Materials
Metformin HCl Mw 165.6 g/mol. Poly (lactic acid) PLA was purchased from Evonik Industries AG (Germany). Poly (vinyl alcohol) (PVA, 87–89% hydrolyzed, average Mw = 30000–70000) was provided by Sigma Aldrich (Barcelona, Spain). High performance liquid chromatography (HPLC) grade acetonitrile, methanol and water were purchased from sigma Aldrich (Barcelona, Spain), Sodium Dodecyl Sulfate (SDS) ultrapure 288.38 g/mol and ammonium acetate 77.08 g/mol were purchased from (Panreac, Barcelona). All other reagents were of analytical grade.
Animals
Fifteen healthy New Zealand white female rabbits were obtained from the Faculty of Veterinary of the University of Murcia (Murcia, Spain). The rabbits were housed in cage in laboratory animal rooms of the laboratory of pharmacy, pharmacology and therapeutics of Veterinary faculty. The animals were kept under a photoperiod of 12 h light/12 h dark. They received standard laboratory chow diet (Nanta, Madrid, Spain) and tap water. All experiments protocols were carried out in accordance with the requirements of applicable national legislation and approved by the Bioethics Committee of the University of Murcia. After completion of the study, the animals were donated to be adopted as pets as they all were healthy after physical examination.
Preparation of Metformin HCl-microparticles
Metformin HCl loaded PLA microparticles were prepared by the W/O/W solvent evaporation method described previously [14]. Briefly, 1 mL of aqueous internal phase was emulsified for 5 min in 10 mL of methylene chloride (containing 200 mg of PLA) with an ultrasound bath (Branson 5510, BioBlock Scientific, Spain) at 135 W output. This primary emulsion was gradually added into 40 mL of a 1.5% PVA aqueous solution and homogenized for 30 min at 700 rpm in order to create the W/O/W emulsion.The Solvent evaporation was achieved at room temperature and atmospheric pressure under a 400 rpm agitation (HeidolphHei-Tec D91126, Germany). Microparticles were obtained after centrifugation of the colloidal suspension for 30 min at 44000 rpm (Centrifuge 5702, Eppendorf Ag, Germany). Drug free microparticles (Placebo MPs) were prepared with the same procedure.
Determination of the encapsulation efficiency (EE)
The amount of metformin entrapped within polymeric microparticles was determined according to an established High-Performance Liquid Chromatography (HPLC) method by measuring the amount of non-encapsulated metformin in the external aqueous solution, which was recovered after centrifugation of microparticles. The assay was repeated using different samples from independent preparations. The encapsulation efficiency (EE) is defined as the amount of metformin present in the microparticles (m1) divided by the initial amount added in the inner aqueous phase (m0) and it is calculated with the following formulas:
Microparticles characterisation
Scanning electron microscopy (SEM)
The morphology of metformin loaded microparticles was investigated by a scanning electron microscopy (SEM) (Zeiss Evo 50, Germany) at a working distance of 9.5 mm and accelerating voltage of 8 KV. The microparticles were prepared by fixing onto carbon adhesive tape and spotter coating with 10 nm Pd/Au layer under vacuum (EVO/LS 15). The microparticles were examined for shape, size and surface characteristic.
Fourier transforms infrared (FTIR) spectroscopy
In order to examine the drug polymer interaction, the FTIR spectra of pure metformin, blank PLA MPs and metformin loaded microparticles were performed from IR Affinity-1 CE spectrophotometer (Shimadzu, Japan). One milligram of samples was finely mixed with 100 mg of purified potassium bromide and pressed in a mechanical die press to form a pellet (90N, 5 min). These pellets were scanned, and spectra were recorded from 400 to 4000 cm-1 at a revolution of 4 cm-1.
X-ray powder diffraction (XRPD)
Pure metformin, blank PLA MPs and metformin loaded microparticles were investigated by wide angle X-ray diffraction (XRPD) technique using X-ray diffractometer (X Per-Pro, PANalytical, Netherland). All the samples were scanned at 40 KV, 30 mA using Cu-Kα radiation (λ=1.54059 Ǻ) at the range of (2θ) from 5 to 80 degree with a scanning speed of 5° min-1.
Differential scanning calorimetry (DSC)
The thermal properties of samples were evaluated by differential scanning calorimetry (DSC-131, Setaram, France). DSC thermograms of pure metformin, blank PLA MPs and metformin loaded MPs were obtained using SETSOFT software. The samples (8-12 mg) were weighted and sealed into aluminium pans; an empty sealed pan was used as a reference. DSC curves were obtained at a heating rate of 10 °C from 30-300 °C.
In vivo Pharmacokinetic study
Fifteen New Zealand healthy white rabbits (n=15) were selected for the pharmacokinetic study. The rabbits were randomly (Excel random numbers tables generator) divided into 3 groups (n=5) in 3 separated individual and marked cages, and were received a single oral dose administration of metformin microparticles suspension (Group A) pure metformin solution (Group B) by gastric intubation using oral feeding needle respectively, and intravenous (IV) of pure metformin solution by injection from the marginal ear vein (Group C). Investigators were not blinded to sample collection or sample analysis at any stage of the study. There is no hypothesis testing involved in this pharmacokinetics study, and therefore a power analysis is not required for estimating sample size as previously described [27]. The microparticles and pure metformin were suspended/dissolved in saline before each administration at a dose of 5mg/kg body weight. At predetermined time points post administration (10, 20, 30, 45, 60, 90, 120 minutes and 4, 6, 8, 10, 24, 34 and 48 hours), blood samples were extracted (0.5 mL) from the marginal ear vein of the rabbits by heparinised syringe and immediately centrifuged (10.000 rpm,10 min) to separate plasma that was frozen at –50 ºC until further analysis.
Plasma analysis
To quantify the drug in plasma rabbits a validated method [28] modified by Carceles-Rodriguez et al. [29] was used. For this, samples were thawed. Then, acetonitrile (200 µL) was added to plasma (200 µL) vortexed for 15 s then shacked in ultrasonic bath for 5 min to allow a complete mixing, followed by centrifugation at 12.000 rpm for 10 minutes to extract metformin. Afterwards, 200 µl of the supernatant was mixed to HPLC mobile phase (ratio of 1:3) transferred into HPLC vials and analyzed. The pharmacokinetic parameters were calculated using a non-compartmental model.
HPLC method
HPLC analysis was performed using a JASCO series HPLC and a JASCO 1575 UV/VIS detector set at a wavelength of 236 nm. The chromatographic separation was achieved by a Kromasil C-18 reverse-phase column (250x4.6 mm of 5µm, (AnálisisVínicos S.L., C. Real, Spain) at 25 ºC. The mobile phase employed was acetonitrile: ammonium acetate (25mM) - dodecyl sulphate sodium (9mM) (pH 7.02) in a volume ratio of 45:55. The flow rate and injection volume of metformin were 1 mL/min and 100 µL, respectively. A standard calibration curve was performed with metformin in plasma. The established linearity range was 100-10000 µg/ L (r > 0.99). The duration of the analysis is 10 min and the retention time of metformin is approximately 5 min.
Pharmacokinetics analysis
The plasma metformin time-concentration data were analyzed by non-compartment methods. The Area Under the Concentration-time Curve (AUC0-∞) was calculated using the linear trapezoidal rule with extrapolation to infinity. Mean Residence Time was calculated as MRT = AUMC/AUC. The systemic clearance as Cl = Dose/AUC. The apparent volume of distribution (area method) and apparent volume of distribution at steady state were calculated as Vz = Dose/ (AUC · lz) and Vss = (Dose ·AUMC)/AUC2, respectively.
Statistical analysis
Descriptive statistical parameters as mean, standard deviation (SD) and coefficient of variation (CV) were calculated. Harmonic means were calculated for the half-lives of elimination and absorption. Kruskal-Wallis test was used to check for normal distribution of parameters and concentrations ranges between animals. The Wilcoxon Rank Sum and Student’s t tests were used to test parameters for significant differences between the different routes of administration. The statistical software used was SPSS Version 19.0 (SPSS Statistic Programme, Chicago, USA). Values of P<0.05 were considered significant.