Most pharmacokinetic studies [1] of antituberculosis drug therapy have used a single blood sample to estimate drug absorption, but a single sample does not provide a reliable measure of total systemic drug exposure. Our objectives were to comprehensively evaluate the pharmacokinetics of commonly used antituberculosis agents in HIV-seropositive patients at different stages of disease (including patients with diarrhea) and to compare the results with those seen in healthy persons.

Methods

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Participants

Forty-eight persons who did not have tuberculosis participated in the study, which was conducted at two medical centers. Participants were 12 healthy volunteers who were not known to be seropositive for HIV infection (group I); 12 HIV-seropositive, asymptomatic patients who had CD4⁺ T-cell counts greater than 300 cells/mm³ (group II); 12 HIV-seropositive, symptomatic patients who had CD4⁺ T-cell counts less than 200 cells/mm³ (group III); and 12 HIV-seropositive, symptomatic patients who had CD4⁺ T-cell counts less than 200 cells/mm³ and persistent diarrhea (defined as more than three loose stools per day for at least 30 days) (group IV). As defined by the Centers for Disease Control and Prevention classification system for HIV infection [9], asymptomatic persons were classified as having stage IA or IIA infection and symptomatic persons were classified as having stage IIIB or IIIC infection. Symptomatic patients had at least one AIDS-defining illness or related illness [9]. Exclusion criteria were age younger than 18 years, pregnancy, results of liver function tests that were more than five times normal, serum creatinine levels greater than 200 µmol/L, neutrophil counts less than 10⁹/L, hemoglobin levels less than 100 g/L, active opportunistic disease, and known hypersensitivity to any of the study medications. The Research Ethics Boards of the Ottawa General Hospital, Ottawa, Ontario, Canada, and the Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada, approved the study. All participants provided written informed consent.

Drug Administration and Blood Sampling

Study participants received 300 mg of isoniazid, 600 mg of rifampin, 1000 mg of pyrazinamide, and 1000 mg of ethambutol, which were administered as individual drugs simultaneously each morning for 3 days. Therapy with other medications was stopped at least 24 hours before the study and for its duration (5 days). After a 10-hour overnight fast, participants received final doses of all medications with 300 mL of water on the morning of day 4. Participants were allowed to eat 3 hours after they received the drugs.

On day 4, venous blood samples were collected immediately before participants were given the drugs and 0.25, 0.5, 0.75, 1.0, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours afterward. Plasma was harvested within 30 minutes of blood collection and stored at –80°C. Ascorbic acid was added to plasma aliquots to prevent oxidation of rifampin.

To measure the absorptive function of the intestines, participants received 25 grams of D-xylose with 400 mL of water 24 hours after they received the final dose of the antituberculosis drugs [10]. Separation from isoniazid was necessary to prevent possible malabsorption of isoniazid by hydrazone formation with D-xylose [11]. Blood samples were collected before the participants received D-xylose and at 0.25, 0.5, 1, 1.5, 2, 3, 4, and 5 hours afterward. Plasma was harvested and stored at –80°C.

Plasma Drug and Pharmacokinetic Analysis

We measured isoniazid, rifampin, and pyrazinamide concentrations with high-performance liquid chromatography according to methods described elsewhere [12-14]. Analytic difficulties precluded the measurement of ethambutol. A colorimetric method was used to measure D-xylose [10].

We compared the plasma drug concentrations with time data using noncompartmental methods. These data included the highest observed drug concentration (C_max), the time to C_max (t_max), the terminal disposition half-life (t_1/2,z), and the area under the plasma concentration-time curve (AUC) over the 24-hour dosing interval.

Outcome Measures

Total systemic drug exposure was measured by the AUC, and peak systemic drug exposure was measured by C_max. Decreases in AUC reflected reduced drug bioavailability rather than increased drug clearance if t_1/2,z was unchanged and corresponding decreases in C_max were noted.

Statistical Analysis

Mean pharmacokinetic variables between groups were evaluated by analysis of variance. The model included the effects of group status and isoniazid acetylator status. Summary statistics for all variables except t (_max) were based on geometric means. The appropriateness of the log-transformation for stabilizing intragroup variances was judged by using the Bartlett test for homogeneity of variance. Three linear orthogonal contrasts were tested to determine the effects of HIV infection (the mean of group I compared with the average of the means of groups II, III, and IV), severity of HIV disease (the mean of group II compared with the average of the means of groups III and IV), and diarrhea (the mean of group III compared with the mean of group IV) on pharmacokinetic variables [15]. A set of contrasts was tested by using orthogonal polynomials to determine whether a linear trend, a quadratic trend, or both was apparent between group order and group geometric mean [15]. The significance level for the test of each contrast was set at 0.05. The association between the AUC of the drugs being studied and that of D-xylose was evaluated by ordinary linear regression.

Results

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Pill counts, questioning of participants, and predosing drug levels showed that all participants seemed to adhere to the dosing regimen. Demographic characteristics are shown in Table 1.

Participants were classified as fast acetylators if they had an isoniazid t_1/2,z of less than 130 minutes [16]. There were 4, 10, 6, and 7 fast acetylators in groups I, II, III and IV, respectively. Analysis of variance showed a significant effect of acetylator status for total drug exposure (AUC) and t_1/2,z. Fast acetylators had shorter half-lives and lower AUC values than did slow acetylators.

Mean pharmacokinetic data for each drug are shown in Table 2. The trend analyses indicated a significant linear decrease in mean AUC with group order for pyrazinamide (P = 0.0002) and a significant linear decrease in mean C_max for rifampin (P = 0.006) and isoniazid (P = 0.046).

Consistent with the trend for decreasing AUC and C_max values from group I to group IV, statistically significant decreases of 18% to 41% in these variables were seen for some of the group contrasts for each drug (Table 2).

Significant positive relations existed between D-xylose AUC₀–5 and pyrazinamide AUC₀–24 (P < 0.005; r² = 0.17) and rifampin AUC₀–24 (P < 0.001; r² = 0.31), but a significant relation did not exist between D-xylose AUC and isoniazid AUC₀–24 for fast (P > 0.2; r² = 0.026) and slow acetylators (P > 0.2; r² = 0.002).

Discussion

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Our pharmacokinetic study showed that total systemic drug exposure was reduced for rifampin by 32% and for pyrazinamide by 24% in persons with HIV infection compared with healthy controls. A significant trend of decreased exposure for pyrazinamide occurred in patients with later-stage HIV disease. The reduction in total exposure probably reflects decreased bioavailability.

Isoniazid was generally well absorbed in HIV-infected patients compared with healthy controls, which may partially explain a lack of correlation with the D-xylose AUC. The 39% decrease in peak exposure and 0.74-hour increase in time to peak exposure suggest that diarrhea reduced the rate of isoniazid absorption in symptomatic patients.

Malabsorption from gastrointestinal malfunction probably contributes to the cause of reduced bioavailability. The significant correlation between the D-xylose AUC and rifampin and pyrazinamide AUC implies an absorptive defect, although the test for D-xylose explained only as much as 31% of the variability in drug AUC. Malabsorption has been suggested elsewhere as the reason for low concentrations of rifampin in serum [17], persistent fever in an HIV-negative man being treated for pulmonary tuberculosis [17], and potentially subtherapeutic levels of rifampin in persons who were infected with both HIV and M. tuberculosis [1, 3, 4]. However, gastrointestinal malfunction may also increase rifampin clearance by reducing its reabsorption in enterohepatic circulation. This may explain why rifampin was associated with the largest decreases in total and peak exposure. Gastrointestinal malfunction is common in HIV-infected patients [18], and subclinical enteropathy in early stages of HIV disease has recently been well described [19].

The clinical significance of our findings for persons infected with both M. tuberculosis and HIV is unclear; none of our patients had tuberculosis or clinical outcomes that could be evaluated. All peak concentrations seen in our study were above the minimum inhibitory concentrations for sensitive organisms [20]: 0.025 to 0.05 µg/mL for isoniazid, 0.005 to 0.2 µg/mL for rifampin, and 12.5 µg/mL for bactericidal concentration of pyrazinamide. However, this finding does not determine whether plasma levels correlate with adequate antituberculosis activity in infected tissue. A substantial reduction in total drug exposure may have clinical consequences during a lengthy course of therapy, especially when this reduction is combined with pre-existing drug heteroresistance, advanced immunodeficiency, and reduced compliance. The few recent case reports [2-4] have suggested that low drug levels in persons with HIV infection may lead to acquisition of drug-resistant M. tuberculosis and therapeutic failures.

Our study demonstrates that HIV-infected patients, especially those with advanced disease, have lower plasma concentrations of one or more antituberculosis drugs, particularly rifampin, compared with healthy controls. However, we do not recommend that prescribed doses of antituberculosis drugs be increased for patients infected with HIV. Studies of patients with active tuberculosis are urgently needed to determine the clinical and microbiological significance of our pharmacokinetic study and to evaluate therapeutic drug monitoring with dose adjustment before recommendations can be made.

Dr. Gallicano and Ms. Seguin: Clinical Investigation Unit, Ottawa General Hospital, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada.

Ms. Swick: 833 Potomac Avenue, Buffalo, New York, 14209.

Dr. Tailor and Mr. Walker: Department of Pharmacy, Sunnybrook Health Sciences Centre, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5, Canada.

Drs. Garber and Cameron: Division of Infectious Diseases, Ottawa General Hospital, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada.

Ms. Oliveras: Department of Biochemistry, Ottawa General Hospital, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada.

Dr. Rachlis: Sunnybrook Medical Centre, Room 226, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5, Canada.