the commercial preparation Vistabel
®Preliminary experiments were performed to assess the feasibility of SDS-PAGE and silver staining methodology to characterise the HSA contained in the commercial BTX A preparation Vistabel®. This is a commercial preparation, manufactured by Allergan, which is identical to Botox®, except that it contains half the mass of BTX A, is therefore less expensive, and is licensed for cosmetic application only (Section 1.4.1). Vistabel® was therefore selected in this experiment as less expensive substitute for Botox®.
Figure 3.9 illustrates the appearance of a polyacrylamide gel loaded (in triplicate) with Vistabel® and BSA (positive control) solutions and processed by SDS PAGE and silver staining methodology. A prominent band with a molecular weight ranging between 52 and 76 kDa was resolved for both solutions loaded. As the molecular weight of HSA and BSA is 66 kDa, the band detected indicated the presence of serum albumin in both samples.
The dense HSA bands obtained indicate that the amount of protein loaded in the gel was too high for the sensitivity of the staining methodology (limit of detection 2-5 ng of protein/well) (http://www.qiagen.com 2011).This sensitive technique was selected to enable detection of quantities of proteins loaded on MDs, as described in the following Section.
Additional protein bands (Figure 3.9) were detected below 52 kDa for both Vistabel® and BSA solution. These bands may be related to impurities in the solutions loaded. Interestingly, no bands between 66 (HSA) and 225 kDa were detected for Vistabel® formulation, probably due to the higher purity of the commercial formulation compared to the BSA solution. The detection of bands with a molecular weight <66 kDa (HSA), suggests possible fragmentation of the protein and/or presence of impurities with a molecular weight smaller than 66 kDa in both formulations (Vistabel® and BSA). The results indicated that the selected methodologies were suitable to detect the HSA contained in BTX A commercial preparations (i.e. Vistabel®) and were therefore adopted for subsequent experiments.
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Figure 3.9: Assessing the feasibility of silver staining methodology to detect HSA, contained in Vistabel®. The Figure depicts the appearance of a polyacrylamide gel with
each well loaded (in triplicate) with ~27 µg of HSA (Vistabel®) and ~4.5 µg of BSA
(positive control). The arrows point to the serum albumin bands (66 kDa). The molecular marker (5 µl) was loaded in the first and the fifth lane (by left). The marker bands with their relative molecular weights are indicated.
3.4.3.2 Comparison of PMDs and NPMDs loading of BTX A from a
‘Botox
®blank’ formulation
HSA loaded on MDs was characterised by SDS-PAGE and silver staining methodologies (Figure 3.10 A, B). Comparison of samples with the standard profile indicated that an approximate dose of HSA ranging between 180 and 2000 ng was loaded on a MD following immersion into a Botox® blank formulation (HSA ~3.3 µg/µl). This suggests that a formulation volume between ~55 and 605 nl could be accommodated onto a MD (i.e ~11 to 120 nl per needle). If this experiment had been performed with the commercial preparation, the concentration of BTX A would have been 5 ng/150 µl (~33 pg/µl). Therefore, a single MD containing 25-300 microneedles would have accommodated 3 µl of Botox® formulation and thus approximately 0.1 ng of BTX A, a dose which is within the therapeutic range (0.1-
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0.25 ng of BTX A per injection site). However, HSA could not be detected in some NPMDs samples (Figure 3.10 B): in NPMDs line 3 and 6 the amount HSA was below the limit of detection, in NPMD line 5 HSA failed to be detected due to improper loading of the sample formulation in the gel). Therefore, the results obtained suggest variability of MD loading.
As suggested by the previous analysis (Section 3.4.2.3), a MD containing 60 microneedles could load therapeutic doses of BTX A. In that case it was assumed that the entire vial of Botox® was dissolved in 50 µl of diluent. However, this experiment required greater volume and therefore was performed using an increased dilution (150 µl). It is reasonable to assume that if a smaller volume (i.e., 50-100 µl) was used to prepare the Botox® blank formulation, detectable doses of HSA may have been loaded on all the studied MDs and thus effective dose loading could have been achieved for all the MDs. However, due to the high level of variation and the semi-quantitative nature of the analysis, accurate predictions cannot be made.
Previous experiments indicated that a Botox® like formulation was successfully loaded on both PMDs and NPMDs (Section 2.4.4). This was confirmed by the present study, as the HSA loaded was detected for the majority of the MDs analysed. Therefore, physicochemical properties of the Botox® formulation are suitable for successful MD loading. The ‘Botox® blank’ formulation employed in the present experiment differed from the so termed ‘Botox® like’ formulation described in Section 2.3.4.1. The latter contained 5 ng of the BTX A model β-gal to mimic the dose of BTX A (5 ng) contained in the commercial preparation Botox®. In the experiment reported in this Section, the BTX A model was not added to the formulation employed as this low mass of protein would not have enabled loading of MDs with detectable quantities of protein.
In conclusion, the result suggested that PMDs and NPMDs with an increased number of microneedles have the capacity to accommodate therapeutic doses of BTX A after only one immersion in a Botox® formulation. However, as previous quantitative loading analysis indicated (Sections 3.4.2.1 and 3.4.2.2), dose reproducibility cannot be guaranteed by using the existing drug loading methodologies.
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Figure 3.10 (A, B): Characterization of HSA loaded on PMDs and NPMDs. The images
depict the appearance of two polyacrylamide gels processed by SDS-PAGE and silver staining methodology. The gels were loaded with HSA standard solutions and samples
solutions generated by PMDs and NPMDs loaded with a ‘Botox® blank’ formulation (HSA
~3.3 µg/µl, NaCl 6 µg/µl). Successful characterization of HSA is indicated by the presence of a band at 66 kDa. The approximate concentration (ng/µl) of the HSA standards loaded on the first 6 lanes (indicated by numbers 1-6) of both of the two gels are the following: 180 (1), 80 (2), 40 (3), 20 (4), 10 (5), 0.9 (6). The approximate mass of HSA in the sample solutions generated from PMDs and NPMDs loading was estimated by multiplying the concentrations of HSA standard 5 and 6 by the volume of diluents (200 µl) used to rinse the MDs after HSA loading. The molecular marker (5 µl) was loaded on the first and last lane (by left) on each gel. The marker bands with their relative molecular weights are indicated. The line in (A) and (B) was drawn to facilitate differentiation between the standards (lanes 1-6, by left) and the samples (lanes 1-6, by right).
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3.5 Conclusion
PMDs employed in the current research project do not have the capacity to load effective doses of BTX A due to the reduced number of microneedles contained in each device. However, PMDs containing 25-300 microneedles would allow loading of therapeutic doses of the existing commercial preparation Botox®, re-constituted with volumes of 50-150 µl.
Microneedle loading of low molecular weight (salbutamol sulphate) and macromolecular compounds (β-gal and HSA) was associated with a significant level of variability. Therefore, PMD loading of reproducible and efficient doses of Botox® would not be guaranteed by using the existing loading methodologies. However, given the large therapeutic window of BTX A, therapeutic dose loading may potentially be achieved by employing robustly engineered loading methodologies and/or specifically tailored MD designs.
Due to absence of statistical difference in the loading capacity of the two microneedle designs studied, PMDs would not increase the dose of BTX A compared to NPMDs. Nevertheless, PMDs may offer the potential to deliver BTX A in a liquid form as well as enable dose monitoring.
Chapter 4