Microemulsions characterization
Rheology of MEs
The rheograms of different formulated MEs (Figure 1) were established by measuring each of the viscosity and shear stress against the shear rate at temperature of 25 oC.
Figure 1: Rheograms of prepared microemulsions by plotting the viscosity and shear stress against the shear rate.
Figure 1 shows that the viscosity is constant and relatively low. Also, the relationship between the shear rate and the shear stress is linear. Hence viscosity of developed MEs are Newtonian fluids.
Droplet size of MEs
The MEs were measured using zeta sizer (Figure 2) without any dilution nor filtration at temperature of 25 oC. The mean and standard deviation of the three measurements for each sample and their composition are represented in table 1.
Figure 2: Droplet size distribution of MEIn2 using zeta sizer
Table1: The composition of different developed MEs and their droplets size and PDIs
|
Insulin (ml)
|
DMSO (ml)
|
IPM (ml)
|
Zeta-avarage
|
PDI
|
MEIn1
|
1
|
0.5
|
2.5
|
15.30±1.7
|
0.886
|
MEIn2
|
0.5
|
0.45
|
2.5
|
100.6±28.3
|
0.4145
|
MEIn3
|
0.5
|
-
|
2.5
|
308.8±120.1
|
0.322
|
The droplets sizes of the three microemulsions were 16.3, 100.6 and 308.8 nm. Hence, these formulated systems are MEs.
HPLC-method:
The HPLC-chromatogram showed a higher retention time of 14.3 min (Figure 3A) for prepared insulin standard from powder in comparison to insulin from solution for injection which showed a retention time of 9.1 min (Figure 3B). However, the linearity of the calibration curves were 99.43 and 99.93 for insulin prepared from powder and for insulin of solution for injection respectively. The expected concretions for detection in Franz diffusion cell were within the concentrations of the calibration curve. However, the ratio of high of the peak of lowest concentration (0.1 iu/ml) to the noise was much higher than 9 [28]. The recovery of insulin from our preparations was 98 ± 2.5.
Figure 3: Chromatogram of insulin of A: insulin solution of rapid injection solution; B: prepared insulin solution using 0.01M HCl.
The flux of insulin using Franz diffusion cell
The stratum corneum in epidermis is the main barrier for hydrophilic drugs therefore it is enough to evaluate the permeability through the epidermis instead of full skin for hydrophilic drugs [29]. The transdermal of insulin was evaluated by calculating the flux through isolated epidermis for DMSO insulin solution as well as for two microemulsions (MEIn1 and MEIn2) of three developed loaded insulin microemulsions. The cumulative penetrated insulin amounts per iu/hr*cm2 are represented against the time in figure 4.
Figure 4: Cumulative transdermally penetrated amounts of insulin over 5 h using DMSO solution plus two different prepared microemulsions (MEIn1 and MEIn2)
The transdermal of Insulin through the epidermis was rapidly and could maintain the steady state only for 5 hr. The recommended applied amount was a bit little (100 µl) and couldn’t keep the steady state for long time (29). However, the flux was calculated from steady state period and the results was as flowing: 0.95 ±0.12, 1.77±0.22 and 0.43±0.04 from DMSO solution, MEIn2 and MEIn3 respectively. The MEs contains DMSO showed higher flux for insulin in comparison to the flux of insulin using DMSO solution. On the other hand, insulin had higher flux via the DMSO solution in comparison to MEs free DMSO. Hence, MEIn2 was used for further studying of in vivo transdermal and oral bioavailability.
The transdermal efficacy of loaded insulin microemulsions in rats:
First the response of insulin was monitored by measuring the blood sugar level over six hours after application of insulin in MEIn2, DMSO solution and water solution over the skin of 4shaved 12 h fasted rats and the results are represented in figure5.
Figure 5: Blood sugar level during 6:30 h in rats (7-18) after transdermal application of insulin using DMSO solution (SR 7-10), MEIn2 (MER 11-14) and water solution of insulin (WR 15-18)
The DMSO solution and the MEIn2 containing insulin caused a slight decrease of blood sugar level where the water solution of insulin didn’t cause a remarkable decease of blood sugar level. When the test was repeated after two days, it was observed that the initial blood sugar level before the study was in lowering. Therefore, the formulations were applied in interval two days and the blood sugar level measured next day after application. These results are represented in figure 6
Figure 6: Blood sugar level during 2weeks in rats (7-18) after transdermal application of insulin every 2 days using DMSO solution (SR 7-10), MEIn2 (MER 11-14) and water solution (WR 15-18)
Figure 6 shows no instantaneous lowering for blood sugar but gradually during the treatment over two weeks. Also, the significant decrease was recorded after 6 days using the DMSO solution where was relatively faster using MEIn2 which was recorded after 4 days. However, no significant change was noted of blood sugar level after using the aqueous solution of insulin.
Efficacy of oral insulin loaded microemulsions
The blood sugar level was monitored over 3 hours after oral application of insulin and the response against the time are represented in figure 7.
Figure 7: Blood sugar level during 3 h is rats (1-6) after oral application of insulin using DMSO solution (Solution R1-3), MEIn2 (Microemulsion R4-6) and water solution (WR)
Figure 7 shows a rapid effect after 30 min of application of insulin orally. The lowering was dependent on the initial sugar level on both of preparations.
Xray measurement
Flowing the ME by X-ray in GI showed that the drug after 15 min is still in the stomach (Figure 8). However, the lower part of the esophagus, sphincter and upper region of the stomach were highly illuminated. Which may refers to primary absorption in this region. However the illumination was detected in the small intestine after 6:30h.
Figure 8: Xray images of orlally injected 0.5 ml ME contianing of iopromide as a contrast agent.