Comparative Bioequivalence Study Of Montelukast In Healthy Males Biology Essay

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Objective: Comparative bioequivalence study of two brands of montelukast was performed in twelve healthy volunteers. Each volunteer was administered 10mg dose of montelukast.

Method: It was randomized, single dose, two-period crossover study with one week washout period. Blood samples were collected before and within 24 hours after drug administration. Plasma concentration of montelukast at different time intervals were determined by validated HPLC method with UV detection at wavelength of 345nm. Various pharmacokinetic parameters were determined from the plasma concentration-time curves of both formulations.

Result: The 90% confidence intervals obtained by analysis of variance were 87-94% for Cmax and 88-97% for AUC0-t, that fell well within the acceptance range of 80-125%. Also, no significant difference (α=0.05, Wilcoxon Signed rank test) were detected between Tmax of both formulations.

Conclusion: The confidence intervals for Cmax and AUC0-• are fully lied within the interval 80-125%, thus two formulations are well tolerated and bioequivalent.

Key words: Bioequivalence, Pharmacokinetics, Montelukast, High Performance liquid, Area under curve, Peak plasma concentration


Montelukast is available in salt form as montelukast Sodium and described chemically as 1-[({(R)-m-[(E)-2-(7-chloro-2-quinolyl)-vinyl]-α-[o-(1-hydroxy-1-methylethyl)phenethyl]-ben-zyl}thio)methyl] cyclopropaneacetate. (Sean, C.S. 2009). It competitively antagonize cysLT1 receptor mediated bronchoconstriction, increased vascular permeability and recruitment of eosinophils. (Tripathi K.D, 2007) It blocks part of the inflammatory process associated with an asthma attack and thus helps to reduce swelling or constriction of airways. It is also used for the treatment of seasonal allergies. Leukotriene receptor inhibitors are compounds of a new pharmacological class for asthma management and their discovery had made a significant impact on treatment strategies of asthma management. (Cylly et al.2003)

Montelukast has rapid onset of action, and produce improvement even after the first day of treatment. Montelukast is also used as an add-on therapy with inhaled corticosteroids in the long term management of asthma also produces good effects. (Laveolitte, 1999). Montelukast is rapidly absorbed after oral administration and reach peak levels 2 to 2.5 hours after administration of the 5 mg tablet and 2 to 4 hours after administration of the 10 mg tablet (Merck, 1998, Cheng et al.1996 and Choors et al.1995. The Montelukast 10mg is approximately 64% bioavailable, regardless of whether it is administered with food. The 5 mg tablet is 73% bioavailable in the fasting state, but bioavailability declines to 63% when it is taken with food. Montelukast is 100% protein bound (Merck 1998, Knorr et al.1998 and Cheng et al.1996).

Montelukast undergoes extensive metabolism in the liver by the cytochrome P450 enzyme system, specifically CYP3A4 and CYP2C9, and is excreted into the bile (Cheng et al.1996, Chiba et al.1997 and Balani et al.1997). The mean plasma half-life of the drug is 2.7 to 5.5 hours. Patients with mild to moderate hepatic dysfunction and evidence of cirrhosis have been shown to have a decrease in metabolism and a resulting increase in AUC of 40% with a prolonged elimination half-life. Despite these effects, dosage adjustment has not been required for patients with liver disease. Dosage adjustments are also not necessary for patients with renal dysfunction.(Merck 1998) Montelukast appears to be well tolerated. In clinical trials, the most common adverse effect reported was headache, occurring in approximately 18% of patients. Rash, dyspepsia, dizziness, and abdominal pain were all reported in less than 2% of patients. It is category B drug and is safe to give pregnant woman. If montelukast is given with Phenobarbital, serum concentration of montelukast will decrease, so dose adjustment is necessary if administered with Phenobarbital (Holland et al.1998). Different gender difference studies were done but it was concluded that it did not affect the pharmacokinetics of montelukast (Haiyng et al. 1996).

Although several pharmacokinetic studies of montelukast have been published, only few studies are done on bioequivalence (Sripalakit., 2010, Knorr et al.,2010). Purpose of this study was to compare a multinational company brand with locally manufacture brand of montelukast. The present study was designed to determine their pharmacokinetic parameters and compare them statistically to evaluate bioequivalence.

Material and Method:


Chemicals and reagents

Montelukast reference standards was obtained through the courtesy of a local Pharmaceutical company. Acetonitrile, methanol and phosphoric acid were purchased from Merck, Germany while heparin was obtained by Hunos Co., Ltd., Sandschin, Korea.


The Hign performance liquid chromatography with autosampler (LC-20A, Shimadzu, Japan), having a reverse phase column (Lichrospher 5 µm RP-18 column (125x4.6 mm), Merck, Germany) and a computer (Pentium IV 333 MHz) with software (LC-10 A for data handling). The other instruments used were: Centrifuge (Labofuge 200 Haraeus Septech, Kendro lab. Products, Germany), pH meter (Metler Toledo, Switzerland), Vortex mixer (Clifton ultrasonic bath, Nickel electric Ltd., England), Weighing balance (Metler Toledo, Switzerland).


Twelve healthy Pakistani male volunteers participated in this study. Volunteers were selected according to inclusion criteria of the study. Age limit of the volunteers range between 19 - 30 years and body weight was more than 50. Medical history, physical examination and routine blood test showed that volunteers were good in health. Volunteers were non smoker and having no history of alcoholism. No OTC drug was allowed 1 week before the study period to avoid the effects of inducing or inhibiting hepatic metabolizing enzyme and the risk of drug interactions. The present study was approved by the Ethical Review Committee, Faculty of bioequivalence, university of veterinary and animal sciences, Lahore Pakistan. All volunteers signed the informed consent forms prior to participating in the present study.

Study products

The study was conducted by using a test product which was a commercially formulated and a reference product which was the research brand of multinational company.

Study Design

A single dose, two treatments, two periods, two sequences, double blind randomized crossover with one-week washout period was used. After overnight fasting, each volunteer was given 10 mg of montelukast tablet. Blood samples of 5ml were collected at pre-dose and at 0.5, 1, 1.5, 2, 2.5, 3, .5, 4, 6, 8, 12 and 24 hours after drug administration and, then, the plasma was separated

immediately by centrifugation. The plasma samples were stored at -80°C and analyzed for montelukast concentration later.

Determination of the plasma concentrations of montelukast sodium

Standard solution:

Standard solutions for plasma determination of montelukast were prepared by dissolving Montelukast in Methanol (1mg/ml). Serial dilutions were then prepared in fresh buffer solution. lank plasma (0.5 ml) was spiked with different amounts of standard solutions in order to prepare plasma standards at the time of analysis.

Sample preparation:

The frozen plasma was allowed to thaw at room temperature just before the extraction procedure; it was then mixed and centrifuged at 10000 rpm for 10 minutes. 4.5 ml of plasma sample was transferred into a test tube, 0.5 ml of different concentration of montelukast standard preparations was added. To 1ml of this solution 0.8ml of acetonitrile was than added and the mixture was vortexes for minutes; then the samples were centrifuged and the supernated solution was transferred to HPLC vials for chromatographic analysis, detection at 345nm by using U.V. absorbance. (Alsara, 2004) . The HPLC system consisted of a C18 column (Hypersil ®, 250 x 4 mm, 5 μm (Agilent Technologies, USA) with column temperature of 30°C.

Validation of the analysis method e.g. specificity, accuracy and precision, lower limit of quantification (LLOQ), linearity, stability, extraction recovery, was performed before using for drug analysis. Standard curves were performed every day of analysis.


Aliquots (20µl) of montelukast reference standard and plasma spiked samples, were injected on C18 column at 30°C, with a flow rate of 2 ml/minute, with run time 10 minutes. The isocratic mobile phase were acetonitrile: potassium diphosphate buffer 0.05M (80:30). pH of potassium diphosphate buffer pH was adjusted to 3.5 by phosphoric acid.

Pharmacokinetic analysis.

The serum concentration vs time curve for each individual animal was fitted with a drug kinetic computer program. Pharmacokinetic parameters were determined using non-compartmental analysis. Peak serum concentration (Cmax) and time to peak serum concentration (Tmax) values were obtained from observed data on the montelukast serum concentration-time curve for each individual. Elimination or terminal rate constant (k) was calculated from the terminal portion of the serum concentration-time curve using least-square regression analysis of the logarithm of concentration versus time. Biologic half-life (T½) was calculated by the following relationship: T½ = 0.693/k.

The area under the concentration-time curve (AUC) was calculated by trapezoidal rule to 24 hours and then extrapolated to infinity by dividing the last experimental concentration by the terminal rate constant (AUC0-∞ = AUC0-24+ C24/k). Area under the first moment of the concentration vs time curve (AUMC) is defined as the area under the curve of the product of time and drug concentration vs time. AUMC0-24, AUMC0-∞, and mean residence time (MRT) were calculated through the following equations:

AUMCt1-tn = [(t2 - t1)(C1t1 + C2t2)/2] + ... + [(tn - tn-1)(Cn-1tn-1 + Cntn)/2]

AUMCtn-∞ = Cnt/k + Cn/k2

AUMCt1-∞ = AUMCt1-tn + AUMCtn-∞

MRT = AUMC0-∞/AUC0-∞

The kinetic parameters used for the bioequivalence comparison between MONT- test and MONT-reference products were Cmax and AUC0-24.

Statistical analysis.

Data from pharmacokinetic analysis are reported as mean ± SEM (n = 12). Student's t-test was used to evaluate the significance of difference between the means of kinetic parameters obtained from the 2 treatmentgroups. P-values <0.05 were considered significant.

The confidence interval was expressed as a percentage relative to the least-square means of the reference treatments. Bioequivalence was to be concluded when 90% confidence intervals were within the acceptable range of 80%-125% for log-transformed primary pharmacokinetic parameters.


As illustrated in Figure , the blank serum had no interfering peak at the retention time of interest. The linearity of the calibration curve for the spiked MONT in serum was studied at the concentration range of 1-1000 ug/mL. The standard curve showed a good linearity over the range of concentrations examined: y = 248x + 2381, r2 = 0.998. The mean ± SD serum concentration of MONT vs time obtained following oral administration of the MONT-test and -reference products (10 mg) to 2 groups of 12 volunteer are plotted in Figure. The metabolite MONT was detected from 4 hours up to 24 hours post-dosing. The Cmax for MONT in Group II treated with MONT-reference product was much higher than that of Group I treated with MONT-test product. However, Tmax was almost the same in both products.

Pharmacokinetic parameter profiles of MONT in each individual animal following oral administration of 2 MONT products are shown in Table. The comparison of the serum kinetic parameter profiles for MONT in the 2 treatment groups is shown in Table. Overall, the disposition kinetic data (Cmax, AUC, and AUMC) for MONT showed that the serum kinetics were markedly same between the 2 products.

Although the MRT values were higher in the MONT-reference group, there was no significant difference between 2 products. Using the ratio of the means of AUC0-24 and Cmax for MONT-test and -reference products for the bioequivalence comparison, it was shown that MONT -test had significantly lower bioavailability than that of MONT-ref-erence. The ratios for AUC-test/AUC-reference and Cmax-test/Cmax-reference were 0.55 and 0.61, respectively.


The pharmacokinetic data (Cmax, Tmax, T½) for MONT-SO following oral administration of MONT-test product in volunteer obtained in the present study were comparable with results reported by Siripalakit et al., 2010 after the administration of MONT at the same dose rate (10 mg) to volunteer. However, the pharmacokinetic profile obtained following oral administration of MONT-reference product was significantly similar from those of MONT-test.

As indicated in Table, Cmax and AUC were significantly equal in MONT-reference product than MONT-test. Accordingly, MONT-reference product had about samebioavailability than MONT-test. Therefore, the difference in bioavailability profiles can be regarded as an important contributing factor for different clinical effectiveness of MONT products in preliminary Studies.

In the present study, the ratio of μT/μR for AUC0-24 and Cmax for oral MONT suspension products were 0.60 and 0.61, respectively, which indicate that they can be assumed to be bioequivalent.


It is concluded that the bioequivalence parameters (Cmax and AUC) obtained after oral administration of MONT-test and MONT-reference were statistically same so both of the drugs are bioequivalent