Comparative Bioavailabilitv Studv of Two Brands of Terbutaline Sulphate Tablets in Healthy Human Volunteers

The purpose of this study was to evaluate the bioavailability of locally produced 2.5 mg terbutaline sulphate tablets (brand A ) relative to a reference product, Bricanyl 2.5 mg tablets (brand 6). The study was a single dose 5 mg randomized crossover one in 15 healthy volunteers in the fasting state. Urine was collected at intervals of 24 h. Total terbutaline excreted in urine as unchanged drug and as conjugates (sulphate and glucuronide) was determined by a developed and validated HPLC method. In-vitro characteristics of both brands were similar. Based on percent of the dose excreted in urine, the oral bioavailability ranged from 33.5% to 75.8% for both brands. Statistics were applied to judge bioequivalence according to USP 24 in-vivo bioequivalence guidance. Results indicated that brand A and B were bioequivalent and hence interchangeable in medical practice.

The bioavailability of oral terbutaline ranges from 25 to 80% in different individuals and is decreased by food.Around 60 % of the absorbed drug, following oral administration, circulates conjugated mainly to sulphate and less to glucuronide, both of which are pharmacologically inactive, and are formed presystemically in gut wall and liver.90% of the absorbed drug including free and conjugated terbutaline is excreted in urine (1-4).These facts coupled with low plasma terbutaline levels in the ng m ~" range (4), suggest that urinary excretion data of total terbutaline could be a possible substitute to blood level in comparative bioavailability studies.
The aim of the present study is to compare the bioavailability of locally produced 2.5 mg terbutaline sulphate tablets with a reference product in healthy volunteers, by measuring total free and conjugated terbutaline excreted in urine, in a single dose crossover study, using a sensitive, rapid and validated HPLC method.

Subjects
Fifteen healthy male volunteers participated in the study after obtaining their signed informed consent.Their age ranged from 21 to 41 years (mean, 31.9), and their mass from 56 to 95 kg (mean, 73.7).They were judged to be healthy by a physician after physical examination and clinical laboratory tests.The study was performed in accordance with the Declaration of Helsinki.The study protocol was approved by the Institutional Review Board and the Risk Involving Human Subject Committee of the FDA.The volunteers were instructed to abstain from taking any medication one week prior to and during the study week.

Materials
The dose administered was 5 mg terbutaline sulphate as either two tablets (brand A), each containing 2.5 mg terbutaline sulphate produced by Arab Drug Co., Egypt, or as two Bricanyl tablets (brand B), each containing 2.5 mg terbutaline sulphate produced by Astra, (Sweden, Batch No TF 225).
Other materials used include an ion-pairing agent for extracting free terbutaline from urine into chloroform, di-(2-ethylhexy1)-phosphate from Sigma Chemicals Co.

Study design
The study was random two-way crossover design (Tablel).Each subject received a single dose of either brand A or B with 200 mL of water after an overnight fast.Subjects continued fasting for further 4 h but drank water regularly.A standard meal was served 4 h after dosing.Urine was collected just before dosing and at 2, 4, 6, 8 and 24 h.Urine volume was recorded and an aliquot was frozen (-18" C for a maximum of 2 weeks).After a period of 7 days, the study was repeated in the same manner to complete the crossover design.No drug could be detected by HPLC in urine collected 24-48 h in a pilot experiment.

Chromatographic conditions
Urine samples were analyzed for terbutaline according to a sensitive selective and accurate high performance liquid chromatography (HPLC) method.
The method of extraction of terbutaline from urine samples was adapted with some modification from a method reported for the extraction of salbutamol from plasma An aliquot of 50 pL of this acidic extract was injected onto the HPLC column.
Recovered standards, 2-6 pg.m ~-' terbutaline sulphate in blank urine were processed with each set of samples.

In vitro studies
Samples of the two brands were tested according to the USP 24 (7) compendial requirement with respect to their mass variation, content uniformity and dissolution profile was performed USP apparatus 1, 100 rpm, 900 mL of water at 37 k 0.5" C) Samples were withdrawn at designated time intervals and assayed by HPLC.Six pL 10 mol L-' HCL were added to each mL sample before injection to ensure peak sharpness.HPLC conditions were as detailed above.

Statistical analysis
Two-way analysis of variance (ANOVA, GLM procedure; using Win Nonlin computer program (8) for crossover design was used to asses the effect of formulation, periods, sequences and subjects within sequence on percent of the dose excreted in urine (9).Sequence effects were tested against the mean square term for subjects within sequence.All other effects were tested against the mean square error term.The difference between two brands was considered statistically significant for p-values equal to or less than 0.05.Parametric 90% confidence intervals (10) based on the ANOVA of the mean difference (A-B) of % terbutaline excreted values were computed.

Results and Discussion
Results of compendial in vitro tests for brand A & B (Table 2) indicated that both brands met specifications required by USP 24.The intra-day precision of the assay (Table 3) was evaluated by replicate (n=3) analyses of urine samples containing terbutaline sulphate at three different concentrations.The relative standard deviation ranged from 1.2 to 8.3%.The interday precision of the assay was assessed by performing several standard curves using three different concentrations over a range of 2-6 pg mL-' in blank urine and were repeatedly analyzed on different days over a period of 32 days.The inter-day relative standard deviation varied from 4.3 to 12.6%.terbutaline profiles following oral administration have been reported (4).
Percent of the dose excreted as unchanged terbutaline and as conjugates in 24 h is shown in Table IV.The values obtained reflect percent absorbed and ranged from 33.5% to 76.6 in agreement with reported value for bioavailability of terbutaline after one application ranging form 25 to 80 % (1, 2).On the basis of the % of dose excreted in the 15 volunteers, the bioavailability of brand A relative to B ranged from 76.9% to 115.5 with a mean of 97.4 % i 12.8 (Table 5).
ANOVA results are given in Table 6.Effects of sequence, period and treatment proved insignificant difference between the two formulations.The 90% confidence interval for the mean difference in percent excreted between A and B fell within the conventional bioequivalence FDA acceptable range of 80-125% (Table 7), and indicated that the two brands were bioequivalent and thus considered therapeutically equivalent.

( 5 )
as terbutaline is very close chemically to salbutamol.The method was developed and validated before the study in our laboratory.Chromatographic conditions were as follows: The mobile phase consisted of 5% acetonitrile and 1% methanol in 0.5% acetic acid adjusted to pH 4.0.Injected sample volume was 50 pi,.flow rate of degassed mobile phase 1 ml.min-' at ambient temperature, UV detector set at 276 nm at an attenuation of 0.04 aufs.No interfering peaks from urine samples was observed.Detection of terbutaline was achieved by monitoring the absorbance at a wavelength of Table I Study design 276 nm; the peak area was measured and the concentration was calculated using the corresponding linear regression equation relating peak areas of the standards to their concentrations.The retention time of terbutaline was 5.3 min.Each analysis required a maximum of 8 min.The method was validated by following international guidelines (Processing of urine samples and standardsFifly pL of 10 mol L-' HCL were added to one mL of each urine sample and the tubes were incubated at 40 O C overnight to hydrolyze terbutaline conjugates (1).Hydrolysed urine samples were neutralized with 50 pL 10 rnol L-' NaOH.One mL of phosphate buffer (0.5 rnol L-', pH 7.2) was added together with 6 mL of 0.1 mol L-I diethylhexyl phosphate in chloroform.The tubes were vortexed for 5 rpm for 20 minutes.The urine layer was discarded and the remaining chloroform layer washed with 3 mL water which was separated and discarded, and the drug in chloroform was extracted into 500 pL 0.5 mol L-' HCL.

Figure I :
Figure I: Dissolution rate of (0) brand A, ( ) brand B tablets according to USP 24 test, each point is the mean of 6 tablets.

Table 2
In vitro data for terbutaline sulphate tablets

Table 3 :
Inter-day and intra-day precision of terbutaline sulphate in blank urineTable 4 gives mean cumulative excretion * SD obtained for each brand at different times.Mean excretion rates are shown in Figure 2. Eight of thirty individuals

Table 6
Analysis of variance applied to percent of the dose excreted in urinein 24 h F = MSlEMSEMS= error mean square Table 7Confidence interval for the difference in percent excreted between brand A *Express as: Mean + t ( SD }