Yield of pellets
The obtained pellets were passed through a suitable sieve to separate oversized (doublets), undersized (fine) pellets, and the weight of the desired pellets was noted. The percentage yield of spherical pellets was calculated with respect to the total weight of the pellets taken for sieving. MFH pellets were manufactured by extrusion and spheronization technique using a roller diameter of 0.2 mm which produced the uniform pellets of defined size and the fines generated during spheronization were removed using #50. The pellets retained on #50 were considered as good pellets for further processing. On the other hand, GMP and PGH pellets were manufactured by extrusion and spheronization technique using roller diameter of 0.3 mm which produced the uniform pellets of defined size, and the fines generated during spheronization were removed using #40. The pellets retained on #40 were considered as good pellets for further processing. Hence, only over-size pellets of specific sieve #50 for MFH and #40 for GMP and PGH were considered to specify the size of pellets. For MFH-SR pellets, sieve #30 was used to remove oversize pellets and sieve #50 to remove fines. The pellets were passed through sieve #30 passed pellets and sieve #50 retained good pellets which are considered to calculate yield. In the case of PG IR pellets, sieve #20 was used to remove oversize pellets and sieve #40 to remove fines. The pellets were passed through sieve #20 and sieve #40 retained good pellets which are considered to calculate yield.
Drug content
Accurately weighed 100 mg pellets of each of the 09 batches were finely powdered using mortar-pestle separately, and the powder was dissolved in methanol using a bath sonication. The solution was then filtered and analyzed using a validated HPLC method with PDA detector (Additional file 1).
In-vitro release study
The in vitro drug release profiles from MFH-SR and PG-IR pellets were determined using a USP type I dissolution apparatus using dissolution medium (0.1 N HCL) maintained at 37 ± 0.5 °C and stirred at 100 rpm and was compared with marketed Glucophage tablet (MFH 500 mg), Amaryl tablet (GMP 1 mg) and Actos tablet (PGH 15 mg). The capsules filled with pellets were placed in the basket, and a study was performed in 900 mL of media (0.1 N HCL) maintained at 37 ± 0.5 °C and stirred at 100 rpm. 0.1 N HCl was used as a release medium for in vitro release study as it is considered a more relevant medium for correlation of obtained results with the in vivo study performed under fasting conditions [12]. The 5 mL of samples were withdrawn at regular intervals (0, 1, 2, 4, 6, 8 and 12 h) for MFH-SR pellets, and at 0, 5, 10, 15, 30, 45 and 60 min for PG-IR pellets. An equal volume of fresh medium was added to the dissolution apparatus. Finally, the solutions were filtered using a 0.45-μm millipore filter and analysed using HPLC (Shimpack ODS C18 250 × 4.6 mm, 5 μm; Mobile phase: Acetonitrile: 0.02 M phosphate buffer pH of 6.3 adjusted with orthophosphoric acid (OPA); Flow rate: 1.0 mL/min; Injection volume: 20μL; Wavelength of 254 nm). This HPLC method is validated (refer to Additional file 1).
The similarity and difference factor is calculated using the following equations,
$$\begin{aligned} & f2 = 50 \times \log \left\{ {\left[ {1 + \left( {1/n} \right)\Sigma \left( { \, R_{t} - T_{t} } \right)^{2} \, } \right]^{ - 0.5} \times 100} \right\} \\ & f1 = \left\{ {\left[ {\Sigma \left| {R_{t} - T_{t} } \right|} \right]/\left[ {\Sigma R_{t} } \right]} \right\} \times 100 \\ \end{aligned}$$
where n = number of time points, Rt = % Active Pharmaceutical Ingredient (API) dissolved of reference product at time point x, Tt = % API dissolved of test product at time point x.
Size distribution analysis
The size distributions of optimized MFH-SR and PG-IR pellets were determined by performing sieve analysis. Briefly, British standard sieves 10, 16, 22, 44 and 60 were taken and arranged in order such that the sieve number 10 (coarser sieve) was on the top, and the sieve number 60 (fine sieve) was at the bottom. Accurately weighed 100 gm of pellets were placed on the stack of sieves, and sieves were shaken for 10 min using a mechanical sieve shaker. Finally, the pellets retained on each sieve were collected separately, weighed, and the mean particle size of pellets was calculated.
Flow properties
The flow properties of optimized batches of MFH-SR and PG-IR pellets were investigated by measuring the angle of repose, bulk density, tapped density and Hausner's ratio in triplicate by standard official methods [13].
Pharmacokinetic study in rabbits
The animal caring and handling and protocols were approved by the animal ethical committee (12/AB/2018). 12 white rabbits (NZW; 06 male and 06 female) weighing 1.5 to 2.0 kg were used for the study. The rabbits were fasted overnight (12 h) before administration of the formulations but had free access to water. The animals were randomly divided into two groups (A and B) with six animals in each group. Rabbits of group A were administered orally with optimized MFH-SR and PG-IR pellets equivalent to dose calculated on a body weight basis. Further, group B received a marketed Glucophage tablet (MFH 500 mg), Amaryl tablet (GMP 1 mg) and Actos tablet (PGH 15 mg) equivalent to dose calculated on a body weight basis [14]. The pellets and tablets were cut with the tablet cutter, and the weight equivalent to the required dose was administered. Briefly, for administration, the product was placed in the smoothly cut (opened) end of a 3 mL syringe (plastic) pushed it ahead with a plunger toward the base of the rabbits tongue for ingestion, followed by a few draughts (nearly 10 mL) of water. A dose of 0.2 mg/kg of GMP, 100 mg/kg of MFH and 3 mg/kg of PGH were administered to each rabbit. Blood samples (0.5 mL) were retrieved by marginal ear vein puncture at different time intervals (0-predose and 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 16 and 24 h post-dosing) for pharmacokinetic analysis. Blood samples were collected in centrifuge tubes containing sodium citrate (3.4% w/v) as an anticoagulant. To separate the plasma, samples were centrifuged for 10 min at 3500 rpm at room temperature, and plasma was collected [15]. Finally, MFH, PGH and GMP were estimated by the liquid chromatography system consisted of solvent delivery (LC10ADVP), controller (LC10ADVP) and column oven (CTO10ASVP) from Shimadzu (Kyoto, Japan). SIL HTC autosampler from Shimadzu (Kyoto, Japan) was used to inject 20µL aliquots of the processed samples on a Peerless basic C18 (33 × 4.6 mm, 5µ particle size) column kept at room temperature. The isocratic mobile phase, a mixture of methanol:water (containing 0.5% formic acid) 8:2 was delivered at 0.6 ml min into the mass spectrometer’s electrospray chamber. Quantification was achieved by MS. All results were presented as mean ± SD values [16].
In vitro stability study
The stability study of the optimized batches of pellets was carried out by storing the formulations in high-density polyethylene bottles for a period of 6 months at accelerated stability conditions (40 ± 2 °C/75% ± 5% RH). At predetermined time intervals (30, 90 and 180 days), the pellets were evaluated in terms of physical appearance, drug content and dissolution profile [17].
Statistical analysis
Data are mentioned as the mean ± standard deviation of three independent experiments. The statistical analysis was performed by using GraphPad Prism software version 5 (GraphPad Software, Inc., La Jolla, CA, USA). The results obtained were analyzed by one-way ANOVA. p < 0.05 was considered statistically significant.