Bioequivalence Study of 1,500 mg Glucosamine Sulfate in Thai Healthy Volunteers

Several clinical studies have indicated that glucosamine sulphate is effective in controlling osteoarthritis (OA) symptoms and disease progression [1-4]. In particular, two randomised, placebo-controlled, double-blind trials of 3-year duration in knee osteoarthritis (OA) patients, showed that this symptom-modifying effect is sustained over long-term treatment courses [5,6]. Moreover, many studies suggested that the drug also has a structure-modifying effect, as assessed by measurement of joint space narrowing using validated techniques on standardised plain radiographs [4,5,7-9]. Another recently completed trial (the GUIDE study) [10], confirmed the symptomatic results described above and indicated that, at the dose of 1500 mg once-a-day, crystalline glucosamine sulphate provided a symptomatic effect that was significantly superior to that observed after the administration of placebo [11]. On the other, some studies did not detect any benefit of glucosamine [12,13].


Introduction
Several clinical studies have indicated that glucosamine sulphate is effective in controlling osteoarthritis (OA) symptoms and disease progression [1][2][3][4]. In particular, two randomised, placebo-controlled, double-blind trials of 3-year duration in knee osteoarthritis (OA) patients, showed that this symptom-modifying effect is sustained over long-term treatment courses [5,6]. Moreover, many studies suggested that the drug also has a structure-modifying effect, as assessed by measurement of joint space narrowing using validated techniques on standardised plain radiographs [4,5,[7][8][9]. Another recently completed trial (the GUIDE study) [10], confirmed the symptomatic results described above and indicated that, at the dose of 1500 mg once-a-day, crystalline glucosamine sulphate provided a symptomatic effect that was significantly superior to that observed after the administration of placebo [11]. On the other, some studies did not detect any benefit of glucosamine [12,13].
The formulation used in several studies [4,7,10] is the original crystalline glucosamine sulphate 1,500 mg once-a-day soluble powder preparation which is a prescription drug in most European and extra-European countries [11]. However, in United States, glucosamine is marketed as a dietary supplement to enhance the repair and synthesis of cartilage and connective tissue. It is reported that the U.S. retail market for nutritional supplements containing glucosamine or chondroitins is more than $1 billion per year; the demand for bulk glucosamine has been growing in excess of 20% annually, and global consumption exceeds 5000 metric tons [14].
Our previous studies [15] show that glucosamine sulfate containing KCl (500 mg capsule) is bioequivalent to glucosamine sulfate containing NaCl (2×250 mg capsules) in terms of rate and extent of absorption. This study is designed to evaluate the quality of the generic sachet formulation of glucosamine sulfate 1,500 mg in compare with the original formulation, Viartril ® -S. The dosage of glucosamine sulfate 1,500 mg for both formulations were administered as a single dose to 26 healthy volunteers under a two-treatment, two-period, and twosequence crossover study design with a minimum of one week washout period. and the purposes of the present study, all healthy volunteers provided written informed consents. They were non-smoking, non-alcoholic, and free from significant cardiac, hepatic, renal, gastrointestinal, and hematological diseases, as assessed by physical examination and the following laboratory investigations: complete blood count, BUN, creatinine, aspartate aminotransferase, alanine aminotransferase, total bilirubin, alkaline phosphatase, fasting blood sugar, serum electrolyte and hepatitis B surface antigen. Urine pregnancy tests were negative in all female volunteers. Volunteers did not have a history of allergy to glucosamine and/or its constituents and did not receive other medicines within 14 days before the first study drug administration.

Study design
Randomized, single dose, fasting, two-period, two-sequence; crossover study with at least one week washout period was conducted. Volunteers were allocated into two equal groups. Each volunteer was assigned to a particular study group using a pre-printed randomization table generated by Microsoft Excel. During each period, the volunteers were admitted to the Siriraj Clinical Research Center, Siriraj Hospital. After overnight fasting for at least 8 hours, they received a single dose of test formulation (1,500 mg sachet) or reference formulation (1,500 mg sachet) along with 240 ml of drinking water. Volunteers continued fasting for 2 and 4 hrs (water and food, respectively) after drug administration.
The subjects were closely observed to assess the adverse events. As test product containing KCl 6.58 mmol /1,500 mg, serum potassium was monitored at pre-dose, 12 and 24 hrs after test and reference products administration.
The study was approved by the independent Ethics Committee of Faculty of Medicine, Siriraj Hospital, Mahidol University prior to commencing and was performed in accordance with the Declaration of Helsinki and Good Clinical Practice Guideline. All subjects were individually given written informed consent prior to starting study procedures.

Sample collection and glucosamine analysis
Nine ml of each blood sample was collected by catheterized venupuncture at forearms from each subject. Sodium heparinized vacutainer tubes were used for sample collection. Thirteen samples were collected: 0 (before the dosing), 10, 20, 30 min and 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h after administration. The blood samples were centrifuged. Then, the plasma fractions were collected and kept at -70 o C until analysis.
Bioanalytical of plasma glucosamine was performed by using a validated high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) method in accordance with the USFDA guidelines [16]. Sample preparation was extracted by liquidliquid extraction technique. Propanolol Hydrochloride was used as an internal standard (10 µg/ml). Briefly, 10 µl of internal standard was added in 500 µl of standard spiked sample (Calibrators and QC sample) and plasma unknown sample and then was mixed for 10 seconds. After well mixed, all samples were added and mixed with 1,000 µl of extract solvent (a mixture of acetronitrile and triethylamine, 3:1 (v/v)). Then, the samples were centrifuged at 10,000 rpm for 10 min. The organic layer was transferred into a new vial. All of organic phase was evaporated to dryness under nitrogen gas. The residual was re-dissolved with a dilution solvent (triethylamine : acetonitrile, 10 : 90 (v/v)) and injected into the LC-MS/MS system. The chromatographic separation was carried out on LC-MS/MS with C18, 2.5 μm (50×3.00 mm i.d.). A mobile phase consisting of acetonitrile and 0.025% formic acid (Gradient condition) was delivered with a flow rate of 0.2 ml/min. Mass spectra were obtained using a Quattro Micro ™ mass spectrometer (Micromass Technologies, UK) equipped with electrospray ionisation (ESI) source in positive mode. The mass transition ion-pair for glucosamine [M+H] + ions was selected as m/z 179.90>161.71 and 179.90>143.70. The mass transition ion-pair for propanolol [M+H] + ions was selected as m/z 260.00>116.00. The data acquisition was ascertained by Masslynx 4.0 software. All validated results of our new developed LC-MS/MS method were found in the acceptable limit criteria of US FDA guidance with exhibit good accuracy and reproducibility. Calibration curve was linearity in the range of 0.05-10 µg/mL. The best linear fit was achieved with a 1/x weighting factor, showing a mean correlation coefficient (r 2 ) ≥ 0.999800. The lower limit of quantification (LLOQ) for the validated assay was 0.05 µg/ml. The limit of detection (LOD) was 0.0075 µg/ ml. Mean recovery of extraction were 89.83-96.99% and 106.45% for glucosamine and internal standard, respectively. The intra-and interassay precision was 0.84%-9.79% and 1.20-3.49%, respectively. The percentage average of intra-and inter-assay accuracy was between 93.65%-102.67% and 97.25%-101.41%, respectively. The stability of glucosamine in plasma during sample processing at room temperature after 6 hours for short term stability and 30 days storage in −70°C for long term stability was within the acceptable limit of standard criteria. The % of variation of glucosamine for post-preparative stability was also showing no significant loss in the quantified values, indicating that samples should be processed within this period of time (10 hours.).

Pharmacokinetic and statistical analysis
A non-compartmental pharmacokinetic model was used to determine the pharmacokinetic parameters of glucosamine. The pharmacokinetic parameters, i.e., AUC 0 → t , AUC 0→∞ , C max , t max , t 1/2 were determined using WinNonlin edition version 3.1. Statistical comparisons between pharmacokinetic parameters of the two products were analyzed using two-way ANOVA with p<0.05 for statistical significance to assess the effect of formulation, periods, sequence, subjects within sequence. The variation in estimation of terminal slope as can be seen in lamda_z or t 1/2 calculation (0.693/lamda_z), the AUC 0→∞ might not be a good parameter to be compared. Moreover, our first previous bioanalytical method was not sensitive enough to detect the concentration of glucosamine. There are many BQL data even also at baseline level. Thus, it may not be possible to obtain reliable AUC 0→∞ parameters. Thus, we did the statistical analysis for AUC 0 → t instead. The 90 percent confidence intervals of the test/reference ratio of C max , and AUC 0 → t using log transformed data were determined. The bioequivalence between the two formulations would be accepted if the 90 percent confidence intervals (CI) of the log transformed C max , and AUC 0 → t of test fell within 80-125% of the original product [17].

Results and Discussion
Twenty-six volunteers (13 males, 13 females) completed the study. Demographic characteristics of subjects among 2 groups seemed similar and shown in Table 1. The average plasma concentrations of at each time point from 26 Healthy Volunteers after administration of the reference and test product are tabulated in Table 2. No significant difference was observed in any of the analyzed pharmacokinetic parameters (  for more log-normal distribution. The generic formulation had C max at 0.99 µg/ml, t max at 1.42 hrs while the original formulation had C max at 1.12 µg/ml, t max at 2.00 hrs (Table 1). Ninety percent CI of the mean ratios (generic/original) of the log transformed of the C max and AUC 0 → t were 93.69% (ranged from 86.68%-113.32%) and 97.73% (ranged from 87.38%-112.62%), respectively. Since the 90% CI for C max and AUC 0 → t fell within the predefined bioequivalence acceptance limits (80-125% of the innovator); the generic and original formulations were considered bioequivalent in terms of the rate and extent of absorption.
The plots of average plasma concentration of glucosamine (ng/ml; mean ± SD) vs time over 24 hrs sampling period after oral administration of 500 mg of the test and reference capsules are presented in Figure 1. It was found that the plasma profiles of the glucosamine concentration of both formulations exhibited closely similar patterns, which were nearly super imposable. The amounts of glucosamine in plasma at pre-dose were detected by the fact that glucosamine is a normal constituent of the extracellular matrix of mammalian articular cartilage and synovial fluid, and, therefore, endogenous concentrations of glucosamine may be present in blood as a result of this and other connective tissue turnover.
Glucosamine was well tolerated. The clinical tolerability was good with both formulations. No serious adverse events were registered in the course of the trial. For effects of potassium contained in test product, most of subjects (87.50%) have normal level of serum potassium (3.5-5.0 mmol/L). Two adverse events of hypokalemia (once after taking   reference product and once after taking test product) and one event of dizziness (after taking test formulation) were reported in 3 volunteers. There were no clinically significant found and these three subjects received appropriate treatment and finally recovered. All of the adverse events were judged to be mild in intensity and were possibly related to the study drug. These events were also reported to Ethics Committee of Faculty of Medicine Siriraj Hospital, Mahidol University.

Conclusions
The bioequivalence study of two formulations of glucosamine sachet between the test product (Flexsa ® -1500) and the reference product (Viartril ® -S Sachet) in 25 healthy male and female volunteers was completed. This study was unbalanced and the ANOVA type III was calculated which demonstrated no significant sequence and treatment effects for all parameters. There was no significant period effect for AUC 0-∞ (obs) but there were significant period effect for C max and AUC 0-24 . Moreover, subject nested in sequence effect was significant for all parameters that may be due to the fact that the bioequivalence was performed in small sample size. Non-parametric Friedman's test for T max was demonstrated no significantly different between both formulations (p>0.05). The 90% confidence interval of the geometric mean ratio (test/reference) of C max , AUC 0-24 and AUC 0-∞ (obs) was not contained within the equivalence criteria of 80.00-125.00% [18]. However, this study showed the high intra-individual CV calculated from ANOVA for C max , AUC 0-24 and AUC 0-∞ that may be due to the fact that glucosamine is endogenous substance which those concentration varied considerably between individuals (high intersubject variability), especially between women [19,20]. Therefore, it can be indicated that glucosamine sulfate is a "highly variable drug" because a within subject variability of ≥ 30% in terms of the ANOVA-CV [21]. Based on the bioequivalence limit of highly variable drugs [22][23][24], it is acceptable for widening of confidence interval from 80.00%-125.00% to 75.00%-133.00% for C max parameter. Therefore, it is reasonable to be accepting that C max was entirely within equivalence limits of 75.00%-133.00%. Moreover, a wider range of AUC may be acceptable [25] since glucosamine occurs naturally in human tissues and results of previous studies show the very well tolerability profile [4][5]. Using this guideline, the 90% CI for AUC 0-24 and AUC 0-∞ (obs) ratios were also within the acceptance bioequivalence range of 75.00%-133.00%. Thus, Based on equivalence limits of USFDA [18], it can be concluded that the test product is not bioequivalent to the reference product in terms of rate and extent of absorption. However, concerning the wider equivalence criteria for highly variable drug [22][23][24], it may be accepted that the test product was bioequivalent to the reference formulation in terms of rate and extent of absorption.