| Pharmacokinetics study is an important means of drug quality assessment, pharmacokinetic parameters of the drugs such as the plasma concentration - time area under the curve (AUC), peak concentration (Cmax) and peak time (tmax) are used to evaluate the quantitative change disciplinarian in vivo. Therefore, pharmacokinetics is very important in the drug report and review, it is also very important in the drug research, development and management.Alendronate sodium is a bisphosphonate drug used to treat Calcium metabolism disorders disease, such as osteoporosis, hypercalcemia, Paget's disease and so on. Alendronate sodium is water-soluble with negative charge; molecular weight is more than 150. The residence time of alendronate sodium is short in plasma, it is selectively accumulated in the skeleton and its oral absorption rate is approximately 0.7%[1]. Therefore, the plasma concentration of Alendronate sodium is very low. Moreover, because the molecule polarity is great, the structure is without chromophore or fluorophore groups, Alendronate sodium is not directly determined with routine UV or fluorescence detector [2],it is difficult to determination of alendronate sodium .Objective: To establish the determination method of alendronate sodium concentration in plasma, study the pharmacokinetic of Chinese healthy male volunteers after 70mg alendronate sodium was taken orally. Pharmacokinetic parameters of two preparations were carried out. In vitro Caco-2 cell model, disintegration and dissolution tests were used to compare the absorption, disintegration and dissolution of two preparations to study why pharmacokinetic parameters of two preparations were significant difference.Methods: alendronate sodium concentration in different media was determined by high performance liquid chromatography (HPLC). The protein precipitation and solid phase extraction were used in pretreatment of the plasma sample. Cell sensitivity to the drug was determined by MTT assay. Caco-2 cell model was established to study drug absorption and transport. In vitro drug disintegration was determined by disintegration and dissolution tests. Contents:1. Alendronate sodium concentration in human plasma was determined by HPLC- fluorescence detection method (HPLC-FLD), and Alendronate sodium concentration in the HBSS solution and deionized water was determined by HPLC-UV detection method. Pamidronate sodium was internal standard(I.S.), column: Kromasil-C18 column (4.6×150 mm, 5μm);mobile phase: methanol-acetonitrile-the mixture of citric acid and pyrophosphate sodium, gradient elution was used, fluorescence detection wavelength: excitation wavelength (λex) was 260 nm, emission wavelength (λem) was 310 nm. UV detection wavelength was 265nm. The quantitative analysis method was verified in various media.2. Clinical trials of Alendronate sodium were two-period randomized crossover trials. Six subjects were divided into two groups according to random numbers. All subjects signed an informed consent and carried out the various inspections according to the provision prior to the trials. Single dose oral administration was taken in the trials. Venous blood at upper limbs were collected at different time points and centrifuged to obtain plasma. The concentration of the alendronate sodium in the plasma after pretreatment was determined by HPLC-FLD method. Pharmacokinetic parameters were calculated and recorded and analyzed. single dose oral administration Cmax,AUC0-t and AUC0-∞data of the test preparation and reference preparation were logarithmically transformed, analysis of variance and two-sided t test (α= 0.05 ) and non-parametric test of Tmax were taken. Pharmacokinetic parameters of two preparations were evaluated.3. The transport and absorption of alendronate sodium were studied on the Caco-2 cell model. Caco-2 cell monolayer model was established, cell single transmembrane resistance and the apparent permeability coefficient of markers and morphology were used to evaluate the integrity of the model. Sensitivity of the alendronate sodium concentration was determined in the MTT test. The known concentration of alendronate sodium Lin was added to Caco-2 cell monolayer based low side (Basolateral, BL) or villous surface side (Apical, AP). Drug transportion from both directions was studied. A certain amount of the biological samples were taken from receive pool respectively at set time points. After pretreatment of sample, alendronate sodium concentration is determined by established HPLC-UV method. Apparent permeability coefficient (Papp) was calculated; data were recorded and statistically analyzed. 4. The disintegration and dissolution test of alendronate sodium. Test methods were in accordance with "Chinese Pharmacopoeia 2005 Edition". 6 tablets from every two preparations were taken and placed into the basket glass tube of the disintegration apparatus respectively, the disintegration apparatus were started, and the disintegration time was recorded successively. In the same way, other 6 tablets from every two preparations were taken and placed into the basket glass tube of the disintegration apparatus respectively. The dissolution medium was 700 mL deionized water, medium temperature was 37℃. Paddle speed was 50 r·min-1. A certain amount of the samples were taken respectively at set time points; after pretreatment of sample, alendronate sodium concentration was determined by established HPLC-UV method., data were recorded and statistically analyzed to calculate their dissolution respectively.Results:1. HPLC-FLD determination method for plasma samples has a high Sensitivity and specificity. The results of method validation meet the requirements of the analysis of biological samples. Endogenous impurity of the plasma samples did not interfered with sample determination. Alendronate sodium and pamidronate sodium (internal standard) of the retention time were 8.9 min and 7.6 min respectively. Linear range of the alendronate sodium was between 1 ng·mL-1 and 100 ng·mL-1(r = 0.9999). The quantification limit was 1 ng·mL-1. The long-term freeze-thaw and repeated freeze-thaws stability of alendronate sodium were good. The within-day precision and day to day precision and accuracy of determination method for alendronate sodium are good. This method was sensitive and accurate, so it could be used in clinical pharmacokinetics and bioequivalence studies.Because the HPLC-UV determination method in HBSS solution and deionized water was simple, rapid, sensitive and specific and linear relations of determination method were good, both media apply to the determination method for the alendronate sodium.2. After single dose oral administration of alendronate sodium test tablets and reference tablets by 6 healthy male volunteers, statistical results of pharmacokinetic parameters: AUC0-t(μg·L-1·h) are 102.06±42.19 and 164.42±50.47 respectively; AUC0-∞(μg·L-1·h) are 129.96±67.84和184.34±55.04 respectively; Cmax(μg·L-1) are 35.48±12.33和55.59±17.02 respectively; tmax(h) is 1.33±0.41 and 1.25±0.59 respectively; t1/2(h)are 2.04±0.66和2.03±0.79 respectively. The statistical results showed that pharmacokinetic parameters of two preparations were significant difference and the relative bioavailability of A preparation was low.3. Transfer volume of two preparations in the Caco-2 cell model was less. The Papp of alendronate sodium were less than 10-7cm·s-1, so absorption of alendronate sodium was poor (absorption rate is less than 1%). Transfer volume between two preparations was not significant difference statistically.4. The HPLC-UV determination method of alendronate sodium in the deionized water meet the requirement of"Chinese Pharmacopoeia" 2005 version provisions", and this method was applied to the dissolution test of alendronate sodium tablets. Test data obtained through the SAS 9.1 software analysis: the disintegration time difference of two formulations was significant .The disintegration time of A preparation was less than that of B preparation; two preparations were dissolved in complete within 15 min; The average dissolution rate of two preparations were not significant difference statistically. But the dissolution difference of six tablets of A preparation were large and significant statistically. The dissolution difference of six tablets of B preparation were small, they were not significant statistically .The results indicate that the dissolution of two preparations, there was still difference.Conclusion:The determination methods of alendronate sodium in plasma and HBSS and deionized water were established in this paper. The sensitivity and good reproducibility of quantitative analytical methods in three media were good, so the method was applied to determine the alendronate sodium concentration accurately and quantitatively.After oral administration of two alendronate sodium preparations by six healthy male Chinese volunteers, pharmacokinetic parameters are analyzed, bioequivalence was evaluated, the results show pharmacokinetic parameters of two preparations were significant difference and the relative bioavailability of A preparation was low.In vitro Caco-2 cell model was established to study the absorption and transport of two preparations to discuss pharmacokinetic parameters of two preparations were significant difference. The results showed that transfer volume of two preparations in the Caco-2 cell model was less, the absorption of two preparations was poor. Transfer volumes between the two were not significant difference statistically. The disintegration test and dissolution test of two alendronate sodium preparations were conducted to study the difference of the disintegration time and dissolution rate between two preparations.The above results show prescription process of two preparations are different that causes the pharmacokinetic parameters of two formulations is significant difference, resulting in disintegration and dissolution of the differences, thereby affecting the drug absorption process in vivo, resulting in differences in pharmacokinetic parameters . At the same time, there may be other factors, which need further study.
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