Iranian Journal of Pharmaceutical Research (2017), 16 (4): 1335-1348 Received: January 2017 Accepted: June 2017
Copyright © 2017 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services
Original Article
Enhanced Dissolution Rate of Tadalafil Nanoparticles Prepared by Sonoprecipitation Technique: Optimization and Physicochemical Investigation Rayehe Teymouri Rada,c, Seyed Alireza Mortazavi a*, Alireza Vatanara b* and Simin Dadashzadeha Pharmaceutics Department, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran. bPharmaceutics Department, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. cStudentsʼ Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran. a
Abstract Nanocrystals of tadalafil, a poorly water-soluble drug, were successfully prepared by sonoprecipitation technique for improving the solubility and dissolution rate. Tween 80 was selected as an efficient surfactant to inhibit aggregation in stabilization of drug nanocrystals. Response surface methodology based on central composite design (CCD) was utilized to evaluate the formulation factors affecting the size of nanosuspensions. Under optimum conditions, relatively spherical nanocrystals with a mean particle size of 358.47 ± 11.95 nm were obtained. FTIR analysis indicated that the precipitated nanoparticles had the same chemical structure as the raw tadalafil. By DSC analysis, no substantial crystalline change was found in the nanocrystals compared with the unprocessed drug. In addition, the dissolution rate of the processed tadalafil nanocrystals in 120 min was significantly increased (3.61-fold) as compared to that of the raw material. Therefore, it was concluded that the sonoprecipitation technique could be a simple and useful technique to prepare poorly water-soluble drug particles with reduction in particle size, a narrow particle size distribution and enhanced dissolution properties. Keywords: Tadalafil; Poorly water-soluble drug; Antisolvent precipitation-ultrasonication method; Nanocrystals; Dissolution enhancement.
Introduction Tadalafil is a potent and selective cyclic guanosine monophosphate specific type (V) phosphodiesterase inhibitor used for the treatment of erectile dysfunction, benign prostatic hyperplasia and pulmonary arterial hypertension (1-3). The chemical structure of tadalafil is shown in Figure 1. Longer duration of action * Corresponding author: E-mail:
[email protected] [email protected]
(approximately 36 h) and minimized potential for vision abnormalities are the most important advantageous of tadalafil in comparison with other drugs in this pharmacological category (4). Since it belongs to class II of classification system, poor aqueous solubility and wettability is an important issue and in spite of good permeability, its bioavailability is limited by solubility and dissolution rate, leads to variability in blood concentrations and irreproducibility in clinical responses (5). Different groups have utilized various formulation strategies for improving the solubility and dissolution rate of tadalafil that the
Tween 80, sodium hydroxide (NaOH), monobasic potassium phosphate (KH2PO4), methanol, ethanol, dimethyl sulfoxide (DMSO), acetone and high-performance liquid chromatography (HPLC) grade acetonitril were obtained from Merck, Germany.
Teymouri Rad R et al. / IJPR (2017), 16 (4): 1335-1348
Deionized water was prepared with Millipore water purification system, Germany. In this method, nanoparticles could be formed in different ways such as pH controlled precipitation, antisolvent precipitation with or without surfactant and sonoprecipitation (18). Sonoprecipitation, a combination of antisolvent precipitation and ultrasoication, is an effective method of controlling the nucleation and crystallization process (19). Ultrasound amplifies the mass transfer when it propagates through a liquid medium, and initiates an important phenomenon Figure 1. Chemical structure of tadalafil (25). known as cavitation. Furthermore, it increases Figure 1. Chemical structure of tadalafil (25). micro-mixing, reduces particle growth and agglomeration, so it is possible to obtain most reported ones include preparation of solid particles with uniform size distribution (20). dispersion by using water soluble polymers and In a study, Xia et al. (21) succeed to prepare copolymers (such as poly(vinyl pyrrolidone) kMethods nitrendipine nanosuspensions with diameter of 30, poly(ethylene glycol) 6000, poloxamer 188 about 209 ± 9 nm with an enhanced dissolution and 407 (6), formation of inclusion complex rate and bioavailability. Also, the same results Preparationand of tadalafil Nanocrystals with ß-cyclodextrins microporous silica (7, by Sonoprecipitation were found in the preparation of carvedilol (22), 8), the use of amorphization methods (such as itraconazole (23), clarithromycin, cefixime, vitrification, cryogenic grinding, ballwere milling, glipizide other groups. Tadalafil nanosuspensions prepared throughnanosuspensions sonoprecipitationbytechnique. The spray drying and freeze drying) (9). However, In this technique, in addition of the type of to the bestexperimental of our knowledge a few have antisolvent stabilizers, process parameters process forstudies the preparation of tadalafiland nanosuspension is illustrated in been conducted in the field of nanoparticles such as the precipitation temperature, the power preparation by2. In antisolvent precipitationinputof and theorganic time length of ultrasonication play Figure a typical procedure, combination two solvents, acetone: DMSO ultrasonication method. an important role (21). Drug particle size reduction has emerged as Formulation parameters such as drug 4 an effective and versatile option for surmounting concentration, stabilizer concentration and solubility issue (10, 11). According to Noyesantisolvent to solvent ratio also need to be Whitney equation, dissolution rate increases optimized because of their important influence when the surface area increases by reducing the on supersaturation degree and nucleation rate size to nanometer (12). (24). Nanosuspension or nanocrystal suspension The aim of this study was to prepare tadalafil is a pure particulate system that is composed of nanosuspension by sonoprecipitation technique submicron (average particle size in the range of and optimize process parameters in order to 200-600 nm) colloidal dispersion with a surfactant enhance solubility and dissolution rate of the as the stabilizer (13). As the first time, in 2000, drug. nanosuspensions have been commercialized in the pharmaceutical market. Increased saturation Materials solubility, increased adhesiveness to surfaces/ The raw tadalafil was purchased from cell membranes and increased dissolution Dr. Reddy’s Pharmaceutical Co., Ltd., India. rate are special properties of nanosuspensions Tween 80, sodium hydroxide (NaOH), monobasic (14). Techniques used to produce drug potassium phosphate (KH2PO4), methanol, nanosuspensions can be classified into two major ethanol, dimethyl sulfoxide (DMSO), acetone groups: top-down and bottom-up technologies and high-performance liquid chromatography (15). One of the most bottom-up promising (HPLC) grade acetonitril were obtained from techniques is nanoprecipitation that is costMerck, Germany. Deionized water was prepared effective, rapid to perform and suited for scaling with Millipore water purification system, up (16, 17). Germany. 1336
Enhanced Dissolution Rate of Tadalafil Nanoparticles
Figure 2. Experimental process to prepare the tadalafil nanoparticles.
Figure 2. Experimental process to prepare the tadalafil nanoparticles.
Methods In the next step, precipitation initiated by dropPreparation of tadalafil Nanocrystals by wise adding of drug solution phase within 5 Sonoprecipitation min. As the nanosuspension emerged, size and Experimental design Tadalafil nanosuspensions were prepared polydispersity index (PDI) were evaluated. through Response sonoprecipitation technique. The Then, the design obtained were surface methodology based on central composite was nanosuspensions employed for experimental process for the preparation of concentrated by centrifugation at 16000 rpm tadalafil evaluation nanosuspension Figure i.e.,fordrug 50 min using an(A), ultracentrifuge of theis illustrated formulationin factors concentration stabilizer (3-30K, 2. In a typical procedure, combination of two Sigma, Germany) and washed three times with (B), antisolvent to solvent volumedeionized ratio (C) and mean Furthermore, particle size (y) the was content of organic concentration solvents, acetone: DMSO (88:12 water. v/v), was selected as water-miscible solvent drug was determined in supernatant by HPLC assessedwater as thewas response. details of designanalysis are shown Table 1. Based on theFinally, the and deionized used The as antisolvent. for incalculation of yield. Tadalafil coarse powder was completely obtained nanoparticle residue was frozen at results of preliminary experiments, the experimental range of each variable was chosen. dissolved in the organic phase and then was -80 °C for 24 h and subsequently lyophilized for ® filtered through a syringewere filter,designed PTFE membrane 48 h ®atsoftware a temperature of 58 °C under The experiments using Design-Expert (ver. 7.0.0, stat-ease , vacuum with pore size of 0.45 µm (Simplepure, USA) by using freeze dryer (Alpha 1-2 LDplus, Christ, to remove theTopossible particulate impurity. Germany). The driedrandomized. powder was then collected USA). reduce systematic errors, the experiments were completely A range of surfactants and polymers (such as and stored in air tight container for further use. By Analysis of collected in datagrades for responses, hydroxypropyl methylcellulose of 6, the relationship linking the main factors 15 and 50 cp, poly (ethylene glycol) 400 and Experimental design and their40 interactions the responses and presented a general form based on 6000, polyoxyl stearate, to poloxamer 188 were and determined Response surfaceas methodology 407, poly (vinyl pyrrolidone) k30, poly (vinyl central composite design was employed for in the following Equation (1). alcohol), sodium carboxymethyl cellulose, evaluation of the formulation factors i.e., drug Tween 80 and sodium lauryl sulfate) were concentration (A), stabilizer concentration (B), (1) screened as stabilizer and finally Tween 80 was antisolvent to solvent volume ratio (C) and mean selected in accordance with the size criteria (data particle size (y) was assessed as the response. � � ����������� � ∑ ������������ � ∑ ������������ Equ 1 not shown). The antisolvent phase was prepared The details of design are shown in Table 1. Based by dispersing a stabilizer in distilled water that on the results of preliminary experiments, the 6 was cooled to 5±1 °C in an ice-water bath and experimental range of each variable was chosen. treated with an ultrasonic probe (Hielscher The experiments were designed using DesignUP400S, 400W, 24 kHz, Germany) at power Expert® software (ver. 7.0.0, stat-ease®, USA). input of 280 W and a cycle of 0.5 per second. To reduce systematic errors, the experiments 1337
concentration (B), antisolvent to solvent volume ratio reverse (C) and mean (y) tadalafil was phase highsizeperformance The concentration ofparticle dissolved
liquid in the chromatography supernatant was (RP-HPLC) determined (Smart using al
assessed as the response. The details of design are shown in Table 1. Based on the
pump,high Smart line Diode liquid array 2800 UV detector,(RP-HPLC) Knauer, Germany) as was re reverse phase performance chromatography (Smart line 1000
results of preliminary experiments, the experimental range of each variable was chosen.
Cheng et a.l (26).2800 EZ UV CHROM software wasGermany) used to record evaluate pump, Smart lineand Diode array detector, Knauer, as wasand reported by
® The experiments Design-Expert software (ver. 7.0.0, stat-ease®, Teymouriwere Rad R designed et al. / IJPR using (2017), 16 (4): 1335-1348
collected during and following analysis. The Cheng and etcompletely a.l (26). EZ CHROM softwarechromatographic was used to record and evaluate USA). To reduce systematic errors, the experiments were randomized.
chroma the data
were completely (26). CHROM was used to record By Analysis of collected data randomized. for responses, the relationship mainEZ factors separation wasthefollowing accomplished usingsoftware a Perfectsil target C18 column (150 ൈ4.6 m collected duringlinking and chromatographic analysis. The chromatographic By Analysis of collected data for responses,
and evaluate the data collected during and analysis. separation(ODS-3, was accomplished usingprotected a Perfectsil C18 MZ, Germany) by atarget guard column (10×4.6mm) which wa column (150 ൈ4.6 mm, 5µm) interactions to the responses were determined The chromatographic separation was in the following Equation (1). and presented as a general form in the following accomplished using a Perfectsil target C18 (ODS-3, with MZ, the Germany) byAcetonitrile a guard column (10×4.6mm) which was(pH packed and 20 mm phosphate buffer 7.0) same Cprotected 18 material. Equation (1). column (150 4.6 mm, 5µm) (ODS-3, MZ, (1) A quadratic ®® model Equation Equation (2) (2) was was fitted fitted toto the the response response using the Design-Expert Design-Expert A quadratic model using the Germany) protected by ®a guard column of 40:60 was used as thethemobile phase. The column was(pH maintained at 25 and 20 mm phosphate buffer with the A quadratic model�Equation (2) was fitted toCthe response using Design-Expert ®same 18 material. ∑ ∑ � � ����������� ������������ � ������������ Equ 1 Acetonitrile (10×4.6 mm) which was packed with the same 7.0) in a ratio (2) was fitted to software. the response using the Design-Expert software. C18 material. Acetonitrile and 20 mM phosphate 60mobile min with the analytical mobile phase before injection. The software. of was Equ used1 asforthe column waswas maintained 6 40:60 equilibrated buffer (pH phase. 7.0) in The a ratio of 40:60 used as at 25 °C and (2) A quadratic model Equation (2) was fitted to the mobile phase. The column was maintained at (2) volume was 20with µL, the and analytical the mobilemobile phase phase was pumped isocratically at a flow r equilibrated min injection. ® for 60 the response using the Design-Expert software. 25 °C and equilibrated for 60 before min with the The injection (2) analytical mobile phase before injection. The ଶ was analyzed The at 283 and the retention theofdrug ଶଶ eluent was 20ߚߚµL, the phase pumped at atime flowofrate 1.0 ߚߚଵଵ ܣଶଶ ܤܣ ܥܣ nm ݕݕൌൌߚߚߚߚଵଵܣܣߚߚଶଶܤܤߚߚଷvolume ܣmL/min. ߚߚଷଷ ܥܥଶmobile ߚߚଵଶ ܤܣ ߚߚwas ܥܣ ଷܥܥ ଵଵ ଶଶ ܤܤand ଷଷ ଵଶvolume ଵଷ injection was 20 µL, isocratically and the mobile ଶଶ ଵଷ ଶ ଶ ଶ was pumped isocratically at a flow rate of ݕൌ ߚ ߚ ܣ ߚ ܤ ߚଷ ܥ ߚଵଵ ܣmin. ߚThe ߚଷଷphase ܥthe process ߚଵଶ ܤܣ ߚଵଷcalculated ܥܣ ଶଶ ܤyield was using thetime Equation ߚଷ ܥ ߚଵଵ ܣଶ ߚଶଶ ܤଶ ߚଷଷ ܥଶ ଵߚଵଶ ܤܣଶ ߚଵଷ ܥܣ ߚߚଶଷ…ܥܤ..Equ mL/min. The eluent analyzed at 283 andwas the retention the3.drug was 9.45 …ܥܤ..Equ 22was of 1.0 mL/min. Thenm eluent analyzed at 283ofnm ଶଷ and the retention time of the drug was 9.45 min. ߚଶଷ …ܥܤ..Equ 2 ߚଶଷ …ܥܤ..Equ 2 min. The yield of the process was calculated using thecalculated Equationusing 3. the The yield of the process was (3) A, Where yy represents represents the the predicted predicted response, response, A, BB and and CC represents represents the the independent independent Where Where y represents the predicted response, A, Equation 3. Where y represents thethe predicted response, A, and B and C represents the independent B and C represents independent variable dicted response, A, B andand C represents thethe independent variable and represents the coefficient. The The three three dimension dimension (3D) (3D) response response surface surface (3) variable ßß represents coefficient. ß represents the coefficient. The three dimension ሺ୭୲ୟ୪ୢ୰୳ିୈ୧ୱୱ୭୪୴ୣୢୢ୰୳ሻ ሺΨሻ ൌ ൈ ͳͲͲ Equ 3 variable and ß represents the coefficient. The using three dimension (3D) response ୭୲ୟ୪ୢ୰୳ surface (3D) response surface graphs were plotted e coefficient. The graphs three dimension (3D) response surface graphs were plotted using origin7.0 7.0 software accordingtotothe theequation. equation. were plotted using origin software according origin 7.0 software according to the equation. ሺ୭୲ୟ୪ୢ୰୳ିୈ୧ୱୱ୭୪୴ୣୢୢ୰୳ሻ ሺΨሻ ൌ equation. ൈ ͳͲͲ Equ 3 graphs were plotted using origin 7.0 software according to the ୭୲ୟ୪ୢ୰୳ gin 7.0 software according to the equation. Physicochemical Characterization of tadalafil nanocrystals Particle morphology Table 1. Numeric variables and their levels investigated in the CCD forpreparation preparation of and the tadalafil Particle size and zeta potential analysis The morphology of tadalafil Table 1. Numeric variables and their levels investigated in the CCD for of The mean particle size (z-average), nanocrystals was evaluated through field Table 1. nanosuspensions. Numeric variables and their levels investigated in the CCD for preparation of tadalafil nanosuspensions. tadalafil nd their levels investigated in the CCD for preparation of 8 polydispersity index (PDI) and zeta potential emission scanning electron microscope (S-4160, tadalafil werenanosuspensions. determined by photon correlation Range Hitachi, Japan). Prior to analysis, the samples Range andlevels levels and Variables Code spectroscopy (PCS) using Zeta-sizer (Nano-ZS, were diluted with 8ultra-purified water to obtain Variables Code Range and levels +1 -1.68 +1.68 -1.68 -1-1 00 +1 +1.68 Range and levels Malvern Instruments, UK). The real refractive a suitable concentration. Then, the samples were Variables Code e index and the imaginary refractive were spread on a sample holder, -1.68 index -1 0 +1 +1.68 dried under vacuum Drugconcentration concentration -1.68 -1Drug 0 +1 AA +1.68 3.2 10 20eventually 30coated with 36.8gold. 3.2 10 20 30 36.8 set (mg/mL) at 1.76 and 0.01, respectively. The z-average and (mg/mL) Drug concentration and PDI values were obtained by A 3.2 averaging 10 of 20 30 36.8 (mg/mL) 3.2 10 20 30 36.8 Stabilizer concentration Stabilizer three concentration measurements. Before the measurement, Fourier transformed infrared spectroscopy B 0.032 0.1 0.2 0.3 0.368 B 0.032 0.1 0.2 0.3 0.368 (%) (%) a small aliquot of nanosuspensions was diluted (FTIR) Stabilizer concentration B 0.032 0.1 0.2 0.3 0.368 (%) 0.032 0.1 Antisolvent/solvent 0.2 of deionized 0.3 0.368 with 5 mL water to have a suitable FTIR technique was applied to determine Antisolvent/solvent C 9.9:1 15:1 22.5:1 30:1 35.1:1 9.9:1 15:1 22.5:1 30:1 35.1:1 volumeratio ratio scattering intensity andCthen sonicated to create the molecular structures of raw tadalafil and volume Antisolvent/solvent C 9.9:1 15:1 22.5:1 30:1 between 35.1:1stabilizer and drug a homogenous suspension. the interaction ratio 9.9:1 15:1 volume 22.5:1 30:1 35.1:1 nanocrystals. FTIR spectra were recorded by Yield of nanoprecipitation process FTIR spectrometer (Nicolet Magna-IRTM, USA) Nanosuspensions were centrifuged (3-30K, within the spectral region of 400 and 4000 cm−1 PhysicochemicalCharacterization Characterizationofoftadalafil tadalafilnanocrystals nanocrystals Physicochemical Sigma, Germany) at 16000 rpm for 50 min. at a resolution of 2 cm−1. The IR spectra were Physicochemical Characterization of tadalafil nanocrystals The concentration of dissolved in the obtained in a KBr disc. ation of tadalafil nanocrystals supernatant was determined using a reverse Particle size and zeta potential analysis Particle size and zeta potential analysis phase high performance liquid chromatography X-ray powder diffraction (XRPD) Particle size and zeta potential analysis al analysis (RP-HPLC) (Smart line 1000 pump, Smart The crystal forms of the samples were The mean meanparticle particlesize size(z-average), (z-average),polydispersity polydispersityindex index(PDI) (PDI)and andzeta zetapotential potential were The line Diode array 2800 UV detector, Knauer, detected using a powderwere X-ray diffractometer The Germany) mean particle size reported (z-average), polydispersity index (IPDS (PDI) and zeta potential were The current and as was by Cheng and et al. II, STOE, Germany). erage), polydispersity index (PDI) and zetacorrelation potential were determined by photon photon correlation spectroscopy (PCS) (PCS) using using Zeta-sizer Zeta-sizer (Nano-ZS, (Nano-ZS, determined by spectroscopy and their interactions to the responses weremain determined presented general form the relationship linking the factorsand and their as afollowing chromatographic
+
determined by photon correlation spectroscopy (PCS) using Zeta-sizer (Nano-ZS, elation spectroscopy (PCS) using Zeta-sizer (Nano-ZS, Malvern Instruments, UK).The The realrefractive refractiveindex indexand and the imaginary refractive index Malvern Instruments, UK). real 1338 the imaginary refractive index Malvern Instruments, UK). The real refractive index and the imaginary refractive index he real refractive index and the imaginary refractive index
7
77 7
study
Enhanced Dissolution Rate of Tadalafil Nanoparticles
voltage using Cu Kαl radiation were 30 mA and 40 kV, respectively. The obtained data were typically collected from 1 to 40 with a step size of 0.06 at a rate of 1/s. the output is given as intensity versus 2θ. Differential scanning calorimetry (DSC) The thermal analysis was performed using differential scanning calorimeter (DSC-60, Shimadzu, Japan). Approximately, 3 mg of each samples was placed in an aluminum pan. The heating and cooling scans were performed from -10 °C to 320 °C at the heating and cooling rates of 10 °C/min in a dry N2 atmosphere. An empty aluminum pan was used as a reference. The melting temperature and enthalpy were calculated from the DSC thermograms.
the integral represents the area under dissolution curve between time zero and t. Saturation solubility The saturation solubility of raw tadalafil and tadalafil nanocrystals were evaluated in two mediums: deionized water and phosphate buffer (pH=7.4) at 25 °C. Excess amounts of lyophilized powder was dispersed in 10 mL of medium and placed on a stirrer for 48 h to ensure that the solubility equilibrium had been reached. The samples were centrifuged and the resulting supernatant was filtered and then analyzed by HPLC. The measurements were repeated three times.
+
Statistical analysis rate experiments of raw tadalafil and tadalafil nanocrystals were carriedThe reported data represented the mean Dissolution rate study value ± standard deviation (SD). Significance of Dissolution rate study The dissolution rate experiments of raw was evaluated using Student t-test and the USP 32 apparatus II (paddle) method. Phosphate buffer (pH difference 7.4) tadalafil and tadalafil nanocrystals were carried one-way ANOVA at the probability level of 0.05 The dissolution rate experiments of raw tadalafil and tadalafil nanocrystals were carried out according to the USP 32 apparatus II (paddle) using SPSS 19 for Windows (SPSS, Chicago, IL, DissolutionThe rate study dissolution medium. stirring speed and the bath temperature were method. Phosphate buffer (pH 7.4) was used Design-Expert® software (ver. 7.0.0, out according to the USP 32 apparatus II (paddle) method. PhosphateUSA) buffer and (pH 7.4) ® Theas dissolution rate experiments of raw tadalafil and tadalafil nanocrystalsstat-ease were carried the dissolution medium. The stirring speed , USA). .0 ± 0.5 was °C,used respectively. Unprocessed tadalafil (2 mg) tadalafil as the medium. The stirring100 speed andand the bath temperature were and thedissolution bath temperature were rpm and out according to the USP 32 apparatus II (paddle) method. Phosphate buffer (pH 7.4) 37.0 ± 0.5 °C, respectively. Unprocessed tadalafil Results and Discussion rpm and 37.0were ± 0.5added °C, respectively. Unprocessed tadalafilmedium. (2 mg) and tadalafil ntaining 2100 mgwas tadalafil) tonanocrystals 900 of speed dissolution usedmg) as theand dissolution medium. ThemL stirring and the bath temperature were (2 tadalafil (containing 2rpmmg tadalafil) were added to 900 mLmLofof Tadalafil nanocrystals (containing 2 mg wereUnprocessed added to 900 dissolution medium.nanocrystals were prepared and and 37.0 ± 0.5 °C,tadalafil) respectively. tadalafil (2and mg) 120 and tadalafil quivalent to 5100 mL were withdrawn after 10, 20, 30, 45, 60, 90 dissolution medium. Then, aliquots equivalent to optimized using sonoprecipitation technique. In Then, aliquots equivalent to2 5mgmL were withdrawn 45,this 60,medium. 90 and 120 nanocrystals (containing tadalafil) were20, added to45, 90010, mL20, of 30, dissolution 5 mL were withdrawn after 10, 30,after 60, 90 method, introduction of the drug solution to iately replaced and with120 themin same volume of phosphate buffer. All of and immediately replaced with the the antisolvent generates high supersaturation min Then, and immediately replaced with thewithdrawn same volume All of aliquots equivalent to 5 mL were after 10,of 20,phosphate 30, 45, 60, buffer. 90 and 120 same volume of phosphate buffer. All of samples and results in fast nucleation rate to produces assed throughminawere 0.45 µm syringe filter and injected into the HPLC replaced with the volume ofand phosphate All number of passed through 0.45µm µmsame syringe filter and ainto large of nuclei, which reduces the samples and wereimmediately passed through aa0.45 syringe filter injectedbuffer. the HPLC injected into the HPLC system for analysis of solute mass for subsequent growth. Growth samples were passed through a 0.45 syringe filter and injectedprofile the HPLC sis of drugsystem concentration. Furthermore, theµmobtained for analysis of drug concentration. Furthermore, the obtained into dissolution profile crystals can be restricted in the drug concentration. Furthermore, thedissolution obtained of nucleating system for analysisprofile of drug concentration. the obtained dissolution profile stabilizer (surfactant or polymer) dissolution data ofnanocrystals the Furthermore, raw tadalafil presence datatadalafil of the rawnanocrystals tadalafil and tadalafil werecompared evaluated and compared using adalafil and were evaluated and using nanocrystals were evaluated andand compared through steric or electrostatic mechanisms (17). dataand of thetadalafil raw tadalafil and tadalafil nanocrystals were evaluated using the dissolution efficiency (DE %) and 90 min. efficiency The measurements compared using thein 30 dissolution In were the repeated case of Tween 80 with CMC of about fficiency (DEthe%) in %) 30 in and min. were repeated dissolution efficiency (DE90 %)The in 30measurements and 90 min. The measurements were (DE 3090and min. The measurements 1.4 repeated 10-2 mol/L (0.6%), hydrophobic moieties three times. This concept was proposed by Khan and Rhodes (27) and is defined as were times. byThis adsorb times. repeated This conceptthree was proposed Khanconcept and Rhodeswas (27) and that is defined as on the hydrophobic drug particles, s concept wasthree proposed by Khan and Rhodes (27) and is defined as prevents crystals from aggregation due to the follows:proposed by Khan and Rhodes (27) and is follows: defined as follows: steric repulsion (28, 29). On the other hand, during the negative-pressure period of the � � ultrasound irradiation, cavitational bubbles are ���� ��� �� �� � �� � Dissolution Efficiency �DE%� ��� ��� E�u� Dissolution Efficiency �DE%� � �� �� ��� E�u� � � that cause powerful shock wave because � ���� � formed ��� � of very rapid collapse. In fact, sonoprecipitation � �� ��� tion Efficiency �DE%� � � � � � ��� E�u� � could act in two ways as induction of primary ���� � denotes 100% dissolution, Where y is the percent of drug dissolved at any time t, y nucleation in particle free solutions and dissolution, Where y is the percent of drug dissolved at any time t, 100 y100 denotes 100% Where y is the percent of drug dissolved at shortening the induction time between and the integral represents the area under dissolution curve between time zero and t. and the integral represents area under dissolution curve between zero and t. any time t, y100the denotes 100% dissolution, and timesupersaturation and the onset of nucleation and
ercent of drug dissolved at any time t, y100 denotes 100% dissolution, Saturation solubility
Saturation solubility
1339
epresents the area under dissolution curve between timenanocrystals zero andwere t. evaluated in The saturation solubility of raw tadalafil and tadalafil
lity
The saturation solubility of raw tadalafil and tadalafil nanocrystals were evaluated in two mediums: deionized water and phosphate buffer (pH=7.4) at 25 °C. Excess amounts
two mediums: deionized water and phosphate buffer (pH=7.4) at 25 °C. Excess amounts of lyophilized powder was dispersed in 10 mL of medium and placed on a stirrer for 48
of lyophilized wassolubility dispersedequilibrium in 10 mL had of medium and placed a stirrer for 48 h to ensurepowder that the been reached. The on samples were
Teymouri Rad R et al. / IJPR (2017), 16 (4): 1335-1348
Table 1. Numeric variables and their levels investigated in the CCD for preparation of tadalafil nanosuspensions. Range and levels Variables
Code
-1.68
-1
0
+1
+1.68
Drug concentration (mg/mL)
A
3.2
10
20
30
36.8
Stabilizer concentration (%)
B
0.032
0.1
0.2
0.3
0.368
Antisolvent/solvent volume ratio
C
9.9:1
15:1
22.5:1
30:1
35.1:1
crystallization (30).
factorial or fractional factorial design with center points, augmented with a group of axial points that let you estimate curvature. The effect of a wide range of dependent variables is predictable by CCD. Drug concentration,
Formulation optimization and statistical analysis A central composite design (CCD) is a
Table 2. Suggested formulation by CCD for preparation of tadalafil nanaosuspention. Run
Levels of factors A
B
C
Size (nm)
1
+1
+1
+1
372.7
2
-1
+1
+1
516.4
3
0
0
+1.68
400.4
4
0
0
0
362.7
5
-1
+1
-1
377.7
6
0
0
-1.68
536.8
7
-1
-1
+1
254.3
8
+1
-1
-1
658.4
9
0
0
0
375.4
10
+1
-1
+1
480.7
11
+1.68
0
0
636.7
12
0
0
0
322.8
13
0
-1.68
0
394.7
14
-1
-1
-1
274.0
15
+1
+1
-1
585.2
16
0
+1.68
0
396.6
17
0
0
0
406.6
18
-1.68
0
0
1138.0
19
0
0
0
345.2
20
0
0
0
387.0
1340
Enhanced Dissolution Rate of Tadalafil Nanoparticles
Table 3. The contribution and significance of different formulation parameters on particle size. Source
Sum of squares
DF
Mean square
F value
Prob>F
Model
74.83
9
8.31
6.07
0.0047
A
7.49
1
7.49
5.47
0.0414
B
22.88
1
22.88
16.72
0.0022
C
0.40
1
0.4
0.30
0.5987
2
28.86
1
28.86
21.09
0.0010
2
B
8.98
1
8.98
6.56
0.0283
C2
0.13
1
0.13
0.094
0.7650
AB
1.53
1
1.53
1.12
0.3151
AC
1.05
1
1.05
0.77
0.4014
BC
0.011
1
0.011
E-003 8.219
0.9296
Residual
13.69
10
1.37
Lack of fit
3.31
5
0.66
0.32
0.8820
Pure error
10.37
5
2.07
Cor total
88.52
19
A
stabilizer concentration and antisolvent/solvent volume ratio were independent variables in the preparation of tadalafil nanosuspension; while, particle size was studied as the dependent variable in a total of 20 experiments (Table 2). The individual and interactive effects of various variables were studied by carrying out the process at different levels of all factors. The particle size of nanoparticles was in the range of 254.3-1138.0 nm and the quadratic model was the best fitted on the data. The obtained model was validated using an ANOVA. As presented in Table 3, the most effective parameters on the size of nanoparticles was stabilizer concentration (B) with the F value of 16.72 (P
