Preparation of ritonavir nanosuspensions by microfluidization using polymeric stabilizers: I. A Design of Experiment approach

    Preparation of ritonavir nanosuspensions by microfluidization using polymeric stabilizers: I. A Design of Experiment approach Alptug Karakucuk, Nevin Celebi, Zeynep Safak Teksin PII: DOI: Reference:

S0928-0987(16)30168-3 doi: 10.1016/j.ejps.2016.05.010 PHASCI 3574

To appear in: Received date: Revised date: Accepted date:

29 January 2016 6 May 2016 10 May 2016

Please cite this article as: Karakucuk, Alptug, Celebi, Nevin, Teksin, Zeynep Safak, Preparation of ritonavir nanosuspensions by microfluidization using polymeric stabilizers: I. A Design of Experiment approach, (2016), doi: 10.1016/j.ejps.2016.05.010

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ACCEPTED MANUSCRIPT Preparation of ritonavir nanosuspensions by microfluidization using polymeric

Alptug Karakucuk, Nevin Celebi*, Zeynep Safak Teksin

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stabilizers: I. A Design of Experiment approach

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Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Technology, Etiler

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06330 Yenimahalle Ankara/ Turkey

* Corresponding author. Tel: +90 312 202 30 49; fax: +90 312 223 50 18

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E-mail addresses: [email protected], [email protected]

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Abstract

The objective of this study was to prepare ritonavir (RTV) nanosuspensions, an anti-HIV protease inhibitor, to solve its poor water solubility issues. The microfluidization method

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with a pre-treatment step was used to obtain the nanosuspensions. Design of

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Experiment (DoE) approach was performed in order to understand the effect of the critical formulation parameters which were selected as polymer type (HPMC or PVP),

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RTV to polymer ratio, and number of passes. Interactions between the formulation variables were evaluated according to Univariate ANOVA. Particle size, particle size distribution and zeta potential were selected as dependent variables. Scanning electron microscopy, X-ray powder diffraction, and differential scanning calorimetry were performed for the in vitro characterization after lyophilization of the optimum nanosuspension formulation. The saturation solubility was examined in comparison with coarse powder, physical mixture and nanosuspension. In vitro dissolution studies were conducted using polyoxyethylene 10 lauryl ether (POE10LE) and biorelevant media (FaSSIF and FeSSIF). The results showed nanosuspensions were partially amorphous and spherically shaped with particle size ranging from 400 to 600 nm. Moreover, 0.1- 0.4 particle size distribution and about -20 mV zeta potential values were obtained. The nanosuspension showed a significantly increased solubility when compared to coarse powder (3.5 fold). Coarse powder, physical mixture, nanosuspension and commercial product dissolved completely in POE10LE; however, cumulative dissolved values reached ~20% in FaSSIF for the commercial product and nanosuspension. The

ACCEPTED MANUSCRIPT nanosuspension showed more than 90% drug dissolved in FeSSIF compared to the commercial product which showed ~50% in the same medium. It was determined that RTV dissolution was increased by nanosuspension formulation. We concluded that DoE approach is useful to

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develop nanosuspension formulation to improve solubility and dissolution rate of RTV.

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Keywords: Ritonavir, Nanosuspension, Microfluidization, DoE, HPMC. 1. Introduction

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The solubility of poorly soluble drugs limits dissolution rate, absorption and bioavailability, and it negatively affects the development of formulations and potential

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drug effects (Gao et. al., 2012). Some specific conventional approaches, such as physical modifications like micronization, using co-solvents, polymorphs, solid dispersions or cyclodextrins and chemical modifications like prodrugs or salt forms have

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been suggested to improve the solubility of poorly soluble drugs (Lanuer and Dressman,

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2000; Gulsun et.al., 2009). However, further approaches are needed because of the disadvantages and limited applications of the conventional methods that can apply only

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to a certain number of drug molecules, drug needs to have some specific physicochemical properties, to have the proper molecular size to fit into the cyclodextrine ring or bioavailability deficiencies of micronization and using high amount of co-solvents cause toxicity problems. Therefore, to overcome these problems, nanocrystal technology has been developed (Rabinow, 2004; Keck and Müller, 2006). The term nanocrystal was first defined at the beginning of 1990’s and the first product, Rapamune®, was commercialized in 2000. Nanocrystal is a technology which provides nanometer range particle sizes with increased surface area to improve saturation solubility and dissolution rate, and hence the bioavailability of Biopharmaceutics Classification System (BCS) Class II and IV drug molecules. The nanometer range particle sizes reduce fed/fasted variability and also inter-subject variability (Müller et.al., 2001). Drug nanocrystals consist of pure drug and they are often prepared in an aqueous medium with polymers or surfactants as stabilizing agents, so they are also called nanosuspensions (Möscwitzer, 2013). Nanosuspensions are prepared with a minimum amount of stabilizing agents, and typically they have particle sizes of between

ACCEPTED MANUSCRIPT 100-1000 nm (Teeranachaideekul et.al., 2008). Surfactants and polymers, or combinations of them, can be used to stabilize nanosuspension systems and they provide steric or electrostatic protection thereby protecting the system from aggregation,

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sedimentation and crystal growth (Ostwald ripening) (Wu et.al., 2011; MeriskoLiversidge and Liversidge, 2008). It is something of a challenge to select the best

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stabilizing agent type and concentration while developing nanosuspension formulations

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(Keck and Müller 2006). In nanosuspension formulations, the active pharmaceutical ingredient (API) to stabilizer ratio can vary from 1:3 to 50:1 (Eerdenbrugh et.al., 2008) or

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1:20 to 20:1 (Dolenc et.al., 2009).

There are two main approaches to produce nanosuspensions, using either top-down or

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bottom-up technologies (Guo et.al., 2013). Wet ball milling or high pressure homogenization processes can be classified as the top-down method and the classical precipitation method is also called a bottom-up method (Müller et.al., 2011). In the

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bottom-up method, drug molecules dissolve in an organic solvent and then an

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antisolvent is added to the solution to precipitate the particles to the nanometer size (Katteboinaa et.al., 2009). One of the top-down method, wet ball milling or pearl milling Elan),

is

one

of

the

most

popular

technology to

produce

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(NanoCrystalTM,

nanosuspensions (Müller et.al., 2011). Milling pearls which are made of zirconium, ceramic or stainless steel rotate at high speed in the macro suspensions to obtain nanosuspensions (Keck and Müller, 2006). The process can be continued for hours or even days. The other top-down method is the high pressure homogenization method has divided into two sub-methods as microfluidization or piston gap homogenization. Microfluidization is performed by a jet stream homogenizer with Z or Y-type chambers (IDD-PTM Technology, SkyPharma). Piston gap homogenization can be processed with aqueous dispersions (Dissocubes®, SkyPharma) or water-free/water reduced media (Nanopure®,

Pharmasol).

Currently

some

combination

technologies

such

as

ARTcrystal®, smartCrsytal® or NanoEdge® are used to overcome challenges encountered with brick dust drugs (Shegokar and Müller 2010; Scholz et.al., 2014). Lyophilization or spray dry processes can be used to obtain dry forms of nanosuspensions to prepare solid dosage forms and provide long term physical and chemical stability (Wu et.al., 2011).

ACCEPTED MANUSCRIPT Design of Experiment (DoE) is a key factor of Quality by design (QbD), and QbD helps researchers to understand the effects of critical formulation and process variables on product quality (Ahuja et.al., 2015). Nanosuspension formulations can be developed

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using statistical approaches and experimental designs to reduce number of experiments, to identify critical process parameters and reveal the interactions between formulation

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variables (Salazar et.al., 2011).

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Ritonavir was chosen as the model drug for this study. It is a BCS Class II anti HIV component (Sinha et.al., 2010). It has very low water solubility and dissolution rate;

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however, it has high permeability.

The objective of this study was to investigate critical formulation attributes and process on

microfluidization

method

influencing

the

quality

of

ritonavir

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parameters

nanosuspension.

Design of experiment (DoE) approach was performed to optimize nanosuspension

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formulation. Polymer type, ratio and number of passes were selected as independent

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variables. Full factorial design was utilized; interaction between formulation variables was investigated and the particle size, the zeta potential and the particle size distribution

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were evaluated as response variables. 2. Materials and methods 2.1.

Materials

The materials used in the study have been obtained in the following way. Ritonavir (RTV) was kindly provided by Mylan Pharmaceuticals, India. (Hydroxypropyl) methyl cellulose (HPMC) (3 cps) was gifted from Colorcon Limited (Istanbul, Turkey). SIF ® Powder was obtained from Biorelevant.com (UK). D(-)-Mannitol and Sodium Dodecyl Sulfate (SDS) were purchased from Merck (Darmstadt, Germany). Polyvinylpyrrolidone K30 (PVP K30) and polyoxyethylene 10 lauryl ether (POE10LE) was purchased from Sigma Aldrich (USA). Other chemicals were HPLC of analytical grade.

2.2.

Preparation of ritonavir nanosuspensions

RTV nanosuspensions were prepared with the microfluidization method. Microfluidics LV1 (Microfluidizer® Processors, USA), a jet stream high pressure homogenizator, was used for this preparation. The RTV ratio was 2% w/w for all formulations. HPMC 3 cps

ACCEPTED MANUSCRIPT and PVP were selected as stabilizing agents. Firstly, HPMC or PVP was dissolved in distilled water under a magnetic stirrer and coarse RTV powder was dispersed in the polymer solution. The final ratios of RTV to polymer were 1:2, 2:1, 1:1 or 4:1. After

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complete wetness of the RTV particles was ensured, in order to prevent Microfluidics LV1 from chamber blockage by reducing particle size (d99) below 84 m pre-treatment

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step was performed using ultraturrax at 10.000 rpm for 10 minutes. Then macro

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suspensions with reduced particle size were transferred into the high pressure homogenizator (microfluidizer) with a Z-type 84 m chamber; different pass numbers (5,

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10, 20 or 30 passes) were applied at 30.000 psi pressure to obtain nanosuspensions. The temperature was carefully controlled in the high pressure homogenization process.

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Finally, nanosuspensions were collected. For the SDS combined formulations, polymer and SDS were dissolved in distilled water and the same process was repeated.

Design of Experiment (DoE)

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2.3.

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A DoE approach was used to determine the optimum nanosuspension formulation. After collecting the nanosuspensions, particle size (PS), particle size distribution (PDI), and

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zeta potential (ZP) values were measured. None of the PVP stabilized formulations showed particle sizes below 1000 nm, and because of that the PVP formulations were excluded from the statistical analysis. For the statistical design, Design Expert® Version 9.0 was used and PS, PDI, ZP were selected as dependent variables while the number of passes on the high pressure homogenization process (5, 10, 20, 30 passes) and the HPMC ratio (0.5%, 1%, 2%, 4%) in formulations were selected as independent variables. 4 2 (4 levels, 2 factors) experiments with randomized designs, three replicates and a total number of 48 experiments were performed. Interaction between formulation variables was analyzed using Univariate ANOVA at p<0.05 as the minimum level of significance using IBM® SPSS® Statistics Version 20.

2.4.

Particle size, particle size distribution and zeta potential

PS, PDI and ZP were measured after the RTV particles were dispersed in the polymer solutions, after the pre-treatment step, and after the high pressure homogenization and

ACCEPTED MANUSCRIPT lyophilization processes. The samples were dropped into the cuvettes at about 10 L and diluted up to 5 mL with distilled water, and measured immediately. The initial particle size and the particle size of the macro suspensions were measured with Symphatec

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HELOS (Symphatec GmbH, Clausthal-Zellerfeld, Germany). Measurement of the PS and PDI values of the RTV nanosuspensions were conducted by photon correlation

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spectroscopy and the zeta potentials of the nanosuspensions were measured by

Short term physical stability

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2.5.

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Malvern ZetaSizer (Malvern Instruments, UK).

Short term physical stability studies of ritonavir nanosuspensions stabilized with HPMC

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were performed at 25 oC. PS values were measured as soon as the nanosuspensions were collected. The measurements were repeated after one day, one week and one-

Sodium dodecyl sulfate combination of optimum nanosuspension formulation

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2.6.

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month of storage times.

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The physical long-term stability of nanosuspensions can be provided using a combination of polymer and surfactant. The ZP value of the optimum nanosuspension formulation of RTV was below -20 mV which is a desired value for long term stability. SDS was used to increase the ZP value. Different SDS ratios such as 0.1%, 0.2%, 0.4%, and 1% were added to the formulations. The proper SDS ratio was selected according to ZP value.

2.7.

Lyophilization of nanosuspensions

The lyophilization process was performed to obtain dry nanosuspensions for the preparation of solid oral dosage forms, such as tablets or hard gelatin capsules. Dry nanosuspensions can also show better long-term stability results. Approximately 2 mL of the nanosuspensions were frozen at -80 oC for 2 hours. The process was continued with freeze drying at -50 oC, 0.021 mbar for 40 hours using Christ Alpha 1-2 LD Freeze Dryer. The lyophilization process can cause uncontrollable increment of particle size, so different amounts of mannitol (0, 1%, 2.5%, 5%, 10%) were added to the formulations as

ACCEPTED MANUSCRIPT a cryoprotectant. The optimum mannitol ratio was selected according to repeated

2.8.

Differential scanning calorimetry (DSC)

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measurements of PS, PDI and ZP values after lyophilization.

DSC was performed to determine the melting points of the samples and also to evaluate

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possible polymorphic transformations of the RTV in the nanosuspensions; Shimadzu

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DSC 60 was used for this purpose. The heating rate of the instrument was 10 oC/min from 25 oC to 300 oC. Each sample was weighed at approximately 2 mg and then placed

Scanning electron microscopy (SEM)

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2.9.

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in aluminum pans, crimped and sealed. The temperature was calibrated with indium.

The morphological analysis of the formulations was carried out by scanning electron microscopy, using Quanta 400F Field Emission at 5-20 kv acceleration voltage. The

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samples were coated with gold-palladium before scanning.

2.10. X-ray powder diffraction (XRPD)

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The XRPD patterns of the ritonavir, the physical mixture and the nanosuspension were collected with a Rigaku Ultima-IV powder diffractometer. All the samples were analyzed from a scanning range angle of 5-60o 2 at the rate of 1o/min at 40 kv.

2.11. Analysis of RTV concentration HPLC or UV/VIS methods were used to determine the RTV concentrations. The methods were validated according to accuracy, precision, repeatability, specificity, detection limit, quantitation limit, linearity and range.

2.11.1.

UV Spectrophotometric method

The UV spectrophotometric method was employed to determine the concentration of RTV in the nanosuspensions, in 0.06M POE10LE for dissolution studies and in distilled water for solubility studies (Shimadzu UV-1700). RTV was dissolved in 0.06M POE10LE for the UV spectrophotometric method. A determined weight of RTV was dissolved in a

ACCEPTED MANUSCRIPT distilled water and methanol was used as a co-solvent. All the samples were analyzed at 240 nm.

HPLC method

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2.11.2.

The HPLC method was used to determine the concentration of RTV in FaSSIF and

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FeSSIF for dissolution studies on the Hewlett Packard 1050 Series. The RTV was

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separated by C18 (RV) Thermo Hypersil Gold 250 x 4.6, 5 m column. The mobile phase consisted of acetonitrile, and 0.05M phosphoric acid (47:53, v/v) was run at 1.0

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mL/min. The injection volume was 25 L. Detection was 235 nm for FaSSIF, and at 248

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nm for FeSSIF.

2.12. Solubility studies

8 mg of RTV, and nanosuspensions or physical mixtures equal to 8 mg of RTV, were

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weighed and dispersed in distilled water. The flasks were shaken for 48h at 37oC. The

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samples were filtered through 0.45 m filters and analyzed by UV spectrophotometer.

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2.13. In vitro dissolution studies In vitro dissolution studies were conducted to make comparisons with Norvir®, coarse powder of RTV, physical mixtures and nanosuspension, using USP Apparatus II (Varian VK-7000). These studies were performed in sink conditions. The samples were put into transparent hard gelatin capsules (number: 0) and dropped into vessels. Norvir® and coarse powder contained 100 mg of RTV. Nanosuspensions and physical mixtures contained equal to 40 mg and 30 mg of RTV, respectively. The dissolution profiles were evaluated by cumulative drug dissolved (%) to time. Each experiment was performed in triplicate and all the samples were 4 mL and filtered through 0.45 m filters for the UV Spectrophotometric method and through 0.22 m filters for the HPLC method before analysis. 2.24 g of SIF® powder was weighed and dissolved in one liter of pH 6.5 buffer and 11.2 g SIF® powder was weighed and dissolved in one liter of pH 5.0 buffer to prepare FaSSIF and FeSSIF media, respectively. A model independent similarity factor (f2) was calculated to determine the similarity of the dissolution profiles. The in vitro dissolution study design is summarized in Table 1.

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TABLE 1

3.1.

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3. Results and Discussion

Particle size, particle size distribution, and zeta potential results

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PS, PDI and ZP values of nanosuspensions were measured at the different numbered

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passes in the microfluidization process. The initial particle sizes of the coarse suspensions (z-average) were about 8.50 m. The pre-treatment step using ultraturrax

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was provided below 84 m particle size for d99 value and it helped to prevent interaction chamber blockage on microfluidics. One pass on the microfluidization process reduced

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particle size to approximately 4 m. The nanosuspensions were obtained after five passes, considering a PS below 1000 nm (Fig. 1 and Fig. 2). According to the PS and PDI results, we decided to continue the statistical analysis and other characterization

FIGURE 2

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FIGURE 1

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nanosuspensions.

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studies with HPMC stabilized nanosuspensions, not with the PVP stabilized

The zeta potential values were found below 20 mV for all nanosuspensions (Table 2). The zeta potential effects, especially the long term stability of nanosuspensions and ideal nanosuspension formulations, have above 20 mV of ZP value in combination with steric stabilization (Wang et.al., 2013). Combinations of stabilizing systems such as polymer-polymer, polymer-surfactant or surfactant-polymer can be used for the stable nanosuspension formulations. In this study, we tried to improve the ZP using a combination of SDS for the final formulation (Section 3.4).

TABLE 2

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3.2.

Effect of formulation variables on physical characteristics of RTV nanosuspensions and final formulation selection

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HPMC ratio and number of passes on microfluidization process were selected as the critical formulation variables. The software Design Expert® 9.0 was used to design

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experiments and optimize the parameters on surface graphs. Univariate ANOVA was

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used to determine the interactions between the formulation variables, and create a

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model equation using IBM® SPSS® Version 20 (Table 3 and Fig. 3).

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TABLE 3

FIGURE 3

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TABLE 5

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TABLE 4

The interaction between percentages of HPMC and number of passes was found significant (p<0.05) for PS and PDI values (Table 4 and Table 5). This means that the particle size was effected by both the HPMC percentage and the number of passes at the same time. Model equation is; Y= a0 + aiXi + ajXj + aijXiXj + 

where Y is the response (PS or PDI), the a’s are parameters whose values are to be determined, Xi is a variable that represent factor number of passes, Xj is a variable that represents factor polymer ratio,  is a random error term and XiXj represents the interaction between Xi and Xj. Equations of the models for PS and PDI;

PS: Y= +889.66 – 156.28Xi – 146.97Xj – 63.08XiXj

ACCEPTED MANUSCRIPT PDI: Y= +0.37 + 0.067Xi – 0.081Xj – 0.044XiXj

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The interaction can be explained by interaction graphs (Fig. 4 and Fig. 5). The optimum formulation was selected according to interaction graphs and also surface graphs for PS

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and PDI values. 20 passes showed better PS results when the RTV to HPMC ratio was

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1:2 (4% HPMC). PDI values were close at the same concentration of HPMC and number of passes. The study was continued with the optimum formulation which was prepared

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with 4% HPMC and 20 passes.

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FIGURE 4

The

Short-term physical stability

short-term

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3.3.

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FIGURE 5

physical stability results were

evaluated after repeating the

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measurements after one day, one week and one month of PS for the HPMC stabilized nanosuspensions (Fig. 6.). HPMC provided good physical stability results at all pass numbers and ratios. The nanosuspensions with ZP values below -20 mV presented proper stability in the short term, which accorded with results of Chao Hong et. al. (2014), who found similar observations for stericly stabilized nanosuspensions. In our study, the particle size of some nanosuspensions decreased when compared to the initial day of measurement, which was not expected, but Chao Hong et.al. (2014), encountered the same situation in their study, and explained it as systemic temperature fall. FIGURE 6

3.4.

Optimization and lyophilization of final nanosuspension

Optimization of the nanosuspensions was completed by using SDS. HPMC provided steric stabilization and in combination with SDS provided better ZP values (Wang et.al., 2013) for long term physical stability, by providing both electrostatic and steric

ACCEPTED MANUSCRIPT stabilization (Dolenc et.al., 2009). The optimum amount of SDS was found after preparing the nanosuspensions with different concentrations of SDS (Table 6. and Table

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7.)

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TABLE 6

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TABLE 7

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The SDS concentration of nanosuspensions did not provide a higher ZP value more than 0.8%. The HPMC and SDS combination seemed to be sufficient for both the steric

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and the electrostatic stabilization of the nanosuspensions. Lyophilization of the nanosuspensions was completed with the optimum formulation, which was RTV: HPMC: SDS (1:2:0.4) at 20 passes.

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Lyophilization of the HPMC stabilized nanosuspensions was performed with and without

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mannitol as a cryoprotectant agent. A cryoprotectant is used to protect nanosuspensions from particle aggregation (Wang et.al., 2013). Four different amounts of mannitol were

formulation.

TABLE 8

TABLE 9

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used (Table 8. and Table 9.) and the results were compared in without mannitol

The PS, PDI and ZP values of nanosuspensions did not change when mannitol was not added. Mannitol free nanosuspension formulations were therefore chosen for further studies. The lyophilized nanosuspensions were also good at re-dispersing in water. Mauludin et.al., (2009), also reported that cryoprotectant free formulations of nanosuspension can be re-dispersed easily. 3.5.

Differential scanning calorimetry (DSC)

The melting point of RTV was found to be 126.29 oC for coarse powder (Fig. 7). DSC curves were used to determine the potential polymorphisms. Physical mixtures of RTV,

ACCEPTED MANUSCRIPT HPMC and SDS did not change the melting point as expected. However, in the nanosuspension formulations the melting point of RTV shifted to 122.44 oC (Fig 8.). This was because the microfluidization process caused particle size reduction in the

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nanometer range (Zhang et.al., 2013). According to the DSC curves, the crystalline state

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of RTV remained unchanged.

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FIGURE 7

Scanning electron microscopy (SEM)

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3.6.

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FIGURE 8

The morphological analysis of RTV was evaluated by SEM (Fig. 9.). The initial shape of RTV particles was that of rods. After HPMC and SDS were added to the formulation, the

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surface of the rod shaped particles seemed smooth. Lyophilized nanosuspension

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exhibited a spherical shape of between 200 and 600 nm particle size. The morphological change of the particles was a result of the high pressure homogenization process,

FIGURE 9

3.7.

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collision and shear forces (Leng et.al., 2014).

X-ray powder diffraction (XRPD) analysis

The XRPD study was performed to understand the crystal properties of the lyophilized nanosuspensions (Fig.10).

Coarse powder RTV exhibited a crystalline state.

Lyophilization and also high pressure homogenization after pre-treatment step effected the crystalline structure (Han et.al., 2014). Lyophilized nanosuspension patterns indicated partially amorphous crystal of RTV in our study.

FIGURE 10

3.8.

Solubility studies

ACCEPTED MANUSCRIPT Particle size in the nanometer range provides an increased curvature in the surface of raw material and it results in an increase in dissolution pressure and hence an enhancement of saturation solubility and dissolution rate, according to the Ostwald-

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Freundlich equation (Ahuja et.al., 2015; Romero et.al., 2015). The solubility studies were conducted in distilled water and the saturation solubility of RTV was found to be

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13.9 μg/mL. Because of the SDS acting as a surface active ingredient, the solubility of

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RTV increased 2.1 fold. The saturation solubility of the lyophilized nanosuspension

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formulation of RTV was enhanced over 3.5 times (Table 10).

3.9.

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TABLE 10

In vitro dissolution studies

The in vitro dissolution studies were conducted using in vitro media consisting of

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POE10LE, FaSSIF and, FeSSIF. FaSSIF media simulates fasting conditions in the

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proximal intestine. FeSSIF media can reflect the effects of lower pH and higher buffer capacity and osmolality, which FaSSIF media does not do. FeSSIF media also provide

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benefits that enable us to understand interactions between the drug and ingested food components (Klein, 2010).

The dissolution profiles of coarse powder RTV, physical mixture, and nanosuspension, as well as the commercial product (Norvir®) in the 0.06M POE10LE, FaSSIF, and FeSSIF are shown in Fig.11. Coarse powder, physical mixture, nanosuspension and Norvir® dissolved completely in POE10LE. However, cumulative dissolved values reached ~20% in FaSSIF for the commercial product and nanosuspension. The nanosuspension showed more than 90% of the drug dissolved in FeSSIF, compared to Norvir® (~50%) in FeSSIF. Also, the solubility in FeSSIF was higher than that in FaSSIF. It was found that RTV solubility was also affected by concentrations of sodium taurocholate and lecithin. The similarity factor (f2) was also determined: the dissolution profiles of nanosuspension and

Norvir®

were

similar

in

POE10LE

and

FaSSIF

(f2=50).

However,

the

nanosuspension and Norvir® did not give similar dissolution curves with f 2=32 for FeSSIF.

ACCEPTED MANUSCRIPT As a result, RTV dissolution was increased by the nanosuspension formulation. The Noyes Whitney equation can help to explain the increased dissolution rate of nanosuspensions. Nanometer particle size provides a significantly increased surface

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area, which results in an improvement of dissolution rate. The particle size of the nanosuspension also affected the diffusion distance (h) which, in turn, can also affect

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the dissolution profile (Sharma et.al., 2015).

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FIGURE 11

4. Conclusion

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RTV nanosuspensions were successfully prepared by a microfluidization method. HPMC stabilized nanosuspensions showed better particle size and particle size distribution results than PVP stabilized nanosuspensions. HPMC stabilized nanosuspensions had

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good short term stability. The Design of Experiment (DoE) approach allowed

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researchers to consider the effect of critical formulation variables on the final product quality. The interaction between number of passes on the microfluidizer and ratio of

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polymer were found to be effective. An optimum formulation was reached after determining the interactions between formulation variables. A combination of SDS with HPMC provided better zeta potential values, which were desired for electrostatic stabilization as well as steric stabilization. A dry nanosuspension was obtained after lyophilization without agglomeration, even in the cryoprotectant-free formulation. The solubility and dissolution rates increased significantly compared to coarse powder. It was determined that RTV dissolution is increased by a nanosuspension formulation and its FeSSIF solubility is higher than its FaSSIF solubility. Even though the enhancement in the solubility and dissolution of RTV was observed, further studies are needed for in vivo performance of ritonavir nanosuspension.

Acknowledgements This study was supported by a grant from The Scientific and Technological Research Council of Turkey (Project No: 113S842, TUBITAK). The authors would like to thank Mylan Lab. for providing ritonavir and Colorcon Ltd. for providing HPMC 3 cps as a gift

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Graphical abstract

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Ritonavir nanosuspensions were prepared by microfluidization using design of

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experiment. Interactions between formulation variables were analysed. Characterization

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of nanosuspensions were evaluated. In vitro dissolution studies showed enhanced dissolution rate due to reduced particle size.

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Table 1. In vitro dissolution study design Dissolution Media FaSSIF

Method

Paddle (USP App II)

Paddle (USP App II)

Paddle (USP App II)

Volume

900 mL

500 mL

500 mL

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37 oC  0.5 oC

RPM

50  4%

50  4%

Time

10, 20, 30, 45, 60, 75, 10, 20, 30, 45, 60, 10, 20, 30, 45, 60, 75,

Intervals

90, 120

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50  4%

37 oC  0.5 oC

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Temperature 37 oC  0.5 oC

FeSSIF

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0.06 M POE10LE

75, 90, 120

Analytical

UV

Method

Spectrophotometric

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(min.)

90, 120

HPLC

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Samples

HPLC

RTV Coarse Powder Physical Mixture Nanosuspension Norvir®

Table 2. Zeta potential values (mV) of the PVP and HPMC stabilized nanosuspensions. Number of Passes

Polymer Type

PVP (%)

HPMC (%)

0.5

1

2

4

0.5

1

2

4

5

-16.4

-14.33

-9.26

-7.59

-7.04

-5.98

-4.86

-3.48

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-13.2

-15.93

-7.44

-9.91

-5.94

-6.72

-5.08

-4.82

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-14.16

-13.9

-9.66

-7.83

-6.07

-5.92

-5.77

-5.07

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-16.3

-17.0

-7.86

-9.34

-6.12

-6.20

-5.51

-4.36

Table 3. Descriptive statistics for the HPMC stabilized nanosuspensions.

PS (nm)

N

Minimum

Maximum

Mean

SD

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536.6

1385.0

936.5

236.3

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0.757

0.367

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ZP (mV)

48

2.90

7.26

5.55

0.954

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PDI

Table 4. Interaction table of the HPMC stabilized nanosuspensions with regard to PS. F

Sig.

Intercept

148.7

Number of Passes

36287,6 .000

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HPMC%

57.1

.000 .000

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HPMC% * Number of Passes 427.1

Table 5. Interaction table of the HPMC stabilized nanosuspensions with regard to PDI.

Intercept

F

Sig.

1464.3 .000

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Source

21.9

.000

HPMC%

12.1

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HPMC% * Number of Passes 6.87

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Number of Passes

Table 6. SDS ratios of the nanosuspension formulations. Formulation F1 F2 F3 F4 F5

RTV (w/w %)

HPMC (w/w %)

SDS (w/w%)

2 2 2 2 2

4 4 4 4 4

0 0,2 0,4 0,8 2

Table 7. PS, PDI and ZP values of the nanosuspensions prepared by different SDS ratios. Formulation F1

Number of Passes 5 10

PS (nm) 868  35.2 1215  29.6

PDI 0.532  0.07 0.485  0.09

ZP (mV) -7.99  0.06 -6.20  0.93

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0.384  0.04 0.381  0.04 0.489  0.005 0.544  0.01 0.297  0.06 0.370  0.08 0.525  0.07 0.651  0.10 0.442  0.03 0.499  0.07 0.673  0.07 0.445  0.03 0.368  0.02 0.366  0.03 0.583  0.134 0.483  0.02 0.256  0.04 0.279  0.04

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F2

603  31.9 527  17.5 1024  95.4 660  29,7 530  10.9 522  1.5 935  144.9 825  38.0 541  14.6 622  31.9 1097  34.3 613  15.5 516  28.1 506  35.7 953  144.9 703  34.8 738  20.6 648  11.7

20 30 5 10 20 30 5 10 20 30 5 10 20 30 5 10 20 30

-4.93  0.44 -6.15  0.51 -15.43  0.43 -16.2  0.53 -16.23  0.47 -17.62  0.21 -17.2  0.85 -18.03  1.47 -17.3  0.67 -19.07  0.75 -21.47  0.85 -19.37  0.25 -21.3  0.85 -19.5  1.19 -18.9  0.85 -18.3  1.26 -21.3  1.17 -21.23  1.10

Table 8. Mannitol ratios of the nanosuspension formulations.

F1 F2 F3 F4 F5

RTV (w/w %) 2 2 2 2 2

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Formulation

HPMC (w/w %) 4 4 4 4 4

SDS (w/w %) 0,4 0,4 0,4 0,4 0,4

Mannitol (w/w %) 0 1 2,5 5 10

Table 9. PS, PDI and ZP values of the nanosuspensions prepared by different mannitol ratios. Formulation Initial F1 F2 F3 F4 F5

PS 562  20.6 563  6.61 679  8.20 535  6.90 668  5.32 534  24.6

PDI 0.368  0.006 0.425  0.04 0.308  0.05 0.421  0.04 0.145  0.01 0.399  0.05

ZP -20.0  0.95 -24.1  0.27 -23.2  0.76 23.1  0.36 -24.8  0.96 -20.0  0.62

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Solubility, μg/mL (mean  S.D)

Sample Coarse powder of RTV

13.91.21

RTV: HPMC: SDS (1:2:0.4) physical mixture

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RTV: HPMC: SDS (1:2:0.4) lyophilized nanosuspension

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48.82.93