DIFFUSION CELL
5.6.1 Introduction
A number of different diffusion cells have been reported (110, 114 - 116, 270) in the literature for the measurement of drug release from topical formulations. Some of these diffusion cells are commercially available and others are laboratory designed (268). This diversity of apparatus has complicated the inter-laboratory comparisons of results and the extrapolation of data to the in vivo situation (268). Chilcott et al. (269) studied the inter- and intra-laboratory variation in diffusion cell measurements from 18 laboratories and reported significantly different results on the in vitro release measurements of methyl paraben through a synthetic membrane. Significantly different statistical results were also reported for similar diffusion cells from different laboratories.
Nearly all the published work on the in vitro release of drugs from semisolids was reported using vertical Franz diffusion cells. An alternative is the USP tablet dissolution apparatus modified to accommodate the European Pharmacopoeia diffusion cell. Figures 1.8 and 1.9 in section 1.4.2 present schematic diagrams of the modified Franz diffusion cell and the
European Pharmacopoeia diffusion cell. In a recent study (100) in this laboratory the release of ibuprofen from various proprietary products through biological and synthetic membranes was greater from the European Pharmacopoeia diffusion cell than the vertical modified Franz diffusion cell, however the data was not corrected for membrane temperature which was different in the two diffusion systems.
The purpose of this study was to compare the more commonly used modified Franz diffusion cell to the European Pharmacopoeia diffusion cell and note any significant differences (p <
0.05, ANOVA) of in vitro release from semisolids while keeping the membrane temperature in both systems constant. Three proprietary 2.5% m/m ketoprofen gel products and a number of extemporaneously manufactured gel formulations were evaluated using both diffusion
5.6.2 Results
The results presented in this study represent data obtained from hplc analysis.
Table 5.11 In vitro release data comparison between Franz and European Pharmacopoeia diffusion cells
Franz diffusion cell European Pharmacopoeia diffusion cell
Formulation
Apparent release constant (μg/cm2/h1/2)
Lag time (minutes)
r2 Apparent
release constant (μg/cm2/h1/2)
Lag time (minutes)
r2
Fastum® Gel 112.0 ± 5.111 3.91 0.9357 154.4 ± 4.319 21.31 0.9748 Ketum® Gel 109.6 ± 2.538 1.10 0.9826 158.6 ± 7.260 18.07 0.9353 Oruvail® Gel 45.63 ± 2.433 28.89 0.9142 81.05 ± 3.859 97.23 0.9304 KET001 139.6 ± 5.109 6.78 0.9577 157.3 ± 7.027 54.81 0.9382 KET002 95.75 ± 2.180 25.67 0.9832 167.2 ± 5.682 44.69 0.9633 KET003 94.47 ± 3.225 34.56 0.9630 108.8 ± 5.914 106.13 0.9111 KET004 104.4 ± 2.694 24.99 0.9785 130.8 ± 6.046 63.28 0.9342 KET005 124.4 ± 4.364 25.17 0.9610 154.4 ± 5.684 59.60 0.9572 KET006 133.3 ± 5.253 39.50 0.9513 158.8 ± 6.083 48.21 0.9538 KET007 86.60 ± 3.744 24.06 0.9419 166.1 ± 5.330 51.10 0.9671 KET008 116.6 ± 4.650 21.62 0.9501 145.0 ± 6.179 46.86 0.9435 KET009 101.5 ± 2.944 38.73 0.9730 169.5 ± 5.696 31.00 0.9641 KET010 96.80 ± 4.047 56.89 0.9455 146.8 ± 5.021 52.99 0.9628 KET011 60.04 ± 5.217 65.90 0.8006 118.9 ± 6.026 81.43 0.9219 KET012 136.1 ± 5.604 21.23 0.9470 160.9 ± 10.33 68.31 0.8803 KET013 7.57 ± 0.7859 103.44 0.7380 - - - KET014 35.29 ± 3.014 62.06 0.8061 83.09 ± 4.320 102.02 0.9181 KET015 43.96 ± 3.143 47.18 0.8556 58.23 ± 3.557 112.78 0.8903 KET016 154.7 ± 9.051 39.70 0.8985 179.0 ± 11.09 80.87 0.8876 KET017 75.56 ± 4.401 85.39 0.8993 138.2 ± 6.902 103.28 0.9239 KET018 142.5 ± 8.035 59.23 0.9051 213.8 ± 7.850 57.27 0.9574 KET019 98.83 ± 6.622 11.34 0.8710 180.4 ± 6.881 20.56 0.9542 KET020 101.4 ± 6.352 115.43 0.8853 160.2 ± 9.997 129.13 0.8861 Table 5.11 summarizes the kinetic data obtained from the linear regression analysis
performed at a 95% confidence interval for both the Franz diffusion cell and the European Pharmacopoeia diffusion cell. For both cells the mean in vitro ketoprofen release across the synthetic membrane increased with the square root of time for all formulations. The linear correlation co-efficient ranges from 0.7380 - 0.9832 for the Franz diffusion cell and 0.8803 - 0.9748 for the European Pharmacopoeia diffusion cell. Although the linear correlation co- efficients expressed by some of the formulations in both diffusion cells were less than 0.9, the slope of each regression line displayed a significant deviation from zero (p < 0.05, ANOVA) indicating conformation to the Higuchi principle. Higher flux values and longer lag times were observed with the European Pharmacopoeia diffusion cell for all formulations. The
lowest flux value was produced by KET013 in the Franz diffusion cell but the concentrations were too small to be detected in the receptor fluid into which the European Pharmacopoeia diffusion cell had been submerged. There was no correlation observed between the two diffusion cells with respect to lag time. KET016 produced the highest flux value of 154.7 ± 9.051 μg/cm2/h½ in the Franz diffusion cell while KET018 produced a flux value of 213.8 ± 7.850 μg/cm2/h½ in the European Pharmacopoeia diffusion cell. KET013 would probably have produced the lowest flux value in the European Pharmacopoeia diffusion cell if quantification was possible by a more sensitive analytical procedure.
The individual diffusion profiles of the proprietary products and all the extemporaneous formulations are presented in appendix II. Group representation of results from Franz diffusion cells and European Pharmacopoeia diffusion cells based on the experimental manipulation of formulations are illustrated below. Figures 5.24 - 5.29 illustrate the
comparison of the Franz diffusion cell and the European Pharmacopoeia diffusion cell for the in vitro release of ketoprofen from various formulations including the proprietary
formulations. The graphs show that the release of ketoprofen was somewhat higher in the case of the European Pharmacopoeia diffusion cells (p < 0.05, ANOVA).
0 25 50 75
0 500 1000 1500
European - Fastum® (SA) European - Ketum® (FR) European - Oruvail® (UK) Franz - Fastum® (SA) Franz - Ketum® (FR) Franz - Oruvail® (UK)
Time (hours) Cumulative amount released (
μ
g/cm2 )0 25 50 75 0
500 1000 1500
European - KET002 European - KET004 European - KET005 European - KET006 European - KET007 Franz - KET002 Franz - KET004 Franz - KET005 Franz - KET006 Franz - KET007
Time (hours) Cumulative amount released (
μ
g/cm2 )Figure 5.25 Effect of different grades of Carbopol® polymers on the release of ketoprofen from Franz diffusion cells and European Pharmacopoeia diffusion cells (n=5)
0 25 50 75
0 500 1000 1500
European - KET001 European - KET002 European - KET003 Franz - KET001 Franz - KET002 Franz - KET003
Time (hours) Cumulative amount released (
μ
g/cm2 )Figure 5.26 Effect of different concentration of Carbopol® Ultrez™ 10 NF polymer on the release of ketoprofen from Franz diffusion cells and European Pharmacopoeia diffusion cells (n=5)
0 25 50 75 0
250 500 750 1000 1250 1500 1750
European - KET002 European - KET015 European - KET016 Franz - KET002 Franz - KET015 Franz - KET016
Time (hours) Cumulative amount released (
μ
g/cm2 )Figure 5.27 Effect of drug concentration on the release of ketoprofen from Franz diffusion cells and European Pharmacopoeia diffusion cells (n=5)
0 25 50 75
0 500 1000 1500
European - KET002 European - KET014 Franz - KET002 Franz - KET013 Franz - KET014
Time (hours) Cumulative amount released (
μ
g/cm2 )0 25 50 75 0
500 1000 1500
European - KET008 European - KET009 European - KET010 European - KET011 Franz - KET008 Franz - KET009 Franz - KET010 Franz - KET011
Time (hours) Cumulative amount released (
μ
g/cm2 )Figure 5.29 Effect of Pemulen® TR1 NF into Carbopol® 980 NF formulations on the release of ketoprofen from Franz diffusion cells and European Pharmacopoeia diffusion cells (n=5) 5.6.3 Discussion
The general setup of the diffusion apparatus was more tedious with the USP dissolution apparatus compared to the Franz apparatus. Approximately 6 l of degassed receptor fluid was required for the USP dissolution apparatus whereas 500 ml was more than enough for a dissolution run time of 72 hours using the Franz diffusion cells. The larger surface area of European Pharmacopoeia diffusion cell required more silicone membrane. The loading of the gel formulation into the European Pharmacopoeia diffusion cell was much easier as compared to the Franz diffusion cell. A direct weighing on a top loader analytical balance sufficed to transfer 500 mg of the gel into the donor chamber of the European Pharmacopoeia diffusion cell but a more tedious procedure was required for transferring 100 mg of gel into the donor chamber of the Franz diffusion cell. It was easier to attain a more uniform spread of the gel in the European Pharmacopoeia diffusion cell compared to the Franz diffusion cell where obtaining a uniform layer was not always possible. The manual manipulation of the receptor phase to avoid air bubbles during the dissolution run became very tedious in the Franz diffusion cells. Since the Franz diffusion cells are made of glass they were more susceptible to breakages while the European Pharmacopoeia diffusion cells were made from
The observed differences in the diffusion rate observed with the European Pharmacopoeia diffusion cell in comparison to the Franz diffusion cell can be explained in terms of the design of the diffusion apparatus, experimental conditions and the intrinsic nature of the gelling agent.
Although Keshary et al. (271) identified some shortfalls with respect to solution
hydrodynamics, mixing efficiency and temperature control in the design of the original Franz diffusion cell, the modified Franz diffusion cell still leaves much to be desired in comparison to the European Pharmacopoeia diffusion cell. A suggestion would be to further extend the water jacket circulating around the receptor chamber in the Franz diffusion cell to the donor chamber in order to maintain the same temperature in both chambers.
The membrane temperature in both diffusion cells was adjusted in order to have the same temperature reading. This was not a problem in the USP dissolution apparatus because when the European Pharmacopoeia diffusion cell is immersed in the receptor fluid, the temperature of the membrane will equilibrate with the temperature of the water bath. This was somewhat of a challenge in the Franz diffusion apparatus. An experiment conducted in this study, on the temperature of the membrane in the donor chamber of the Franz diffusion cells,
confirmed that a membrane temperature of 32 ± 0.5°C was only possible when the
temperature of the heating element was set to 37 ± 0.5°C. Two problems were encountered as a result of this temperature manipulation. There was a substantial amount of receptor fluid evaporation through the sampling port from the receptor chamber of the Franz diffusion cell and there was some heat loss from the plastic tubing connecting the pump to the Franz diffusion cells. The evaporation from the receptor chamber meant that the lost fluids had to be replaced with fresh degassed receptor fluid which inevitably reduces the concentration of the permeant in the receptor chamber. More serious though with regards to the evaporation of receptor fluid is the formation of air bubbles on the underside of the membrane which reduces diffusion. The problem is exacerbated during the overnight run times when the Franz diffusion cell could not be manually tipped to remove the air bubbles even though the open
The loss of receptor fluid containing diffused drug was inevitable during complete emptying of the Franz diffusion cell at sampling times. This may have resulted in inaccurate drug quantification thus producing lower diffusion profiles as compared to those obtained from the European Pharmacopoeia diffusion cell. The non-continuous process of the Franz diffusion cell may result in absolute sink conditions not being maintained during the entire diffusion run especially after 8 hours. The concentration of the drug in the receptor fluid increases with time. This increase decreases the concentration gradient of the drug between the donor chamber and the receptor chamber resulting in lower drug diffusion before the next sampling time. In the case of the European Pharmacopoeia diffusion cell, absolute sink conditions were maintained at all times due to the large volume of the receptor phase and the continuous process which allowed for minimum drug loss during sampling times. Reference to the diffusion profile of KET016 in appendix II shows that a nearly superimposable profile was observed until about 24 hours for both diffusion cells after which the rate of release from the Franz diffusion cell began to decrease. Sink conditions were not maintained in the Franz diffusion cells after 24 hours.
Another possible reason to explain the high release from the European Pharmacopoeia
diffusion cell compared to the Franz diffusion cell is the nature of the gelling agent employed in the manufacture of the gels. Carbopol® polymers, as with most other gelling agents, exhibit an intrinsic property known as thixotropy. This is where the gel has the ability to exhibit gel-sol transitions when subjected to external conditions such as increase in shear rate or an increase in temperature (129). In the European Pharmacopoeia diffusion cell, the temperature may be high enough for the gel to change into a slightly viscous liquid which will increase the kinetic energy of the drug within the formulation and therefore increase the rate of diffusion into the receptor fluid. An increase in temperature is associated with an increase in kinetic energy and a decrease in activation energy. This finding may not be the same for that obtained from the Franz diffusion cell. Increasing the temperature in the Franz diffusion apparatus would lead to an inevitable temperature loss through the plastic tubing and thus the temperature of the donor chamber would not be high enough to change the physical state of the gel under evaluation. Thixotropy would therefore not occur under such experimental conditions and fewer drug molecules will diffuse into the receptor phase.
5.6.4 Conclusion
In product development, the large volume of the European Pharmacopoeia diffusion cell does not allow for the detection of very small quantities of diffused drug whereas it may be more than adequate to compare dissolution profiles of established finished products. Conversely, the Franz diffusion cells will be the best diffusion cell to employ in the initial stages of product development because the small volume produces high concentrations of the diffused drug which can easily be quantified. Formulations containing a large amount of drug may show reduced diffusion over long sampling times due to sink conditions not being
maintained.
5.7 COMPARISON OF DIFFUSION STUDIES OF KETOPROFEN BETWEEN