INTRODUCTION

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We previously reported our experience with the granule dosage form (Paser; Jacobus Pharmaceuticals, Princeton, NJ) of p-aminosalicylic acid (PAS) (1). In that report, we presented data from healthy volunteers who took the granules with food, and data from patients with multidrug-resistant tuberculosis (MDR-TB) who received PAS granules three times daily. Because multiple daily doses are not practical in all settings, particularly for directly observed treatment (DOT) programs conducted by public health services, less frequent dosing of PAS would be desirable (2). We describe herein our experience with twice-daily and once-daily dosing of PAS granules.

	METHODS

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The study protocol followed the guidelines of the Helsinki Declaration of 1975 and its amendments, and was approved by the Institutional Review Board at the National Jewish Medical and Research Center. Written informed consent was obtained from each patient before the study.

Patients

Twelve patients admitted to the National Jewish Medical and Research Center for the treatment of MDR-TB participated, six in each treatment arm. The studies were performed sequentially. After completing the twice-daily regimen, a data analysis was performed, and it showed that sufficient PAS was present in the blood after twice-daily dosing to attempt a once-daily regimen.

PAS was prescribed by the attending physicians for all patients as part of their multidrug regimens. By necessity, companion drugs were allowed to vary across the 12 patients according to the susceptibility pattern of each patient's organism. None of the concurrent antimycobacterial drugs has been shown to have drug-drug interactions with PAS. In the first study, PAS granules were dosed 4 g twice daily (10 A.M. and 8 P.M.) for 8 d. All doses were taken with an acidic beverage to prevent early release of the drug in the stomach (1). On Day 8, blood samples were scheduled to be collected in plain red-top vacuum tubes at 4, 8, and 12 h after the morning dose. In the second study, PAS granules were dosed once daily (10 A.M.) for 8 d, followed by twice-daily dosing thereafter. Again, all doses were taken with an acidic beverage. On Day 8, blood samples were scheduled to be collected at 6, 12, and 24 h. During the twice-daily dosing phase, blood samples were collected at 6 and 10 h post-dose. Blood samples were allowed to clot, centrifuged, and the serum harvested and frozen within 1 h of collection.

After completing Study 1, we adopted a twice-daily dosing regimen for all subsequent patients receiving PAS granules. An additional 40 inpatients were treated with PAS granules 4 g twice daily as part of routine practice (third study). Therapeutic drug monitoring was performed to verify the adequacy of the doses, and the data were recorded and summarized (5).

Self-Rating Scales

Subjective tolerance of PAS was determined using visual analog scales rating gastrointestinal discomfort, bloating or fullness in the stomach, nausea, vomiting, and diarrhea. The form contained five vertical demarcations, the left-most indicating no symptoms and the right-most indicating severe symptoms. Subjects were instructed to circle the vertical mark that most closely fit their perception of the given symptom. Subjects were asked to complete these evaluations before dosing, and at 1, 2, 4, and 8 h after dosing on Day 1 and Day 8.

Sample Analysis

All serum samples were analyzed using high-performance liquid chromatography (HPLC) with the following equipment: a Waters model 510 pump and model 680 gradient controller and solvent select valve (Milford, MA), a Spectra Physics model 8875 fixed-volume autosampler (San Jose, CA), a McPherson model FL-750A fluorescence detector (Acton, MA), a Macintosh IIci computer (Apple Computers, Inc., Cupertino, CA), and the Rainin Dynamax HPLC data management system (Woburn, MA). The standard curves for PAS were 1.0 to 100.0 µg/ml. The best fits of the standard curves were achieved using a weight of 1/Y². The recovery of PAS from serum was 94% (range, 91 to 97%). Testing for PAS within sample precision produced a percent coefficient of variation (CV) of 2.0%. Testing for PAS day-to-day assay precision produced a coefficient of variation of 2.4% (low: 0.1% at 1 µg/ml; high: 5.0% at 10 µg/ml).

Pharmacokinetic Analysis

The data were analyzed using noncompartmental methods. The observed maximal serum concentration (Cmax) and the time at which it occurred (Tmax) were determined by inspection of the serum concentration versus time graphs.

Statistical Analysis

The mean, standard deviation, median, and range were calculated for each parameter. All analyses were performed using JMP software (Cary, NC), version 3.2.2. Because of the small sample size and the use of discontinuous data (Tmax), medians and ranges are reported.

	RESULTS

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Study 1 Patients

The six inpatients included two Asian women, one white woman, two Hispanic men, and one white man. The median age was 40 yr (range, 18 to 58 yr) and the median weight was 58 kg (range, 56 to 70 kg). The patients received 4 g PAS, or a median 69 mg/kg.

Study 2 Patients

The six inpatients included one Asian woman, one white woman, one Asian man, one black man, and two white men. The median age was 37 yr (range, 19 to 64 yr) and the median weight was 63 kg (range, 53 to 73 kg). The patients received 4 g PAS, or a median 64 mg/kg.

Most samples were collected at the scheduled times. In a few cases, it was not possible to obtain all of the samples at the scheduled time points due to intervening clinical events. Table 1 shows that PAS granules dosed 4 g twice daily maintained PAS serum concentrations well above 1 µg/ml for the entire dosing interval. This is the typical minimal inhibitory concentration (MIC) for PAS against Mycobacterium tuberculosis (6). Subject 3 had the lowest serum concentrations. The pattern suggests that the first two measurements were residual PAS from the previous dose, and that the late rise in concentration was due to absorption of the study dose. A similar pattern may be seen with Patient 5. The median Cmax was 31.95 µg/ml, and the median Tmax was 4.2 h. Table 2 shows the twice-daily dosing data for three of the six patients in Study 2. PAS serum concentrations also were maintained above 1 µg/ ml in these patients. The median Cmax was 25.80 µg/ml, and the median Tmax was 7.3 h. The remaining three patients from Study 2 were discharged from the hospital before repeat blood draws could be performed.

With the once-daily regimen, there is no accumulation of PAS from previous doses available to maintain serum concentrations above the MIC. Also, in contrast to the twice-daily dosing data, the once-daily dosing data in Table 3 show gaps in the antimycobacterial coverage, with PAS serum concentrations falling below the MIC at some point between the 12- and 24-h serum concentrations. Once-daily doses produced a median Cmax of 23.40 µg/ml, and a median Tmax of 6.0 h.

The PAS granules were generally well tolerated. In Study 1, one patient noted "mild" diarrhea 1 h after the first dose. Another patient noted "moderate" bloating or fullness in the stomach and "moderate" diarrhea 8 h after the first dose, and "moderate" bloating and "severe" diarrhea throughout Day 8. A third subject noted "mild" bloating 8 h after the first dose, and "mild" diarrhea 4 h post-dose on Day 8. In Study 2, one patient noted "moderate" bloating 1 h after the first dose. Another patient noted this complaint at all time points on Day 1 and Day 8. A third patient complained of "mild" nausea 2 h after the Day 1 dose. No serious adverse events were noted.

Study 3 Patients

We evaluated the data from 40 patients (14 females and 26 males) of all races dosed twice daily with 4 g PAS granules. The median age was 39 yr (range, 20 to 88 yr), the median weight was 59 kg (range, 32 to 58 kg), and the median dose was 68 mg/kg (range, 48 to 126 mg/kg). A total of 88 serum concentrations were collected between 4 to 8 h post-dose, with a median concentration of 24.83 µg/ml (range, 0 to 90.57 µg/ ml). Sixty-seven serum concentrations were collected from 9 to 12 h post-dose, with a median concentration of 20.58 µg/ml (range, 0 to 90.51 µg/ml). Only two samples in each time grouping were 0 µg/ml, and only a total of five others were less than 1 µg/ml.

	DISCUSSION

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Maximum serum concentrations are generally achieved about 6 h after doses of the PAS granules (1). Their sustained release characteristics allow for intermittent dosing. Because PAS is bacteriostatic and because it lacks a post-antibiotic effect, it may be desirable to maintain serum concentrations above the typical MIC for M. tuberculosis of 1 µg/ml throughout the dosing interval (7). This may be true particularly when the companion drugs are weak, such as ethionamide and cycloserine.

PAS granules produced a relatively flat serum concentration versus time curve when dosed 4 g twice daily. Concentrations with this regimen remain above 1 µg/ml for the entire dosing interval for Study 1. Under routine clinical conditions, very similar results were seen in the 40 additional patients. Only nine of 155 serum concentrations (6%) were below the typical MIC. A few patients showed high serum concentrations (80-90 µg/ml). Such higher concentrations may be seen with immediate release PAS sodium tablets (not currently available in the United States), or when patients consume a basic food or beverage near the time of dosing of PAS granules. This can lead to a more rapid release of PAS from the granules in the stomach, and can be associated with nausea. In general, however, these higher PAS concentrations appear to be well tolerated. Patients should take PAS with sufficient acidic beverage to wash down all of the granules (typically 4-8 ounces of fruit juice), and they must not chew the granules. Mild diarrhea is often self-limited over the first 1-2 wk of treatment. A couple of doses of an anti-motility agent can be effective in terminating the diarrhea. Warn patients that empty granules may appear in their stool.

PAS granules dosed 4 g once daily produced serum concentrations below 1 µg/ml for part of the dosing interval, which in theory may be less effective. The intrinsic half-life of PAS is 45-60 min (7). Beginning at the time when PAS absorption stops, PAS serum concentrations decline rapidly. However, that time cannot be known, and there were insufficient late serum concentrations in these studies with which to determine the exact time when PAS serum concentrations fell below the MIC. Given the low 12-h values for four of the six subjects, this probably occurred closer to 12 h than to 24 h. It remains unknown whether these gaps in coverage will result in suboptimal clinical performance. A new study of 8 g PAS given once daily is under way. If more sustained serum concentrations can be achieved, such dosing would further facilitate DOT.

	CONCLUSIONS

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The granule form of PAS was well tolerated with both dosing regimens. The twice-daily regimen produced inhibitory concentrations of PAS throughout the dosing interval. Because this regimen is easier to administer than three daily doses, we have adopted twice-daily dosing of PAS granules for our patients with MDR-TB.