3.3.1 Nanosuspensions
Fenofibrate formulations
The release of differently viscous fenofibrate nanosuspensions from micro-osmotic pumps was tested in 50 mL of water. Besides the horizontal position, the bottom-up or head-up position was investigated by fixing the pumps with sewing-cotton at the cover of the beaker glasses to achieve these positions within the release medium. Water was used as release medium instead of biorelevant 0.9% isotonic saline solution. Hereby a possible nanosuspension particle agglomeration in electrolyte solution was avoided, which could influence the formulation release behavior.
Compound A nanosuspension
In-vitro delivery of compound A nanosuspension was tested in 15 mL of isotonic saline solution in horizontal position to mimic in-vivo conditions regarding electrolyte presence.
[127I]iFF nanosuspensions
In-vitro release of [127I]iFF nanosuspensions was tested in 15 mL of MilliQ water in case of formulations A and B and in 15 mL of isotonic saline solution in case of formulation C and D, as the latter both were considered for in-vivo testing.
All vials with pumps were stored for at 40°C for7 days. Sampling was done after specific time points between 1 h and 168 h: 80% of media was exchanged starting with sample points after 9 h, whereas from 1 h to 9 h only 4 mL were withdrawn from release medium.
At the beginning of the release experiments lower sample volumes were taken, as due to an expected lag-time of formulation release only small amounts of substance were considered to be present in release medium. Replacing 80% of the medium was important to imitate the transport of the nanosuspension particles or dye molecules from the implantation site in-vivo. For the concentration of samples between 1 h to 9 h prior to HPLC/UV analysis, a rotational vacuum concentrator type RVC 2-33 IR by Martin Christ (Osterode am Harz, Germany) was utilized. Water was evaporated at a rotational speed of 1000 min-1 at 10 mPas pressure for 4 h. For the subsequent HPLC/UV analysis of samples, the dried solids were reconstituted with 1 mL of solvent. All release experiments were done in triplicate.
3.3.2 Methylene blue and fluorescein sodium formulations
Methylene blue solutions of various osmolalities were prepared by dissolving methylene blue in MilliQ water at 5 mg/mL and subsequently adding particular amounts of K2HPO4 to
3.3. CONDITIONS FOR IN-VITRO ALZET® PUMP RELEASE reach the required osmolality. The influence of osmolar differences between formulation and media solutions were investigated with values of ∆1200, ∆500, ∆300, ∆200 and
∆-162 mOsm/kg. “∆” is denoting the value of formulation osmolality minus osmolality of the release medium. A negative prefix depicts a higher osmolality in the environmental release medium in contrast to the solution formulation in pump. Fluorescein sodium solutions were prepared from 20.5 mg/mL up to 200 mg/mL in MilliQ water and osmolality was adjusted with the API concentration to the required level. Investigated osmolar differences between pump load and medium were equal to the methylene blue solutions.
For comparison purposes, a 5 mg/mL fluorescein sodium solution was prepared, thereby adjusting the osmolality with K2HPO4 to ∆1200 mOsm/kg. Release experiments with higher viscous solutions were done with methylene blue 5 mg/mL in water. For one set of pumps 10% of HPMC was added as viscosity increasing excipient; for the other one 1.1%
HEC was stirred into the dye solution for 2 hours; osmolality was adjusted by K2HPO4 to
∆0 mOsm/kg. Both polymers were utilized in release experiments, as they were able to create a similar solution viscosity in spite of the different concentrations, but develop a different formulation density. 15 mL of isotonic saline solution served as release medium.
Pumps filled with higher viscous methylene blue solutions were additionally tested regarding the pump position influence on delivery. For the impact of the surfactant DOSS on the release of formulation, a 5 mg/mL fluorescein sodium solution with 0.1% (w/v) DOSS and K2HPO4 to reach ∆0 mOsm/kg or ∆-250 mOsm/kg was utilized.
3.3.3 Concentration measurements via HPLC/UV
API and model compound concentrations in formulations and in release media after delivery from micro-osmotic pumps were determined by HPLC on a LaChrom Elite system and UV detection (VWR Hitachi, Darmstadt, Germany) in an eluent gradient mode. Eluent compositions, detector wavelengths and type of columns used for each compound are displayed in Tab. 3.3.1.
3.3.4 Mathematical examination of formulation release curves
Interpretation of in-vitro release graphs of nanosuspensions or dye solutions was done with the Origin® 6.1 software. R2- values of linear regression analysis of graphs plotted against the time were determined for each release graph to evaluate the linear model fit, as shown in equation (7). For calculation of R2 the method of least squares was used and data points of the mean values of three pumps served as calculation basis. Root-mean-square errors (RMSE) of single graphs were calculated to determine the differences between predicted values of linear regression and experimental values. Additionally, the R2 values of first order
Table 3.3.1: Summary of HPLC/UV analytical conditions for determination of API and dye concentra-tions. Dimensions of columns: XDB-C18 3.5 µm, 4.6 x 150 mm; EclipsePlus C18 and Zorbax SB-C8: 3.5 µm, 4.6 x 50 mm.
API/model
compound Column type Detection
wavelength [nm] Eluent composition
Captopril Agilent® XDB-C18 220
A: 95% ACN/5% water/0.01%
TFA, pH 2.9; B: ACN/water reversed
Compound A Agilent® EclipsePlus
C18 268
A: 95% ACN/5% water/0.1%
TFA, pH 1.9; B: ACN/water reversed
DPPH Agilent® EclipsePlus
C18 320
A: 95% ACN/5% water/0.01%
TFA, pH 2.9; B: ACN/water reversed
Fenofibrate/iFF Agilent® XDB-C18 288
A: 95% ACN/5% water/0.01%
TFA, pH 2.9; B: ACN/water reversed
Fluorescein sodium Agilent® Zorbax
SB-C8 220
A: 95% ACN/5% water/0.1%
TFA, pH 1.9; B: ACN/water reversed
Griseofulvin Agilent® XDB-C18 214
A: 95% ACN/5% water/0.01%
TFA, pH 2.9; B: ACN/water reversed
Methylene blue Agilent® Zorbax
SB-C8 292
A: 95% ACN/5% water/0.1%
TFA, pH 1.9; B: ACN/water reversed
kinetics model were determined for all osmotic pump delivery experiments having a linear R2 value below 0.95, which is displayed in equation (8).
Wt=k0·t (7)
Wt =W0·(1−e−k1·t) (8) whereWtis the amount of released API or model compound,W0 the amount of substance present in the osmotic pump at t=0, k0/k1 present the linear/first order proportionality constants and t the time. Reduced Chi2 values of the ratio of residual of sum squares and number of freedom degrees were calculated. For release graphs showing no appropriate linear fit against the time t, graphs were additionally plotted against the square root √
t and R2 and RMSE values were determined.
3.4. ELECTRON PARAMAGNETIC RESONANCE IMAGING OF DPPH