1. Determination of Vismodegib by
Gradient Reversed-Phase High-Performance Liquid Chromatography
Hiep X. Nguyen, Ajay K. Banga
Mercer University College of Pharmacy, Atlanta, GA 30341
RESULTS
INTRODUCTION
The purpose of the study is to develop and validate a gradient reverse-phase high-performance liquid chromatography method for measurement of vismodegib to be used in transdermal in-vitro permeation studies.
Vismodegib was determined to be stable in ethanol after storage at room temperature for 24 h, at 40C for 30 days and at – 200C for 60 days. Stability assessments indicated that vismodegib was also stable in PBS, PG, PEG 400, PBS:PEG 400 (50:50 v/v) and PBS:EtOH (75:25 v/v) at room temperature for 24 h and at 40C for 30 days. *
Fig. 2. Representative chromatographs: (A) Blank ethanol; (B) Vismodegib solution in ethanol (50 µg/mL)
Table. 2 Determination of LOD and LOQ
LOD and LOQ
Slope
Y - Intercept
STD 1
23707
STD 2
29629
STD 3
29376
-943.9
Average (S)
SD (σ)
344.65
0.04 (µg/mL)
0.13 (µg/mL)
Fig. 1. Chemical structure of vismodegib
{* indicates statistical difference from other group (Single factor ANOVA, p value < 0.05)}
Fig. 3. Amount of vismodegib in porcine ear skin
METHODOLOGY
Analytical Method:
Column: 5 µ micron Phenomenex SU Luna C x 4.60 mm
Temp: Ambient temperature.
Run time: 15 min
Detection wavelength: 266 ± 1.65 nm.
Mobile phase: Acetonitrile (Mobile phase A) : DI water containing 0.05 % (v/v) trifuoroacetic acid (Mobile phase B) (Table 1)
Flow rate: 1.0 mL/min
Injection volume: 10 µl. *
Table. 3 Intra-day and inter-day accuracy and precision
Nominal conc. (µg/mL)
Determined mean conc. (µg/mL)
Mean % accuracy
Precision (%)
Intra-day (n=6)
0.25
0.35
140.98
5.40
2.5
2.36
94.53
1.37 25
25.29
101.17
0.39
Inter-day (n=12)
140.11
5.85
2.40
96.17
5.11
24.41
97.62
3.83
Table. 1 HPLC gradient composition
Time (min)
% Mobile Phase A
% Mobile Phase B 10 90 8 80 20 13 15
{* indicates statistical difference from other group (Single factor ANOVA, p value < 0.05)}
Fig. 4. Amount of vismodegib in tapes and skin
CONCLUSIONS
The method is specific, precise and reliable.
The regression line equation is capable to statistically, reliably predict the drug concentration in the range 0.1–50 µg/mL.
The study for the first time presented the solubility of vismodegib in 10 mM phosphate buffered saline, propylene glycol, polyethylene glycol 400, 10 mM phosphate buffered saline:ethanol (75:25 v/v), 10 mM phosphate buffered saline:polyethylene glycol 400 (50:50 v/v).
The stability assessment revealed vismodegib was stable in ethanol at room temperature for 24h, at 40C for 30 days and at –200C for 60 days and in these solvents after the storage at room temperature for 24h and at 40C for 30 days.
The in vitro permeation study by vertical Franz diffusion cells indicated that no drug entered the receptor chamber during 52-hour experiment.
Solubility Study: To prepare the solubility samples, vismodegib was added in excess to each solvent (PBS, PEG 400, PG, PBS:EtOH (75:25 v/v), PBS:PEG 400 (50:50 v/v)). Following shaking, samples were centrifuged and the supernatant was filtered .
Table. 4 Solubility of vismodegib in solvents (n=3)
Solvent
Saturation solubility (µg/mL)
PBS
3.56 ± 0.52 PG
± 55.43
PEG 400 ±
PBS:PEG 400 (50:50 v/v)
± 10.85
PBS:EtOH (75:25 v/v)
39.06 ± 1.29
Stability Study: The stability of vismodegib in ethanol was evaluated on 3 standard concentrations (5; 25 and 50 µg/mL) (n=3) after storage at room temperature for 24h; at – 40C for 30 days and – 200C for 60 days. Vismodegib saturated solutions of solubility samples in PBS, PG, PEG 400, PBS:EtOH (75:25 v/v), PBS:PEG 400 (50:50 v/v) (n=3) were subjected to – 40C in a freezer for 30 days and room temperature for 24h.
REFERENCES
[1]. De Smaele E, Erretti E, Gulino A., “Vismodegib, a small-molecule inhibitor of the hedgehog pathway for the treatment of advanced cancers”, Curr Opin Investig Drugs, 11(6), (2010).
[2]. Sivamani RK, Stoeber B, Liepmann D, Maibach HI, “Microneedle penetration and Injection past the stratum corneum in humans”, J Dermatolog Treat, 20(3), (2009).
Permeation Study: The In-vitro permeation study of vismodegib 7 mg/mL in propylene glycol through hair-cut, fat-removed porcine ear skin using PermeGear V6 station vertical Franz diffusion cell (n=4) was used to measure the rate and extent of vismodegib transport across the skin and drug retention in skin