DEPOSITION OF NANOSUSPENSIONS OF ACTIVE PHARMACEUTICAL INGREDIENTS ON CARRIERS

Abstract

The present invention provides a method for preparing a pharmaceutical composition of a pharmaceutical ingredient (API) which is loaded on a carrier and stabilized therethrough. In particular, the present invention relates to a composition of a poorly soluble nanoparticulated API on a carrier in the dry state and which is processed as pharmaceutical formulation of said API with improved release profile and bioavailability.

Claims

1. A method for producing a pharmaceutical composition characterized by the following steps a) an active ingredient is brought into suspension in a solvent or a solvent mixture, b) the prepared suspension is milled at a temperature below 0° C. to a mean particle diameter of the active ingredient of less than 200 nm, c) the resulting suspension is mixed with a carrier material and d) the solvent is removed and the active ingredient is adsorbed on the carrier.

2. Method according to claim 1, characterized in that the active ingredient is a poorly soluble and/or low bioavailable ingredient of substance classes BCS class II or IV.

3. Method according to claim 1, characterized in that the active ingredient is selected from the group of acidic or basic agents.

4. Method according to claim 1, characterized in that in step a) the active ingredient is suspended in water.

5. Method according to claim 1, characterized in that in step a) the suspension is stabilized by the addition of at least one stabilizer selected from the group Hydroxypropylmethylcellulose (HPMC) and sodium dioctylsulfosuccinate (DOSS).

6. Method according to claim 1, characterized in that in step d) the solvent is removed by freeze drying.

7. Method according to claim 1, characterized in that in step c) the suspension is mixed with a silica gel as carrier material.

8. Method according to claim 1, characterized in that in step c) the suspension is mixed with a silica gel as carrier, having a specific surface area in the range of about 1 m.sup.2/g to about 600 m.sup.2/g (BET measurement) and an average pore size of about 2 to 600 nm.

9. Method according to claim 1, further comprising granulation, capsule filling or tableting.

10. Pharmaceutical formulation containing a pharmaceutical composition obtainable by a method according to claim 1.

11. Pharmaceutical formulation according to claim 10, wherein the pharmaceutical composition has an improved release profile.

12. Pharmaceutical formulation of claim 10, formulated in powders, capsules, granules, coated granules, tablets or coated tablets.

13. The method according to claim 1, wherein in b) the prepared suspension is milled at a temperature below 0° C. to a mean particle diameter of the active ingredient in a range from 60 to 160 nm,

14. The method according to claim 1, wherein in step c) the suspension is mixed with a silica gel as carrier, having a specific surface area in the range of about 3 m.sup.2/g to about 500 m.sup.2/g (BET measurement) and an average pore size of about 6 to 500 nm.

Description

LIST OF FIGURES

[0064] FIG. 1: shows schematically a possible procedure and an assembly of devices for performing a wet bead nano-milling process.

[0065] FIG. 2: shows the DSC curve of FF_29062016_SLC_500_001, plotted next to the DSC curve of pure fenofibrate. The endothermic melting peak of pure, crystalline fenofibrate is clearly visible at about 80° C.

[0066] FIG. 3: shows the Comparison of the API releases (loaded silica carriers+fenofibrate), 50 mg API, 1000 mL SGFsp+0.1% SDS, 75 rpm, Mean value [mg/L]+standard deviation [mg/L]

[0067] FIG. 4: Results of comparison of formulation achieved using Fenofibrate nanosuspension versus Fenofibrate (crystalline)

[0068] FIG. 5: Comparison of results achieved by loading amorphous API in presence of organic solvents (preparation as described before) versus loading by nanosuspension (still crystalline API)

[0069] FIG. 6: Release data of the nano-milled particles of loaded Kieselgel SI 5000 batch (FF_29062016_SI_5000_001) are compared with data of loaded nano-milled suspension of third batch of the Parteck® SLC 500 (FF_29062016_SLC_500_003).

[0070] FIG. 7: The dissolution of the nano-milled drug without stabilizer applied to a carrier whereby Parteck® SLC 500 and Kieselgel SI 5000 showed a very similar release property with or without stabilizer

[0071] FIG. 8: shows the DSC curve of crystalline itraconazole, along with the curves of the nanosuspension-loaded batches of Parteck® SLC 500 and Kieselgel SI 5000

[0072] FIG. 9: shows the results of the batch, ICZ_16092016_SLC_2, which releases with a maximum concentration of approx. 3 mg/L and substantial faster than itraconazole crystalline sample compared.

[0073] In the following the present invention is shown by different experiments and examples. The results of these experiments are explained in detail, discussed and evaluated. These additional embodiments illustrate the general applicability of the principle of the invention and, therefore, as well as the examples, are included in the disclosure of the present invention.

Examples

[0074] The present description enables the person skilled in the art to apply the invention comprehensively. Even without further comments, it is assumed that a person skilled in the art will be able to utilize the above description in the broadest scope.

[0075] Practitioners will be able, with routine laboratory work, using the teachings herein, to prepare active ingredients comprising formulations as defined above in the new process.

[0076] The invention described may be further illustrated by the following examples, which are for illustrative purposes only and should not be construed as limiting the scope of the invention in anyway.

[0077] If anything is still unclear, it is understood that the publications and patent literature cited should be consulted. Accordingly, these documents are regarded as part of the disclosure content of the present description.

[0078] For better understanding and in order to illustrate the invention, examples are given below which are within the scope of protection of the present invention. These examples also serve to illustrate possible variants. Owing to the general validity of the inventive principle described, however, the examples are not suitable for reducing the scope of protection of the present application to these alone.

[0079] Furthermore, it goes without saying to the person skilled in the art that, both in the examples given and also in the remainder of the description, the component amounts present in the compositions always only add up to 100% by weight, volume or mol-%, based on the composition as a whole, and cannot exceed this, even if higher values could arise from the percent ranges indicated. Unless indicated otherwise, % data are % by weight, volume or mol-%, with the exception of ratios.

[0080] The temperatures given in the examples and the description as well as in the claims are always in ° C.

[0081] Methods (See in the Following Text):

TABLE-US-00002 Loss on drying Differential Scanning Calorimetry Release Determination of content by HPLC Determination of content by H.sub.1-NMR Light micrographs (only for FF_2906_SLC_500_001)

[0082] Drying Loss (IR Balance) [0083] Device: Mettler PM 400; Mettler LP16 (Mettler Toledo GmbH, Gießen, Germany) [0084] Weighing: 0.3 g (Minimum) [0085] Temperature: 105° C. [0086] Method: 0-100% [0087] Constance: 1 Digit/10 s [0088] Aluminum dish: ME-13865 [0089] No. of determinations: 3

[0090] The loss on drying should ideally be below 1% for release. If the drying loss is higher, it may be necessary to dry again.

[0091] Light Micrographs [0092] Device: Light Microscope Zeiss Stemi 2000-C (Carl Zeiss AG, Oberkochen, Germany)  Camera Power Shot A640 (Canon Germany GmbH, Krefeld, Germany  Cold light lamp CL1500 ECO (Carl Zeiss AG, Oberkochen, Germany) [0093] Measuring Software: Axio Vision Rel. 4.8 (Carl Zeiss AG, Oberkochen, Germany) [0094] Slides: 76×26×1 mm; ISO 8037/1; edges 90° ground, pre-cleaned, without mat edge  (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany)

[0095] The substance to be measured is evenly distributed on the slide and the lighting conditions and sharpness adjusted until the desired display is achieved.

[0096] Differential Scanning Calorimetry [0097] Device: Mettler Toledo DSC 3+ (Mettler Toledo, Gießen, Germany),  STARe-Excellence-Software (Mettler Toledo, Gießen, Germany) [0098] Weighed quantity: 2-4 mg for 40 μL aluminum crucible  30-40 mg for 100 μL aluminum crucible [0099] Atmosphere: 50.0 mL/min N.sub.2 [0100] Temperature range: 25-350° C. [0101] Heating rate: see in the following [0102] No. of determinations: at least 2

[0103] Type of Heating:

[0104] Program 1: (“40-100/5K”)

[0105] Continuous heating of the sample from 30° C. to 120° C. with a heating rate of 5 K/min.

[0106] Program 2: (“40-100/30K”)

[0107] Continuous heating of the sample from 30° C. to 120° C. with a heating rate of 30 K/min.

[0108] Program 3: (“40-60/10K_60iso_5min_60-90/2K_Alu40_N2”)

[0109] Continuous heating of the sample from 40° C. to 90° C. with a heating rate of 2 K/min including temperature maintenance phase of 5 min at 60° C.

[0110] Program 4: (“25-100/5K_(Alu100_N-2)”)

[0111] Continuous heating of the sample from 25° C. to 100° C. with a heating rate of 5 K/min.

[0112] Program 5: (“25-85/5K_85_5min_85-50/5K_(Alu100_N-2)”)

[0113] Continuous heating of the sample from 25° C. to 85° C. with a heating rate of 5 K/min, keeping the temperature for 5 min, cooling the sample from 85° C. to 50° C. with a cooling rate of 5 K/min.

[0114] Release of Active Ingredient (Sotax 1 and 2) [0115] Device: Sotax 1 and 2,  Release apparatus: Sotax AT 7smart (Sotax AG, Lörrach, Germany),  Photometer Agilent 8453 (Agilent Technologies, Waldbronn, Germany) [0116] Number of vessels: 3 or 6 [0117] Method: Paddle [0118] Medium: SGFsp+0.1% sodium dodecyl sulfate [0119] Amount of medium: 1000 mL [0120] Temperature of medium: 37° C. [0121] Rotation: 75 rpm [0122] Duration: 2 h [0123] Time of sampling: 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105, 120 min [0124] Final spin: no [0125] Cuvette layer thickness: 5 mm [0126] Wavelength: 288 nm [0127] Dose of active ingredient: 50 mg [0128] Drug loading: about 16% [0129] Filter removal station: GF/D 2.7 μm [0130] Sample volume: 2.5 mL

[0131] Each sample is collected in a test tube with the automatic sampler. The samples are then measured offline by HPLC determination (see Method Determination of content by HPLC).

[0132] Determination of Content by HPLC [0133] Device: HPLC-system LaChrom® Elite (Hitachi Europe GmbH, Düsseldorf, Germany) [0134] Detector: UV Detector L-2400 VWR Hitachi (Hitachi Europe GmbH, Düsseldorf, Germany) [0135] Autosampler: Autosampler L-2200 VWR Hitachi (Hitachi Europe GmbH, Düsseldorf, Germany) [0136] Column: LiChroCART® 125-4, LiChrospher® 100 RP-18e (5 μm) [0137] Eluent: Acetonitrile/Milli-Q water/trifluoroacetic acid (700:300:1) [0138] Wash solution sampler: Acetonitrile/Milli-Q-Wasser (1:1) [0139] Column oven—temperature: 50° C. [0140] Injection volume: 25 μL [0141] Wave length—detector: 288 nm [0142] Flow rate: 2.0 mL/min (isocratic) [0143] Duration—run: 5 min [0144] Sequence: XXXXXXXX_01_PK_Fenofibrate_Disso_Nanosuspensionen 1. Seq [0145] Method: XXXXXXXX_01_PK_Fenofibrate_Method [0146] Filter—sample preparation: Whatman™ Anotop™ 10, 0.02 μm, Cat.-No.: 6809-1002

[0147] Prior to filling into vials, each sample from the release is first filtered with a syringe with Luer-Lock connection and above filters for sample preparation to retain any particles of the nanosuspension and eliminate a systematic error. Such a systematic error can be found in majority of scientific papers and patents as most evaluation do not carefully remove still nano-milled particle from the samples by using appropriate filters. Only soluble API content should be detected.

[0148] For evaluation of the HPLC results, the saturation concentration is determined by fixed lab-method and the release of crystalline fenofibrate from a lab test done before (online determination) is taken.

[0149] Determination of Content by H.sub.1-NMR [0150] Method: 1H-NMR spectroscopy [0151] Condition: DMSO-d6 [0152] Measurement mode: content [%]

[0153] The measurement is made by an external analysis order. For this purpose, a sample tube is filled up to half and sent to the appropriate place. The result is given in % content.

[0154] Particle Size Determination (Zetasizer Nano ZS) [0155] Device: Zetasizer Nano ZS (Malvern Instruments Ltd, Herrenberg, Germany) [0156] Amount: few drops (diluted, non-turbid solution) [0157] Dispersing medium: desalinated water (viscosity: 0.8872 cP) [0158] Measuring range: 0.3 nm-10 μm [0159] Measuring time some minutes [0160] Temperature: 25° C. [0161] Equilibration time: 60 s [0162] Number of measurements: 6×12 measurements [0163] Method of measurement: Size measurement (Number) [0164] Measurement angle: 173° Backscatter (NIBS default) [0165] Active ingredient: Fenofibrate (RI: 1,547*; Absorption: 0.01) [0166] Data processing: general purpose [0167] Type of cuvettes: DTS0012—Disposable sizing cuvette [0168] Cuvette: 10×10×45 mm Polystyrol/Polystyrene (REF: 67.754; Sarstedt AG & Co, Numbrecht, Germany) [0169] Evaluation: Formation of an average of at least 3 determinations [0170] (*source: http://www.lookchem.com/Fenofibrate/)

[0171] The sample is filled into a cuvette (preferably 40 μL cuvette) up to the mark of the Zetasizer and measured. If the results are not “good” (see “Expert Advise”), repeat the measurement with a more dilute sample. The sample should be slightly cloudy at most, in order to exploit the optimal working range of the Zetasizer.

[0172] General Information “Nano-Milling”

[0173] Wet-Milling:

[0174] The milling is to be carried out with the Dyno®-Mill Research Lab (Willy A. Bachofen Maschinenfabrik, Muttenz, Switzerland) [0175] Filling volume: 60-200 mL [0176] Grinding balls: SiLi ZYP 0.2-0.3 mm [0177] Weighing of grinding balls: 200 g [0178] Stirring speed: 2000-4000 rpm [0179] Temperature of cooling liquid: −10° C.

[0180] Loading of Two Carrier Parteck® SLC 500/Kieselgel SI 5000 with Different Pore Sizes [0181] Devices: Heidolph RZR 2102 control Laboratory stirrer (Heidolph Instruments, Schwalbach, Germany) Head stirrer with stirring blade [0182] Weighing (silica): approx. 10 g [0183] Weighing (suspension): approx. 10 g (and approx. 20 g) [0184] Stirring speed: 70 rpm [0185] Type of cannula: 0.80×120 mm BL/LB [0186] Dosing speed: approx. 2 g/min

[0187] Parteck® SLC 500 or Kieselgel SI 5000 is loaded by uniform application of the nanosuspension using a 10 mL syringe with Luer-Lock cap and cannula. The carrier material is in a beaker in which the stirrer fits straight into it. During application, stirring is continued with the stirrer. If the mixing of the carrier material is not complete, the height and immersion depth of the stirrer can be changed manually (for example, by lifting/lowering of the beaker).

[0188] Other Devices: [0189] Magnetic stirrer: IKA®-Werke GmbH & Co. KG, Staufen, Germany

TABLE-US-00003 TABLE 1 Materials: Material Origin Fenofibrate BEC Chemicals Provate Ltd., Ind. Hypromellose (HPMC) (Pharmacoat Shin Etsu Chemical Co., Ltd. 603) Dioctylsulfosuccinate-Natrium = Aldrich Chemistry DOSS Parteck ® SLC 500 Merck KGaA, Darmstadt Kieselgel SI 5000 Merck KGaA, Darmstadt (synthesis) Milli-Q-Wasser Merck KGaA, Darmstadt

[0190] The Parteck® SLC 500 is a silica gel with a specific surface area of 500 m.sup.2/g (BET measurement) and an average pore size of 6 nm.

[0191] The Kieselgel SI 5000 comes from a silica synthesis of Merck KGaA by using addition and melting of NaCl to change pore size of the carrier. It has a specific surface area of 3 m.sup.2/g (BET measurement) and has an average pore size of 500 nm.

[0192] To stabilize the nanosuspension, HPMC and DOSS are added to the suspension medium. Without these stabilizing agents, the fenofibrate nanosuspension produced might be prone to rapid formation of aggregates and build-up of larger particles due to greatly increased surface effects, such as electrostatic attraction and dissolution rate (Ostwald ripening). [0193] SGFsp: simulated gastric fluid sine pepsin [0194] SDS: sodium dodecyl sulfate

[0195] Reasons for the Experiments:

[0196] The aim of the following experiments is to find out whether an improvement in the release of the sparingly soluble active ingredient is achieved by a impregnation loading method when the active ingredient is applied in the form of a nanosuspension where the API is suspended as nano-particles but still in crystallin state. The carrier used for this purpose is Parteck® SLC 500 and fenofibrate as the active ingredient.

[0197] In addition, Kieselgel SI 5000 is used as support material and loaded with the crystalline fenofibrate nanosuspension. Here, the influence of the pore size on the release of fenofibrate is investigated.

[0198] Most important during HPLC analyzes is the appropriate filtration of samples. Prior to filling into vials, each sample from the release is first filtered with a syringe with Luer-Lock connection and above filters for sample preparation to retain any particles of the nanosuspension and eliminate a systematic error. Such a systematic error can be found in majority of scientific papers and patents as most evaluation do not carefully remove still nano-milled particle from the samples by using appropriate filters. Only soluble API content should be detected.

[0199] Carrying Out the Experiments:

[0200] At the beginning of the experiments, a fenofibrate suspension (see Method of Experiment 1 A) is prepared which is stabilized with HPMC and DOSS (dioctylsulfosuccinate sodium) and then nanomilled (see Methods Nano-milling in the following). The nanosuspension obtained is stored in the refrigerator at temperatures between 2 and 8° C.

[0201] Using the impregnation method (as described in the following examples), the carriers Parteck® SLC 500 and Kieselgel SI 5000 are loaded in a ratio of 1:1 or 2:1 (w/w) with the fenofibrate nanosuspension finding out best loading ratio but to see if higher loading is possible as well without impact of loading amount. The drying is then carried out by freeze-drying (see Method “Nano-milling” in the following). Since Parteck® SLC 500 has hygroscopic properties, all batches produced are stored in the desiccator over orange gel.

TABLE-US-00004 TABLE 2 Overview of the experiments for dissolution measurement and comparative experiments Active Experiment Carrier ingredient Comments FF_2906 — Fenofibrate Fenofibrate- Nanosuspension FF_2906_SLC_500_001 Parteck ® Fenofibrate SLC 500 FF_2906_SLC_500_002 Parteck ® Fenofibrate SLC 500 FF_2906_SLC_500_003 Parteck ® Fenofibrate SLC 500 FF_2906_SI_5000_001 Kieselgel Fenofibrate SI 5000

[0202] Method

[0203] Experiment 1

[0204] A) Preparation of the Suspension

[0205] 5 g of HPMC and 0.2 g of DOSS are placed in a beaker and are dissolved in 154.88 g of deionized water by stirring with a magnetic stirrer for about 40 minutes. When the substances are dissolved, 80.0 g of the received solution are placed in a beaker and 20 g of fenofibrate are added and suspended in the solution by stirring (450 rpm) for 10 minutes. [0206] Theoretical fenofibrate content: 20% (w/w)

[0207] A′) Determination of the Saturation Concentration (for Analytical Measurement)

[0208] For the preparation of the saturated solution, the magnetic stirrer of IKA®—Werke GmbH & Co. KG, Staufen, Germany, is also used here.

[0209] For the preparation of the saturated fenofibrate solution, which is needed to determine the saturation concentration, 1 tablespoon of fenofibrate is suspended in 200 ml of solution (SGFsp+1% SDS). This suspension is heated to a temperature of 40° C. and stirred at 300 rpm for at least 24 hours, here for 72 hours. The beaker is covered with Parafilm during this time. Subsequently, the saturation concentration is measured by HPLC determination.

[0210] Method

[0211] Nano-Milling

[0212] The suspension (Experiment 1 A) is filled into the hopper of the mill and the milling process is started at 2000 rpm. The particle size is checked every 5 minutes (see some results of every 15 minutes/Table 3) via Dynamic Light Scattering. If necessary, the stirring speed can be increased to 3000 or 4000 rpm. Overall, the milling process should not exceed a time of 2 hours.

TABLE-US-00005 TABLE 3 Particle size of nano-milled fenofibrate as function on milling time Time d(10) d(50) d(90) Median [min] [μm] [μm] [μm] [μm] 0 27.2 101.5 267.1 94.9 15 0.088 0.150 1.029 0.158 30 0.079 0.131 0.206 0.129 45 0.077 0.131 0.198 0.125 60 0.076 0.131 0.189 0.120

[0213] B) Applying the Active Ingredient on to the Silica Carrier [0214] a) approx. 10 g of suspension are applied to 10 g of Parteck® SLC500 with stirring, resulting in a loading of about 16.4%. [0215] b) approx. 20 g of suspension are applied to 10 g of Parteck® SLC500 with stirring, resulting in a loading of about 27.5%.

[0216] C) Freeze-Drying of Resulting Loaded Carriers [0217] Device: Freeze Dryer Gamma 2-16 LSC (Christ Gefriertrocknungsanlagen Martin Christ, Osterode, Germany) [0218] Cooling: water cooling

[0219] The loaded, moist products from a) and b) are freeze-dried under the following conditions in a beaker:

TABLE-US-00006 TABLE 4 a) Program 1: “Nano-milling” Temperature Time [° C.] [h] Freezing −45° C. to −35° C. ~6 First drying −35° C. ~25 Second drying 0° C. 1 +25° C. 18 Total 50

TABLE-US-00007 TABLE 5 b) Program 2: “Nanosus PK” Temperature Time [° C.] [h] Freezing −45° C. 8 Main drying −35° C. 30 Second drying 0° C. 4 +25° C. 18 Total 60

[0220] The samples are placed into the freeze dryer for freeze drying and the freeze dryer is closed.

[0221] Water is used for cooling (first the drain is turned on, only then the inlet!). Then the desired program is started. After freeze-drying, the drying loss of the product should be determined as described. If drying is insufficient, further drying is carried out

[0222] D) Analysis of the Product Obtained [0223] a) DSC/XRD studies: check of the physical state of the API [0224] b) HPLC/NMR studies: determination of the drug content on the silica [0225] c) release->offline, samples over 0.2 μm PTFE filter (comparison with nanosuspension before adding silica) [0226] d) stability study

TABLE-US-00008 TABLE 6 The batches are produced with the following amounts: Amount of Theor. Amount of suspension content Batch no. Silica [g] [g] [%] FF_29062016_SLC_500_001 10.00 10.00 16.6 FF_29062016_SLC_500_001_a 10.00 20.00 28.6 FF_29062016_SLC_500_002 10.06 9.84 15.7 FF_29062016_SLC_500_002_a 10.00 20.00 28.6 FF_29062016_SLC_500_003 10.00 9.93 16.6 FF_29062016_SLC_500_003_a 10.00 20.00 28.6 FF_29062016_SI_5000_001 9.99 9.87 16.5 FF_29062016_SI_5000_001_a 10.00 20.00 28.6

[0227] Experiment 2

[0228] (Nano-Milling without Viscosity Enhancer) [0229] A) Preparation of the suspension

[0230] 0.1 g of DOSS is dissolved in 79.9 mL of deionized water. When the substance is dissolved, 20 grams of fenofibrate are suspended in the solution. The suspension is filled into the hopper of the mill and the milling process is started at 2000 rpm. The particle size is checked every 5 minutes via Dynamic Light Scattering. If necessary, the stirring speed can be increased to 3000 or 4000 rpm. Overall, the milling process should not exceed a time of 2 hours. [0231] B) Applying the active ingredient to the silica carrier [0232] a) approx. 10 g of suspension are applied to 10 g of Parteck® SLC500 with stirring, resulting in a loading of about 16.6%. [0233] b) approx. 20 g of suspension are applied to 10 g of Parteck® SLC500 with stirring, resulting in a loading of about 28.6%. [0234] C) Freeze-drying of resulting loaded carriers [0235] The loaded, moist products from a) and b) are freeze-dried under the same conditions as in Experiment 1. [0236] D) The analytical evaluation of the products obtained is carried out in the same manner as in Example 1.

[0237] Experiment 3 [0238] (without viscosity enhancer and without stabilizer) [0239] A) Preparation of the suspension [0240] 20 g of fenofibrate are suspended in 80 mL of deionized water. The suspension is filled into the hopper of the mill and the milling process is started at 2000 rpm. The particle size is checked every 5 minutes via Dynamic Light Scattering. If necessary, the stirring speed can be increased to 3000 or 4000 rpm. Overall, the milling process should not exceed a time of 2 hours. [0241] B) Applying the active ingredient to the silica carrier [0242] a) approx. 10 g of suspension are applied to 10 g of Parteck® SLC500 with stirring, resulting in a loading of about 16.6%. [0243] b) approx. 20 g of suspension are applied to 10 g of Parteck® SLC500 with stirring, resulting in a loading of about 28.6%. [0244] C) Freeze-drying of resulting loaded carriers [0245] The loaded, moist products from a) and b) are freeze-dried under the same conditions as in Experiment 1. [0246] D) The analytical evaluation of the products obtained is carried out in the same manner as in Example 1.

[0247] Evaluation of the Experiments:

[0248] Assessment and comparison of the results obtained

[0249] FF_29062016_SLC_500_001:

[0250] This batch is dried according to Program 1 “Nano-milling” as described above. Since there was a disruption during the drying over the weekend, the program was canceled after 66 hours in the main drying.

[0251] The drying loss is on average at about −0.99%.

[0252] FF_29062016_SLC_500_002/FF_29062016_SI_5000_001:

[0253] These batches have been dried together in Program 2 “Nanosus_PK”.

[0254] The drying loss after freeze-drying is on average for FF_29062016_SLC_500_002 [0255] −3%

[0256] and for

[0257] FF_29062016_SI_5000_001 −0.13%.

[0258] FF_29062016_SLC_500_003:

[0259] This batch has been dried in Program 1 “Nano-milling”. Since the drying loss (n=1) according to the program is −12.98%, the batch is dried once more in Program 2 “Nanosus_PK”.

[0260] The drying loss after drying is −1.01%.

[0261] Results: Optical Assessment

[0262] FF_29062016_SLC_500_001

[0263] After loading of Parteck® SLC 500 with the fenofibrate nanosuspension, the support material is slightly clumped. After freeze-drying, these lumps remain. But they are easy to crush with a spatula. An influencing factor in this context may be the metering rate during loading. Since by manual dosing, fluctuations in the dosing rate can occur here. The remainder is loose powder which is of fine consistency.

[0264] FF_29062016_SLC_500_002

[0265] Also, in the second batch the loading of Parteck® SLC 500 with fenofibrate nanosuspension leads to the formation of smaller lumps. However, they are smaller in relation to those of the first batch. These lumps also can be easily crushed with a spatula.

[0266] FF_29062016_SLC_500_003

[0267] As well as in the other batches of the loading of Parteck® SLC 500, some clumps are formed after the impregnation, which can easily be crushed with a spatula. Under the light microscope, all nano-milled loaded Parteck® SLC 500 particles are only recognizable as blurred structures. The applied nanosuspension is neither recognizable nor visible fenofibrate crystals have been formed prove successful loading of nano-milled particles distributed on carrier.

[0268] FF_29062016_SI_5000_001

[0269] Comparable as during loading of Parteck® SLC 500, the loading of the Kieselgel SI 5000 produces some lumps which may have different sizes. But in comparison to loaded Parteck® SLC 500, here the remaining loose powder is floury-like_

[0270] FF_29062016_SLC_500_001_a and Further Experiments (=20 g Loadings)

[0271] The optical assessment of FF_29062016_SLC_500_001_a and the further samples loaded with 20 g nano-milled suspension for comparison reasons, results in similar powder properties as 10 g loadings. Materials are partly clumped together, but samples were easy to transfer to flowable powder important for further processes as tableting. Based on the optical assessment we follow up with the analytical evaluation of the materials loaded with 10 g suspension only.

[0272] Particle Size Distribution of the Nanosuspension

[0273] As described above, the particle size distribution is measured with: Zetasizer Nano SZ. Samples are stored for comparison and the stability of the suspension after nano-milling is examined (Table 7).

TABLE-US-00009 TABLE 7 Process 2 weeks 10 weeks (t.sub.0) (t.sub.1) (t.sub.2) Mean [nm] 126.94 128.35 142.20 Std. Dev. [nm] 15.70 16.10 40.71 d(10) [nm] 105.70 106.40 56.20 d(50) [nm] 126.00 127.50 129.00 d(90) [nm] 147.80 149.00 236.00

[0274] Due to the greatly increased specific surface of the particles of the nanosuspension, the solution processes may accelerate in this suspension, so that the fenofibrate dissolves faster. This can lead to the growth of larger particles, while smaller particles completely dissolve. This effect is called Ostwald ripening.

[0275] But in this experiment, the effect is small. This means the growth of suspension crystals is slow and could be seen after 10 weeks storage. Within 2 weeks they grow on average within acceptable range to use the nanosuspension over a longer period. So, the nanosuspension is sufficiently well stabilized by the use of HPMC and DOSS.

[0276] Determination of Content by H.sub.1-NMR

TABLE-US-00010 TABLE 8 The H.sub.1-NMR-API content evaluation showed following values: Theoretical Measured content content batch [%] [%] FF_29062016_SLC_500_001 16.6 13.6 FF_29062016_SLC_500_002 15.7 13.5 FF_29062016_SLC_500_003 16.6 13.0 FF_29062016_SI_5000_001 16.5 15.0

[0277] The actual measured content is up to 3.5% below the theoretical content. There may be many reasons for this deviation: for example, already during nano-milling a decrease in fenofibrate content may occur when the suspension is transferred to the nanomill. When transfer is done always a small remainder suspension stays in the transport vessel. It is possible that, despite shaking, a certain amount of fenofibrate crystals has settled there, which remain in the vessel during pouring. Since all batches have a reduced content, it is probably a systematic error.

[0278] Differential Scanning Calorimetry (Evaluation of API Morphology)

[0279] (DSC Measurements)

TABLE-US-00011 TABLE 9 Evaluation if amorphous/crystalline morphology: amorphous/ batch carrier crystalline FF_29062016_SLC_500_001 Parteck ® SLC 500 crystalline FF_29062016_SLC_500_002 Parteck ® SLC 500 crystalline FF_29062016_SLC_500_003 Parteck ® SLC 500 crystalline FF_29062016_SI_5000_001 Kieselgel SI 5000 crystalline

[0280] The referring results are to be found under the corresponding sub-items for the DSC measurement.

[0281] Identification of the Melting Peaks of Fenofibrate

[0282] FIG. 2 shows the DSC curve of FF_29062016_SLC_500_001, plotted next to the DSC curve of pure fenofibrate. The endothermic melting peak of pure, crystalline fenofibrate is clearly visible at about 80° C. It can be seen that the melting peak of the fenofibrate nanosuspension on Parteck® SLC 500 is significantly less pronounced and shifted to lower temperature. While the fenofibrate peak of pure fenofibrate sets in sharply, the charged Parteck® SLC 500 is more likely to have only a “dent” in the curve. It is still crystalline on the Parteck® SLC 500.

[0283] The DSC curve of FF_29062016_SLC_500_001 also shows a slight melting point depression of the fenofibrate. Since there is not pure fenofibrate in the sample, both the hydroxypropylmethyl cellulose used and also the DOSS can lower the melting point.

[0284] In addition, a “masking” of the heat transfer by the Parteck® SLC 500 could take place, so that a defined, clear melting peak is concealed.

[0285] Dissolution Measurements: Batches of Loaded Parteck® SLC 500

[0286] FIG. 3 shows the Comparison of the API releases (loaded silica carriers+fenofibrate), 50 mg API, 1000 mL SGFsp+0.1% SDS, 75 rpm, Mean value [mg/L]+standard deviation [mg/L].

[0287] In the release study, the Parteck® SLC 500 batches 1 and 3 show that the fenofibrate nanosuspensions release the active substance comparably well. Both achieve the saturation concentration of approx. 15 mg/L after only 5 minutes, which is significantly faster as it is by dissolving pure crystalline fenofibrate. Crystalline fenofibrate reaches the saturation concentration after 60 minutes. Overall, the saturation concentration is only slightly exceeded with the nano-suspension of Parteck® SLC 500, but also with the crystalline active ingredient. As expected, (as the morphology of API was measured to be still crystalline and not amorphous) there is no improvement in the solubility by nanoparticulate API loaded Parteck® SLC 500 compared to fenofibrate not loaded on a carrier. Very much favorable in comparison between pure API and nanosuspension loaded on carrier is that the loaded Parteck® SLC 500 batches have a greatly increased dissolution rate in the beginning.

[0288] Comparison of Results Achieved Using Fenofibrate Nanosuspension Versus Fenofibrate (Crystalline) is Shown in FIG. 4.

[0289] Comparison of Results Achieved by Loading Amorphous API in Presence of Organic Solvents (Preparation as Described Before) Versus Loading by Nanosuspension (Still Crystalline API) (FIG. 5)

[0290] In contrast to the Parteck® SLC 500, which is loaded with a nanosuspension (and API is due to physical milling still crystalline), the release of organically loaded Parteck® SLC 500 (API is loaded in amorphous morphology) shows a significantly higher, initial increase in concentration (so-called supersaturation). This reaches its maximum after 15 minutes at about 47 mg/L. This is followed by a decrease in concentration with asymptotic approximation to 25 mg/L after 90 minutes.

[0291] Compared to the organic loading, the two samples nanosuspension loaded Parteck® SLC 500 reaches its maximum concentration in 5 minutes after release. After reaching the maximum, the concentration remains constant, in contrast to organic loading; this maximum is slightly above the saturation concentration of the fenofibrate.

[0292] The maximum concentration of the organically loaded Parteck® SLC 500 was 47 mg/L; the highest released concentration in nanoparticulate loaded Parteck® SLC 500 was only 25 mg/L. Positive in this context, however, is the lack of recrystallization with a decrease in the concentration of nano-milled fenofibrate loaded, Parteck® SLC 500.

[0293] Systematic error can be found in majority of scientific papers and patents as most evaluation do not carefully remove during analytical evaluation still nano-milled particle from the samples by using appropriate filters. Only soluble API content should be detected otherwise API concentration detected above solubility of API in crystalline state and wrong conclusions are based on often.

[0294] In FIG. 6 the releases of the nano-milled particle loaded Kieselgel SI 5000 batch (FF_29062016_SI_5000_001) are compared with the same amount of nano-milled suspension loaded third batch of the Parteck® SLC 500 (FF_29062016_SLC_500_003).

[0295] Conclusions from these Comparative Measurements

[0296] The aim of the experiment was to verify the release of the model drug fenofibrate by nanomilling and subsequent loading of the suspension onto Parteck® SLC 500 (app. 6 nm pore diameter measured) and compare it's dissolution properties versus with the same procedure loaded Kieselgel SI 5000 carrier, with pore diameter in the range of 500 nm.

[0297] It was found by this experiment, that the use of a fenofibrate nanosuspension which is applied onto a Kieselgel SI 5000 support, has a significantly faster dissolution rate than crystalline, micronized fenofibrate. A supersaturation or faster dissolution is not observed in comparison to loaded Parteck® SLC 500 even a little smaller total dissolution could be measure using the Kieselgel SI 5000 loaded sample.

[0298] The stabilization and release of the API seems to result from the surface and pore nature of the Parteck® SLC 500 as well as Kieselgel SI 5000 and not from the pore diameter.

[0299] Further experiments and measurements must confirm these results, for example with itraconazole.

[0300] Further experiments without Addition of Stabilizers

[0301] In previous studies HPMC and DOSS have been used as a stabilizer and viscosity enhancer for nanosuspension production. In order to test if favorable material of nano-milled loaded carrier, without additional stabilizer is possible to prepare in order to be able to use such not so complex materials in final administration forms, the nanosuspension is loaded onto Parteck® SLC 500 and Kieselgel SI 5000 and the release profile is investigated.

[0302] As previously described, a fenofibrate suspension is prepared but without the addition of DOSS as stabilizer. The resulting suspension is then nanomilled. The nanosuspension obtained is stored in the refrigerator at a temperature between 2 and 8° C.

[0303] The carriers Parteck® SLC 500 and Kieselgel SI 5000 are each loaded in a ratio of 1:1 (w/w) with the fenofibrate nanosuspension using the impregnation method. Subsequently, the drying is carried out by freeze-drying. Since Parteck® SLC 500 has hygroscopic properties, all batches produced are stored in the desiccator over orange gel.

[0304] Then the release of the active ingredient from the samples and the loss on drying of the samples is determined.

[0305] For these measurements, samples are prepared with a theoretical fenofibrate content of approximately 18.0% (w/w).

[0306] The loading of the carriers is carried out as described in “Loading of Parteck® SLC 500/Kieselgel SI 5000”.

[0307] Here, three loadings were made with Parteck® SLC 500 and one Kieselgel Si 5000 loading, always using the prepared nanosuspension without addition of stabilizer (Table 10).

[0308] The batches are produced with the following weights:

TABLE-US-00012 TABLE 10 Silica Suspension batch [g] [g] FF_HPMC_SLC_1 10.00 10.09 FF_HPMC_SLC_2 10.02 9.99 FF_HPMC_SLC_3 10.00 10.06 FF_HPMC_SI.sub.— 10.04 9.99

[0309] Results:

[0310] Preparation of Nanosuspension without Stabilizers

[0311] The preparation of a nanosuspension, without stabilizers DOSS or viscosity enhancer HPMC, only with fenofibrate and MilliQ water is only with a short milling time possible thus commercial process may not feasible to establish. It comes to the floating of the drug and the mill clogged. The addition of HPMC (hypromellose=Pharmacoat 603) allows nano-milling even the suspension foams a little bit more as with addition of DOSS.

[0312] Weighing's for Nanomilling without any Stabilizers: [0313] 20.03 g Fenofibrate [0314] 80.0 g MilliQ water

[0315] Weighing's for Nanomilling with HPMC: [0316] 19.99 g Fenofibrate [0317] 2.52 g HPMC/Pharmacoat 603 [0318] 77.51 g MilliQ water

[0319] Drying Loss

TABLE-US-00013 TABLE 11 loss on drying batch [% w/w] FF_HPMC_SLC_1 2.15% FF_HPMC_SLC_2 1.98% FF_HPMC_SLC_3 1.06% FF_HPMC_SI_1 1.12%

[0320] The drying loss of the samples is just over 1%. The drying loss of the samples was not determined directly after freeze-drying, but only a few days later. Therefore, it can be assumed that despite the storage in the desiccator, the dry loss has increased slightly. Since the values are under 3% self imposed mark, no subsequent drying of the samples was carried out.

[0321] Release of Nano-Milled Fenofibrate without Stabilizer Loaded on Carrier

[0322] The dissolution of the nano-milled drug without stabilizer applied to a carrier is better compared to the crystalline drug (not milled). Fenofibrate nano-milled (without stabilizer) loaded Parteck® SLC 500 and Kieselgel SI 5000 as carrier enables a faster release as the pure crystalline drug. The use of formulation without stabilizers in final administration forms as tablets or capsules is favorable, as no additional influence or interference of stabilizer with API has to be considered during the development or clinical phases. API nano-milled formulations reported so far are containing stabilizer resulting in more complex administration forms without easy prediction of influence of additives.

[0323] The dissolution of the nano-milled drug without stabilizer applied to a carrier as Parteck® SLC 500 and Kieselgel SI 5000 showed a very similar release property with or without stabilizer (FIG. 7). In both cases the nano-milled drug loaded carrier showed faster drug release as the pure crystalline drug.

[0324] In summary it is found that nano-milling without the addition of any stabilizers is possible, however, the active substance floats on top and no further workable nanosuspension is obtained. By adding a small amount of HPMC, “floating” can be prevented and the production of a nanosuspension is possible. In all cases the release of samples of the nano-milled drug loaded on the carriers was faster (in approx. 10 minutes) in comparison with the pure crystalline drug samples that do not reaches the max. solubility possible even after the 2 hours tested.

[0325] Comparison with Nanosuspensions with Itraconazole (Representative Example for Weak Bases) as Active Ingredient

[0326] In the same way as in the previous experiments, an itraconazole nanosuspension is prepared here, which is applied to Parteck® SLC 500. It is to be investigated if an improvement of the release can be achieved by the application of a nanosuspension.

[0327] The results obtained are compared with the results of the fenofibrate nanosuspension.

[0328] In comparison, Kieselgel SI 5000 is loaded with the crystalline itraconazole nanosuspension. Here, the influence of the pore diameter on the release of the itraconazole nanosuspension will be investigated. In addition, goal is to verify analytical results of itraconazole loaded carrier achieved, with the fenofibrate loaded carrier, to confirm conclusion that release of API nano-milled loaded carrier of different API is faster as crystalline API without milling and is independent from pore-diameter.

[0329] Procedure

[0330] At the beginning of the experiments, an itraconazole suspension is prepared, which is stabilized with hydroxypropylmethyl cellulose (HPMC) and DOSS and then nano-milled. The nanosuspension obtained is stored in the refrigerator between 2 and 8° C.

[0331] Using the impregnation method, the carriers Parteck® SLC 500 and Kieselgel SI 5000 are loaded in a ratio of 1:1 (w/w) with the itraconazole nanosuspension. The drying takes place in freeze-drying (see program “Nanosus_PK”). As drying in the program “Nanosus_PK” is not sufficient, it is dried again (program: Nanosus_PK_modified). Due to the hygroscopic properties of the Parteck® SLC 500, all batches produced are stored in the desiccator over orange gel.

TABLE-US-00014 TABLE 12 Batch overview batch carrier Drug (API) ICZ_16092016_SLC_1 Parteck ® SLC 500 Itraconazole ICZ_16092016_SLC_2 Parteck ® SLC 500 Itraconazole ICZ_16092016_SLC_3 Parteck ® SLC 500 Itraconazole ICZ_16092016_SI_1 Kieselgel SI 5000 Itraconazole

[0332] Performed Measurements

TABLE-US-00015 All batches Loss on drying Differential Scanning Calorimetry Release Determination of content by HPLC Determination of content by H.sub.1-NMR

[0333] The measurements and determinations are carried out in the same way and with the same equipment and means as previously described.

TABLE-US-00016 TABLE 13 Used Materials Product Origin Itraconazole Metrochem API Private limited Hypromellose (HPMC) Shin Etsu Chemical Co., Ltd. (Pharmacoat 603) Dioctylsulfosuccinate Aldrich Chemistry sodium = DOSS Parteck ® SLC 500 Merck KGaA Kieselgel SI 5000 Merck KGaA (Synthesis) Milli-Q Water Merck KGaA

[0334] To stabilize the nanosuspension, hydroxypropylmethyl cellulose (HPMC) and DOSS are added to the suspension medium. It could be expected that without DOSS as stabilizing agent, the produced itraconazole nanosuspension would likely rapidly tend to aggregate and build up larger particles due to greatly increased surface effects, such as electrostatic attraction and dissolution rate (Ostwald ripening).

[0335] 1.)

[0336] Preparation of Itraconazole Suspensions: [0337] Itraconazole 19.870 g [0338] HPMC 2.496 g [0339] DOSS 0.500 g [0340] Milli-Q water 77.020 g [0341] Equipment: magnetic stirrer (IKA®-Werke GmbH & CO. KG, Staufen, Germany) [0342] Rotation speed: 600 rpm [0343] Temperature: off [0344] Others: stirring fish, beaker, spatula

[0345] HPMC and DOSS are weighed into VWR screw-cap glass (250 mL) and supplemented with Milli-Q water to 80.016 g (weighed in, see above) to prepare the itraconazole suspension for nanomilling. The mixture is stirred with the magnetic stirrer and with the stirring fish for about 2 hours until completely dissolving. The screw jar is then closed.

[0346] The next day, shortly before milling, itraconazole is weighed. With stirring, the itraconazole is added and suspended for about 10 minutes. Subsequently, the suspension is milled in the nanomill. [0347] Theoretical Itraconazole content: 19.89% (w/w)

[0348] 2.)

[0349] Prepare Saturated Itraconazole Suspension in SGFsp: [0350] Itraconazole about 200 mg [0351] SGFsp 100 mL [0352] Equipment: magnetic stirrer (IKA®-Werke GmbH & CO. KG, Staufen, Germany) [0353] Rotation speed: 450 rpm [0354] Temperature: 40° C. (thermostat) [0355] Others: stirring fish, beaker, tablespoon [0356] Time: 72 h

[0357] Approximately 200 mg of itraconazole are added to 100 mL SGFsp and suspended at 40° C. at 450 rpm for 72 h. The beaker is screwed during this time. Subsequently, the saturation concentration is measured by HPLC.

[0358] Nano-Milling [0359] Equipment: Dyno®-Mill Research Lab (Willy A. Bachofen AG—Maschinenfabrik, Muttenz, Switzerland) [0360] Weighed amount: 100 g [0361] Time: 30 min [0362] Milling balls: 55.0 mL zirconium oxide balls (SiLi ZYP 0.2-0.3 mm, Sigmund Lindner GmbH, Warmensteinach, Germany) [0363] Temperature: −2° C. (cryostat) [0364] Rotation speed: 3000 Upm [0365] Taking samples: t=0, 10, 20, 30 min

[0366] To prepare the nanosuspension, the suspension to be placed in the hopper of the nanomill and the milling process is started. The temperature of the cryostat should be around 2° C. during milling. At the defined times, a few drops of the suspension are removed from the feed hopper using a disposable pipette and the particle size is measured by means as described above. When the desired particle size is reached, the grinding process is finished. The measurement of the particle size takes place at regular intervals and the particle size determination is carried out according to the methods described above.

[0367] Loading of Parteck® SLC 500/Kieselgel SI 5000 with Liaconazole and Production of Following Batches

TABLE-US-00017 TABLE 14 The loading is carried out as described above and the batches are produced using the following amounts: Silica Suspension batch [g] [g] ICZ_16092016_SLC_1 10.00 10.00 ICZ_16092016_SLC_2 10.06 9.84 ICZ_16092016_SLC_3 10.00 9.93 ICZ_16092016_SI_1 9.99 9.87

[0368] Freeze-Drying of Resulting Loaded Carriers [0369] Device: Freeze Dryer Gamma 2-16 LSC (Christ Gefriertrocknungsanlagen Martin Christ, Osterode, Germany) [0370] Cooling: water cooling

[0371] The loaded, moist products from table 14 are freeze-dried under the following conditions in a beaker:

TABLE-US-00018 TABLE 15 a) Program 1: “Nanosus PK” Temperature Time [° C.] [h] Freezing −45° C. 8 First drying −35° C. 30 Second drying 0° C. 4 +25° C. 18 Total 60

TABLE-US-00019 TABLE 16 b) Program 3: “Nanosus PK modified” Temperature Time [° C.] [h] Freezing −45° C. 5 Main drying −35° C. 30 Second drying 0° C. 4 +25° C. 18 Total 57

[0372] The samples are placed into the freeze dryer for freeze drying and the freeze dryer is closed.

[0373] Water is used for cooling (first the drain is turned on, only then the inlet!). Then the desired program is started. After freeze-drying, the drying loss of the product should be determined as described. If drying is insufficient, further drying is carried out.

[0374] Since the drying loss of all batches after drying with program 1 is about −10%, they are dried again. Program 3: Nanosus_PK_modified is used for this purpose. After repeating the drying, the drying loss is again measured and was below 3%. The drying loss of the Kieselgel SI 5000 is thereafter only −2%.

[0375] The drying loss is determined as described above. The drying loss should ideally be below 3% for release. If the drying loss is higher, a further drying may be necessary.

[0376] Differential Scanning Calorimetry is Carried Out as Described Before, but According to Program 1: [0377] Methods: Program 1: (“25-200/5K_(Alu100_N-2)”)  Continuous heating of the sample from 25° C. to 200° C. at a heating rate of 5 K/min

[0378] Release of active ingredient is determined using the same device as applied before (Sotax 1 and 2; Freisetzungsapparatur Sotax AT 7smart (Sotax AG, Lörrach, Germany)

[0379] SGFsp is used as the medium and the release determinations are carried out at a wavelength of 225 nm. Each sample is collected in a test tube with the automatic sampler. Subsequently, the content of the samples is determined offline by HPLC. Advantageously the nano-milled loaded samples do not float on the release medium like the crystalline active substance but are better wetted.

[0380] Determination of Content by HPLC [0381] Device: HPLC-system LaChrom Elite (Hitachi Europe GmbH, Düsseldorf, Germany) [0382] Detector: UV Detector L-2400 VWR Hitachi (Hitachi Europe GmbH, Düsseldorf, Germany) [0383] Autosampler: Autosampler L-2200 VWR Hitachi (Hitachi Europe GmbH, Düsseldorf, Germany) [0384] Column: Chromolith Performance RP-18e 100-4.6 mm (OB1108048) [0385] Eluent: Acetonitrile/TBAHS-Buffer (1.7 g/L)/Methanol (450:450:100) [0386] Wash solution—sampler: Acetonitrile/Milli-Q-Wasser (1:1) [0387] Column oven—temperature: 25° C. [0388] Injection volume: 15 μL [0389] Wave length—detector: 225 nm [0390] Flow rate: 2.0 mL/min (isocratic) [0391] Duration—run: 7 min [0392] Sequence: XXXXXXXX_01_PK_Itaconazole_Disso_Nanosuspensionen 1. Seq [0393] Method: XXXXXXXX_01_PK_Itaconazole_Method [0394] Filter—sample preparation: Whatman™ Anotop™ 10, 0.02 μm, Cat.-No.: 6809-1002

[0395] Prior to filling into vials, each sample from the release is first filtered with a syringe with Luer-Lock connection and above filters for sample preparation to retain any particles of the nanosuspension and eliminate a systematic error.

[0396] For evaluation of the HPLC results, the saturation concentration is determined and the release of crystalline Itraconazole from the experiment is taken.

[0397] H.sub.1-NMR: [0398] Method: 1H-NMR spectroscopy [0399] Conditions: DMSO-d6 [0400] Measurement modus: content [%]

[0401] Results:

[0402] Particle Size Distribution of the Nanosuspension

[0403] The particle size distribution is measured with a Zetasizer Nano SZ.

[0404] The particle size is measured immediately after the preparation of the nanosuspension.

[0405] The particle size remains almost constant over a longer period, the crystals in suspension grow only very slowly. This means that the nanosuspension is sufficiently well stabilized by the use of HPMC and DOSS.

[0406] Optical Assessment of the Batches Produced

[0407] ICZ_16092016_SLC_1

[0408] After loading the Parteck® SLC 500 with the itraconazole nanosuspension, the support material is slightly clumped. After freeze-drying, these lumps remain. They are easy to be divided with the spatula. The remainder, loose powder is of fine consistency. The color is white as that of the starting substance.

[0409] ICZ_16092016_SLC_2

[0410] The loading of the Parteck® SLC 500 also leads to the formation of smaller lumps in the second batch. As with the first batch, the lumps can be easily crushed with the spatula.

[0411] ICZ_16092016_SLC_3

[0412] Just like the other batches, the Parteck® SLC 500 also has some lumps that can be easily crushed with the spatula after impregnation.

[0413] ICZ_16092016_SI_1

[0414] Similar as the loading of the Parteck® SLC 500, the loading of the Kieselgel SI 5000 produces some lumps of different sizes. Compared to Parteck® SLC 500, the loose, remaining powder is floury-like.

[0415] Determination of Content by H.sub.1-NMR

TABLE-US-00020 TABLE 17 By the external H.sub.1-NMR content determination of the samples from the different batches shows the following active ingredient contents: Theoretical Measured content content batch [%] [%] ICZ_16092016_SLC_1 16.08 13.3 ICZ_16092016_SLC_2 16.09 13.2 ICZ_16092016_SLC_3 16.20 12.6 ICZ_16092016_SI_1 16.21 15.2

[0416] DSC Measurement

TABLE-US-00021 TABLE 18 physical status amorphous/crystalline batch excipient crystallinity ICZ_16092016_SLC_1 Parteck ® SLC 500 crystalline ICZ_16092016_SLC_2 Parteck ® SLC 500 crystalline ICZ_16092016_SLC_3 Parteck ® SLC 500 crystalline ICZ_16092016_SI_1 Kieselgel SI 5000 crystalline

[0417] This FIG. 8 shows the DSC curve of crystalline itraconazole, along with the curves of the nanosuspension-loaded batches of Parteck® SLC 500 and Kieselgel SI 5000. The endothermic melting peak of the pure itraconazole, starting at approx. 166° C., is clearly visible. The melt peaks of the nanosuspension batches on Parteck® SLC 500 and Kieselgel SI 5000 are much less pronounced and is shifted to lower temperatures. Instead of a sharp melting peak of itraconazole a broadened melting peak occurs for the loaded batches which begins earlier, this means at about 155° C.

[0418] Since the nanosuspension is not just pure itraconazole, both the hydroxypropylmethyl cellulose used and DOSS can cause a melting point depression.

[0419] In addition, a “masking” of the heat transfer could take place through the silica supports used, so that a defined, clear melting peak is covered. The active ingredient is found in crystalline form in all samples.

[0420] Release of Active Ingredient Itraconazole Loaded on Different Carrier

[0421] FIG. 9 shows the results of the batch, ICZ_16092016_SLC_2, which releases with a maximum concentration of approx. 3 mg/L and substantial faster than itraconazole crystalline sample compared. The saturation solubility is exceeded by about 0.5 mg/L due to challenges in analytical evaluation.

[0422] While crystalline itraconazole floats on the release medium, all samples of loaded Parteck® SLC 500 batches drop rapidly to the bottom of the vessel after adding. Only a small part of the sample floats on the surface of the medium.

[0423] Analytical results of itraconazole nano-milled loaded on different carrier supported evaluation of fenofibrate nano-milled loaded carrier reported before. In all case API (representative of API natures) loaded on different carrier results in substantial faster release in comparison to the pure crystalline API.