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KAUNAS UNIVERSITY OF MEDICINE FACULTY OF PHARMACY

DEPARTAMENT OF PHARMACEUTICAL TECHNOLOGY AND SOCIAL ORGANIZATION

Edita Eidukaityt MASTER THESIS

INCORPORATION OF PELLETS AND MICROGRANULES INTO PRESSED AND LYOPHILIZED TABLETS, RESPECTIVELY

Master thesis is accomplished at a Departament of Pharmaceutical Technology, Medical University of Gdansk

Head of thesis: prof. hab. dr. Małgorzata Sznitowska- Departament of Pharmaceutical Technology, Medical University of Gdansk, prof. Dr. Vitalis Briedis- Departament of Pharmaceutical Technology and social orgnization, Kaunas University of Medicine

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Content

1. Introduction………..……4

1.1.The importance of multiparticulate dosage forms………...…………4

1.2.The aim of the study………....4

1.3.The tasks of the study……….……….5

2. Literature review……….……..6

2.1. Pellets and microparticles – characteristics and methods of preparation………..6

2.1.1. Pellets...6

2.1.2. Microcapsules...8

2.1.3. Microspheres...10

2.1.4. Lipospheres...11

2.1.5. Microgranules...13

2. 2. The influence of pellet core composition and coating film type on the release of active substance ...14

2. 3. Lyophilized tablets... ...18

2.3.1. Lyophilization process...19

3. Experimental part………...21

3.1. Incorporation of microgranules with selegiline hydrochloride into lyophilized tablets………..………21

3.1.1. Reagents, materials and equipment...21

3.1.2. Methods ...23

3.1.2.1. Microgranules formation...23

3.1.2.1.1. Preparation of microgranules type XPS1...23

3.1.2.1.2. Preparation of microgranules type XPS3...23

3.1.2.1.3. Coating of the microgranules (type XPS3P preparation)...23

3.12.2. Visual inspection of microgranules ...24

3.12.3. Evaluation of selegiline hydrochloride content in the cross-linked microgranules ...24

3.1.2.4. The release of selegiline hydrochloride from cross-linked microgranules...24

3.1.2.5. Preparation of lyophilized tablets containing microgranules with selegiline hydrochloride...24

3.1.2.6. Visual inspection of lyophilized tablets ...25

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3.1.2.7. Release test of selegiline hydrochloride from lyophilized tablets...25

3.1.2.8. Quantitative analysis of selegiline hydrochloride by HPLC method...26

3.2. Incorporation of floating pellets into pressed tablets – tableting of floating pellets………...28

3.2.1. Reagents, materials and equipment...28

3.2.2. Preparation of floating pellet cores with verapamil hydrochloride by extrusion- spheronization method...30

3.2.3. Coating of pellet cores with Eudragit NE 40 D...31

3.2.4. Tableting of floating pellets with verapamil hydrochloride by using impact tableting machine...32

3.2.5. Analysis of pellets and tablets...34

3.2.5.1. Measurement of film thickness...34

3.2.5.2. In vitro drug release test from pellets...34

3.2.5.3. Tablets appearance and size...34

3.2.5.4. Content of VH in tablets...34

3.2.5.5. Determination of single tablet mass uniformity...35

3.2.5.6. Evaluation of tablets resistance to crushing (hardness of tablets)...35

3.2.5.7. Evaluation of tablets friability...35

3.2.5.8. In vitro drug release test from tablets and flotation start time of pellets……….35

4. Results and discussion………...36

5. Conclusions...54

6. Acknowledgments………..55

7. References...56

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1. Introduction

1.1. The importance of multiparticulate dosage forms

Multiparticulate dosage form contains actives divided into many individual units, so-called subunits, each exhibiting some desired characteristics. These subunits usually are microparts such as microcapsules, microspheres, lipospheres, microgranules or larger particles - pellets.

Multiparticulate dosage forms are more expensive to manufacture and develop, but despite of it are widely used in pharmaceutical formulations. They are more reliable in their biopharmaceutical behaviour and considered to provide pharmacokinetic advantages compared with monolithic dosage forms [27].

When compared with single-unit dosage forms, oral multiparticulate drug-delivery systems offer biopharmaceutical advantages:

Microparticles can be used to prepare pharmaceutical formulations composed of incompatible drugs or to obtain delivery systems with different release profiles [16].

Can be divided into desired doses without formulation and process changes.

Possibility to produce modified release dosage forms as very significant means of drug delivery nowadays [16, 27].

More even and predictable distribution and transportation in the gastrointestinal tract, which is fairly independent of the nutritional state [26]. When taken orally multiparticulates generally disperse freely in the gastrointestinal tract, thus maximize drug absorption, reduce peak plasma fluctuation, minimize side effects and reduce inter- and intrapatient variability [15].

Are less susceptible to dose dumping than the reservoir-type, single unit formulations [4].

These dosage forms have several disadvantages, like the risk of tampering with capsules or the rupturing of the coating during compression resulting in a loss of the modified drug-release properties [27].

1.2. The aim of the study

With regard to the final dosage form, the multiparticulates can be filled into hard gelatin capsules or be incorporated into tablets. The compression of multiparticulates into tablets is becoming more popular. The advantages of tableting multiparticulates include a reduced risk of tampering and fewer difficulties in oesophageal transport when compared with capsules. Large

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volume tablets generally have a higher patient compliance than capsules; higher dose strength could be administered with tablets. Tablets with pellets can be prepared at lower cost when compared to pellet-filled capsules because of higher production rate of tablet presses. The expensive control of capsule integrity after filling is also eliminated. In addition, tablets allow a more flexible dosing regimen [3, 39].

The aim of the study was to obtain multiparticulate prolonged release drug delivery systems by incorporating microgranules with selegiline hydrochloride (SCh) and pellets with verapamil hydrochloride (VH) into lyophilized and pressed tablets, respectively. Also it was assumed to preserve primary dosage form properties unchanged in final drug form. Controlled SCh release of microgranules was achieved by cross - linking pectin with 20% ZnSO4 solution, whereas controlled VH release from pellets was gained by coating it with methacrilic acid copolymer – Eudragit NE 40 D in a fluidized-bed coater.

Preparing lyophilized buccoadhesive tablets with microgranules and compressed oral tablets with floating pellets and maintaining the same drug release profile for the resulting tablets as for the microgranules or pellets, respectively, was the goal of the study.

1.3. The tasks of the study

The tasks of the study were as following:

1. To obtain microgranules with SCh by two different methods of preparation;

2. To incorporate these microgranules into lyophilized tablets;

3. To test and compare release profiles of microgranules before and after incorporation;

4. To prepare floating pellets with VH by extrusion-spheronization method;

5. To coat pellets by fluidized-bed method with Eudragit NE 40;

6. To tablet these floating pellets with various tableting excipients by means of single stroke machine with 12 kN and 18 kN compression force;

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2. Literature review

The main types of multiparticulate dosage forms (pellets, microcapsules, microspheres, lipospheres, and microgranules) are discussed in this chapter.

2.1. Pellets and microparticles – characteristics and methods of preparation

2.1.1. Pellets

Pellets are uniformly sized spherical granules, which range in size from 0.5 – 1.5 mm and in some applications may be as large as 3.0 mm. This drug form can contain 10 – 90% of active pharmaceutical ingredient (API) and usually is obtained by coating the cores, wet granulation, hot extrusion or extrusion-spheronization method. The last mentioned method is applied mostly [17] (Fig. 1).

Fig. 1. Pellets, prepared by extrusion-spheronization method (interspace on the scale=1 mm)

Preparation of pellets by extrusion and spheronization offers numerous advantages over other methods:

Ease of operation;

High throughput with low wastage;

Narrower particle size distribution;

Production of pellets with low friability;

Production of pellets that are suitable for film coating;

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More sustained and better controlled drug-release profile when compared with other techniques [15].

The major advantage is the ability to incorporate high levels of active ingredients without producing exclusively large particles.

The main steps of the process are [33]:

1. Dry mixing of ingredients to achieve homogenous powder dispersion;

2. Granulation (wet massing) to produce a sufficiently plastic wet mass;

3. Extrusion to form rod-shaped particles of uniform diameter;

4. Spheronization to round off these rods into spherical particles;

5. Drying to achieve the desired final moisture content;

6. Screening (optional) to achieve the desired narrow size distribution.

Different steps, parameters and equipment used in the process are summarized in Fig. 2 [15].

Fig. 2. Flow diagram showing different steps, process parameters and equipment involved in extrusion and spheronization to produce spherical modified release pellets [15]

Powder dry mixing Granulating

liquid

Mixer Wet mixing

Granulator type Granulation liquid Mixing time

Extruder Extrusion

Spheronizer Spheronization

Dryer Drying

Coater Coating

Extruder type Extrusion speed Screen opening size

Extrusion temperature

Spheronizer type Plate type Plate speed Spheronization time

Spheronizer load

Dryer type Drying temperature

Coating solution

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dosing. This simplification in the therapy improves compliance and leads to a decrease in overall costs. Controlled release dosage forms have essentially been implemented in the treatment of hypertension, inflammatory processes, obstructive pulmonary, Parkinson’s diseases [12].

2.1.2. Microcapsules

Microcapsules are kind of capsules, which size varies from 5 to 1000 m, commonly they have a diameter between 100 – 500 m (fig. 3).

Fig. 3. SEM photograph of microcapsules (bar=5 m) [20]

They consist of an active agent or core material which is surrounded by coating or shell.

The core can contain solid, liquid (solution, suspension, emulsion) or gaseous substance. The mass of the core usually works out 30 – 90% of the all microcapsule mass. Depending on the core composition microcapsules have spherical or irregular, close to core substance, shape. A wide range of core materials have been encapsulated, including pharmaceuticals, adhesives, agrochemicals, live cells, active enzymes, flavors, fragrances and inks. Most microcapsule shell materials are synthetic, but natural ones are also used. Gelatin, arabic gum, shellac, colophony, ethyl cellulose, carboxymethyl cellulose, methylcellulose, cellulose nitrate, cellulose acetate, polyvinyl alcohol, polyamides, polyoxyethyl glycol, polypropylene, polyvynilpirolidone are applied mostly. The mixtures of polymers for example ethylcellulose and methylcellulose are also used frequently. Depending on the kind of core, the substance passes through the film faster or slower. The films may be stiff, elastic or porous [23, 45].

Methods of preparation I Chemical methods

Coacervation. There are two types of coacervation: simple and complex. Only one colloid, e. g., gelatin in water, it is used in the simple coacervation and the cores – solid or liquid

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substances – are suspended or emulsified in the water solution of shell material. The associated water from around the dispersed colloid is removed by adding agents with a greater affinity for water, such as alcohols, salts or by changing environmental parameters: temperature, pH, concentration. The dehydrated molecules of polymer tend to aggregate with surrounding molecules to form coacervate (it is coacervation in aqueous media). Soluble and insoluble in water substances can be encapsulated using coacervation in nonaqueous media. The substances can not be soluble in the organic solvent, in which the process is accomplished. Shell materials also must be insoluble in the organic solvent, for this reason usually cellulose derivates (cellulose nitrate, cellulose acetate, ethylcellulose) are applied. The coacervation comes when reciprocal organic solvent, miscible with polymer solution, is added [9, 23].

Complex coacervation involves the use of more than one colloid. This process occurs with reciprocal neutralization of two oppositely charged polymers. Method is based on the ability of cationic charged gelatin and negatively charged polymer (for example arabic gum or acacia) to interact in water and form a liquid, polymer rich phase called a coacervate. This method is rather used to encapsulate water-immiscible liquids [9, 45].

Polymer – polymer incompatibility. Polymer – polymer incompatibility occurs because two chemically different polymers dissolved in a common solvent are incompatible and do not mix in solution (e.g.ethyl cellulose and polyethylene in hot (80oC) cyclohexane). These polymers form two separate liquid phases: one phase is rich in polymer which acts as the capsule shell (in this case ethyl cellulose), the other is rich in the second, incompatible polymer. This polymer causes formation of two phases and it is not designed to be part of the final capsule shell. Then the core material (small particles) is dispersed in two-phase system. Since the ethyl cellulose is more polar than polyethylene, it adsorbs on core material and forms a thin coating. When the system is cooled to room temperature the ethylcellulose precipitates forming solid microcapsules [45].

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In situ polymerization. In situ polymerization is closely related to IFP. The difference is that with in situ encapsulation processes, no reactive agents are added to the core material.

Polymerization occurs on the continuous-phase side of the interface formed by the dispersed core material and continuous phase. Polymerization of reagents located there produces relatively low molecular weight prepolymer. As this prepolymer grows in size, it deposits onto the surface of the dispersed core material being encapsulated where polymerization with cross-linking continues to occur thereby generating a solid capsule shell [45].

II Physical methods

Spray drying. Microencapsulation by spray drying is based on two steps: emusification or dispersion of the core substance in the polymer solution and removal of the solvent by a hot stream of air. The core material is generally water-immiscible. The shell material normally is water soluble polymer like gum arabic or a modified starch [45, 28].

Fluidized bed method. Fluidized bed coaters suspend solid particles in a moving gas stream, usually air. A liquid coating formulation is sprayed onto the individual particles. Freshly coated particles are moved into a zone where the coating formulation is dried either by solvent evaporation or cooling. This coating and drying sequence is repeated until a desired coating thickness is achieved [45].

Rotational suspension separation. During this process, core material dispersed in a liquid shell formulation is fed onto a rotating disk. Individual core particles coated with a film of shell formulation are flung off the edge of the rotating disk along with droplets of pure coating material. The shell mass is solidified, e.g., by cooling, and discrete microcapsules are produced.

The excess of pure shell material also solidify, but it is collect in a discrete zone away from the capsules [23, 45].

2.1.3. Microspheres

Microspheres are monolithic, porous spheres, composed of various polymers, in which active pharmaceutical substances are dispersed or dissolved. The size of microspheres varies from 1 to 500 m (fig. 4). Although the size and the shape sometimes are very close to microcapsules, essential difference is composition of microspheres. Microcapsules possess active pharmaceutical ingredient in liquid or solid form, enclosed in polymeric membrane. Meanwhile in the microspheres API is incorporated in polymeric matrix. Therefore, microspheres might be called micrometric matrix systems.

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Fig. 4. SEM picture of microspheres (bar=10 µm) [14]

The most suitable polymeric materials for producing microspheres are synthetic hydroxyacids polyesters: polylactic acid (PLA) and polylacticglycolic acid (PLGA). These polymers undergo biodegradation to natural products of metabolism - lactic and glycolic acids.

The main method of preparation of these drug delivery systems is solvent evaporation.

This technique is based on removing the hydrophobic polymer solvent by evaporation [29].

Polymeric material is dissolved in a volatile organic solvent. The API is then dispersed or dissolved in the organic solution. In the following step, a dispersing phase, consisting of nonsolvent of the polymer and immiscible with the organic solvent also containing appropriate tensioactive substance, is added by continuous mechanical stirring. The solvent evaporates after diffusing through the continuous phase and the result is creation of solid microspheres.

Microspheres also can be produced by polymer-phase separation, spray-drying, milling methods or methods using fluids under supercritical conditions [2].

2.1.4. Lipospheres

Lipospheres are a new type of lipid based drug delivery system developed for parenteral or topical action of bioactive compounds. They are composed of solid hydrophobic fat core

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Fig. 5. Stereomicrograph of lipospheres (bar=100 m) [46]

The internal core contains the bioactive compound dissolved or dispersed in the solid-fat matrix. The lipospheres system has been used for the controlled delivery of various types of drugs including anti-inflammatory compounds, local anaesthetics, antibiotics and anticancer agents. They have also been used successfully as carriers of vaccines and adjuvants.

Lipospheres have several advantages over the other drug delivery systems: better physical stability, low cost ingredients, ease of preparation and scale-up, high dispersability in aqueous medium, high entrapment of hydrophobic drugs, controlled particle size, extended release of incorporated drug after a single injection from a few hours to several days.

The main hydrophobic core constituents are tricaprin, trilaurin, tristearin, stearic acid, ethyl stearate, hydrogenated vegetable oil. The phospholipids used to form the surrounding layer of lipospheres are pure-egg phosphatidylcholine, soybean phosphatidylcholine, dimyristoyl phosphatidylglycerol and phosphatidylethenolamine.

Liposphere formulations are prepared by a solvent or melt process. In the melt method, the API is dissolved or dispersed in the melted solid carrier and then a hot buffer solution is added at once with the phospholipid powder. The hot mixture is homogenized for about 2-5 min using homogenizer or ultrasound. The milky formulation is rapidly cooled by immersing the flask with mixture in an acetone-dry ice bath while homogenization is continued to yield a uniform dispersion of lipospheres.

In the solvent technique, the active agent, the solid carrier and phospholipid are dissolved in an organic solvent (e.g.acetone, ethyl acetate, ethanol or dichlormethane). The solvent is evaporated; the resulting solid is mixed with warm buffer solution. Mixing is continued until a homogenous dispersion of lipospheres is obtained [11].

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2.1.5. Microgranules

Microgranules are compacted powder particles with a size range between 200 and 500 m [50] (fig. 6). In order to obtain required release profile microgranules can be coated. It is difficult to prepare microgranules of a proper size and density using conventional dry/wet granulation or the other methods. Usually microgranules, prepared by these methods, have bigger size and lower density than the required ones, e.g. for coating or incorporation into lyophilized tablets.

Fig. 6. Microgranules (bar=100 m)

During the process in a fluid-bed apparatus, the granulation powder is kept in suspension by an appropriate air flow, while the granulation fluid is simultaneously sprayed. The resulting product has an even shape, but is very porous and has a low density. Granules are therefore unsuitable for subsequent coating, as they are inclined to break. Furthermore, the material to be subjected to a fluid-bed coating process must be made up of particles of sufficient density to avoid the agglomeration phenomenon. Otherwise, the particles tend to occupy the upper section of the apparatus, are not subject to the normal movement inside the apparatus, and thus do not receive an appropriate gradual coating.

A conventional mixer-granulator consists of a vessel, which may be of varying shape, equipped with an agitator that keeps the powder moving while the granulation fluid is being

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A high-shear mixer-granulator is made up of a vessel in which the mixture to be granulated is introduced that is equipped with a mixer and a mill that rotate with a normal mixer motion.

Since the mixer and the mill have variable and adjustable speeds, they ensure densification and preparation of the granulate in shorter times as compared to conventional granulators.

It has now been found that, using high-shear mixer-granulators and operating within specific critical ranges of the parameters that control the granulation process, it is possible to obtain a microgranulate of a size smaller than 500 m. It is an object of the present invention to provide a size distribution, density, surface and shape of the particles produced that makes them particularly suitable for preparing the final drug forms, e.g. coating and for suspension in low density fluids [35].

The other method, which can be used for preparation of microgranules, is modified wet granulation. Wet granulation generally involves the wetting of a mixture of dry primary powder particles using a granulating fluid. The fluid contains a solvent which must be volatile so that it can be removed by drying, and be non-toxic. Typical liquids include water, ethanol and isopropanol, either alone or in combination [50]. In a modified granulation method, applied for preparing microgranules with selegiline, as granulating liquid aqueous zinc sulphate solution was used. Bivalent ions such as zinc, calcium etc. cross-link the polymer (in this case – pectin). This modification served as a good mean for achieving prolonged drug release.

2.2. The influence of pellet core composition and coating film type on the release of active substance

In order to obtain a medication with modified drug release quality, it is important to choose appropriate constituents of the core and the coating formulation. As the film is commonly responsible for controlled release action, in most studies the main attention is paid exactly for the influence of coating type on releasing of active substances. Dissolution tests are performed under in vitro conditions, but to optimize a dosage form the in vivo investigations are also necessary.

During the study of in vitro and in vivo dissolution of theophylline from pellets, coated with Eudragit L, the in vivo liberation of theophylline was studied in rabbits. Finally, it was concluded, that there was no great difference in the maximum values (all the pellets gave cmax >

10 µg/ml) between the uncoated and the Eudragit L-coated pellets, but a significant shift in tmax

was found for both Eudragit-coated pellets. The difference was 4h as compared with the uncoated preparation (fig. 7). This result exhibits a relationship between the in vitro dissolutions (3 h), which confirms the reliability of the in vitro dissolution method. The slower decreases in

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theophilline concentration in the cases of the Eudragit L (64 µm) and Eudragit L (85 µm) films demonstrate the effectiveness of the enteric coating process [30].

Fig. 7. Plasma level of theophylline after administration in rabbits [30].

In the study of Hu et al. [21], the influence of surface modification by talc, the effects of Eudragit types and ratios, as well as the correlation between in vitro release and in vivo absorption were investigated in detail in metformin hydrochloride (MH) sustained release pellets. Three pellets formulations were prepared: formulation 1 (F-1): coated with Eudragit NE30D, resulting in 10% coat loading; formulation 2 (F-2): coated with Eudragit L30D-55:

Eudragit NE30D (1:20), resulting in 7% coat loading; formulation 3 (F-3): coated with Eudragit L30D-55: Eudragit NE30D (1:20), resulting in 10% coat loading. The relative bioavailability of the sustained release pellets was studied in 12 healthy volunteers after oral administration in a fast state using a commercially available immediate release (IR) tablet (Glucophage) as a reference. The results suggest that talc modification effectively controls drug release and avoids drug dumping. The in vivo bioavailability showed varying sustained-release characteristics for the coated pellets when compared with IR MH tablets (fig. 8).

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Fig. 8. Mean plasma metformin concentration-time profiles of three sustained MH pellets and IR MH tablets [21]

When coated with a blend of Eudragit L30D-55 and Eudragit NE30D (1:20) to a loading weight of 7% or 10%, pellets exhibited excellent sustained-release effects and high relative bioavailability. Restricted delivery of metformin hydrochloride to the small intestine from differently coated pellets resulted in increased relative bioavailability and a sustained release effect. The adoption of several different pH dissolution media (0.1 M HCl, distilled water and phosphate buffer (pH 6.8) established a better relationship between the in vitro release and in vivo absorption of the sustained-release pellets.

Zhou et al. [51] tested the bioavailibilty of ibuprofen from pellets based on microcrystalline wax (Lunacera P® and Lunacera M®) and starch derivatives. This matrix system provides a flexible drug delivery system, whereby the drug release rate depends on the type and the concentration of the hydrophobic and the hydrophilic component. During the studies three pellets formulations were prepared, which had the following composition: F-1:

ibuprofen–waxy maltodextrin (WMD)–Lunacera P® and Lunacera M® mixture (ratio 3/7) 60/15/25 (w/w/w); F-2: ibuprofen-waxy maltodextrin-Lunacera P 60/15/25 (w/w/w). Both formulations were filled into hard gelatine capsules. Formulation F-3 (ibuprofen–drum dried corn starch (DDCS) –Lunacera P® 30:40:30 (w:w:w)) pellets were compressed into tablets.

Pellets (F-1) formulated with the wax having the highest melting range gave the slowest drug release rate while F-3 pellets failed to form a sustained release matrix system as 90% of the dose was released within 45 min of the dissolution test. In vivo evaluation was performed with

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healthy human volunteers. The plasma profiles also indicated that the absorption of ibuprofen depended on the composition of the matrix pellet formulation. From these in vivo studies it can be concluded that the bioavailability of pellet formulations based on the combination of microcrystalline waxes and starch derivatives can be adjusted by means of varying the type and the content of both the waxes and the starch derivatives. Pellets with a sustained as well as an immediate drug release could be formulated using the wax–starch delivery system.

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2.3. Lyophilized tablets

The main method for obtaining this drug form is freeze-drying or lyophilization. It is a process in which water is sublimated from the product after freezing [10]. The main advantage being that pharmaceutical substances can be processed at non-elevated temperatures that enables drying labile substances, excluding the action of enzymes and microorganisms [13]. Final product can be stored in a dry state with relatively few shelf life stability problems. Another reason for applying this method is that freeze-dried forms offer more rapid dissolution than other available solid products. The lyophilization process imparts a glassy amorphous structure to the bulking agents and, sometimes, to the drug, thereby enhancing the dissolution characteristics of the formulation. After placing it in the mouth, these dosage forms immediately disperse/dissolve in the saliva and are then swallowed in the normal way without the need for water. This is very important especially in the paediatric and geriatric patients, who have difficulty swallowing tablets and hard gelatine capsules. Also lyophilized tablets offer a convenience during travels.

The bioavailability of a drug from fast dispersing formulations may be even grater than observed for standard dosage forms. Furthermore, side-effects may be reduced, if they are caused by first- pass metabolites [43]

The use of lyophilization however is strongly limited by the long time and handling required for processing. Freeze-drying is also energy- intensive process, having limited amount of materials processed for each batch. Other major disadvantages of the final dosage forms include the lack of physical resistance and limited ability to accommodate adequate concentrations of active [10].

Lyophilized tablets are prepared by sublimation of solutions after dosing it to blisters.

Polymers and sugars that give for lyophilized tablet appropriate structure are applied in this process as excipients (table 1).

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Table 1. The main excipients applied for preparation of lyophilized tablets [43]

Kind of substance Sample Properties

Binders Gelatine, alginates, gums (xanthan, arabic), cellulose derivatives, povidone

Determine the structure of tablets Have influence on it hardness Ensure amorphous structure Make easier suspending of active substance particles

Fillers Mannitol, sorbitol, saccharose, lactose, glucose, maltodextrins, erytritol,

lactitol

Commonly forms the main tablets mass

Fill the structure created by polymers

Have sweet flavour Sweeteners Aspartame, sodium saccharine Mask bitter taste of API

Aroma Mostly mint, cherry, orange or vanilla Make drug application more attractive

2.3.1. Lyophilization process

Generally the complete freeze-drying process comprises three stages: freezing, primary drying, and secondary drying [44].

Freezing

Freezing is an efficient desiccation step where most of the solvent, typically water, is separated from the solutes to form ice. As freezing progresses, the solute phase becomes highly concentrated and is termed the “freeze concentrate.” By the end of freezing, the freeze concentrate usually contains only about 20% of water (w/w), or less than 1% of total water in the solution before ice formation. The freezing stage typically takes several hours to finish. In this step, it is important to freeze the material at a temperature below the eutectic point of the material. Since the eutectic point occurs at the lowest temperature where the solid and liquid phase of the material can coexist, freezing the material at a temperature below this point ensures that sublimation rather than melting will occur in the following steps.

Primary drying

Primary drying, or ice sublimation, begins whenever the chamber pressure is reduced and the shelf temperature is raised to supply the heat removed by ice sublimation. During primary

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isotherms. In this phase, the temperature is raised even higher than in the primary drying phase to break any physico-chemical interactions that have formed between the water molecules and the frozen material. Usually the pressure is also lowered in this stage to encourage sublimation.

After the freeze drying process is complete, the vacuum is usually broken with an inert gas, such as nitrogen, before the material is sealed.

Tablets obtained by lyophilization can also be applied as buccal drug delivery systems.

These formulations may prove to be an alternative to the conventional oral medications as they can be readily attached to the buccal cavity retained for a longer period of time and removed at any time. In order to achieve buccal adhesive drug delivery systems quite a wide variety of bioadhesive substances can be used including such polymers as pectin, gelatine, sodium CMC, HPMC, sodium alginate, xanthum gum, acacia. Bioadhesive formulations use polymers as the adhesive component. These formulations are water soluble and when in a dry form attract water from the biological surface and this water transfer leads to a strong interaction with mucous membrane. In order to retain drug delivery, polymer can be transformed into insoluble form those prolonging the release of active substance [42]. This may be performed using bivalent ions, e.g. Zn, Ca etc, which cross-link the polymer and makes it insoluble [48].

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3. Experimental part

3.1. Incorporation of microgranules with selegiline hydrochloride into lyophilized tablets

3.1.1. Reagents, materials and equipment

Reagents

Gelatin powder pure, POCh, Gliwice, Poland

Pectin classic (type CU 701), Herbstreith & Fox, Neuenburg, Germany Selegiline hydrochloride (series 03/1-6), Dipharma, Milan, Italy Sodium citrate, POCh, Gliwice, Poland

Sodium carboxymethylcellulose (type 7HXF), Hercules, Wilmington, USA Zinc sulfate × 7H2O, POCh, Gliwice, Poland

Water purified through ion change and reversed osmosis (system Elix 3), Millipore, Bedford, USA

Materials

PCV blisters – gift from Polpharma, Starogard Gdanski, Poland

Equipment

Freeze-dryer – Alpha 2-4, Christ, Osterode am Harz, Germany

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- Microscope – type B1 223A (Motic, Welzlar, Germany), equipped with digital camera – type GP-KR 222, Panasonic, Osaka, Japan

HPLC set (Merck Hitachi, Darmstad, Germany):

- integrator – model D-2500A - detector – UV-Vis – L-4250 - pump – type L-6200A

Vibration mill – type KM1, Heinz – Janetzki, Germany Coffee grinder (type 651), Z. S. P. Niewiadow, Poland

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3.1.2. Methods

3.1.2.1. Microgranules formation

In order to obtain prolonged-release microgranules containing selegiline hydrochloride (SCh) two different methods were applied. In the first method SCh was introduced in powdered form (granulate type XPS1), while in the second one SCh was added to pectin in form of 20%

aqueous solution (granulate type XPS3). The exact composition of these granules is listed in table 2.

Table 2. Ingredients utilized for preparing cross-linked microgranules containing SCh Amount [g]

Ingredient

XPS1 XPS3

Pectin 4.0 4.0

Purified water 13.3 3.3

Selegiline hydrochloride 2.0 (powder form) 10.0 (20% solution)

20% ZnSO4 solution 13.3 13.3

3.1.2.1.1.Preparation of microgranules type XPS1

The weighed amount of pectin was transferred to a mortar and 13.3 g of water was added by continuous mixing. The mixture was left for 30 min for swelling of pectin. Then 2.0 g SCh was added and mixed using a pestle. The pectin was cross-linked by dripping 20% ZnSO4

solution to the mixture of powders. During the cross-linking process, large granules were formed, which were transferred to a dryer (35 ºC) for 12 h.

In order to obtain microgranules, the dried large granules were milled in a vibration mill and sieved with 3 sieves set (200, 100 and 45 m).

3.1.2.1.2.Preparation of microgranules type XPS3

Four grams of pectin was transferred to mortar and 3.3 g of water was added. The mixture

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solution, while microgranules were stirred. For 1.0 g of microgranules 3.3 g of 2% pectin solution were added. The coated microgranules were dried as described above.

3.1.2.2. Visual inspection of microgranules

The shape, surface and colour of the microgranules was observed using optic microscope equipped with digital camera. The images of microgranules were analyzed by means of Multi Scan (version 12.07) program.

3.1.2.3. Evaluation of selegiline hydrochloride content in the cross-linked microgranules

Three samples (about 50 mg) of each microgranules batch were dissolved in 10 ml of 2%

sodium citrate solution using magnetic stirrer. The content of SCh in the solution was determined by HPLC method. Before testing the solution was diluted 100-fold with HPLC mobile phase.

Composition of the phase and the analysis parameters are listed in section 3.1.2.8.

3.1.2.4. The release of selegiline hydrochloride from cross-linked microgranules

Three samples, approximately 50 mg of the cross-linked microgranules (corresponding to about 10 mg of selegiline hydrochloride), were analyzed. The test was accomplished in water bath shaker, using following conditions:

medium: 20 ml of purified water

temperature: 37ºC ± 1ºC;

shaking amplitude: 10 (apparatus scale) shaking speed: 70 c.p.m.

After 1, 2, 4, 6, and 24 h a 0.5 ml sample of the medium was taken. After diluting the samples 10-fold with HPLC mobile phase, the concentration of SCh was measured by HPLC (the parameters are described in section3.1.2.8.).

3.1.2.5. Preparation of lyophilized tablets containing microgranules with selegiline hydrochloride

For preparation of the freeze-dried tablets two different formulations of polymers as matrix forming agents were used. Microgranules of type XPS1 were incorporated in the pectin matrix, while microgranules of type XPS3 and XPS3P - in the Orabase®. Both solutions - 2% solution of

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pectin and Orabase (2.0 g pectin, 2.0 g sodium caramellose and 2.0 g of gelatin in 94.0 g of water) were prepared by dissolving polymers in warm (60 ºC) water and mixing with a magnetic stirrer.

Lyophilization was performed after dosing of the solutions and microgranules to blisters by two methods:

a) the blisters were filled with 0.35 g polymer solution, then microgranules were placed on the polymer solution layer and the blisters were filled up with polymer solution to 0.70 g;

b) microgranules were suspended in the polymer solution and then the blisters were filled up with 0.70 g of this suspension.

The composition of the mixture, transferred into blister cavity, was set that each lyophilized tablet contained 10 mg of SCh.

The filled blisters were transferred to a freeze-dryer and the lyophilization process was accomplished as shown in the table 3.

Table 3. Freeze-drying parameters

Stage Shelf temperature [ºC] Duration time [h] Chamber pressure [mbar]

Freezing - 45 2.5 -

Primary drying

- 20 - 5 + 5 + 20

2.5 14 3 2

0.08 0.08 0.08 0.08

Secondary drying + 30 1 - 2 0.08

3.1.2.6. Visual inspection of lyophilized tablets

The surface, edges, porosity and mechanical properties of the tablets were evaluated. The diameter was measured using a slide calliper. Photos of freeze-dried tablets were also made by a

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3.1.2.8. Quantitative analysis of selegiline hydrochloride by HPLC method

The quantitative analysis of SCh was performed by reverse phase HPLC method using mobile phase consisting of buffer and acetonitril 80:20 (v/v). The composition of the buffer was as follows: 0.1 mol/l ammonium dihydrogenphosphate and 0.08 % triethylamine (pH of the buffer was adjusted to 3.1 with phosphoric acid).

HPLC parameters:

volume injected: 20 µl;

flow rate: 1 ml/min;

column: Lichrospher RP-18 (250 mm, 5 m) analytical wavelength: 215 nm,

retention time: about 10 min;

For calculation of SCh concentration the calibration curve was used. For this purpose the stock solution (containing 1 mg/ml of SCh) was prepared and the following dilutions were made:

1, 5, 10, 20 and 50 g/ml. The linear relationship between the concentration and peak area is shown in fig. 9.

y = 12.7994x + 0.7688 R2 = 1.0000

0 100.000 200.000 300.000 400.000 500.000 600.000 700.000

0 10 20 30 40 50 60

ug/ml area

Fig.9. Selegiline hydrochloride calibration curve ( =215 nm)

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The concentration of SCh in the analyzed solutions C (µg/ml) was calculated using the following equation:

C = (A – 0.7688) / 12.7994, where:

A – peak area (in thousands).

The sample chromatogram obtained for a standard solution (50 µg/ml) is presented in fig.

10.

Fig. 10. Chromatogram of a standard selegiline hydrochloride solution (50 µg/ml).

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3.2. Incorporation of floating pellets into pressed tablets – tableting of floating pellets

3.2.1. Reagents, materials and equipment

Reagents

Aqueous dispersion of methacrilic acid copolymer – Eudragit NE 40 D, Röhm, Pharma Polymers - Darmstadt, Germany

Calcium hydrophosphate · 2H2O, Merck – Darmstadt, Germany Calcium hydrophosphate anhydrous, Merck, Darmstadt, Germany

Cross-linked polyvidon – Kollidon CL, BASF – Ludwigshafen, Germany Hydrochloric acid – 0.1 mol/l solution, P.O.Ch. – Gliwice, Poland

Lactose and polyvidon (96.5:3.5) for direct tableting – Ludipress LCE, BASF – Ludwigshafen, Germany

Lactose for direct tableting – Tablettose 80, Meggle – Walsenburg, Germany Macrogol 6000 S, Fluka Chemie AG – Buchs, Switzerland

Magnesium stearate, Riedel-de Hean, Seelze, Germany Mannitol, P.O.Ch. – Gliwice, Poland

Microcrystalline cellulose and guar gum – Avicel CE-15, FMC Europe, Bruccels, Belgium

Microcrystalline cellulose, particle size 100 µm – Avicel PH 102, FMC Europe, Brussels, Belgium

Microcrystalline cellulose, particle size 50 µm – Avicel PH 101, FMC Europe, Brussels, Belgium

Modified starch for direct tableting – Starch 1500, Colorcon – Dartford, UK

Monohydrated lactose and corn starch (85:15) for direct tableting – StarLac, BG Excipients &Tech. – Walsenburg, Germany

Povidone K-30, ICN Biomedicals – Aurora, Ohio, USA

Powdered cellulose, particle size 200 µm – Viva Pur 200, Rettenmeier & Söhne – Rosenberg, Germany

Powdered cellulose, particle size 70 µm – Arbocel 290, Rettenmeier & Söhne – Rosenberg, Germany

Sodium hydrocarbonate, Farm Impex – Gliwice, Poland

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Sorbitol, BASF - Ludwigshafen, Germany Talc, Ph. Eur.

Verapamil hydrochloride, Recordati – Mediolan, Italy Water purified through ion change and reversed osmosis

Equipment

Analytical electronic balance WAX 62 RPT 0246, Radwag – Radom, Poland Apparatus for granulation and coating Uni-Glatt, Dresden, Switzerland

Apparatus for water purification and ion exchange, system Elix 3, Millipore, Bedford, USA

Automatic hardness tester TBH 20, Erweka, Hensenstamm, Germany Electronic balance WPS 600/C RPT 9553, Radwag – Radom, Poland

Extruder model 25, Caleva – Dorset. UK Friabiliator TAP, Erweka – Frankfurt, Germany

High shear mixer Cucina, Philips – Budapest, Hungary

Microscope – type B1 223A (Motic, Wetzlar, Germany), equipped with digital camera – type GP-KR 222, Panasonic, Osaka, Japan

Ph. Eur. paddle apparatus, Erweka DT 800, Frankfurt, Germany Sieves 80, 100µm, 1.0 and 1.25 mm, Retsch, Germany

Single - stroke tablet press machine EK0, Korsch – Berlin, Germany, integrated with compression force measurement system UCT 5882/S, Spais – Gdansk, Poland

Spectrophotometer UV VIS V 530, Jasco – Tokyo, Japan Spheronizator 120 MP, Caleva – Dorset. UK

Tachometer, Caleva – Dorset, UK

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3.2.2. Preparation of floating pellet cores with verapamil hydrochloride by extrusion-spheronization method

Pellets were prepared by extrusion-spheronization method. The composition of pellet cores is showed in table 4.

Table 4. The composition of pellet cores.

Substance Composition of granulated mass (g) Floating pellet cores (dry mass)

Verapamil hydrochloride 20.5 20.0

Sodium hydrocarbonate 20.5 20.0

Avicel PH 101 46.4 45.3

Lactose 12.6 12.2

Povidone K-30 - 2.5

VH (verapamil hydrochloride) and excipients were dry mixed in a high shear mixer (Philips H 7720/06, Budapest, Hungary) for 2-3 min. The mixture was granulated using a granulation fluid (~50 g of 5% Polividon K-30 solution for 100 g of granulation mass) to achieve the appropriate level of moisture content for extrusion and spheronization. The wet granulation mixture was extruded through the sieve of 1.2 mm using the extruder model 25 (Caleva – Dorset, UK). The rotation speed of extruder carried out 30 rpm.

About 20 g portions of the wet extrudate were immediately introduced into the spheronizator’s chamber (Caleva - Dorset, UK). Spheronization process parameters were as follows:

the pressure of incoming air 2.0 bar;

the speed of spheronizator disk 1500-1550 rpm;

spheronization time 1.5 min.

Obtained pellets were dried in the dryer at 40ºC for 24 h. In order to achieve desired narrow size distribution (1.00 – 1.25 mm) the dried pellets were then separated using 1.00 mm and 1.25 mm sieves and stored in screw-capped, high-density polyethylene bottles.

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3.2.3. Coating of pellet cores with Eudragit NE 40 D

Different methods and coating agents can be combined to achieve a specific release profile.

In this case pellets cores were coated with coating mixture based on Eudragit® NE 40 D. The composition of coating mixture is showed in the table 5.

Coating mixture was prepared according to the following steps: the appropriate amount of Eudragit NE 40 D was introduced to a beaker with a magnetic stirrer. Next, approximately 2 g portions of talc were added during stirring process (before this procedure talc was sieved through a sieve size 80 m). At the same time Macrogol 6000 S was dissolved parallel in the water earlier and added to the beaker. Eventually all mixture was stirred for 2 h. At last coating mixture was perfused through the sieve size 200 m.

Table 5. The composition of coating mixture.

Substance Content (%)

Eudragit NE 40 D 43.2

Talc (ø 80 m) 6.9

Macrogol 6000 S 2.6

Purified water 48.6

Core coating was prepared by fluidized-bed/bottom-spray technique using Uni-Glatt apparatus (Glatt Systemtechnik, Dresden, Germany). The process parameters were as follows:

incoming air temperature 40 °C;

outgoing air temperature 30 °C;

incoming air pressure 6 bar, air pressure in spray nozzle 2 bar;

peristaltic pump feeding rate 3 ml/min.

At once 200 g of pellet cores were given in for coating and also about 130 ml of coating

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3.2.4. Tableting of floating pellets with verapamil hydrochloride by using impact tableting machine

The composition of tableting masses, which were used for compression using the laboratory single stroke Korsch tablet press (Korsch EK0, Berlin, Germany), is shown in tables 6 – 9. The masses were prepared in such way, that one 550 mg tablet contained 40 mg of VH. For tableting 12 mm diameter round punches were used. The matrix was filled up manually. The tableting mass was prepared for about 60 tablets in each batch.

Table 6. Tableting mass composition [%]; formulations I – IV.

Formulation Substance

I II III IV

Floating pellets with VH 38.2 38.2 38.2 38.2

Avicel PH 101 13.5 - - 51.3

Avicel PH 102 - 13.5 51.3 -

Mannitol 37.8 37.8 - -

Kollidon CL 9.5 9.5 9.5 9.5

Magnesium stearate 1.0 1.0 1.0 1.0

Table 7. Tableting mass composition [%]; formulations V – VIII.

Formulation Substance

V VI VII VIII

Floating pellets with VH 38.2 38.2 38.2 38.2

Avicel PH 102 13.5 13.5 - 13.5

Tablettose 80 37.8 - - -

Ludipress LCE - 37.8 - -

Arbocel 290 - - 51.3 -

D-Sorbitol - - - 37.8

Kollidon CL 9.5 9.5 9.5 9.5

Magnesium stearate 1.0 1.0 1.0 1.0

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Table 8. Tableting mass composition [%]; formulations IX – XII.

Formulation Substance

IX X XI XII

Floating pellets with VH 38.2 38.2 38.2 38.2

Avicel PH 102 13.5 13.5 13.5 13.5

Polividon K-30 37.8 - - -

Star Lac - 37.8 - -

Starch 1500 - - 47.3 -

Calcium hydrophosphate 2H2O

- - - 37.8

Kollidon CL 9.5 9.5 9.5 9.5

Magnesium stearate 1.0 1.0 1.0 1.0

Table 9. Tableting mass composition [%]; formulations XIII – XVI.

Formulation Substance

XIII XIV XV XVI

Floating pellets with VH 38.2 38.2 38.2 38.2

Avicel PH 102 13.5 13.5 13.5 13.5

Calcium hydrophosphate anhydrous

37.8 - - -

Vivapur 200 - 37.8 - -

Avicel CE-15 - - 37.8 -

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3.2.5. Analysis of pellets and tablets

3.2.5.1. Measurement of film thickness

In order to determine the film thickness twenty randomly selected floating pellets were coss-sectioned. Film thickness was measured using microscope (Motic, Wetzlar, Germany) equipped with digital camera (Panasonic, Osaka, Japan), connected to PC. For survey of film thickness software image analysis Multi Scan program v. 12.07 (Computer scanning systems, Warsaw, Poland) was applied.

3.2.5.2. In vitro drug release test from pellets

Dissolution studies of VH (verapamil hydrochloride) pellets (equivalent to 40 mg VH) were conducted using the Ph. Eur. paddle apparatus, Erweka DT 800 (Erweka, Frankfurt, Germany). Test specifications were as follows: USP I paddle rotating at 75 rpm with 750 ml of hydrochloric acid (0.1 mol/l) maintained at temperature of 37 ± 0.5 °C as dissolution medium and accurate amount floating pellets corresponding to 120 mg VH. The 5 ml samples were withdrawn at 1 h time intervals during a 6 h time period, and the volume was immediately replaced with a fresh medium. The concentration of VH in the samples, after 5-fold dilution, was analyzed spectrophotometrically (Jasco V-530, Jasco Corporation, Tokyo, Japan) at a wavelength 278 nm.

3.2.5.3. Tablets appearance and size

Tablets were estimated visually especially paying attention on the surface and edge.

Tablets thickness and diameter was measured using a slide calliper with 0.1 mm precision.

3.2.5.4. Content of VH in tablets

10 randomly selected one series tablets were pulverized in mortar. By means of analytical balances four accurate samples of 550 mg (what correspond tablet mass) were weighted. Every sample was dissolved in 100 ml of 0.1 mol/l hydrochloric acid in volumetric flask. The flask content was shaken throughout 6 h at 37°C ± 0.5°C and then incubated in the same temperature during 18 h. The 5 ml samples were withdrawn using a pipette, which ends with a glass filter.

The concentration of VH in the samples, after 5-fold dilution, was analyzed spectrophotometrically at a wavelength 278 nm.

The content of VH in each tablet should be comprised between 85 and 115% of average content.

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3.2.5.5. Determination of single tablet mass uniformity

Single tablet mass uniformity determination was performed according to Eur. Ph. 5.

3.2.5.6. Evaluation of tablets resistance to crushing (hardness of tablets)

Tablets resistance to crushing was determined by using automatic hardness tester type TBH 20 (Erweka, Hensenstamm, Germany). Resistance to crushing was evaluated for 10 randomly selected tablets.

3.2.5.7. Evaluation of tablets friability

The tablets’ friability was performed according to Eur. Ph. 5.

3.2.5.8. In vitro drug release test from tablets and flotation start time ofpellets

The test was performed according to p. 4.2. Six samples were examined; each of them contained three randomly selected tablets. During in vitro test, the process of tablet disintegration in appropriate beakers was observed. Consequently, by means of stop-watch, the time of pellets flotation start was measured. As pellet flotation start time was considered the moment, in which almost all pellet agglomerates undergo disintegration into individual floating pellets.

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