Eur J Drug Metab Pharmacokinet DOI 10.1007/s13318-013-0158-5
ORIGINAL PAPER
Preparation and in vitro/in vivo evaluation of felodipine nanosuspension Bhanu P. Sahu • Malay K. Das
Received: 22 March 2013 / Accepted: 3 October 2013 Ó Springer-Verlag France 2013
Abstract The purpose of this study was to develop a nanosuspension of a poorly soluble drug felodipine by nanoprecipitation to achieve superior in vitro dissolution and high oral absorption in vivo in rats. Felodipine nanosuspensions were prepared by precipitation with ultrasonication method using polyvinyl alcohol (PVA) and hydroxy propyl methyl cellulose (HPMC) as stabilizers. The particle size of nanosuspension with PVA was 60–200 nm, while with HPMC is 300–410 nm. The in vitro dissolution and pharmacokinetics of optimized nanosuspensions were studied after oral administration in male wistar rats. The results showed significant improvement during in vitro dissolution and in vivo plasma level. Dissolution studies of lyophillised nanoparticles showed that up to 93.0 % dissolved in 2 h. In the in vivo evaluation, nanosuspension exhibited significant increase in AUC0–24, Cmax and decrease in tmax. The findings revealed that particle size reduction can influence felodipine absorption in gastrointestinal tract and nanosuspension can enhance oral bioavailability of felodipine in rats. Keywords Felodipine Nanosuspension Oral bioavailability Nanoprecipitation
B. P. Sahu (&) GIPS, Gauhati University, Hathkhowapara, Azara, Guwahati 781017, India e-mail: [email protected] M. K. Das Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, India
1 Introduction Felodipine is a dihydropyridine calcium channel blocker widely used as a potent anti-hypertensive drug (Saltiel et al. 1988). However, due to its poor solubility (solubility \0.5 mg/l), the oral bioavailability of felodipine is very poor of only 15 % (Karavas et al. 2005; Diez et al. 1991; Abrahamsson et al. 1994; Wingstrand et al. 1990). Due to its poor bioavailability it is found only as sustained-release dosage form and there is no conventional oral dosage form in present market. Several attempts have been made to increase its dissolution (Kerc et al. 1991; Kim et al. 2005; Lee et al. 2003). However, most of these techniques require a large amount of additives limiting their use from the safety perspective. Recently, the nanosuspension technology has been successfully applied as a safe formulation to tackle the formulation issue of several poorly soluble drugs. Nanosuspensions are carrier-free colloidal drug delivery system containing minimum additives (Keck and Muller 2006; Gao et al. 2008; Dong-Han et al. 2005). These preparations have several advantages and results in considerable increase in drugs saturation solubility (Mu¨ller and Peters 1998; Douroumis and Fahr 2006). The preparations are much homogenous, have good dispersity and scale up features (Horn and Rieger 2001; Rogers et al. 2004; Xiong et al. 2008). The methods of preparation of nanosuspenisons are simple and universal in approach (Mo¨schwitzer et al. 2004). Nanosuspension preparation can be prepared either by top–down or bottom–up processes. The top–down processes involves particle size reduction of large drug particles into smaller particles using various techniques such as media milling (Van Eerdenbrugh et al. 2007) microfluidization and high pressure homogenization. However, all these processes involve high energy input and are highly inefficient. In the bottom-up approach, the drug is
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dissolved in an organic solvent and is then precipitated on addition of an anti-solvent in the presence of a stabilizer. The precipitation method results in smaller size and homogenous particles (Li et al. 2007). Besides, it may lead to amorphous drug nanoparticles which have higher saturation solubility and dissolution rate (Kesisoglou et al. 2007; Mu¨ller and Peters 1998). Various adaptations of this approach include: (1) solvent–anti-solvent method; (2) supercritical fluid processes (Pathak et al. 2006; Reverchon 1999); (3) spray drying; (4) emulsion–solvent evaporation; and (5) ultrasonication (Luque de Castro and PriegoCapote 2007; Patravale et al. 2004; Rabinow 2004; LouhiKultanen et al. 2006). Previously, we developed a stable felodipine nanosuspension using precipitation–ultrasonication technique and studied its physical characterization and optimization (Sahu and Das 2013). Homogenous stable nanoparticles were obtained. In the present study, the optimized felodipine nanosuspensions were evaluated for in vitro and in vivo oral bioavailability in rats. To investigate the enhancement in oral bioavailability of the nanosuspension, plain felodipine suspension was used as a control. The aim of the present study was therefore to prepare stable felodipine nanosuspension by precipitation–ultrasonication technique and investigate the effectiveness of particle size reduction on drug dissolution rate as well as oral bioavailability of felodipine nanosuspension in rats.
Das 2013). The required amount of drug was completely dissolved in ethanol as water-miscible solvent. Different concentrations of drug in solvent (20, 40, 60, 80,100 mg/ml) were used. The obtained drug solution was then injected into the water containing the stabilizer under stirring followed by ultrasonication. The freshly prepared nanosuspension was centrifuged at 20,000 rpm for 20 min at 5 °C temperature using cool ultracentrifuge. After the centrifugation, the supernatant was replaced with 5 ml of stabilizer solution. The solid residue was redispersed and the final drug content was adjusted to respective drug concentration (drug weight/ volume of nanosuspension) using an appropriate volume of stabilizer solution. The nanosuspensions were then lyophilized. 3.2 Size measurement and zeta potential analysis The particle size and the polydispersity Index (PI) of the particles were measured by dynamic laser light scattering after suitable dilution (Zetasizer Ver. 6.11 Malvern). The measurement was done at 25 °C at a scattering angle of 908. The zeta potential of the preparations was also measured using the clear disposable Zeta cell for zeta potential analysis by electrophoretic mobility method (Zetasizer Ver. 6.11 Malvern). 3.3 Topography of nanoparticles
2 Materials and methods 2.1 Materials Felodipine was kindly provided by Glenmark Pharmaceutical Laboratories (Mumbai, India). Ethanol (Rankem, Ranbaxy Fine Chemicals Ltd., RFCL, New Delhi, India) was commercially obtained. Disodium hydrogen phosphate (Merck Specialities Pvt Ltd., Mumbai, India), potassium di hydrogen ortho phosphate (Merck Specialities Pvt Ltd., Mumbai, India), Sodium di hydrogen phosphate (Merck Specialities Pvt Ltd., Mumbai, India), Hydroxy Propyl Methyl Cellulose (Hi-Media Pvt Ltd., Mumbai, India), Poly Vinyl Alcohol (Hi-Media Pvt Ltd., Mumbai, India) were commercially obtained. Deionised water was used for all experiments (Deionizer, MAC, CA-50 5).
3 Methods 3.1 Preparation of nanoparticles Felodipine nanoparticles were produced by precipitation– ultrasonication technique described previously (Sahu and
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The morphology and size of the nanoparticles were measured by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). 3.4 Transmission electron microscopy (TEM) The particle size and morphology were observed with transmission electron microscopy analysis (Veerareddy et al. 2012) using transmission electron microscope (JEM2100, 200 kV, Jeol, Japan). 3.5 Scanning electron microscopy (SEM) Particle morphology was observed using scanning electron microscopy (SEM) JSM-6360 (JEOL Inc., Japan). The samples small drop of the suspension was air dried followed by oven drying and were fixed on an SEM stub using double-sided adhesive tape and coated with Au at 20 mA for 6 min through a sputter coater (Ion sputter JFC 1100). A scanning electron microscope with a secondary electron detector was used to obtain digital images of the samples at an accelerating voltage of 15 kV (Yuancai et al. 2010; Sahu and Das 2013).
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3.6 X-ray diffraction studies (XRD) The effect on crystallinity of precipitated felodipine nanoparticles (NS1) was studied by X-ray diffraction of formulation and pure drug using XRD-6,000 diffractometer (Shimadzu, Japan). The powder was placed in a glass sample holder. CuK radiation was generated at 30 mA and 40 kV. Samples were scanned from 108 to 908 with a step size of 0.028 (Martine et al. 1998; Sahu and Das 2013). 3.7 Fourier transforms infrared spectroscopy (FT-IR) Drug excipient interactions were studied by FTIR spectroscopy (Hai-Xia et al. 2009). The spectra were recorded for felodipine, Polyvinyl Alcohol (PVA) and the dried nanoparticles (NS1). Samples were prepared in KBr discs (2 mg drug in 8 mg KBr) with a hydrostatic press at a force of 8 t cm-2 for 2 min. The scanning range was 450–4,000 cm-1 and resolution was 2 cm-1. 3.8 Differential scanning calorimetry (DSC) The DSC analysis of pure drug, PVA and the NS1 was carried out using Mettler Toledo (Model SW 810) to evaluate any possible drug–excipients interaction. Samples (5.5–8 mg) were weighed accurately using a single pan electronic balance and heated in sealed aluminum pan at a rate of 5 °C/min from 25 to 450 °C temperature range under a nitrogen flow of 35 ml/min (Hany et al. 2009; Sahu and Das 2013). 3.9 Lyophilisation of nanosuspensions The nanosuspensions were kept in deep freezer (IIC Industrial Corporation, India) for 24 h at -30 °C for freezing the concentrated nanosuspensions. The freezed suspension was then transferred to laboratory freeze dryer (Laboratory Freeze Dryer, IINSTIND, IIC Industrial Corporation, India) and allowed the temperature to attain -35 °C in which condition vacuum is attained. The preparation is then kept in this condition for 4–5 h daily for 5 days for freeze drying of the nanosuspensions under vacuum to obtain lyophilized nanoparticles. 3.10 Determination of saturation solubility To compare the solubility of the felodipine before and following nanoprecipitation process, saturation solubility was determined. An excess amount of lyophillised nanosuspension containing equivalent felodipine (20 mg) was dispersed into 20 ml of water and placed on a shaking water bath for 48 h to ensure that the solubility equilibrium had been reached. The samples were centrifuged and the
resulting supernatant was analyzed by UV spectrophotometer at 363 nm (Xia et al. 2010). 3.11 Physical stability study Three temperature conditions were applied in the stability study of the nanosuspensions: 4 °C (refrigerator), room temperature and 40 °C (stability chamber). Physical stability of the nanosuspensions was evaluated after 6 months of storage. The measurement of particle size was performed and settling behavior by visual examination (Van Eerdenbrugh et al. 2008). 3.12 In vitro dissolution The lyophilized nanoparticles and pure drug were filled in gelatin capsules (Size 1, Yarrow Chem Products, Mumbai, India) and dissolution was studied using a basket type dissolution apparatus (Tablet Dissolution Tester, TDT-06T, Electrolab, Mumbai, India) at 100 rpm in PB6.5 (Phosphate Buffer pH 6.5) used as dissolution medium for 2 h. 3.13 In vivo studies in rats 3.13.1 Animals Eighteen white Male Wistar rats (body weight 200 ± 50 g) of equal size (n = 6) were randomly divided into three groups. Food was withdrawn 12 h prior to the in vivo study with water provided ad libitum and no food was allowed after dosing until the end of the study (after 24 h). All experimental procedures were performed as per the ethics and regulations of animal experiments CPSCEA, India. 3.14 Study design The in vivo studies compared the pharmacokinetics of lyophilized felodipine nanosuspension based on PVA (NS1) and HPMC (NS2) with a control drug powder filled in hard gelatin capsules. These were given orally in single doses of 20 mg drug. Doses were established depending on the linearity of pharmacokinetic parameters in rat and the necessary conditions requested by the analytical method. A non-blind, three treatments, randomized, parallel design was used. The control capsules were hard gelatin capsules (size no. 1) each filled with 100 mg of a blend of 20 mg felodipine with 80 mg of a 7:1 mixture of lactose and microcrystalline cellulose. Blood samples (0.2 ml) were withdrawn by retro orbital puncture at the following time points: 0 (predose) 0.25, 0.5, 1, 2, 3, 4, 8, 12 and 24 h after administration of a treatment. Plasma was obtained by centrifugation at 4,000 r/min for 15 min and then it was
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Eur J Drug Metab Pharmacokinet Fig. 1 Chromatogram of felodipine and internal standard in plasma sample
deep-frozen at -70 °C until required for analysis (AboulEinien 2009). 3.15 Chromatographic conditions Felodipine was analyzed using HPLC system consisting of waters 2,695–Alliance L05SM4395M model separations module with Millennium32 software, auto injector and waters 2,996 photodiode array detector. Separation was carried out using a waters symmetry C18 column (25 cm 9 4.5 mm, 5 lm). Mobile phase consisting of an acetonitrile, methanol and phosphate buffer 0.05 M (40:20:40) adjusted to pH 3.0 with phosphoric acid was used at a flow rate of 1.5 ml/min. Mobile phase was filtered (Millipore system, 0.2 lm) under vacuum and degassed. Chromatographic separation was performed at 238 nm. Total run time for the analysis was 10 min.
nylon membrane filters and portions of the filtrates (20 ll) were then injected onto the column. A blank plasma sample, without the addition of felodipine was treated in the same way. A standard curve was constructed by plotting the ratio of peak area of felodipine to the internal standard against the drug concentrations in plasma. All assays were performed in triplicate. The retention time of felodipine and internal standard was 7.454 and 5.161, respectively. Figure 1 shows a chromatogram of felodipine containing diclofenac as internal standard in plasma. The LOD and LOQ of the method were determined by replicate injections of serially diluted drug. The LOD was defined as a minimum signal-to-noise ratio of 3. The LOQ was determined by taking three times of S/N ratio of LOD. LOD and LOQ were found to be 0.41 and 1.25 lg/ml, respectively. A linear response across the full range of concentrations from 2.5 to 25.0 (2.5, 5.0, 6.25, 12.5, 25.0) lg/ml (r2 = 0.994) was obtained.
3.16 Standard solutions 3.17 Plasma analysis Blank plasma samples (0.1 ml) were spiked with felodipine methanolic stock solution to obtain serial concentrations of the drug. Diclofenac sodium has been used as an internal standard and its concentration was kept constant in all samples at 10 lg/ml. Aliquots of 100 ll of spiked plasma samples were extracted with 500 ll toluene. The mixtures were shaken overnight then centrifuged for 10 min at 3,000 r/min. Supernatant was evaporated under nitrogen at 35–45 °C under reduced pressure until dryness, reconstituted the dried residue with 300 ll of mobile phase and vortexed. Although the boiling point of toluene is 110 °C, it can be evaporated at much lower temperature of 35–45 °C by heating under reduced pressure in a vacuum condition. Toluene has been selected as solvent because of its efficient extraction of felodipine from Plasma (Durol and Greenblatt 1997). These were passed through 0.45 lm
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The plasma samples obtained from the rats after receiving nanosuspensions NS1, NS2 and control were assayed as described above without the addition of felodipine. 3.18 Validation of analytical method The HPLC method of extraction and subsequent analysis were validated by observing the extraction efficiency of the method, precision and accuracy of the method. 3.19 Extraction efficiency The extraction efficiency for felodipine and internal standard from plasma were experimentally determined using the method explained above. Extraction efficiency was
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determined by comparison of the peak areas of the spiked plasma samples (2.5, 5, 12.5 lg/ml) with those of unextracted felodipine solution of similar concentrations (Durol and Greenblatt 1997).
terminal log-linear phase by log-linear regression analysis. The apparent terminal elimination half-life (t1/2) was calculated as t1/2 = 0.693/k. 3.24 Statistical analysis
3.20 Inter- and intra-day accuracy and precision Replicates of six were produced to evaluate intra-day and inter-day accuracy and precision. Accuracy was determined as the absolute value of the ratio of the back-calculated mean values to their respective nominal values and expressed as percentage. Precision was determined by the percentage coefficient of variation (% CV) for QC concentrations.
Statistical significance in the differences of means between the nanosuspensions and control was determined by Student’s t test. Statistical analyses of pharmacokinetic parameters were performed by applying a two-way analysis of variance (ANOVA). Differences in mean values were considered statistically significant at a P value B0.05. The software, Graph Pad Prism (version 6.01 Graph Pad software Inc.), was used to perform the statistical analysis.
3.21 Accuracy 4 Results and discussion The accuracy of the method was demonstrated through determination on samples in 3 concentrations of 2.5, 5.0 and 12.5 lg/ml in triplicates. Standard solutions were injected onto the column and from the chromatograph measured the area due to felodipine peak. Calculated the amount of felodipine found in each of these solutions using calibration curve and compared for accuracy. 3.22 Precision The precision of the method was observed through injection reproducibility. For injection reproducibility, six injections of the same standard preparations were made and the relative standard deviation for the replicate injections was calculated. The mean and standard deviation and the relative standard deviation were then calculated for the six replicate injections. 3.23 Pharmacokinetic analysis Pharmacokinetic characteristics from plasma data following administration of the three treatments were estimated for each rat. The observed maximal drug concentration (Cmax) and its time (tmax) were estimated from the plasma concentration–time curve. The area under the curve, AUC0–24 (lg h/ml), was calculated using the trapezoidal rule from zero time to time of the last blood sample (24 h). The elimination rate constant (k) was estimated from the
4.1 Preparation of nanosuspension Felodipine nanoparticles were produced by precipitation– ultrasonication technique. The aqueous phase containing a suitable stabilizer has been used as the anti-solvent and ethanol having good solubility of felodipine has been used as solvent. The results of optimized formulation for PVAand HPMC-based nanosuspenison are shown in Table 1. The nanosuspensions can be stabilized by electrostatic or steric stabilization or a combination of both. However, steric stabilization is more advantageous than electrostatic stabilization as the latter may be lost in the variable pH condition of the GIT and is effected by electrolytes. The stability of sterically stabilized nanosuspensions depends on the property of the drug-like enthalpy and logP as well as the hydrophobicity of the stabilizer (Matteucci et al. 2007; George and Ghosh 2013). Also the choice of an ideal stabilizer is influenced by the degree of hydrophobicity of the drug itself. And it has been concluded from various studies that ionic stabilization with high zeta potential is not a requisite for stability of nanosuspension. In fact, the stabilizer–drug interaction is a stronger factor than surface charge itself. A similar hydrophobicity should result in better surface coverage thereby providing better steric stabilization. Hence, polymeric non-ionic stabilizer HPMC and PVA of moderate hydrophobicity were selected for the steric stability of the felodipine nanosuspensions. HPMC
Table 1 Effect of drug concentration and surfactant concentration on mean particle size, and polydispersity index of optimized formulations Formulation code
Diffusing drug concentrations (mg/ml)
PVA (% w/v)
HPMC (% w/v)
Z average diameter (nm) ± SD
Polydispersity Index (PI)) ± SD
NS1 NS2
60 40
0.75 –
– 0.5
95.97 ± 12.5 362.1 ± 22.5
0.494 ± 0.026 0.624 ± 0.041
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Fig. 3 SEM photomicrograph of PVA-based felodipine nanoparticles. Scale bar 0.2 lm
Fig. 2 TEM photomicrograph of PVA-based felodipine nanoparticles. Scale bar 100 nm
electron micrographs revealed that the particles were spherical and homogeneous as shown in Fig. 2. 4.4 Scanning electron microscopy (SEM)
and PVA were used at various concentrations (0.1, 0.25, 0.5, 1.0 %). 4.2 Size measurement and zeta potential analysis The formulations containing HPMC as stabilizer showed particle size in the range of 320–410 nm at various drug concentrations. The formulations were homogeneous as indicated by polydispersity index of 0.5 ± 0.1. The average particle diameter was found to decrease further in formulations containing PVA as stabilizer at concentrations of 0.5 %.The particle size distributions were found to be more uniform as the average particle size of PVAbased felodipine nanoparticles were found to be in the range of 60–330 nm and the polydispersity index narrowed down to 0.3–0.5. The zeta potential of the nanoparticles was found to be negative and in the range of 5–18 mV. The negative charge may be due to ionization of the carboxyl group in an aqueous environment. However, adsorption of non-ionic stabilizers (HPMC and PVA) results in an increase in the thickness of the diffuse double layer, and hence, a lower zeta potential (Sahu and Das 2013). 4.3 Transmission electron microscopy (TEM) A transmission electron microscopic study was carried out for the optimized formulation to observe the physical properties of precipitated nanoparticles. Transmission
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The drug particles precipitated with the PVA as stabilizer show spherical in shape and the size ranges from 60 to 200 nm as shown in Fig. 3. The particles are discrete and uniform in size and there is no sign of agglomerations. The drug particles precipitated with the HPMC as stabilizer are slightly bigger and the size ranges from 100 to 300 nm (Sahu and Das 2013). 4.5 Saturation solubility The results of saturation solubility of pure drug and lyophilized felodipine nanosuspension revealed a saturation solubility of 19.17 and 582.32 lg/ml, respectively. The saturation solubility of felodipine as nanosuspension is 30-fold higher than that of pure felodipine. In the present study, particle size of felodipine has been reduced and hence, increased the saturation solubility due to increasing the surface area of the reduced particles. 4.6 Differential scanning calorimetry (DSC) The DSC analysis of pure drug, PVA and the NS1 was carried. The pure drug felodipine showed a melting point peak at 145 °C. The DSC thermogram, however, showed that after precipitation its melting point was decreased to 132 °C, indicating reduced crystallinity. The results are shown in Fig. 4. The DSC analysis of pure drug, HPMC and dried nanoparticles based on HPMC were done
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Fig. 4 DSC thermograms of (redline) pure drug felodipine (blueline) PVA and (greenline) precipitated felodipine nanoparticles (color figure online)
felodipine displayed the presence of numerous distinct peaks at 10.198, 16.518, 20.478, 23.278, 24.518, 25.378, 26.458, and 32.658, which suggested that the drug was of crystalline form. The PVA stabilized precipitated nanoparticles samples showed the similar diffraction pattern but with lower peak intensity, suggesting the crystallinity of precipitated nanoparticles decreased during the precipitation process. Similar results have been obtained and previously reported with HPMC-based formulation (Sahu and Das 2013). 4.8 Fourier transforms infrared spectroscopy (FT-IR)
Fig. 5 X-ray diffraction patterns of (blue line) pure felodipine drug (red line) precipitated PVA-based felodipine nanoparticles (color figure online)
similarly. The results have been reported previously (Sahu and Das 2013). 4.7 X-ray diffraction studies (XRD) The representative X-ray diffraction patterns of the pure felodipine powder and oven-dried nanosuspensions (NS1) are shown in Fig. 5. The figures indicated the changes in the drug crystal structure. The X-ray patterns of the pure
Infra red spectra of Felodipine and PVA and the precipitated nanoparticles (NS1) were comparable and the peaks of felodipine in the formulation are of lower intensity than pure drug. The characteristic peaks of pure were found to be intact in the physical mixture as well as the formulation which indicates the absence of PVA and drug interactions. The results have been shown in Fig. 6. Similar results have been obtained and previously reported with HPMC-based formulation (Sahu and Das 2013). 4.9 Physical stability study The particle size of the formulations after 6 months was observed. The formulations at 4 8C remained stable,
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Eur J Drug Metab Pharmacokinet Fig. 6 FTIR spectra’s of a pure drug felodipine b PVA c precipitated PVA stabilized felodipine nanoparticles
Table 2 Physical nanosuspensions Formulations
FF3
stability
Storage temperature conditions 8C 4
evaluation
Initial particle size (±SD, n = 3) 108.5 ± 4.6
RT 4
the
felodipine
Particle size after 3 months (±SD, n = 3) 104.5 ± 2.6 106.4 ± 3.6
40 FF6
of
118.5 ± 2.4 310.7 ± 13.8
295.4 ± 14.1
RT
302.6 ± 12.6
40
354.2 ± 16.2
whereas that at room temperature showed slight increase in particle size. In the case of HPMC formulations, only those stored at higher temperatures (40 8C) exhibited an increase
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in mean particle size. The increase in size in the HPMC formulation stored at 40 8C was attributed to dehydration of the HPMC chains and subsequent loss of protection of the nanoparticles. The particle size data are given in the Table 2. Sedimentation was observed in all the conditions but the preparations were easily redispersed on shaking (Sahu and Das 2013). 4.10 In vitro dissolution The in vitro dissolution of a drug was determined for the control and formulation (NS1 and NS2) filled capsules. Optimized nanosized felodipine showed a dramatic increase in rate and extent of dissolution compared with coarse powder. The lyophilised optimized formulations
Eur J Drug Metab Pharmacokinet Table 3 Pharmacokinetic parameters of felodipine nanosuspension NS1, NS2 and control formulation in rats (mean ± SD, n = 6)
Fig. 7 Drug dissolution profile of optimized lyophilized formulations and control (Standard error bar, n = 6)
NS1 and NS2 showed 93 and 74 % drug dissolution in 120 min, respectively, from the filled capsules as compared to 38 % of pure drug. The results are shown in Fig. 7. This enhanced dissolution may be due to the increased saturation solubility resulted due to increase in surface area of drug. 4.11 Pharmacokinetic studies The HPLC method and extraction process were well validated. The extraction efficiency was found to be of 96.5, 93.5, and 92.2 % for felodipine for concentration of 2.5, 5.0 and 12.5 %, respectively, and 94.2 ± 1.2 % for internal standard. Efficient extraction of felodipine from plasma was obtained using the method explained using toluene and
Formulation
NS1
NS2
Control
AUC0?24 (lg h/ml)
48.34 ± 3.78
37.40 ± 3.56
20.48 ± 1.27
Cmax (lg/ml)
4.171 ± 0.0175
3.627 ± 0.18
1.093 ± 0.004
tmax (h) k
1.0 ± 0.25 0.058
1.0 ± 0.33 0.057
2.0 ± 0.5 0.056
t1/2 (h)
11.8 ± 2.1
12.09 ± 1.9
12.6 ± 1.7
showed that extraction decreases with increasing drug concentration. The accuracy and precision of the process was also established and found to be satisfactory. Relative standard deviation (RSSD) value for six injection of the same concentration was found to be 1.44 indicating precision of the injector as well as the method. The plasma drug concentration–time profiles and pharmacokinetic parameters of felodipine resulted from the oral administrations of the nanosuspensions and control in wistar rats are presented in Fig. 8 and Table 3, respectively. The control preparation used in this study was felodipine powder filled in hard gelatin capsules. No marketed felodipine product was used as a control because felodipine is commercially available only in the form of sustainedrelease tablets and no felodipine formulation with enhanced drug dissolution is available commercially. Felodipine powder filled in hard gelatin capsules has been used as a control treatment in bioequivalence studies on the coenzyme Q10 (Weis et al. 1994; Zaghloul et al. 2002). The mean plasma concentration–time curves following administration of capsules filled with lyophilized
Fig. 8 Average plasma drug concentration versus time profiles after oral administration of lyophilized felodipine nanosuspensions NS1, NS2 and control formulation (mean ± SD, n = 6)
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nanosuspensions NS1, NS2 and a control capsule containing felodipine powder are shown in Fig. 8. The shape of the plasma concentration–time curve of nanosuspensions was found to be markedly different from that of control. The plasma drug concentration profile of nanosuspensions showed significant improvement in drug absorption than the reference formulation. The Cmax values of nanosuspensions NS1 and NS2 were approximately 3.8-fold and 3.3-fold greater than that of control preparation, respectively. The AUC0?24 values of nanosuspensions were approximately 2.4 and 1.8-fold greater than that of control preparation. The improved oral bioavailability of felodipine may be due to the rapid absorbtion of felodipine from gastrointestinal wall due to the significantly improved dissolution rate by the reduced particle size with increased surface area (Hintz and Johnson 1989; Bernhard and Muller 1999). The mean pharmacokinetic parameters, Cmax, tmax, AUC0–24, Ke and t1/2, are reported in Table 3. The mean Cmax values were found to be 4.171 ± 0.017 and 3.627 ± 0.18(lg/ml) for nanosuspensions NS1 and NS2, while control showed a value of 1.093 ± 0.004 lg/ml. The mean tmax values were 1 and 2 h for nanosuspension and control, respectively. The differences between the treatments NS1 and NS2 with the control for Cmax and tmax were statistically significant. The shortened tmax obtained for nanosuspensions could be attributed to the enhanced dissolution resulting in rapid drug absorption. The mean area under the curve reflecting the total amount of absorption in 24 h, AUC0–24, was estimated for all treatments and was found to be statistically significantly different (P \ 0.05) between the nanosuspensions and control. Statistical analysis of the half-life indicated a nonsignificant difference between results from nanosuspensions (11.8 ± 2.1 h and 12.09 ± 1.9) and pure drug (12.6 ± 1.7 h). Based on these results, it can be concluded that formulation of felodipine as nanosuspensions results in rapid and enhanced absorption of felodipine which could be explained by the improved saturation solubility and thereby dissolution of drug.
5 Conclusion From the study, it can be concluded that the oral absorption of felodipine increased significantly in rats when formulated as nanosuspensions. This enhancement in oral bioavailability may be due to the enhanced dissolution resulted due to increase in surface area with reduction in size. Hence, felodipine nanosuspension prepared by precipitation with sonication technique may be suitable to increase the oral bioavailability of poorly soluble felodipine.
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Eur J Drug Metab Pharmacokinet Louhi-Kultanen M, Karjalainen M, Rantanen J, Huhtanen M, Kallas J (2006) Crystallization of glycine with ultrasound. Int J Pharm 320:23–29 Luque de Castro MD, Priego-Capote F (2007) Ultrasound-assisted crystallization (sonocrystallization). Ultrason Sonochem 14:717–724 Martine LV, Laurence F, Young-Il K, Maurice H, Philippe M (1998) Preparation and characterization of nanoparticles containing an antihypertensive agent. Eur J Pharm Biopharm 46:137–143 Matteucci ME, Brettmann BK, Rogers TL, Elder EJ, Williams RO, Johnston KP (2007) Design of potent amorphous drug nanoparticles for rapid generation of highly supersaturated media. Mol Pharm 4:4782–4793 Mo¨schwitzer J, Achleitner G, Pomper H, Muller RH (2004) Development of an intravenously injectable chemically stable aqueous omeprazole formulation using nanosuspension technology. Eur J Pharm Biopharm 58:615–619 Mu¨ller RH, Peters K (1998) Nanosuspensions for the formulation of poorly soluble drugs I. Preparation by a size-reduction technique. Int J Pharm 160:229–237 Pathak P, Meziani MJ, Desai T, Sun Y-P (2006) Formation and stabilization of ibuprofen nanoparticles in supercritical fluid processing. J Supercrit Fluids 37:279–286 Patravale VB, Date AA, Kulkarni RM (2004) Nanosuspensions: a promising drug delivery strategy. J Pharm Pharmacol 56:827–840 Rabinow BE (2004) Nanosuspensions in drug delivery. Drug Discov 3:785–796 Reverchon E (1999) Supercritical antisolvent precipitation of microand nanoparticles. J Supercrit Fluids 15:1–21 Rogers T, Gillespie IB, Hitt JE, Fransen KL, Crowl CA, Tucker CJ, Kupperblatt GB, Becker JN, Wilson DL, Todd C, Elder EJ (2004) Development and characterization of a scalable controlled precipitation process to enhance the dissolution of poorly water-soluble drugs. Pharm Res 21:2048–2057 Sahu BP, Das MK (2013) Nanosuspension for enhancement of oral bioavailability of felodipine. Appl Nanosci. doi:10.1007/s13204012-0188-3
Saltiel E, Ellrodt AG, Monk JP, Langley MS (1988) Felodipine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in hypertension. Drugs 36:387–428 Van Eerdenbrugh B, Froyen L, Martens JA, Blaton N, Augustijns P, Brewster M, Van den Mooter G (2007) Characterization of physico-chemical properties and pharmaceutical performance of sucrose co-freeze-dried solid nanoparticulate powders of the anti-HIV agent loviride prepared by media milling. Int J Pharm 338:198–206 Van Eerdenbrugh B, Van den Mooter G, Augustijns P (2008) Topdown production of drug nanocrystals: nanosuspension stabilization, miniaturization and transformation into solid products. Int J Pharm 364:64–75 Veerareddy PR, Poluri K, Sistla R, Chaganty S, Vennishetty VK (2012) Formulation development and comparative pharmacokinetic evaluation of felodipine nanoemulsion in SD rats. Am J Pharm Tech Res 2:931–945 Weis M, Mortensen SA, Rassing MR (1994) Bioavailability of four oral coenzyme Q10 formulations in healthy volunteers. Mol Aspects Med 15:273–280 Wingstrand K, Abrahamson B, Edgar B (1990) Bioavailability from felodipine extended-release tablets with different dissolution properties Int. J Pharm 60:151–156 Xia D, Quan P, Piao H, Sun S, Yin Y, Cui F (2010) Preparation of stable nitrendipine nanosuspensions using the precipitation– ultrasonication method for enhancement of dissolution and oral bioavailability. Eur J Pharm Sci 40:325–334 Xiong R, Lu W, Li J, Wang P, Xu R, Chen T (2008) Preparation and characterization of intravenously injectable nimodipine nanosuspension. Int J Pharm 350:338–343 Yuancai D, Wai KN, Jun H, Shoucang S, Reginald BHT (2010) A continuous and highly effective static mixing process for antisolvent precipitation of nanoparticles of poorly water-soluble drugs. Int J Pharm 386:256–261 Zaghloul AA, Gurley B, Khan M (2002) Bioavailability assessment of oral coenzyme Q10 formulations in dogs. Drug Dev Ind Pharm 28:1195–1200
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ORIGINAL PAPER
Preparation and in vitro/in vivo evaluation of felodipine nanosuspension Bhanu P. Sahu • Malay K. Das
Received: 22 March 2013 / Accepted: 3 October 2013 Ó Springer-Verlag France 2013
Abstract The purpose of this study was to develop a nanosuspension of a poorly soluble drug felodipine by nanoprecipitation to achieve superior in vitro dissolution and high oral absorption in vivo in rats. Felodipine nanosuspensions were prepared by precipitation with ultrasonication method using polyvinyl alcohol (PVA) and hydroxy propyl methyl cellulose (HPMC) as stabilizers. The particle size of nanosuspension with PVA was 60–200 nm, while with HPMC is 300–410 nm. The in vitro dissolution and pharmacokinetics of optimized nanosuspensions were studied after oral administration in male wistar rats. The results showed significant improvement during in vitro dissolution and in vivo plasma level. Dissolution studies of lyophillised nanoparticles showed that up to 93.0 % dissolved in 2 h. In the in vivo evaluation, nanosuspension exhibited significant increase in AUC0–24, Cmax and decrease in tmax. The findings revealed that particle size reduction can influence felodipine absorption in gastrointestinal tract and nanosuspension can enhance oral bioavailability of felodipine in rats. Keywords Felodipine Nanosuspension Oral bioavailability Nanoprecipitation
B. P. Sahu (&) GIPS, Gauhati University, Hathkhowapara, Azara, Guwahati 781017, India e-mail: [email protected] M. K. Das Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, India
1 Introduction Felodipine is a dihydropyridine calcium channel blocker widely used as a potent anti-hypertensive drug (Saltiel et al. 1988). However, due to its poor solubility (solubility \0.5 mg/l), the oral bioavailability of felodipine is very poor of only 15 % (Karavas et al. 2005; Diez et al. 1991; Abrahamsson et al. 1994; Wingstrand et al. 1990). Due to its poor bioavailability it is found only as sustained-release dosage form and there is no conventional oral dosage form in present market. Several attempts have been made to increase its dissolution (Kerc et al. 1991; Kim et al. 2005; Lee et al. 2003). However, most of these techniques require a large amount of additives limiting their use from the safety perspective. Recently, the nanosuspension technology has been successfully applied as a safe formulation to tackle the formulation issue of several poorly soluble drugs. Nanosuspensions are carrier-free colloidal drug delivery system containing minimum additives (Keck and Muller 2006; Gao et al. 2008; Dong-Han et al. 2005). These preparations have several advantages and results in considerable increase in drugs saturation solubility (Mu¨ller and Peters 1998; Douroumis and Fahr 2006). The preparations are much homogenous, have good dispersity and scale up features (Horn and Rieger 2001; Rogers et al. 2004; Xiong et al. 2008). The methods of preparation of nanosuspenisons are simple and universal in approach (Mo¨schwitzer et al. 2004). Nanosuspension preparation can be prepared either by top–down or bottom–up processes. The top–down processes involves particle size reduction of large drug particles into smaller particles using various techniques such as media milling (Van Eerdenbrugh et al. 2007) microfluidization and high pressure homogenization. However, all these processes involve high energy input and are highly inefficient. In the bottom-up approach, the drug is
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dissolved in an organic solvent and is then precipitated on addition of an anti-solvent in the presence of a stabilizer. The precipitation method results in smaller size and homogenous particles (Li et al. 2007). Besides, it may lead to amorphous drug nanoparticles which have higher saturation solubility and dissolution rate (Kesisoglou et al. 2007; Mu¨ller and Peters 1998). Various adaptations of this approach include: (1) solvent–anti-solvent method; (2) supercritical fluid processes (Pathak et al. 2006; Reverchon 1999); (3) spray drying; (4) emulsion–solvent evaporation; and (5) ultrasonication (Luque de Castro and PriegoCapote 2007; Patravale et al. 2004; Rabinow 2004; LouhiKultanen et al. 2006). Previously, we developed a stable felodipine nanosuspension using precipitation–ultrasonication technique and studied its physical characterization and optimization (Sahu and Das 2013). Homogenous stable nanoparticles were obtained. In the present study, the optimized felodipine nanosuspensions were evaluated for in vitro and in vivo oral bioavailability in rats. To investigate the enhancement in oral bioavailability of the nanosuspension, plain felodipine suspension was used as a control. The aim of the present study was therefore to prepare stable felodipine nanosuspension by precipitation–ultrasonication technique and investigate the effectiveness of particle size reduction on drug dissolution rate as well as oral bioavailability of felodipine nanosuspension in rats.
Das 2013). The required amount of drug was completely dissolved in ethanol as water-miscible solvent. Different concentrations of drug in solvent (20, 40, 60, 80,100 mg/ml) were used. The obtained drug solution was then injected into the water containing the stabilizer under stirring followed by ultrasonication. The freshly prepared nanosuspension was centrifuged at 20,000 rpm for 20 min at 5 °C temperature using cool ultracentrifuge. After the centrifugation, the supernatant was replaced with 5 ml of stabilizer solution. The solid residue was redispersed and the final drug content was adjusted to respective drug concentration (drug weight/ volume of nanosuspension) using an appropriate volume of stabilizer solution. The nanosuspensions were then lyophilized. 3.2 Size measurement and zeta potential analysis The particle size and the polydispersity Index (PI) of the particles were measured by dynamic laser light scattering after suitable dilution (Zetasizer Ver. 6.11 Malvern). The measurement was done at 25 °C at a scattering angle of 908. The zeta potential of the preparations was also measured using the clear disposable Zeta cell for zeta potential analysis by electrophoretic mobility method (Zetasizer Ver. 6.11 Malvern). 3.3 Topography of nanoparticles
2 Materials and methods 2.1 Materials Felodipine was kindly provided by Glenmark Pharmaceutical Laboratories (Mumbai, India). Ethanol (Rankem, Ranbaxy Fine Chemicals Ltd., RFCL, New Delhi, India) was commercially obtained. Disodium hydrogen phosphate (Merck Specialities Pvt Ltd., Mumbai, India), potassium di hydrogen ortho phosphate (Merck Specialities Pvt Ltd., Mumbai, India), Sodium di hydrogen phosphate (Merck Specialities Pvt Ltd., Mumbai, India), Hydroxy Propyl Methyl Cellulose (Hi-Media Pvt Ltd., Mumbai, India), Poly Vinyl Alcohol (Hi-Media Pvt Ltd., Mumbai, India) were commercially obtained. Deionised water was used for all experiments (Deionizer, MAC, CA-50 5).
3 Methods 3.1 Preparation of nanoparticles Felodipine nanoparticles were produced by precipitation– ultrasonication technique described previously (Sahu and
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The morphology and size of the nanoparticles were measured by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). 3.4 Transmission electron microscopy (TEM) The particle size and morphology were observed with transmission electron microscopy analysis (Veerareddy et al. 2012) using transmission electron microscope (JEM2100, 200 kV, Jeol, Japan). 3.5 Scanning electron microscopy (SEM) Particle morphology was observed using scanning electron microscopy (SEM) JSM-6360 (JEOL Inc., Japan). The samples small drop of the suspension was air dried followed by oven drying and were fixed on an SEM stub using double-sided adhesive tape and coated with Au at 20 mA for 6 min through a sputter coater (Ion sputter JFC 1100). A scanning electron microscope with a secondary electron detector was used to obtain digital images of the samples at an accelerating voltage of 15 kV (Yuancai et al. 2010; Sahu and Das 2013).
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3.6 X-ray diffraction studies (XRD) The effect on crystallinity of precipitated felodipine nanoparticles (NS1) was studied by X-ray diffraction of formulation and pure drug using XRD-6,000 diffractometer (Shimadzu, Japan). The powder was placed in a glass sample holder. CuK radiation was generated at 30 mA and 40 kV. Samples were scanned from 108 to 908 with a step size of 0.028 (Martine et al. 1998; Sahu and Das 2013). 3.7 Fourier transforms infrared spectroscopy (FT-IR) Drug excipient interactions were studied by FTIR spectroscopy (Hai-Xia et al. 2009). The spectra were recorded for felodipine, Polyvinyl Alcohol (PVA) and the dried nanoparticles (NS1). Samples were prepared in KBr discs (2 mg drug in 8 mg KBr) with a hydrostatic press at a force of 8 t cm-2 for 2 min. The scanning range was 450–4,000 cm-1 and resolution was 2 cm-1. 3.8 Differential scanning calorimetry (DSC) The DSC analysis of pure drug, PVA and the NS1 was carried out using Mettler Toledo (Model SW 810) to evaluate any possible drug–excipients interaction. Samples (5.5–8 mg) were weighed accurately using a single pan electronic balance and heated in sealed aluminum pan at a rate of 5 °C/min from 25 to 450 °C temperature range under a nitrogen flow of 35 ml/min (Hany et al. 2009; Sahu and Das 2013). 3.9 Lyophilisation of nanosuspensions The nanosuspensions were kept in deep freezer (IIC Industrial Corporation, India) for 24 h at -30 °C for freezing the concentrated nanosuspensions. The freezed suspension was then transferred to laboratory freeze dryer (Laboratory Freeze Dryer, IINSTIND, IIC Industrial Corporation, India) and allowed the temperature to attain -35 °C in which condition vacuum is attained. The preparation is then kept in this condition for 4–5 h daily for 5 days for freeze drying of the nanosuspensions under vacuum to obtain lyophilized nanoparticles. 3.10 Determination of saturation solubility To compare the solubility of the felodipine before and following nanoprecipitation process, saturation solubility was determined. An excess amount of lyophillised nanosuspension containing equivalent felodipine (20 mg) was dispersed into 20 ml of water and placed on a shaking water bath for 48 h to ensure that the solubility equilibrium had been reached. The samples were centrifuged and the
resulting supernatant was analyzed by UV spectrophotometer at 363 nm (Xia et al. 2010). 3.11 Physical stability study Three temperature conditions were applied in the stability study of the nanosuspensions: 4 °C (refrigerator), room temperature and 40 °C (stability chamber). Physical stability of the nanosuspensions was evaluated after 6 months of storage. The measurement of particle size was performed and settling behavior by visual examination (Van Eerdenbrugh et al. 2008). 3.12 In vitro dissolution The lyophilized nanoparticles and pure drug were filled in gelatin capsules (Size 1, Yarrow Chem Products, Mumbai, India) and dissolution was studied using a basket type dissolution apparatus (Tablet Dissolution Tester, TDT-06T, Electrolab, Mumbai, India) at 100 rpm in PB6.5 (Phosphate Buffer pH 6.5) used as dissolution medium for 2 h. 3.13 In vivo studies in rats 3.13.1 Animals Eighteen white Male Wistar rats (body weight 200 ± 50 g) of equal size (n = 6) were randomly divided into three groups. Food was withdrawn 12 h prior to the in vivo study with water provided ad libitum and no food was allowed after dosing until the end of the study (after 24 h). All experimental procedures were performed as per the ethics and regulations of animal experiments CPSCEA, India. 3.14 Study design The in vivo studies compared the pharmacokinetics of lyophilized felodipine nanosuspension based on PVA (NS1) and HPMC (NS2) with a control drug powder filled in hard gelatin capsules. These were given orally in single doses of 20 mg drug. Doses were established depending on the linearity of pharmacokinetic parameters in rat and the necessary conditions requested by the analytical method. A non-blind, three treatments, randomized, parallel design was used. The control capsules were hard gelatin capsules (size no. 1) each filled with 100 mg of a blend of 20 mg felodipine with 80 mg of a 7:1 mixture of lactose and microcrystalline cellulose. Blood samples (0.2 ml) were withdrawn by retro orbital puncture at the following time points: 0 (predose) 0.25, 0.5, 1, 2, 3, 4, 8, 12 and 24 h after administration of a treatment. Plasma was obtained by centrifugation at 4,000 r/min for 15 min and then it was
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Eur J Drug Metab Pharmacokinet Fig. 1 Chromatogram of felodipine and internal standard in plasma sample
deep-frozen at -70 °C until required for analysis (AboulEinien 2009). 3.15 Chromatographic conditions Felodipine was analyzed using HPLC system consisting of waters 2,695–Alliance L05SM4395M model separations module with Millennium32 software, auto injector and waters 2,996 photodiode array detector. Separation was carried out using a waters symmetry C18 column (25 cm 9 4.5 mm, 5 lm). Mobile phase consisting of an acetonitrile, methanol and phosphate buffer 0.05 M (40:20:40) adjusted to pH 3.0 with phosphoric acid was used at a flow rate of 1.5 ml/min. Mobile phase was filtered (Millipore system, 0.2 lm) under vacuum and degassed. Chromatographic separation was performed at 238 nm. Total run time for the analysis was 10 min.
nylon membrane filters and portions of the filtrates (20 ll) were then injected onto the column. A blank plasma sample, without the addition of felodipine was treated in the same way. A standard curve was constructed by plotting the ratio of peak area of felodipine to the internal standard against the drug concentrations in plasma. All assays were performed in triplicate. The retention time of felodipine and internal standard was 7.454 and 5.161, respectively. Figure 1 shows a chromatogram of felodipine containing diclofenac as internal standard in plasma. The LOD and LOQ of the method were determined by replicate injections of serially diluted drug. The LOD was defined as a minimum signal-to-noise ratio of 3. The LOQ was determined by taking three times of S/N ratio of LOD. LOD and LOQ were found to be 0.41 and 1.25 lg/ml, respectively. A linear response across the full range of concentrations from 2.5 to 25.0 (2.5, 5.0, 6.25, 12.5, 25.0) lg/ml (r2 = 0.994) was obtained.
3.16 Standard solutions 3.17 Plasma analysis Blank plasma samples (0.1 ml) were spiked with felodipine methanolic stock solution to obtain serial concentrations of the drug. Diclofenac sodium has been used as an internal standard and its concentration was kept constant in all samples at 10 lg/ml. Aliquots of 100 ll of spiked plasma samples were extracted with 500 ll toluene. The mixtures were shaken overnight then centrifuged for 10 min at 3,000 r/min. Supernatant was evaporated under nitrogen at 35–45 °C under reduced pressure until dryness, reconstituted the dried residue with 300 ll of mobile phase and vortexed. Although the boiling point of toluene is 110 °C, it can be evaporated at much lower temperature of 35–45 °C by heating under reduced pressure in a vacuum condition. Toluene has been selected as solvent because of its efficient extraction of felodipine from Plasma (Durol and Greenblatt 1997). These were passed through 0.45 lm
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The plasma samples obtained from the rats after receiving nanosuspensions NS1, NS2 and control were assayed as described above without the addition of felodipine. 3.18 Validation of analytical method The HPLC method of extraction and subsequent analysis were validated by observing the extraction efficiency of the method, precision and accuracy of the method. 3.19 Extraction efficiency The extraction efficiency for felodipine and internal standard from plasma were experimentally determined using the method explained above. Extraction efficiency was
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determined by comparison of the peak areas of the spiked plasma samples (2.5, 5, 12.5 lg/ml) with those of unextracted felodipine solution of similar concentrations (Durol and Greenblatt 1997).
terminal log-linear phase by log-linear regression analysis. The apparent terminal elimination half-life (t1/2) was calculated as t1/2 = 0.693/k. 3.24 Statistical analysis
3.20 Inter- and intra-day accuracy and precision Replicates of six were produced to evaluate intra-day and inter-day accuracy and precision. Accuracy was determined as the absolute value of the ratio of the back-calculated mean values to their respective nominal values and expressed as percentage. Precision was determined by the percentage coefficient of variation (% CV) for QC concentrations.
Statistical significance in the differences of means between the nanosuspensions and control was determined by Student’s t test. Statistical analyses of pharmacokinetic parameters were performed by applying a two-way analysis of variance (ANOVA). Differences in mean values were considered statistically significant at a P value B0.05. The software, Graph Pad Prism (version 6.01 Graph Pad software Inc.), was used to perform the statistical analysis.
3.21 Accuracy 4 Results and discussion The accuracy of the method was demonstrated through determination on samples in 3 concentrations of 2.5, 5.0 and 12.5 lg/ml in triplicates. Standard solutions were injected onto the column and from the chromatograph measured the area due to felodipine peak. Calculated the amount of felodipine found in each of these solutions using calibration curve and compared for accuracy. 3.22 Precision The precision of the method was observed through injection reproducibility. For injection reproducibility, six injections of the same standard preparations were made and the relative standard deviation for the replicate injections was calculated. The mean and standard deviation and the relative standard deviation were then calculated for the six replicate injections. 3.23 Pharmacokinetic analysis Pharmacokinetic characteristics from plasma data following administration of the three treatments were estimated for each rat. The observed maximal drug concentration (Cmax) and its time (tmax) were estimated from the plasma concentration–time curve. The area under the curve, AUC0–24 (lg h/ml), was calculated using the trapezoidal rule from zero time to time of the last blood sample (24 h). The elimination rate constant (k) was estimated from the
4.1 Preparation of nanosuspension Felodipine nanoparticles were produced by precipitation– ultrasonication technique. The aqueous phase containing a suitable stabilizer has been used as the anti-solvent and ethanol having good solubility of felodipine has been used as solvent. The results of optimized formulation for PVAand HPMC-based nanosuspenison are shown in Table 1. The nanosuspensions can be stabilized by electrostatic or steric stabilization or a combination of both. However, steric stabilization is more advantageous than electrostatic stabilization as the latter may be lost in the variable pH condition of the GIT and is effected by electrolytes. The stability of sterically stabilized nanosuspensions depends on the property of the drug-like enthalpy and logP as well as the hydrophobicity of the stabilizer (Matteucci et al. 2007; George and Ghosh 2013). Also the choice of an ideal stabilizer is influenced by the degree of hydrophobicity of the drug itself. And it has been concluded from various studies that ionic stabilization with high zeta potential is not a requisite for stability of nanosuspension. In fact, the stabilizer–drug interaction is a stronger factor than surface charge itself. A similar hydrophobicity should result in better surface coverage thereby providing better steric stabilization. Hence, polymeric non-ionic stabilizer HPMC and PVA of moderate hydrophobicity were selected for the steric stability of the felodipine nanosuspensions. HPMC
Table 1 Effect of drug concentration and surfactant concentration on mean particle size, and polydispersity index of optimized formulations Formulation code
Diffusing drug concentrations (mg/ml)
PVA (% w/v)
HPMC (% w/v)
Z average diameter (nm) ± SD
Polydispersity Index (PI)) ± SD
NS1 NS2
60 40
0.75 –
– 0.5
95.97 ± 12.5 362.1 ± 22.5
0.494 ± 0.026 0.624 ± 0.041
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Fig. 3 SEM photomicrograph of PVA-based felodipine nanoparticles. Scale bar 0.2 lm
Fig. 2 TEM photomicrograph of PVA-based felodipine nanoparticles. Scale bar 100 nm
electron micrographs revealed that the particles were spherical and homogeneous as shown in Fig. 2. 4.4 Scanning electron microscopy (SEM)
and PVA were used at various concentrations (0.1, 0.25, 0.5, 1.0 %). 4.2 Size measurement and zeta potential analysis The formulations containing HPMC as stabilizer showed particle size in the range of 320–410 nm at various drug concentrations. The formulations were homogeneous as indicated by polydispersity index of 0.5 ± 0.1. The average particle diameter was found to decrease further in formulations containing PVA as stabilizer at concentrations of 0.5 %.The particle size distributions were found to be more uniform as the average particle size of PVAbased felodipine nanoparticles were found to be in the range of 60–330 nm and the polydispersity index narrowed down to 0.3–0.5. The zeta potential of the nanoparticles was found to be negative and in the range of 5–18 mV. The negative charge may be due to ionization of the carboxyl group in an aqueous environment. However, adsorption of non-ionic stabilizers (HPMC and PVA) results in an increase in the thickness of the diffuse double layer, and hence, a lower zeta potential (Sahu and Das 2013). 4.3 Transmission electron microscopy (TEM) A transmission electron microscopic study was carried out for the optimized formulation to observe the physical properties of precipitated nanoparticles. Transmission
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The drug particles precipitated with the PVA as stabilizer show spherical in shape and the size ranges from 60 to 200 nm as shown in Fig. 3. The particles are discrete and uniform in size and there is no sign of agglomerations. The drug particles precipitated with the HPMC as stabilizer are slightly bigger and the size ranges from 100 to 300 nm (Sahu and Das 2013). 4.5 Saturation solubility The results of saturation solubility of pure drug and lyophilized felodipine nanosuspension revealed a saturation solubility of 19.17 and 582.32 lg/ml, respectively. The saturation solubility of felodipine as nanosuspension is 30-fold higher than that of pure felodipine. In the present study, particle size of felodipine has been reduced and hence, increased the saturation solubility due to increasing the surface area of the reduced particles. 4.6 Differential scanning calorimetry (DSC) The DSC analysis of pure drug, PVA and the NS1 was carried. The pure drug felodipine showed a melting point peak at 145 °C. The DSC thermogram, however, showed that after precipitation its melting point was decreased to 132 °C, indicating reduced crystallinity. The results are shown in Fig. 4. The DSC analysis of pure drug, HPMC and dried nanoparticles based on HPMC were done
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Fig. 4 DSC thermograms of (redline) pure drug felodipine (blueline) PVA and (greenline) precipitated felodipine nanoparticles (color figure online)
felodipine displayed the presence of numerous distinct peaks at 10.198, 16.518, 20.478, 23.278, 24.518, 25.378, 26.458, and 32.658, which suggested that the drug was of crystalline form. The PVA stabilized precipitated nanoparticles samples showed the similar diffraction pattern but with lower peak intensity, suggesting the crystallinity of precipitated nanoparticles decreased during the precipitation process. Similar results have been obtained and previously reported with HPMC-based formulation (Sahu and Das 2013). 4.8 Fourier transforms infrared spectroscopy (FT-IR)
Fig. 5 X-ray diffraction patterns of (blue line) pure felodipine drug (red line) precipitated PVA-based felodipine nanoparticles (color figure online)
similarly. The results have been reported previously (Sahu and Das 2013). 4.7 X-ray diffraction studies (XRD) The representative X-ray diffraction patterns of the pure felodipine powder and oven-dried nanosuspensions (NS1) are shown in Fig. 5. The figures indicated the changes in the drug crystal structure. The X-ray patterns of the pure
Infra red spectra of Felodipine and PVA and the precipitated nanoparticles (NS1) were comparable and the peaks of felodipine in the formulation are of lower intensity than pure drug. The characteristic peaks of pure were found to be intact in the physical mixture as well as the formulation which indicates the absence of PVA and drug interactions. The results have been shown in Fig. 6. Similar results have been obtained and previously reported with HPMC-based formulation (Sahu and Das 2013). 4.9 Physical stability study The particle size of the formulations after 6 months was observed. The formulations at 4 8C remained stable,
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Eur J Drug Metab Pharmacokinet Fig. 6 FTIR spectra’s of a pure drug felodipine b PVA c precipitated PVA stabilized felodipine nanoparticles
Table 2 Physical nanosuspensions Formulations
FF3
stability
Storage temperature conditions 8C 4
evaluation
Initial particle size (±SD, n = 3) 108.5 ± 4.6
RT 4
the
felodipine
Particle size after 3 months (±SD, n = 3) 104.5 ± 2.6 106.4 ± 3.6
40 FF6
of
118.5 ± 2.4 310.7 ± 13.8
295.4 ± 14.1
RT
302.6 ± 12.6
40
354.2 ± 16.2
whereas that at room temperature showed slight increase in particle size. In the case of HPMC formulations, only those stored at higher temperatures (40 8C) exhibited an increase
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in mean particle size. The increase in size in the HPMC formulation stored at 40 8C was attributed to dehydration of the HPMC chains and subsequent loss of protection of the nanoparticles. The particle size data are given in the Table 2. Sedimentation was observed in all the conditions but the preparations were easily redispersed on shaking (Sahu and Das 2013). 4.10 In vitro dissolution The in vitro dissolution of a drug was determined for the control and formulation (NS1 and NS2) filled capsules. Optimized nanosized felodipine showed a dramatic increase in rate and extent of dissolution compared with coarse powder. The lyophilised optimized formulations
Eur J Drug Metab Pharmacokinet Table 3 Pharmacokinetic parameters of felodipine nanosuspension NS1, NS2 and control formulation in rats (mean ± SD, n = 6)
Fig. 7 Drug dissolution profile of optimized lyophilized formulations and control (Standard error bar, n = 6)
NS1 and NS2 showed 93 and 74 % drug dissolution in 120 min, respectively, from the filled capsules as compared to 38 % of pure drug. The results are shown in Fig. 7. This enhanced dissolution may be due to the increased saturation solubility resulted due to increase in surface area of drug. 4.11 Pharmacokinetic studies The HPLC method and extraction process were well validated. The extraction efficiency was found to be of 96.5, 93.5, and 92.2 % for felodipine for concentration of 2.5, 5.0 and 12.5 %, respectively, and 94.2 ± 1.2 % for internal standard. Efficient extraction of felodipine from plasma was obtained using the method explained using toluene and
Formulation
NS1
NS2
Control
AUC0?24 (lg h/ml)
48.34 ± 3.78
37.40 ± 3.56
20.48 ± 1.27
Cmax (lg/ml)
4.171 ± 0.0175
3.627 ± 0.18
1.093 ± 0.004
tmax (h) k
1.0 ± 0.25 0.058
1.0 ± 0.33 0.057
2.0 ± 0.5 0.056
t1/2 (h)
11.8 ± 2.1
12.09 ± 1.9
12.6 ± 1.7
showed that extraction decreases with increasing drug concentration. The accuracy and precision of the process was also established and found to be satisfactory. Relative standard deviation (RSSD) value for six injection of the same concentration was found to be 1.44 indicating precision of the injector as well as the method. The plasma drug concentration–time profiles and pharmacokinetic parameters of felodipine resulted from the oral administrations of the nanosuspensions and control in wistar rats are presented in Fig. 8 and Table 3, respectively. The control preparation used in this study was felodipine powder filled in hard gelatin capsules. No marketed felodipine product was used as a control because felodipine is commercially available only in the form of sustainedrelease tablets and no felodipine formulation with enhanced drug dissolution is available commercially. Felodipine powder filled in hard gelatin capsules has been used as a control treatment in bioequivalence studies on the coenzyme Q10 (Weis et al. 1994; Zaghloul et al. 2002). The mean plasma concentration–time curves following administration of capsules filled with lyophilized
Fig. 8 Average plasma drug concentration versus time profiles after oral administration of lyophilized felodipine nanosuspensions NS1, NS2 and control formulation (mean ± SD, n = 6)
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nanosuspensions NS1, NS2 and a control capsule containing felodipine powder are shown in Fig. 8. The shape of the plasma concentration–time curve of nanosuspensions was found to be markedly different from that of control. The plasma drug concentration profile of nanosuspensions showed significant improvement in drug absorption than the reference formulation. The Cmax values of nanosuspensions NS1 and NS2 were approximately 3.8-fold and 3.3-fold greater than that of control preparation, respectively. The AUC0?24 values of nanosuspensions were approximately 2.4 and 1.8-fold greater than that of control preparation. The improved oral bioavailability of felodipine may be due to the rapid absorbtion of felodipine from gastrointestinal wall due to the significantly improved dissolution rate by the reduced particle size with increased surface area (Hintz and Johnson 1989; Bernhard and Muller 1999). The mean pharmacokinetic parameters, Cmax, tmax, AUC0–24, Ke and t1/2, are reported in Table 3. The mean Cmax values were found to be 4.171 ± 0.017 and 3.627 ± 0.18(lg/ml) for nanosuspensions NS1 and NS2, while control showed a value of 1.093 ± 0.004 lg/ml. The mean tmax values were 1 and 2 h for nanosuspension and control, respectively. The differences between the treatments NS1 and NS2 with the control for Cmax and tmax were statistically significant. The shortened tmax obtained for nanosuspensions could be attributed to the enhanced dissolution resulting in rapid drug absorption. The mean area under the curve reflecting the total amount of absorption in 24 h, AUC0–24, was estimated for all treatments and was found to be statistically significantly different (P \ 0.05) between the nanosuspensions and control. Statistical analysis of the half-life indicated a nonsignificant difference between results from nanosuspensions (11.8 ± 2.1 h and 12.09 ± 1.9) and pure drug (12.6 ± 1.7 h). Based on these results, it can be concluded that formulation of felodipine as nanosuspensions results in rapid and enhanced absorption of felodipine which could be explained by the improved saturation solubility and thereby dissolution of drug.
5 Conclusion From the study, it can be concluded that the oral absorption of felodipine increased significantly in rats when formulated as nanosuspensions. This enhancement in oral bioavailability may be due to the enhanced dissolution resulted due to increase in surface area with reduction in size. Hence, felodipine nanosuspension prepared by precipitation with sonication technique may be suitable to increase the oral bioavailability of poorly soluble felodipine.
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