Clozapine, an effective atypical antipsychotic drug has a very poor oral bioavailability (< 27%) due to first pass metabolism. Clozapine solid lipid nanoparticles were prepared using different lipids (Compritol ATO 888, GMS and Precirol ATO 5) by solvent emulsification diffusion technique. The effect of type and concentration of lipid and emulsifiers on particle size, zeta potential, entrapment efficiency and in vitro steric stability of SLNs was studied. In vitro release studies were performed using dialysis bag method. SLN formulations of clozapine having a mean size range of 385.5 ± 3.5 to 655.2 ± 2.6 nm and zeta potential range of -13.1±3.9 to -27.3±1.3 mV were developed. More than 70% of clozapine was entrapped in SLNs. The release pattern of drug was found to follow Weibull and Higuchi equations.
Keywords: In vitro release, particle size, zeta potential.
Solid lipid particles have been considered as promising drug carrier systems and as alternative to other colloidal carriers like emulsions, liposomes, polymeric micro and nanoparticles (Muller et al. 1995). SLNs combine the advantages of other colloidal carriers and avoid some of their disadvantages. SLNs are colloidal drug carrier systems in the size range of 50-1000 nm and composed of biocompatible and biodegradable materials capable of incorporating lipophilic and hydrophilic drugs and are proposed for different administration routes such as oral, topical (Kunisawa et al. 2000), ophthalmic, subcutaneous and intramuscular injection (Reithmeier et al. 2001) and particularly for parenteral administration (Cavalli et al. 1997; Yang et al. 1999). Clozapine is an effective atypical antipsychotic therapeutically effective in both the positive and negative symptoms of schizophrenia and is a unique drug for treating patients with schizophrenia that is refractory to other neuroleptics. Clozapine undergoes extensive first pass metabolism with a bioavailability of 27% (Jann, 1991). The present work was designed with the objective of investigating the effect of type of lipid, stabilizer and their concentration on SLN formation and drug release. The SLNs were characterized for particle size, zeta potential entrapment efficiency and in vitro release profiling. Electrolyte-induced flocculation was performed to study the in vitro steric stability.
Materials and methods
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Clozapine was a gift sample from RKG Pharma Pvt Ltd., Faridabad, India. Compritol was obtained from Colorcon Asia Ltd, Goa, India. GMS and Precirol were supplied by Lupin Pharmaceuticals Inc., Pune, India. Poloxamer 188 (Pluronic F 68) and dialysis membrane-50 (molecular weight cutoff 12,000) were purchased from HiMedia and Polysorbate 80 from Merck Specialities Pvt Ltd., Mumbai. The other chemicals were of analytical reagent grade.
Partitioning behavior of clozapine in various lipids
Fifty milligrams of clozapine was dispersed in a mixture of melted lipid (1 g) and 5 ml of hot phosphate buffer (PB, pH 7.4) and shaken for 30 min on a hot water bath. Aqueous phase was separated after cooling by centrifugation and analyzed for drug content using UV- Spectrophotometer (UV-1601, Shimadzu, Japan) at 214 nm against a suitable blank. The partition coefficient (PC) of clozapine in lipid/PB pH 7.4 was calculated using following equation:
PC = (Ci - Ca) / Ca
Where, Ci = the initial amount of clozapine added, and Ca = the concentration of clozapine in PB (pH 7.4).
Preparation of clozapine SLNs
The SLNs were prepared by solvent emulsification diffusion technique described by Battaglia et al with minor modifications (Battaglia et al. 2007). Briefly, solvent ethyl acetate and water were mutually saturated for 10 min at the temperature at which lipid dissolves in the solvent in order to ensure initial thermodynamic equilibrium of both liquids. Typically, the lipid (Table 1) was dissolved in 1000 µl of water-saturated solvent and this organic phase was emulsified with 20 ml of the solvent-saturated aqueous solution containing 0.5 to 1% of stabilizer using a mechanical stirrer (Remi Motors Pvt Ltd., Mumbai, India) at 2000 rpm for 5 min at the temperature (70° C) where lipid remains solubilised in the solvent. Coarse hot oil-in-water emulsion so obtained was ultrasonicated using Sonopuls ultrahomogenizer (BANDELIN, Berlin, Germany) for 5 min and allowed to cool to room temperature. SLNs were prepared using different type and concentrations of lipids (Compritol, GMS and Precirol) and stabilizers (Tween 80 and poloxamer 188). Blank SLNs were prepared in a similar way without drug for comparison studies.
Characterization of SLNs
Measurement of size and zeta potential of SLNs
Size and zeta potential of SLNs were measured by photon correlation spectroscopy (PCS) using Malvern Zetasizer. Samples were diluted appropriately with the aqueous phase of the formulation for the measurements.
In vitro Steric Stability
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In vitro steric stability was determined by electrolyte-induced flocculation test. Sodium sulfate solutions ranging from 0 M to 1.8 M were prepared in a 16.7% w/v sucrose solution (Subramanian et al. 2003). An appropriate volume of SLN dispersion was made upto 5 ml using sodium sulfate solutions of varying concentrations (0 - 1.8 M) to obtain a final concentration of 1 mg/ml lipid. The absorbance of the resulting dispersions was measured within 5 minutes at 400 nm using a UV-visible spectrophotometer (Shimadzu-1601, Japan) against a respective blank.
The aqueous SLN suspension was filtered to isolate SLNs from aqueous phase and dried at 40°C. 50 mg of SLNs was then heated with 5 ml of methanol in which the drug is soluble and shaken in order to extract the drug in the solvent. The solvent was diluted with water to analyze spectrophotometrically at 214 nm using UV-visible spectrophotometer (Shimadzu-1601, Japan) against a suitable blank. The entrapment efficiency was determined using the formula:
EE (%) = (amount of drug incorporated X 100)/ amount of drug initially used.
In vitro drug release studies
In vitro release of clozapine from SLNs was evaluated using a dialysis bag diffusion technique. Dialysis bags were filled with 5 ml of SLN dispersion and immersed in 500 ml dissolution medium (0.1 N HCl- pH 1.2, phosphate buffer pH 7.4, or double distilled water) using a dissolution rate test apparatus with a paddle rotation of 50 rpm. Aliquots of 5 ml samples were withdrawn from the medium and replaced with the same volume of fresh dissolution medium every time. The samples were estimated spectrophotometrically at 214 nm using UV-visible spectrophotometer (Shimadzu-1601, Japan) against a suitable blank. The UV spectrophotometric method for clozapine was validated. The calibration curve was linear in the working range of 2-12 µg/ml (r2 =0.9938).
Selected formulation, CP2, was stored at controlled condition of relative humidity (RH) and temperature in a stability chamber (Remi Instruments Ltd., Mumbai). At the end of stability studies the SLNs were evaluated for in vitro drug release and drug content.
The data obtained from the dissolution studies was statistically analyzed by one way ANOVA followed by Tukey method. A probability value of p<0.05 was considered as statistically significant.
Results and discussions
Partitioning behavior of clozapine
Partition coefficient of clozapine was found to be 56.25 ± 9.25, 38.18 ± 3.74 and 28.91 ± 9.25 for Compritol, Precirol and GMS. Compritol and Precirol are mixtures of mono- di- and tri-glycerides and hence form less perfect crystals with many imperfections, offering space to accommodate the drug while lipid matrix consisting of similar molecules (eg glyceryl monostearate) form perfect crystals with few imperfections leading to drug expulsion (Wissing et al. 2004).
Size and zeta potential of SLNs
The particle size, PI and zeta potential of the SLNs are summarized in Table 2. Slight increase in particle size was observed on incorporation of drug into particles. On shifting from Tween 80 to poloxamer 188, the particle size of SLNs also increased, this was found to be in contrast to that observed by Varshosaz et al with pentoxifylline SLNs. (Varshosaz et al. 2009). A decrease in particle size was observed with decrease in lipid concentration.
The polydispersity index (PI) is a measure of dispersion homogeneity and ranges from 0 to 1. Values close to zero indicate a homogeneous dispersion, while those greater than 0.3 indicate high heterogeneity (Yegin et al. 2006). Thus, PI indicated good homogeneity for formulation PP1 and blank Compritol and GMS SLNs. While, high heterogeneity was observed for formulations GT5, PT1 and blank Precirol SLNs.
All formulations possessed negative zeta potential. SLNs of Compritol had the highest negative zeta-potential (-27.3 mV) due to the highly negative charge distributed at the surface of the SLNs, while the negative value of zeta-potential decreased to -23.7 mV when Compritol was replaced with Precirol and GMS (-19.0 mV). Also the strong negative charge provides a basis for the overall high stability of the suspension (Yegin et al. 2006).
In vitro steric stability
The electrolyte-induced flocculation technique helps in determining the resistance of SLNs to aggregation against the flocculating agents. SLNs stabilized by Tween 80 or poloxamer 188 showed a gradual increase in flocculation with the increase in electrolyte concentration (Figure 1). SLNs started to flocculate when concentration of sodium sulfate exceeded 0.3 M.
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It has been reported that coating the particulate carrier systems with hydrophilic surfactants, provides steric stability by rendering a hydrophilic surface, which prevents opsonisation in blood (Huang et al. 1993), thus enhances their circulation half life after intravenous administration (Porter et al. 1992). Excess electrolyte concentration distorts the steric barrier around the particle resulting in flocculation of the particles, with a corresponding increase in optical turbidity of the particle dispersion, which can be measured by the absorbance of the dispersion at 400 nm (Vivek et al. 2007).
As shown in Figure 2a entrapment efficiency was found to marginally decrease with the increase in Tween 80 concentration. This may be due to increased solubility of clozapine in aqueous phase as percentage of Tween 80 increased, as entrapment efficiency of drug depends on solubility of drug in the lipid melt as well as on the amount of surfactant (Tiyaboonchai et al. 2007). The decrease was significant for each lipid used during the formulation. SLNs stabilized by poloxamer 188 showed little variations to the concentration of surfactant on EE, except in formulation with Precirol where a marginal decrease in EE was observed (Figure 2b). The entrapment of drug in lipid matrix (SLN's) is determined by several factors like solubility of drug in lipid melt, chemical and physical structure of solid matrix, and polymorphic state of lipid material.
In vitro release studies
Effect of pH of dissolution medium
About 72.93 ± 4.1, 93.16 ± 4.12 and 101 ± 2.41% of clozapine was released within 2 h from SLNs of Compritol (CT1), GMS (GT1) and Precirol (PT1) respectively, in 0.1 N HCl (pH 1.2). In contrast, slow release was observed in double distilled water and phosphate buffer (pH 7.4) (data not shown). Clozapine is easily soluble in acid medium while the solubility is reduced in distilled water and phosphate buffer pH 7.4. This solubility behavior of clozapine in different pH of solution accounts for the observed variation in drug release in dissolution media with different pH.
Thus, pH of dissolution medium has a significant (p < 0.05) influence on the release rate of clozapine from SLNs significantly. The data gives comprehensive information on the route of dosage form. The prepared nanosuspension can be administered parenterally, alternatively the freeze dried product can be administered by oral route. The dissolution data can be used as a tool for selecting the route of administration based on therapeutic need of patient. While parenteral administration provides long action of clozapine, oral administration could be explored for faster action of the clozapine, however a detailed study of enhancement in bioavailability is required.
Effect of type of lipids
Clozapine release from SLNs was dependent on the type of the lipid present in the matrix and was in the following order Precirol SLNs > Compritol SLNs > GMS SLNs as shown in Figure 3. Slow release of clozapine from SLNs suggests that clozapine was homogenously dispersed in lipid matrix (Venkateswarlu et al. 2004). No significant difference (p>0.05) in release was observed between SLNs of Compritol and GMS when stabilized by Tween 80 while a significant difference in the release pattern between them (p < 0.05) was evident when poloxamer 188 was used as stabilizer.
Effect of lipid concentration
In vitro drug release profiles from SLNs with different lipid concentration are shown in Figure 4. In case of GMS and Compritol SLNs, any increment in lipid concentration was found to cause a decrease in drug release. However no significant difference in the release (p>0.05) was observed on increasing the concentration of Precirol in SLNs.
Effect of stabilizer concentration
Percentage of clozapine released from the SLNs stabilized by different concentrations of Tween 80 is shown in Figure 5. In case of SLNs stabilized by Tween 80, the drug release was found to increase with stabilizer concentration. However, when poloxamer 188 was used to stabilize SLNs with GMS and Compritol matrix, no significant effect on drug release was observed. While in Precirol SLNs a significant effect on drug release was observed.
In order to determine the release model which best describes the pattern of drug release, the in vitro release data was fitted to zero order and first order equations. The correlation coefficient (R2) value was used as criteria to choose the best model to describe drug release from SLNs. The R2 values indicated that drug release from SLNs can be best described according to first order kinetics. The values of 'n' in Peppas model indicated that most of the formulations followed Fickian diffusion (n < 0.5) except formulations CT2 and CP2 which indicates anomalous transport. Clozapine release from almost all SLNs followed Weibull and Higuchi equations as the regression coefficient was towards linearity. The shape factor 'b' characterizes the curves as one with steeper initial slope then consistent with the exponential as it was <1 for all formulations (Table 3).
MDT values were used to characterize drug release rate from SLNs and indicated the drug release retarding efficiency of the lipids as shown in Table 3.
At the end of stability studies, EE of Compritol SLNs (CP2) was significantly (p<0.05) lowered by 2.9 ± 0.46%, while the drug release was found to be similar till sixth hour but at the eigth hour a significant increase was observed (p<0.05). Transitions of dispersed lipid from metastable forms to stable form might occur slowly on storage due to small particle size and presence of emulsifier that may lead to drug expulsion from SLNs (Westesen et al. 1993; Bunjes et al. 1995). Therefore, lowered EE may be due to drug expulsion due to lipid modification.
Solvent emulsification diffusion technique is suitable to produce SLNs of 385-655 nm size ranges with reasonable stability. Clozapine, a lipophilic drug can be successfully loaded in solid lipid nanoparticles consisting of Compritol, GMS and Precirol matrix. In vitro release of clozapine followed diffusion mechanism described by Weibull and Higuchi equations. Further evaluation of the prepared systems in vivo like pharmacokinetic, pharmacodynamic and tissue distribution studies will go a long way in establishing the therapeutic efficacy of prepared SLNs.