Discussion

4.1. IN-VITRO CHARACTERIZATION AND RELEASE OF VARIOUS FORMULATIONS FROM ALZET^® PUMPS the cell, the titration process was characterized by a phase of exothermic and subsequent endothermic heat signals for both types of surfactant (Fig. 4.1.5 e and f). Blackett and coworkers observed similar kinetics of evolved/absorbed heat signals after titration of oleic acid in salbutamol sulfate suspension in a non-polar liquid [175]. They hypothesized, that an exothermic surfactant adsorption within the first injections of oleic acid was overlapped by signals of the subsequent endothermic desagglomeration of particles. With increasing surfactant concentrations the heat outcome tended to a net endotherm one, which was also seen in the current case. However, the heat outcomes in the results were very small.

In addition, the repetition of the DOSS titration into griseofulvin suspension (Fig. 4.1.5 c) showed large differences in ∆H values between the first and second experiment, indicating that the observed differences in ∆H between beginning and at saturation level of the experiment were of lower relevance.

0,000,050,100,150,200,250,300,35 -0,6

-0,4 -0,2 0,0 0,2 0,4 0,6

a

DOSS --> compound A

µmol surfactant in cell

H[kcal/molofinjectant]

0,00 0,05 0,10 0,15 0,20

-0,6 -0,4 -0,2 0,0 0,2 0,4 0,6

b

NaDC --> compound A

µmol surfactant in cell

H[kcal/molofinjectant]

0,000,050,100,150,200,250,300,35 -0,6

-0,4 -0,2 0,0 0,2 0,4 0,6

c

DOSS --> griseof ulvin (1 st

experiment)

DOSS --> griseof ulvin (2 nd

experiment)

µmol surfactant in cell

H[kcal/molofinjectant]

0,00 0,05 0,10 0,15 0,20

-0,6 -0,4 -0,2 0,0 0,2 0,4 0,6

d

NaDC --> griseof ulvin

µmol surfactant in cell

H[kcal/molofinjectant]

0,00 0,05 0,10 0,15 0,20

-0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0

e NaDC --> griseof ulvin

Htot

[kcal/molinjectant]

µmol surfactant in cell

0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,35 0,40 -0,8

-0,4 0,0 0,4 0,8 1,2 1,6 2,0

f

DOSS --> griseof ulvin (1 st

experiment)

DOSS --> griseof ulvin (2 nd

experiment)

Htot

[kcal/molinjectant]

µmol surfactant in cell

Figure 4.1.5: Integrated heat signals of DOSS and NaDC titration into compound A and griseofulvin wet-milled microsuspension particles at 35°C (data are shown after subtraction of heat outcome of DOSS/NaDC titration into water)(a-d). DOSS interaction with griseofulvin was conducted twice under same conditions (c). Cumulative heat outcome after NaDC and DOSS titration onto griseofulvin particles (e+f).

4.1. IN-VITRO CHARACTERIZATION AND RELEASE OF VARIOUS FORMULATIONS FROM ALZET^® PUMPS However, the settling velocity of the particle front was about 300 µm in 22 h, which is quite a low value, but could be resolved by the multiple light scattering analysis. Therefore, this technique is a suitable method for the detection of destabilization phenomena at an early stage, by comparison of different nanosuspension formulations regarding sedimentation and agglomeration behavior. Disadvantages of this method are the need for large sample volumina (>12 mL) and the lacking possibility to accelerate gravity in contrast to e.g. the LUMiFuge^® analysis [181]. Within the investigation of stabilizer-API surface interactions on an thermodynamic level by means of ITC, the goal was to correlate nanosuspension formulation stabilities with thermodynamic interactions of drug surfaces with stabilizer molecules. Based on different adsorption enthalpy values obtained for various surfactant-drug substance combinations, evidence about the surfactant-API interaction should be given.

As the ITC method is a sensitive and accurate method for the calorimetric determination of the interaction between two reagents [182], this technique requires only low amounts of reagents in syringe and in the sample cell, which has an overall volume of 1.5 mL.

Previously, ITC was utilized for the determination of vesicle surfactant interactions [183], inclusion of guest molecules into cyclodextrins [184], micellization processes [185] and adsorption of molecules onto nano/microparticles. Dimova and coworkers determined adsorption enthalpies deriving from the interaction of different double hydrophilic block copolymers with calcite crystal particles of 10 µm in diameter. The adsorption process was depicted by exothermic signals at the beginning of the titration and followed by a plateau at a value of 0 kJ/mol injectant [186]. Similar heat kinetics curves could be observed with the titration of acylated insulin onto polystyrene beads with a hydrophobic surface [187] or with the titration of PEG onto cationic silica nanoparticles [174]. However, in the current study, the direct utilization of API nanoparticles without any stabilizer was not possible due to the strong agglomeration tendencies of griseofulvin and compound A nanosuspension particles. For this, micronized material was used as template. During the injection of surfactants into an API microsuspension, the isothermal titration calorimetry records the total heat which is evolved or absorbed after the contact of both reagents.

This heat includes besides dilution effects of stabilizers, demicellization and the desired adsorption process onto the microparticles. A possible demicellization can be excluded when using surfactants in concentrations below their CMC. The dilution effect was subtracted by conducting control experiments with surfactant titration into pure water. However, the last calculation will be slightly inexact, as the amount of stabilizer titrated into the suspension and adsorbed onto the particles is not longer available for the dilution process, which reduces the concentration of the surfactant in solution [175]. This particularly counts, when the heat outcome is very low. Within the conducted titration experiments of DOSS and NaDC into griseofulvin or compound A microsuspension, as obtained from the

manufacturer or after further size reduction by wet milling, determined ∆H values were indeed low and partially below the signal amplitudes of the control dilution processes. This could be caused by weak interactions of surfactant and stabilizer molecules paired with the low particle surface of microsuspensions. Lindman and coworkers investigated previously the amplitude of heat signals for human serum albumin adsorption onto propyl/butylacrylamide copolymeric nanoparticles with increasing diameters [188]. They found a decreasing of signal amplitudes between particles of 70 nm and 700 nm, which was based on the low total surface area available on the larger particles. In the current study, titration of DOSS and NaDC into griseofulvin suspension after further particle downsizing with wet milling and concentration of suspensions resulted in slightly higher signals, but with increased variabilities between repetitions, making a relevance of the observed effect questionable.

Moreover, a second weak, with heat connected process was overlapping with the adsorption kinetics: The disintegration of agglomerates during stabilizer titration, which was previously shown to be an endothermic process [175]. This highlights, that for the correct ITC analysis knowledge has to be present about all in the sample cell occurring thermal events.

Nevertheless, as the heat outcomes of the titration of NaDC and DOSS into griseofulvin microsuspension was similar in amplitude, no conclusion could be drawn regarding a more favorable adsorption of stabilizer. Within the use of compound A suspension as surface, no visible adsorption pattern was obtained. From the results it can be concluded, that a prediction of nanosuspension stability with regard to the thermodynamic interaction between utilized API and stabilizers is challenging. Moreover, the results indicate, that ITC has its limits in investigating the adsorption of surfactant molecules onto the analyzed API microparticle surfaces.

4.1.2 In-vitro release of nanosuspensions and solutions from