peak concentrations of PI drug. Results from the six patients that provided specimens collected at the optimal time for measuring peak and trough concentrations of their respective protease inhibitors are outlined in Table 2Up (patients 5–10) and graphically in Fig. 2 . Four of the six patients (patients 5, 6, 8, and 10) were on indinavir-containing regimens, patient 7 was receiving nelfinavir, and patient 9 was being treated with a saquinavir-based regimen. All patients except one enrolled in the controlled arm of the study were on their initial regimen. Patient 6 was on a salvage regimen consisting of indinavir, stavudine, didanosine, and hydroxyurea. He was thought to be nonadherent and had an HIV-1 RNA value of 6721 copies/mL. All five patients on initial antiretroviral therapy had an estimated adherence of >85%. Four of the five had HIV-1 RNA values below 400 copies/mL. Patient 9 had HIV-1 RNA values just above the limit of detection, i.e., 496 copies/mL; this patient was on saquinavir.



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  		Figure 2. Ranges of serum concentrations reported previously [Refs. (2,3,15,26,27,31–45,55,56] for monotherapy.

Previously reported ranges of peak values, shown in orange, and trough values, shown in green, compared with the ranges of values observed in the 12 subjects enrolled in this study (peak serum concentrations in red and trough concentrations in blue). P, peak values; T, trough values; blue text, responding subjects; red text, nonresponding subjects; NP, serum concentrations collected at a time that would not represent either the anticipated peak or trough time.

Our patient population represents too few subjects taking didanosine, nelfinavir, nevirapine, ritonavir, and saquinavir to make any useful observations relating patient adherence and response to serum antiretroviral concentration for those drugs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Precise and sensitive assays adequate to assess antiretroviral blood serum concentrations have been developed to monitor therapy for HIV-1 infection. The assays described here demonstrated imprecision (CVs) in the range of 4–15% at serum concentration adequate to assess trough concentrations of delavirdine, indinavir, lamivudine, nelfinavir, nevirapine, ritonavir, and saquinavir. Because didanosine, stavudine, and zidovudine have short half-lives and are administered less than once each half-life, serum concentrations just before the next dose are below the detection limit of these assays. However, all of the assays were able to detect the concentration expected atthe end of one half-life. Assay linearity for all drugs studied here is adequate to measure peak concentration with imprecision (CVs) ranging from 1% to 8%.

From a list of 256 drugs,4 several significant assay interferences were noted. Diazepam and midazolam would increase the apparent concentration of encainide, the internal standard used in the assay for delavirdine, indinavir, nelfinavir, ritonavir, and saquinavir. The effect would be apparent decreases in the measured concentrations of these drugs. Pentoxifylline would increase the apparent concentration of tegafur, the internal standard for the didanosine, lamivudine, and stavudine assays. The effect would be apparent decreases in the measured amounts of these drugs.

The assay for indinavir would experience interference producing increased apparent values from alprazolam, carbamazepine, chlordiazepoxide, clonazepam, flunitrazepam, griseofulvin, methaqualone, methoxypsoralen, nafcillin, nitrazepam, oxazepam, thiopental, and triazolam. Nelfinavir would show an apparent increase in the presence of metoprolol. Cisapride, haloperidol, loxapine, medazepam, and prazepam would induce an artifactual increase in ritonavir. Saquinavir would show an apparent increase in the presence of clozapine and flurazepam.

The assay for delavirdine would experience interference producing increased apparent values from disopyramide, flunitrazepam, metoclopramide, methylclonazepam, methylnitrazepam, N-desmethyldiazepam, temazepam, thiopental, and trazodone. Phenacetin and penicillin V would artifactually increase the apparent concentration of nevirapine.

Hydrochlorothiazide and sulfapyridine would cause an apparent increase in zidovudine concentration. Lamivudine would be artifactually increased in the presence of cefotetan and ceftizoxime, and didanosine would be artifactually increased in the presence of ceftizoxime and metronidazole. Ceftizoxime would also cause an artifactual increase in measured stavudine concentration.

Drug-drug and drug-food interactions as well as metabolic and pharmacokinetic variabilities can influence serum drug concentrations, and it is possible to draw the wrong conclusion about a given serum drug concentration if all confounding factors are not taken into consideration. Our pilot study, limited as it was by the small number of patients, illustrated some of this dilemma. For example, patient 10 (Table 2Up ), adherent to treatment regimen and with a good virological response, had a trough concentration of the PI below the detection limit, which might suggest inadequate dose. On the other hand, patient 6, nonadherent, on salvage regimen, and failing therapy, had a trough concentration of PI within the reference range, whereas the peak concentration was low.

Because of the limited number of patients involved in this study, data presented here do not demonstrate that response is related to achieving a specific serum concentration. The observations here do suggest that measurable concentrations occur at the times predicted based on studies of monotherapy. Fletcher et al. (10) presented data that showed such a relationship for zidovudine. Evaluation of a larger population of patients, under way at this time, will be needed to draw a more definitive conclusion about the relationship of specific serum drug concentrations to therapeutic success. It does seem reasonable that measurement of serum antiretroviral concentrations can be used as an adherence indicator, recognizing that this evaluation indicates only that the patient administered the drug recently and does not confirm long-term adherence.

Measurement of the serum concentrations of antiretroviral drugs is technically feasible. Our study shows that the assays developed using HPLC were able to measure serum antiretroviral drug concentrations with clinically relevant sensitivity and precision. Data presented here demonstrate that antiretroviral serum concentrations can be measured in a clinical environment. The usefulness of serum drug monitoring to assess adherence is limited because the serum concentration may reflect drug administrated within the past 24 h but does not confirm that the patient has been taking the medications days or weeks before blood samples were obtained. Interference can be expected from a limited number of coadministered drugs. Timing of specimen collection coordinated with dose increased the complexity of this study because the peak or trough concentrations of each drug occur at different times. Additional studies involving a larger number of patients are under way, and more definitive clinical relationships can be stated when this study is completed.


   Acknowledgments
 
This work was carried out with approval of the Mayo Clinic Investigational Review Board.


   Footnotes
 
4 The following drugs were evaluated for interference in the antiretroviral assays: acetaminophen, acetazolamide, acetoacetic acid, acetohexamide, N-acetylprocainamide, acetylsalicylic acid, allobarbital, alprazolam, amantadine, amiodarone, amitriptyline, amobarbital, amoxapine, amoxicillin, amphetamine, ampicillin, antipyrine, aprobarbital, aztreonam, baclofen, barbituric acid, bendroflumethiazide, benzocaine, benzoylecgonine, benzthiazide, bisacodyl, bupropion, butabarbital, butalbital, caffeine, carbamazepine, carbamazepine-10,11-epoxide, carisoprodol, cefazolin, cefotaxime, cefoxitin, ceftazidime, ceftizoxime, ceftriaxone, cefuroxime, cephalexin, chloramphenicol, chlordiazepoxide, chlorimipramine, 8-chlorotheophylline, chlorothiazide, chlorpheniramine, chlorpromazine, chlorpropamide, chlorzoxazone, cimetidine, ciprofloxacin, ciproheptadine, cisapride, clonazepam, clonidine, clozapine, cocaine, codeine, compazine, cotinine, coumarin, cyclobenzaprine, cyclosporine, cyclothiazide, cyheptamide, dapsone, demoxepam, desethylamiodarone, desipramine, N-desmethyldiazepam, N-desmethylsertraline, O-desmethylvenlafaxine, dextromethorphan, diazepam, dibucaine, diclofenate, dicloxacillin, dicumarol, dicyclomine, diflumsal, diltiazem, diphenhydramine, disopyramide, disulfiram, doxepin, encainide, ß-estradiol, ephedrine, ethosuximide, felbamate, fenoprofen, fentanyl, flecainide, 5-flucytosine, flufenamic acid, flunitrazepam, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, furosemide, gabapentin, ganciclovir, gemfibrozil, glipizide, glutethimide, glybenclamide, griseofulvin, guaifenesin, halazepam, haloperidol, haloperidol metabolite, heroin, hexabarbital, hydralazine, hydrochlorothiazide, hydrocodone, hydroflumethiazide, hydromorphone, 9-hydroxyrisperidone, hydroxyzine, ibuprofen, imipenem, imipramine, indapamine, indomethacin, isoniazide, itraconazole, ketoconazole, ketoprofen, lamotrigine, levorphanol, lidocaine, lorazepam, loxapine, maprotiline, meclofenamic acid, medazepam, mefenamic acid, meperidine, mephentermine, meprobamate, metformin, methadone, methapyrilene, methaqualone, metharbital, methoxyphenamine, methoxypsoralen, methsuximide, methylclonazepam, methylnitrazepam, methylphenidate, methylsalicylate, methyprylon, metoclopramide, metoprolol, metronidazole, mexiletine, midazolam, morphine, mycophenolic acid, nabumetone, nafcillin, naltrexone, naproxen, nefazodone, nicotine, nifedipine, nitrazepam, nitroglycerin, norchlorimpramine, nordoxepin, norfluoxetine, nortriptyline, norverapamil, noscapine, orphenadrine, oxacillin, oxazepam, oxycodone, paroxetine, penicillin G, penicillin V, pentazocine, pentobarbital, pentoxifylline, phenacetin, phenazopyridine, phencyclidine, phenformin, pheniramine, phenobarbital, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylethylamine, phenylpropanolamine, phenytoin, placidyl, prazepam, prednisone, primidone, probucol, procainamide, promazine, promethazine, propafenone, propoxyphene, propranolol, protriptyline, pseudoephedrine, pyrazinamide, quetiapine, quinidine, quinine, ranitadine, retrovir, risperidone, salicylic acid, secobarbital, sertraline, strychnine, succinimide, sulfadiazine, sulfamethoxazole, sulfapyridine, sulfisoxazole, sulindac, temazepam, theobromine, theophylline, thiocyanate, thiopental, thioridazine, thiothixene, tiagabine, ticarcillin, ticlopidine, tocainide, tolazamide, tolbutamide, tolmetin, topiramate, trazodone, triazolam, trichlormethiazide, trifluoperazine, trimethoprim, trimipramine, trioxsalen, tripelennamine, venlafaxine, verapamil, vigabatrin, warfarin, zolpidem, zomepirac.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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