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Keywords:

  • children;
  • low dose;
  • pharmacokinetics;
  • pyrazinamide;
  • spectrophotometer;
  • tuberculosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. REFERENCES

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Pyrazinamide is recommended in doses varying from 15 to 40 mg kg−1. The most commonly used average daily dose is 25 mg kg−1. Its use is associated with dose dependent hepatotoxicity.

• Lower doses are not used because of lack of pharmacokinetic data especially in children. There is only one detailed study of pyrazinamide in children at a dose of 35 mg kg−1.

WHAT THIS STUDY ADDS

• This is the first study evaluating serum concentrations of pyrazinamide in children at a dose of 15 mg kg−1 which is on the lower side of the recommended dose. The study also compared the serum concentrations and pharmacokinetics achieved with this dose with the widely used dose of 25 mg kg−1 in children suffering from tuberculosis.

• The pharmacokinetics and pharmacodynamic indices of pyrazinamide were comparable with the 25 and 15 mg kg−1 doses.

AIMS

To evaluate the pharmacokinetics and pharmacodynamic indices of pyrazinamide at doses of 15 and 25 mg kg−1 in children suffering from tuberculosis.

METHODS

Twenty children with tuberculosis received pyrazinamide at a single dose of 25 mg kg−1 (group I) and 15 mg kg−1 (group II). Serial blood samples were collected and the drug concentrations were analyzed spectrophotometrically. The pharmacokinetic parameters were calculated and the duration of time for which pyrazinamide concentrations in serum remained above the pyrazinamide inhibitory concentrations of 20 μg ml−1 and 25 μg ml−1 was studied.

RESULTS

The mean peak serum concentration was 42.4 ± 3.3 μg ml−1 (95% CI ± 6.5) and 38.6 ± 3.9 μg ml−1 (95% CI ± 7.7) in groups I and II, respectively. The elimination half-life was 9.3 ± 1.3 h and 10.5 ± 2.3 h (P = 0.6) and clearance was 0.06 ± 0.01 l h−1 kg−1 and 0.04 ± 0.01 l h−1 kg−1 (P = 0.08) in groups I and II, respectively. Pharmacokinetic parameters and PKPD indices were comparable with both the doses.

CONCLUSIONS

The study indicates that comparable serum concentrations of pyrazinamide are attained with 25 mg kg−1 and 15 mg kg−1 doses in children. The elimination half-life was longer and volume of distribution greater in children than in the adult population.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. REFERENCES

Children with tuberculosis (TB) represent 6–40% of all TB cases globally. Hence, the World Health Organization has identified certain research priorities in childhood TB and one of the main elements is to evaluate the pharmacokinetics of antitubercular drugs under different conditions in children [1].

Pyrazinamide (PZA) is recommended in a dose range varying from 15 to 40 mg kg−1 by various scientific bodies [2–4]. In India, under the Revised National Tuberculosis Programme the dose of PZA recommended is 25 mg kg−1 for daily dosing and 35 mg kg−1 for intermittent, thrice a week dosing [5]. These recommendations are based on studies of PZA in adults, mostly carried out at an average dose of 30 mg kg−1[6]. Hepatotoxicity is the most important and serious side-effect associated with the use of pyrazinamide. Incidence of PZA associated hepatotoxicity has been shown to be dose dependent [7].

Rational use of drugs necessitates that their disposition be carefully evaluated in the population in which the drug has to be used [8]. However there are few pharmacokinetic studies of PZA in children [9]. PZA is not routinely prescribed at lower doses due to lack of pharmacokinetic and efficacy data in children at these lower doses.

The effectiveness of antimicrobial agents may be described as a function of the concentration of antimicrobial agents and area under the curve vs. minimum inhibitory concentration (MIC) of the drug for the microorganisms and the time for which the concentration remains above the MIC [10]. Hence this study was conducted to compare the serum concentrations of PZA at doses of 25 mg kg−1 and 15 mg kg−1 in children suffering from TB and to evaluate the pharmacodynamic indices for PZA, i.e. Cmax : MIC, AUC : MIC and Time > MIC.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. REFERENCES

The Institute's Ethics Committee approved this randomized, parallel group, open label study conducted in children suffering from TB, attending the Outpatient Pediatric Clinic of Lok Nayak Hospital, New Delhi, India. The parents or guardians of all the patients gave written informed consent.

Study patients

Twenty patients, newly diagnosed with pulmonary TB or tubercular lymphadenitis, aged 5–12 years (10 male, 10 female) were enrolled. Diagnosis of TB was based on relevant history, clinical examination, tuberculin test, chest X-ray and when possible, fine needle aspiration cytology for acid-fast bacilli.

Baseline laboratory evaluation was done for haematological, hepatic and renal functions in all the patients. Only patients with values within the normal range for these tests were included. Patients suffering from severe TB requiring hospital admission or any concomitant hepatic disease, renal disease, diabetes, seizures, any other serious illness and patients on any other medication were excluded from the study.

Study protocol

The patients were admitted to the paediatric ward 1 day prior to the study and randomized into two groups. Group I (mean age 9.9 years, height 132 cm and weight 27.7 kg) received 25 mg kg−1 PZA and group II (mean age 7.7 years, height 119 cm and weight 21.5 kg) received 15 mg kg−1 PZA. After an overnight fast, the patients were administered a single dose of PZA at 07.00 h. A standard breakfast and lunch were given 3 h and 6 h after administration of PZA, respectively. Regular antitubercular treatment was started after collection of all the blood samples.

Venous blood samples were collected predose and 1, 2, 4, 6, 8, 12 and 24 h after PZA administration. Within 4 h, the deproteinized serum was stored at −20°C until PZA estimation.

Assay method

Pyrazinamide was estimated by the spectrophotometric method of Subbammal et al.[11]. This method is specific for PZA estimation as pyrazinoic acid, a major metabolite of PZA which could interfere with the estimation of the parent compound, is removed by passing the serum through a vertical column of Dowex 1–8 chloride, 200–400 mesh. This prevents the elution of pyrazinoic acid. A 1 ml sample of deproteinized serum extract was applied to the column. The column was washed with distilled water until two 5 ml eluates were collected. To each eluate, 0.5 ml of 2 n NaOH and 0.5 ml of freshly prepared nitritopentacyanoferroate (0.2%) was added; 10 min later the intensity of the resulting yellow orange colour was read on a LKB ultrospec 4050 UV visible scanning spectrophotometer equipped with an Apple 11e computer. The sum of the optical density for two eluates was regarded as the optical density of the serum.

Prior to the above, the absorption of PZA was observed in water and serum over the wavelength ranging from 460 to 510 nm. The maximum absorption was observed at 490 nm which was then chosen for estimation of the test samples. Standard curves were obtained in deionized water and in pooled donor serum over a concentration range of 0–50 μg ml−1. The recovery of PZA from serum samples was 100%. The lower limit of detection was 5 μg ml−1. A linear relationship was obtained between percentage absorbance and PZA concentration over the concentration range tested. The correlation coefficient for the standard curve in pooled healthy serum was 0.98 ± 0.01 for a concentration range of 5–50 μg ml−1. The interassay coefficient of variation for replicate estimations at the lower limit of detection of 5 μg ml−1 was 17.9% and at 50 μg ml−1 it was 7.7%. Control standards of PZA in serum and water were run concurrently with the test samples on each day.

Data analysis

A single open compartment model was used to interpret the serum concentration of PZA. The bioavalability of orally administered PZA has been observed to be 100% by Ellard [12] and the same was assumed for the calculation of clearance and volume of distribution in our study. Pharmacokinetic calculations were done as described earlier [9].

The MIC for PZA varies from 6.2 to 50 μg ml−1. The average MIC of PZA is considered as 20 μg ml−1 since it inhibits most strains of Mycobacterium tuberculosis[13]. A serum concentration of PZA above 25 μg ml−1 has been associated with a prolonged duration of antimycobacterial effect [13]. Hence 20 μg ml−1 and 25 μg ml−1 were selected to calculate the ratios of Cmax : MIC, AUC : MIC and Time > MIC.

The results are expressed as mean ± SEM. Student's t-test for unpaired data was used for calculations. P < 0.05 was considered significant at a confidence interval of 95%.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. REFERENCES

All the subjects completed the study protocol. Eleven patients were diagnosed to have pulmonary TB and nine were suffering from tubercular lymphadenitis. In both the groups, patients were comparable in their demographic profile.

Figure 1 summarizes the changes in serum concentration with time. At 6 h the concentration had fallen to 27.1 ± 3.9 μg ml−1 and 24.4 ± 3.0 μg ml−1 in group I and group II, respectively. The serum concentration declined gradually thereafter to 6.3 ± 1.8 μg ml−1 and 5.7 ± 1.7 μg ml−1, respectively, at 24 h.

image

Figure 1. Mean ± SEM serum concentrations of pyrazinamide over 24 h in group I (25 mg kg−1; continuous line) and group II (15 mg kg−1; dotted line)

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The pharmacokinetic parameters are shown in Table 1. The Cmax and the AUC were higher, as expected, in group I. However, the difference between the two groups was not statistically significant. The t1/2, λz, CL and V were also comparable.

Table 1.  Pharmacokinetic parameters (mean (SEM)) of group I (25 mg kg−1) and group II (15 mg kg−1)
 Group IGroup II95% CI of difference
  1. AUC, area under the serum concentration–time curve; AUC(0,24 h), area under the serum concentration–time curve in 24 h; CL, apparent clearance; Cmax, peak serum drug concentration; λz, elimination rate constant; MIC, minimum inhibitory concentration; tmax, time to achieve peak serum concentration; t1/2, elimination half-life; V, volume of distribution.

Cmax (μg ml−1)42.4 (3.3)38.6 (3.9)−6.9, 4.6
tmax (h)1.7 (0.2)1.8 (0.1)−0.5, 0.3
AUC(0,24 h) (μg ml−1 h)453 (67)385 (43)−99, 236
AUC (μg ml−1 h)561 (99)515 (89)−235, 327
t1/2 (h)9.3 (1.3)10.5 (2.3)−6.7, 4.2
λz (h−1)0.09 (0.01)0.10 (0.02)−0.6, 0.4
V (l kg−1)0.67 (0.08)0.46 (0.07)−0.02, 0.4
CL (l h−1 kg−1)0.06 (0.01)0.04 (0.01)−0.004, 0.04
Cmax : MIC (20 μg ml−1)2.1 (0.2)1.9 (0.2)−0.4, 0.7
Cmax : MIC (25 μg ml−1)1.7 (0.1)1.5 (0.2)−0.3, 0.6
AUC : MIC (20 μg ml−1)22 (11)19 (7)−5, 12
AUC : MIC (25 μg ml−1)18 (9)15 (5)−4, 9
Time (h) > MIC (20 μg ml−1)5.9 (2.5)5.4 (3.0)−2, 3
Time (h) > MIC (25 μg ml−1)4.9 (2.8)3.8 (3.0)−2, 4

The mean serum concentrations were maintained above 25 μg ml−1 for 6 h in group I and for 4 h in group II. Four patients in group I and one patient in group II had serum concentrations above 25 μg ml−1 for more than 8 h, while five patients in group II had serum concentrations maintained above 25 μg ml−1 for more than 6 h.

The mean serum concentrations were maintained above 20 μg ml−1 until 8 h in group I and until 6 h in group II. Five patients in group I and four patients in group II had concentrations above 20 μg ml−1 for 8 h. The Cmax : MIC, AUC : MIC and Time > MIC were comparable for both the doses (Table 1).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. REFERENCES

It is recognized across the world that research and drug development specifically for children has been a neglected area [14]. Drug therapy in children requires special consideration of dose calculation [8]. However dosing regimens in children are often based on studies carried out in adults.

There are very few studies on blood concentrations and pharmacokinetics of PZA in children. There is only one detailed study in children at a dose of 35 mg kg−1[9] and a population pharmacokinetic study at a dose of 25 mg kg−1[15]. In both the studies, pharmacokinetic estimates in children were significantly different from those in adults.

This is the first detailed pharmacokinetic study of PZA in children at the doses of 25 mg kg−1 and 15 mg kg−1. Given the dose dependent incidence of PZA related hepatotoxicity, such data at lower doses would help in dose optimization in children.

The serum concentrations achieved with the two doses were comparable over the 24 h study period. The Cmax and AUC in our study were comparable with those seen in adults at proportionate doses [6]. The tmax has been shown to be higher in 30% of children with delayed absorption as compared with adults [9, 15]. However in our study the tmax (up to 2 h) was comparable with that in adults [6]. The absorption of PZA was not delayed. The elimination half-life was longer than that reported in adults (6.1–9.6 h) [6] and similar to that previously reported in children (9–10 h) [9].

As has been suggested, an inverse relationship exists for age and apparent volume of distribution. The volume of distribution in our study appears to be lower in comparison with previously reported data in children (0.86 l kg−1 at a PZA dose of 35 mg kg−1) [9]. The CL observed in our study was comparable with the previously reported clearance of 0.05 l h−1 kg−1 for children [9] and 0.06 l h−1 kg−1 for adults [6].

As reported earlier [9], a wide interindividual variation in the pharmacokinetics of PZA was observed. This is a pilot pharmacokinetic study with multiple sampling points, conducted in children. Hence for ethical reasons, the sample size was small. However, the distribution of patients was normal.

Since the mechanism of action of PZA is only partly understood and allows no predictions as to whether the mycobacterial killing is concentration-dependent or time-dependent, the optimum range of ratio of Cmax : MIC, AUC : MIC and Time > MIC which would be effective for the antimicrobial action of PZA have not been clearly defined. However, considering an MIC90 as 10 μg ml−1, the ratio of Cmax : MIC and AUC : MIC at a PZA dose of 27 mg kg−1 was 3.8 ± 0.6 and 52 ± 10, respectively [6, 16]. These are below the recommended values [16]. The Cmax : MIC and AUC : MIC ratio as calculated in our study at MIC of 20 μg ml−1 were below the recommended values for an MIC90 of 10 μg ml−1 in adults [16]. The ratios were further decreased when calculated for an MIC of 25 μg ml−1. Since the Cmax : MIC and AUC : MIC ratios of 3.8 ± 0.6 and 52 ± 10 at a PZA dose of 27 mg kg−1 have been substantiated by relatively poor bactericidal activity in vivo[16], it suggests that 25 mg kg−1 and 15 mg kg−1 may not be the appropriate doses for use in children. The suitability of PZA for intermittent therapy is based on higher serum concentrations being achieved with a large intermittently administered dose than with a smaller more frequently administered dose [17]. However, the higher concentration achieved with larger doses also increases the pH range in which the organisms would be inhibited [18].

Hence both for daily use and for intermittent therapy whether decreasing the dose of PZA further below 27 mg kg−1 provides clinical benefit or not, needs to be evaluated especially when PZA is administered as part of combination therapy and correlated with in vivo observations.

This study has certain limitations. The reported values of MIC of PZA vary over a wide range depending upon the pH of the medium, type of medium, size of innoculum and the strain of tubercle bacilli used [13]. Also it is an in vitro parameter that has been used while in vivo concentrations fluctuate over a wide range due to differences in absorption and elimination rates.

In conclusion, this study is the first attempt to compare the serum concentrations and pharmacokinetics of PZA at 25 mg kg−1 and 15 mg kg−1 in children suffering from tuberculosis. The elimination half-life was longer and volume of distribution was greater than in adults.

However this was a study using PZA alone and more large scale studies need to be conducted to calculate PKPD parameters of PZA when administered at lower doses in combination therapy for TB in children.

Competing interests: None declared.

The authors gratefully acknowledge the gift of pyrazinamide powder from the Biotech division of Cadila Pharmaceuticals Ltd, India.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. REFERENCES
  • 1
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