Potential of Sustained Release Microparticles of Metformin in Veterinary Medicine: An in Vivo Pharmacokinetic Study of Metformin Microparticles as Oral Sustained Release Formulation in Rabbits.


 Background: Metformin hydrochloride a biguanide derivative has been widely used in the treatment of type 2 diabetes in humans. In veterinary medicine, metformin has been increasing his potential in different species as equids for insulin dysregulation, dogs and cats with diabetes. It is a highly soluble hydrophilic drug, shows incomplete absorption from the gastrointestinal tract and the absolute bioavailability is 40-60 % with a short biological half-life of 1.5-1.6 h in humans. In this study, to improve its efficacy a sustained-release microparticles of metformin was developed by loading within poly lactic acid (PLA) polymer followed by an in vivo pharmacokinetics study in rabbits. Results: Pharmacokinetic study in rabbits showed the sustained-release characteristic from the prepared microparticles with delayed time to reach maximum concentrations Tmax, decreased Cmax, increased Mean Residence Time (MRT) and half-life compared to the pure drug solution. Physicochemical characterization suggested that PLA and metformin hydrochloride interacted within the microparticles via hydrogen bonds and that the drug was transformed to an amorphous state. Conclusions: The The pharmacokinetics parameters resulted in delayed Tmax, increased MRT and t1/2, decreased Cmax of metformin from microparticles that show promise for prolonged/sustained release of metformin after oral administration in different animal species affected by insulin disorders. PLA microparticles provided sustained release of the drug, and these systems can be useful as drug carriers for hydrophilic drugs in long term disease treatment such as diabetes.

decreased C max of metformin from microparticles that show promise for prolonged/sustained release of metformin after oral administration in different animal species affected by insulin disorders. PLA microparticles provided sustained release of the drug, and these systems can be useful as drug carriers for hydrophilic drugs in long term disease treatment such as diabetes.

Background
Diabetes mellitus is a chronic problem in humans characterised by high blood glucose levels induced by; a de cient secretion of insulin, anomalous of insulin action or both. It is a main factor of developing health complications such as cardiovascular disease, neuropathy, nephropathy and eye disease, leading to retinopathy and blindness. Diabetes is classi ed into three main types; gestational diabetes, type 1diabetes and type 2-diabetes. The latest one represents 90% of all the cases [1].
In veterinary medicine, diabetes affects to different animal species, it is more frecuently reported in dogs and cats [2], and rarely in other animal species like rabbits [3]. In equids, insulin dysregulation is the primary endocrine disorder in equine metabolic syndrome (EMS) affecting also some equids with pituitary pars intermedia dysfunction (10-22% prevalence) [4,5]. There are several reports about the use of the hypoglycemic drug metformin in clinical cases in different animal species [2,4]. However, its e cacy is controversial mainly due to its low oral bioavailability [2,4].
Metformin hydrochloride (metformin, Fig. 1.A) a biguanide derivative of galegine alkaloid, is used as a rst line treatment for type 2 diabetes. In the presence of insulin, it acts by lowering the blood glucose concentration mainly through the inhibition of the gastrointestinal glucose absorption and the hepatic glucose production, increasing the glucose uptake and insulin sensitivity in muscle [6]. Metformin a highly soluble hydrophilic drug with low permeability via biological membrane belongs to Biopharmaceutics Classi cation System class III drug (BCS).The low permeability of this drug results in poor drug absorption which is the rate limiting step in attaining suitable bioavailability. After oral administration, metformin is incompletely absorbed from the gastrointestinal tract. It has 40-60% absolute bioavailability with a short biological half-life (1.5-1.6 h) [7]. To maintain effective plasma concentration, it should repeatedly be taken at high doses (2.5 g/day) with a consequent of serious gastrointestinal side effects [8]. Furthermore, metformin is required to be taken for a long period by patients.
In such therapeutic treatment, the development of polymeric controlled drug delivery systems (CDDSs) is an important strategy to overcome the above limitations. The purpose of these systems is to reduce side effects, sustain drug release and increase the bioavailability of drugs at a controlled rate for a sustained time (days, weeks or months) [9]. Thereby the drugs concentration is maintained between the therapeutic windows over a speci ed period of time. In this context, microparticles formulated with polymers, are intensively used in designing CDDSs for a various therapeutic drugs [10,11].
Poly lactic acid (PLA, Fig. 1.b) is a hydrophobic polymer belongs to the α-hydroxyl acid family. As a biodegradable and biocompatible, this polymer is widely used for many applications in pharmaceutical and medical areas because its decomposition products are not toxic and are easily excreted from human organism [12,13]. Several methods for the preparation of CDDs microparticles from PLA polymer were reported in the literature. 8 Because of the easy preparation process, solvent evaporation method (double emulsion) is mostly used to encapsulate water soluble drugs.
The literature does not indicate studies involving microparticles containing metformin, using PLA as polymer. Recently, an optimisation of metformin encapsulation/formulation in PLA microparticlesin terms of encapsulation e ciency was investigated by using response surface design [14]. However, in vivo bioavailability of the prepared microparticles was not done. It is of important to investigate the in vivo performance of the formulated microparticles formulation. For this purpose, this study was conducted to investigate the in vivo metformin sustained release and the pharmacokinetics parameters of PLA microparticles after oral administration to rabbits and compared to pure drug solution administered by oral and IV routes.

Encapsulation e ciency and characterization of PLA microparticles
Metformin HCl was encapsulated within PLA microparticles by double emulsion solvent evaporation method. Many batches were prepared; the percentage of encapsulation e ciency of metformin calculated was 76 ± 2.72%.
The obtained microparticles were analyzed by scanning electron microscopy (SEM). SEM micrograph of the PLA encapsulated metformin is shown in Fig. 2. The microparticles had almost spherical shapewith smooth surfaces with particles adhered to the surface. In addition to surface morphology characteristic, it is interesting to note that the size of microparticles is variable, it is ranged from ~ 1 to 55 µm. SEM images also suggested that metformin was dissolved in the microparticles, since no individualized drug crystals observed.
Fourier transforms infrared (FTIR) spectroscopy FTIR spectroscopy was used to investigate the encapsulation process of metformin loaded microparticles. FTIR spectra of pure metformin, blank PLA MPs and metformin loaded microparticles are shown in Fig. 3. The FTIR spectrum of pure metformin (Fig. 3a) shows characteristic peaks at 3390, 1633 and 1045 cm − 1 corresponds to the stretching of primary N-H, stretching of C = N, and stretching of C-N respectively. For the blank MPs (Fig. 3b) four characteristic peaks were observed at 1764, 2993 − 2943 and 1087 which are attributed to C = O stretching of the ester group, asymmetric and symmetric vibration of the CH3 group -CH 3 (asym), CH 3 (sym) and C-O ester bond.As shown in the microparticles spectra ( Fig. 3c), the intensity of the peak of C = O ester groups of PLA at 1764 cm − 1 after metformin loading was reduced, this is due to hydrogen bond formation between carbonyl of ester of PLA and hydrogen of amine groups of metformin (Fig. 1). It is reported that the elongation of C = O band to C-O-H band after hydrogen bond formation resulted in shift of absorption band to lower wave number. These results con rm that metformin was successfully encapsulated in PLA microparticles. X-ray powder diffraction (XRPD)

Differential scanning calorimetry (DSC)
In addition to IRTF and XRD, DSC analysis was performed to obtain more results about the physical state of metformin in PLA microparticles and intermolecular interactions between the drug and the polymer. The DSC curves of samples were recorded during the rst heating process Fig. 5. The metformin DSC curve exhibited a very sharp endothermic peak at ~ 232 °C that indicates its crystalline nature. DSC curve of the blank PLA (unloaded) microparticles showed a broad peak at ~ 178 °C, while this peak is shifted to 168 °C in metformin loaded PLA microparticles. No peak of metformin in the loaded microparticleswas recorded. The water soluble metformin has interaction with the PLA via the electrostatic force between NH3 + and COO − , consequently changes in the thermal behaviour of both metformin and PLA polymer are occurred [16]. This result indicates that metformin is dispersed or dissolved into the polymer matrix during microencapsulation process.

In vivo Pharmacokinetic studies in rabbits
The in vivo study was conducted to assess the pharmacokinetics of metformin loaded PLA microparticles after three routes of administration (intravenous solution, oral solution and oral PLA microparticles). The mean plasma concentration-time pro les after a single administration at the same dose (5 mg / kg) to rabbits of the IV, oral solutions and the oral microparticles of metformin are illustrated in Fig. 6. Quanti cation of metformin in plasma after 24 h was not detected by HPLC. No adverse events were observed during the study.
After oral administration of pure metformin solution, the drug was absorbed gradually to reach a maximum of absorption at a T max of 110 min with a higher C max of 3.19 ± 0.16 µg/ml followed by a decrease to ~ 1 µg/mL within 300 minutes. In contrast, the metformin concentration in plasma was maintained at low levels within ~ 600 min post administration of metformin-PLA-MPs, showed lower C max (1.36 ± 0.05 µg/ml) and higher T max (138 min) than that of metformin oral solution, this may be attributed to the delayed release of metformin from PLA microparticles.
The pharmacokinetic parameters derived from plasma concentration-time data for the different routes are summarized in Table 1. The results indicated that the Mean Residence Time (MRT) was prolonged with PLA microparticles loaded with drug (369.08 min) as compared to MRT of pure metformin solution given by IV and oral route respectively (156.52 min, 254.59 min).This longer MRT enables to maintain the concentration of metformin for prolonged period in rabbit plasma.
Regards the half-life time (t 1/2 ), it was noted that thet 1/2 of metformin loaded PLA microparticles following oral administration (223.30 ± 21.30 min) was higher in comparison to the t 1/2 obtained from pure metformin solution administered by oral (153.78 ± 4.19 min) and iv (139.61 ± 4.39 min) routes, respectively.
This result indicates that incorporation/encapsulation of metformin in the PLA microparticles achieved high half lifetime.
The AUC 0−∞ after oral administration of metformin PLA microparticles was 617.88 ± 12.92 µg/ml/min, while AUC 0−∞ of pure metformin solution was 882.85 ± 23.90 µg/ml/ min and 2440.77 ± 68.71 µg/ml/min after oral and IV administration respectively. The oral bioavailability of metformin MPS is calculated to be 25.59 ± 0.46%. Signi cantly low AUC 0−∞ of metformin PLA microparticles indicates low release of metformin from the developed microparticles as compared to oral solution. In the case of the PLA microparticles, the decrease in AUC 0−∞ and the low C max value could be explained by a further decrease in the diffusion of metformin from the PLA microparticles which limit the release of the drug.
It was also observed that the values of other pharmacokinetics parameters such as V d (2606.33 ± 233.54 mL/Kg), Cl (8.09 ± 0.17 mL/min/kg) and MAT of metformin encapsulated in PLA microparticles (206.95 ± 31.91 min) were higher than the pharmacokinetics parameters observed for pure metformin solution after oral and intravenous administration (Table 1).

Discussion
The oral route is by far the preferred route of drug administration for many dosages forms. However, many anti-diabetic drugs suffer from low bioavailability following oral administration especially BCS class III like metformin due to its high solubility and low permeability [17]. Therefore, microencapsulation into hydrophobic polymer microparticles systems is an effective strategy to achieve sustained release over time and enhance drug bioavailability in human and veterinary medicine.
In a previous study, we showed the successful preparation of poly (lactic acid) microparticles loaded metformin using the double emulsion (w/o/w) solvent evaporation method. In this method, the effect of microencapsulation parameters such as the amount of metformin, pH of the external aqueous phase, PVA concentration and the stirring rate on the encapsulation e ciency, microparticles size and zeta potential were evaluated and optimized using response surface methodology by Box-Behnken Design [14]. In the present study after microparticles characterization, the potential of poly (lactic acid) microparticles loaded metformin to sustain the in vivo release carried out on rabbits under standard fed conditions.
Metformin loaded PLA microparticles, under the conditions reported previously, exhibited high encapsulation e ciency (76 ± 2.72%) and spherical shape with no porous surfaces. In the FTIR diagrams, some variations in the absorption bands could be observed befDore and after loading metformin onto the microparticles. These changes could be resulted from the physicochemical interactions between the microparticles and metformin to form metformin-loaded microparticles during the process of microencapsulation [18]. The solid state characteristic is important in controlling drug release. High energy is required in the crystalline states to separate the molecules that led to low aqueous solubility and consequently low physiological bioavailability, while the amorphous state need low energy to separate molecules and drug solubility and bioavailability is superior [10]. SEM images, XRD and DSC results indicate that metformin was dissolved in the microparticles with the presence of intermolecular interactions between the drug and the polymer, and the drug was encapsulated in the microparticles in a non-crystalline state. The crystallinity of metformin and PLA disappeared after microencapsulation because of the fast diffusion of dichloromethane to the outer phase during evaporation step, thus inhibits the formation of crystalline structure [15].
Microencapsulation of metformin in the PLA microparticles caused a change in the pharmacokinetics of the drug in the rabbit model used in this study. C max , T max and Bioavailability (F) re ect the in vivo absorption rate of drug. In our study, metformin concentration in plasma was maintained at a low level for 24 h after oral administration of metformin-PLA-MPs, demonstrating slow released metformin which may provide an e cient therapeutic concentration for a prolonged period of time. The peak plasma concentration (C max = 1.36 ± 0.05 µg/ml) for metformin-PLA-MPs is 2.34-fold lower than the peak plasma concentration of oral administration of pure metformin solution. In addition, the time (T max ) needed to reach the maximum plasma concentration was signi cantly delayed in the metformin-PLA-MPs group which may be due to the low absorption of metformin in the cells of the gastrointestinal truck. This type of behaviour is expected to PLA microparticles and it has been found for extended release formulations. The mean value of bioavailability (F) obtained in the oral solution and microencapsulated administration groups were 36.19% and 25.59%, respectively. This value is similar to bioavailability of metformin in diabetic rabbits (36.73%) [19] and rats (34.1%) [20], lower than in cats (48%) [21] and humans (50-60%) [22]. However, signi cantly higher than in horses (3.9%) [23]. The F value of the metformin-PLA-MPs formulation (25.59%) is promising showing the characteristic sustained-release of metformin of extended release formulations.
Particle size and size distribution could in uence the in vivo dissolution rate as well as the oral bioavailability of a drug. As shown from the SEM image (Fig. 2), PLA microparticles obtained had a large particle size that leads to slower degradation rate of the polymer matrix. As a result, a delayed of the drug diffusion through PLA microparticles occurs, this may explain the lower C max and the AUC 0−∞ of the metformin-PLA-MPs compared to the oral solution [24].
The nature of the drugs to encapsulate is an important factor which in uences the release of drug from polymer matrix [25]. The experimental nding by IRTF and DSC suggest the presence of interaction between protonated metformin and carboxylic end group of PLA which affect the drug release and led to a slow release rate over time. Our results are consistent to those obtained by Proikakis et al. [25]. Besides, the amorphous state of metformin in the microparticles could enhance the release of the drug; however, it is not the case in our study. This could be due to the slow degradation of PLA polymer [26].
Based on these ndings, it could be concluded that a promising extended-release PLA microparticles of the highly water soluble drug, metformin, was successfully designed. However, because of the low bioavailability of metformin, future works are needed to improve the bioavailability of the PLA microparticles by evaluating the in uence of PLA microparticle's properties on the release of metformin.

Conclusions
Metformin hydrochloride loaded into PLA microparticles was prepared according to their individual optimised formulations developed in our previous study. The obtained microparticles were spherical with heterogeneous size distribution. The pharmacokinetics parameters resulted in delayed T max , increased MRT and t 1/2 , decreased C max of metformin from microparticles that show promise for prolonged/sustained release of metformin after oral administration in different animal species affected by insulin disorders. Low bioavailability is the consequence of slow release of metformin from PLA microparticles. Therefore, further work is needed to investigate the importance of microparticle size distribution on the release from PLA matrix and the release mechanism of these sustained-release microparticles.

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]. Brie y, 1 mL of aqueous internal phase was emulsi ed for 5 min in 10 mL of methylene chloride (containing 200 mg of PLA) with an ultrasound bath (Branson 5510, BioBlock Scienti c, 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 e ciency (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 nonencapsulated 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 e ciency (EE) is de ned as the amount of metformin present in the microparticles (m 1 ) divided by the initial amount added in the inner aqueous phase (m 0 ) 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 xing 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 A nity-1 CE spectrophotometer (Shimadzu, Japan). One milligram of samples was nely mixed with 100 mg of puri ed 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 Xray 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] modi ed 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 ow 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 (AUC 0-∞ ) was calculated using the linear trapezoidal rule with extrapolation to in nity. 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 V z = Dose/ (AUC · l z ) and V ss = (Dose ·AUMC)/AUC 2 , respectively.

Statistical analysis
Descriptive statistical parameters as mean, standard deviation (SD) and coe cient 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 signi cant 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 signi cant.