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2036 Afr. J. Pharm. Pharmacol.
well to anti-TB medications found lower than expected
drug levels of isoniazid and rifampin in many patients with
adequate clinical response (Mcllleron et al., 2006; Narita
et al., 2001; Holland et al., 2009; Holdiness, 1984). In the
present study, we measured serum concentrations of
anti-tuberculosis drugs (INH, RIF, EMB and PZA) using
HPLC method at 2 and 6 h after drug ingestion. The aims
of the present study were to determine the prevalance of
low serum concentrations of antituberculosis drugs and to
identify the determinants of the serum concentrations of
these drugs in TB patients.
MATERIALS AND METHODS
Study subjects and patients
The present study was performed at a tuberculosis referral center,
Samsun (Turkey) Chest Diseases and Thoracic Surgery Hospital
and all the patients were informed about the study and they gave
their consent to this investigation. 49 adult patients enrolled in the
study between January 2011 to June 2011. All the patients were
bacteriologically confirmed as active pulmonary tuberculosis. Study
subjects had to have received daily standart anti-tuberculosis
drugs: Isoniazid (INH) (daily oral 300 mg), rifampicin (RIF) (daily
oral 600 mg), EMB (weight adjusted daily oral 1000 or 1500 mg)
and PZA (weight adjusted daily oral 1500 or 2000 mg) for at least 7
days before serum drug level measurements. The same trade
marks of drugs were used in all study subjects and the antituberculosis
drugs were single drug products rather than fixed-dose
combinations. Patients with any of the following were excluded from
the study: HIV co-infection, diabetes mellitus, a history of diarrhea,
hepatic, gastrointestinal or renal disease, regular alcohol
consumption and any medication that could affect drug serum
concentrations (e.g. antacids and theophylline) .
Sample preparation
The specific drugs were administered to patients under fasting
conditions and directly observed therapy by the nurses. Venous
blood samples were obtained in an EDTA tube, 2 h after drug
ingestion to estimate peak serum levels of the four drugs and
second samples were taken after 6 h to rule out the possibility of
delayed absorption. After collection, blood samples were
centrifuged at 4000 rpm for a period of 15 min immediately. Plasma
was frozen after centrifugation and stored at -20°C until analysis
and transferred to the reference laboratory on dry ice according to
published recommendations (Holdiness, 1984).
High performance liquid chromatography (HPLC)
Samples were deproteinized by trichloroacetic acid for INH and by
acetonitrile for EMB, RIF and PZA. Solutions were injected to HPLC
system with an LC-20AT pump and 20AC-HT autosampler
(Shimadzu, Japan); results were analysed by using reversed phase
technique, SPD-20A UVdetector and a VP-ODS(150 × 3.9 mm)
Nova-pak C18 4 um (Waters) column (Moussa et al., 2002). The
gradient elution created using 10 mmol potasium hidrojen
phosphate (pH 6.24). Mobile phase A was 10 mmol potasium
hydrogen phosphate and mobile phase B was acetonitrile for INH,
RIF and PZA analysis. Samples were eluted at a flow rate of 0.8
ml/min. EMB mobile phase consisted methanol and deionized water
(70/30) and flow rate was 1,0 ml/min. The limit of quantitation
values in this system for INH, RIF, EMB and PZA were 2.0, 1.5, 1.4
and 1.6 µg/ml, respectively.
Reference serum levels of anti-tuberculosis drugs
INH, RIF, EMB and PZA reach peak serum concentrations
approximately 2 h after ingestion and may delay in malabsorption.
Using published reference ranges, low 2 h drug levels were defined
as follows: INH < 3 µg/ml (300 mg/day); RIF < 8 µg/ml (600
mg/day); EMB < 2 µg/ml (weight adjusted daily dose; 25 mg/kg) and
PZA < 20 µg/ml (weight adjusted daily oral dose; 25 mg/kg). The
patients were treated with oral and standart dose of drugs.
Therapeutic serum levels for INH, RIF, EMB and PZA were 3 to 6, 8
to 24, 2 to 6 and 20 to 50 µg/ml, respectively (Heysell et al., 2010;
Peloquin, 2002).
Statistical analysis of determinants
Statistical analysis was performed using SPSS 15.0 for Windows
(SPSS Inc., Chicago, IL, USA). Variabbles are represented as
mean ± SD (standart deviation) when normally distributed and as
medians (range) in other cases. The potential determinants were
age, sex, smoking status, serum albumin, heamoglobin and drug
dose per kilogram of body weight for all drugs, calculated creatinine
clearance (CCr) for EMB, as it requires renal elimination.
Independent samples T-test was used to compare drug plasma
levels with sex and smoking behaviour variables. The other
variables were statistically analysed with Pearson correlation.
Statistical significance was accepted at P