Materials and methods

2.2.1 Materials

The drug substance praziquantel was provided by PCAS, Limay, France. Eudragit EPO and L were supplied by Röhm GmbH, Pharma Polymers, Darmstadt, Germany.

Aluminium-monostearate, light liquid paraffin, phosphate buffer pH 6.8 and buffer pH 3 were purchased from Fluka AG, Buchs, Switzerland. Polysorbate 20 (Tween 20), sodium chloride and buffer pH 5 were obtained from Riedel-de Haën AG, Seelze, Ger-many. Solutol HS 15 was provided by BASF, Ludwigshafen, GerGer-many. Magnesium stearate was supplied by Faci Metalest, S.L., Zaragoza, Spain.

The solvents ethanol absolute, n-hexane, cyclohexane, hydrochloric acid, sodium hydroxide solution and acetone were used from Merck Inc. Darmstadt, Germany.

Demineralised water was used from the laboratory tap prepared in-house.

2.2.2 Coacervation

The ratio of drug to polymer for coacervation was set at 70:30 to ensure a thick enough coating around the drug particles for taste masking (Voigt, 2006). Due to the very poor wettability of praziquantel in water a detergent (Tween 20) was needed.

For coacervation by pH-shift 5 g Eudragit EPO were dissolved in 100 ml buffer pH 5 by sonication. 11.7 g unmicronized praziquantel and 0.1 g Tween 20 were added to the polymer solution and stirred with a paddle agitator at 500 rpm (Eurostar dig-ital, Ika-Werke GmbH, Staufen, Germany) until the suspension was homogeneous.

Afterwards 100 ml of 0.1 N sodium hydroxide solution were added with 1 drop/3 seconds. The product was filtered and dried at 60 ^◦C and 300 mbar for one hour (Salvis Trockenschrank, Typ KVTS11, Reussbühl, Switzerland).

Coacervation was further tried using a solvent/non-solvent system. 5.8 g prazi-quantel were suspended in 100 ml water using a detergent (0.1 g Tween 20). 50 ml of a 5% ethanolic Eudragit EPO-solution were added drop by drop while continuously stirring with a paddle agitator at 500 rpm. The product was filtered and dried un-der an extractor hood. For another process according to Okor (1990), Eudragit EPO (0.3 g) and praziquantel (0.6 g) were dissolved in 10 ml ethanol. Excess non-solvent containing a flocculating agent, 80 ml of 0.1 M sodium chloride solution in water, was added gradually (1 drop/3 seconds) with continuous stirring (600 rpm). The sticky polymer-drug precipitate was collected by filtration, washed with water to remove the salt and dried under an extractor hood.

For determination of the drug content in the products the coacervates were ground in a mortar and an appropriate amount was dissolved in 50.0 ml water containing 5% Tween 20. After sonication and agitation with a magnetic stirrer (IKAMAG^®RET S8, Ika-Werke GmbH, Staufen, Germany) until complete dissolution the solutions were filtered (0.45 µm filter, Millipore Millex-HV, Billerica, USA) and measured spectrophotometrically at 263 nm (Spectrophotometer Lambda 2, Perkin Elmer AG, Schwerzenbach, Switzerland).

2.2.3 Solvent evaporation

The drug/polymer ratio was set at 1:3 because the active ingredient is embedded in a polymer matrix and therefore more polymer is needed than for coating where only a thin polymer film is applied around the drug particles. Different formulations were used for the production of microspheres. The exact compositions are displayed in Tab. 2.1.

Component Microspheres A Microspheres B Microspheres C Microspheres D

Praziquantel 1.25 g/20.4% 1.25 g/20.4% 0.7 g/22.6% 0.7 g/22.2%

Al-stearate 1.13 g/18.4% 1.13 g/18.4% -

-Mg-stearate - - 0.3 g/9.7% 0.35 g/11.1%

Eudragit L 3.75 g/61.2% - -

-Eudragit E - 3.75 g/61.2% 2.1 g/67.7% 2.1 g/66.7%

Table 2.1:Compositions of Eudragit microspheres

Solvent evaporation was first tried with Eudragit L (microspheres A) because this polymer has a higher glass transition temperature than Eudragit EPO and is therefore easier to handle. Eudragit L (3.75 g) was dissolved in acetone (30.3 ml) with the addition of water (0.94 ml) whilst stirring with a magnetic stirrer and praziquantel (1.25 g) was added. Aluminum-monostearate (1.13 g) was dispersed in light liquid paraffin (125 ml). The solution of Eudragit L and praziquantel was then poured into the light liquid paraffin at 800 rpm and stirred at room temperature (25^◦C) for 24 h.

After sedimentation of the particles, they were washed three times with cyclohexane on a fluted filter and subsequently dried in a vacuum oven (200 mbar, 25^◦C) for 24 h.

Solvent evaporation using Eudragit EPO and praziquantel was first performed as described above but without the addition of water which is not necessary to dis-solve Eudragit EPO in acetone (microspheres B).

In order to improve this formulation the method was changed according to Bo-gataj et al. (1991). The main changes were the use of magnesium-stearate as dispersing agent, which is better suitable for the soft Eudragit E than for Eudragit L, and the tem-perature adjustment before and during the experiment. Briefly, Eudragit EPO (2.1 g) and praziquantel (0.7 g) were dissolved in 11 ml acetone and magnesium-stearate (0.3 g or 0.35 g; microspheres C and D, respectively) was added. After homoge-neously mixing, this dispersion was poured into light liquid paraffin (80 ml) which had been previously cooled to 4^◦C. During the experiment the paraffin was heated in a water bath to approximately 45 ^◦C and stirred with a three-blade stirrer (500 rpm, 4 h). Afterwards the product was filtered, washed with n-hexane and dried (200 mbar, 3 h).

Content measurements of the microspheres were performed by HPLC in ace-tonitrile. Solutions were prepared with an approximate drug concentration of 100 ppm, filtered (0.45 µm filter, Millipore Millex-HV, Billerica, USA) and determined by

HPLC according to the method described in the following chapter.

2.2.4 HPLC method

An HPLC-method was used for quantitative analysis of the active ingredient. For this purpose, an Agilent LC 1100 apparatus (Agilent Technologies, Basel, Switzerland) was employed. The column (250 mm length and 4.6 mm internal diameter) was packed with Nucleosil 5 µm, C18 (Macherey-Nagel, Düren, Germany) and maintained at ambient temperature. The elution medium consisted of a mixture of 0.05% phosphoric acid and acetonitrile and was kept constant. The flow rate was 1.1 ml/min during the whole analyze-run. A volume of 10 µl of the sample solution was injected per run with an auto-sampler. The samples were detected and analyzed with UV-light at 215 nm.

2.2.5 Particle size distribution

Particle size was determined by the polydisperse method using the Mastersizer X (Malvern Instruments Ltd., Malvern, UK) with a dry powder feeder unit, a range lens of 1000 mm and a beam length of 10 mm. Measurements were run in triplicate and results are reported as volumetric mean diameter D(4, 3).

2.2.6 Scanning electron microscopy

Scanning electron microscopy (SEM) was used to study the size, morphology and especially the surface of different Eudragit microspheres. Prior to examination, sam-ples were gold sputter-coated to render them electrically conductive. The SEM pho-tographs were recorded using a Philips XL 30 ESEM (Philips Electron Optics, Eind-hoven, The Netherlands). Different magnifications were applied to gain overall and detailed impressions.

2.2.7 Dissolution studies

Drug dissolution was performed using the paddle method according to USP 30 spec-ification. As dissolution apparatus a Sotax AT 7 (Sotax AG, Allschwil, Switzerland) was employed. Paddle speed was set at 100 rpm and temperature at 37 ± 0.5 ^◦C.

The medium for drug dissolution from the coacervates consisted of water containing 5%(w/w) Tween 20. The microspheres prepared by solvent evaporation were tested in two media: water with 1.5%(w/w) 1 N HCl containing 5%(w/w) Tween 20 and phosphate buffer pH 6.8 containing 5%(w/w) Tween 20 or Solutol HS 15. 900 ml medium was used and the particles were weighed to reach a maximum concentration of 100 ppm praziquantel. All tests were performed in triplicate. Samples were taken after 1, 3, 5, 10, 15, 30 and 60 min, filtered and the drug content was determined by UV spectroscopy or by HPLC according to the method described in Chapter 2.2.4.

2.2.8 Stability measurements

To detect any changes in the microspheres formulation during storage stability of the microspheres was tested at standard conditions: 25 ^◦C/60% relative humidity (RH), 30 ^◦C/65% RH and 40 ^◦C/75% RH. Samples were kept for three and six months and were then characterized by dissolution studies in phosphate buffer pH 6.8 with 5% Solutol HS 15 as described in the previous chapter; the only difference was that only 600 ml medium were used because there was not enough substance to reach a concentration of 100 ppm in 900 ml medium.