Preparation, characterization, stability and in vitro-in vivo evaluation of pellet-layered Simvastatin nanosuspensions

Abstract

The objective of the present study was to develop stable pellets-layered Simvastatin (SIM) nanosuspensions with improved dissolution and bioavailability. The nanosuspensions were prepared with 7% HPMC, antioxidant 0.03% butylated hydroxyanisole and 0.2% citric acid (m/v) by low temperature grinding. After that, SDS with SIM was in a ratio of 1:5 (m/m), was evenly dispersed in the nanosuspensions. Then, they were layered on the surface of sugar pellets. The mean particle size of the SIM nanosuspensions was 0.74 µm, and 80.6% of the particles was below 1 µm in size. The pellets could re-disperse into nanoparticle status in the dissolution medium. In 900 mL pH 7.0 phosphate solutions, the dissolution of the layered pellets was better than that of commercial tablets. Also, nearly 100% of the drug dissolved from the pellets within 5 min under sink conditions. During the stability studies, SIM pellets exhibited good physical and chemical stability. The relative bioavailability of SIM and Simvastatin β-hydroxy acid (SIMA) for nanosuspensions layered pellets compared with commercial tablets was 117% and 173%, respectively. The bioavailability of SIMA was improved significantly (p < 0.05), confirming the improvement of bioavailability. Thus, the present study demonstrates that the pellet-layered SIM nanosuspensions improved both the dissolution and bioavailability of SIM.

Keywords: Simvastatin, pellets, nanosuspensions, bioavailability, dissolution, stability

Introduction

Simvastatin (SIM) (chemical structure shown in Figure 1A) is a cholesterol-lowering agent, which is widely used to treat hypercholesterolemia. After oral administration, SIM is converted to the corresponding, β-hydroxy acid (SIMA) (chemical structure shown in Figure 1B), which is a potent inhibitor of HMG-CoA reductase. This enzyme catalyzes the conversion of HMG-CoA to mevalonate, which is an early and rate-limiting step in the biosynthesis of choles- terol1,2. SIM has a low solubility and high permeability, its solubility being 6.3 × 10-3 g/L at 25°C pH 1–73. According to the Biopharmaceutical Classification System (BCS), SIM is a class II drug. Literature reports show that 30–40% of the newly developed drugs are poorly water soluble and, consequently, they have a poor bioavailability and there is a high dropout rate during their development4,5. Consequently, the solubility of SIM is the limiting factor for bioavailability.

Numerous methods have been described to improve drug solubility in the literatures such as reducing the particle size to increase the surface area6, the formation of water-soluble complexes7, the use of pro-drug and drug derivatization8, and the transformation from a crystalline form to an amorphous one. However, formation of water-soluble complexes or drug derivatization may influence the therapeutic effect of SIM. In addition, SIM, which is susceptible to slow oxidative degradation9, is different from common poorly water-soluble drugs. Due to the highly ordered structure of the crystalline spherulites, the oxygen diffusion rate is significantly lower in the crystalline form than in the amorphous form10. Accordingly, particle size reduction was chosen to increase the solubility of SIM.

Nanosizing can be used to increase the solubility, dis- solution rate and oral absorption of poorly water-soluble compounds. It is a robust approach with the possibility of scale-up as exemplified by several marketed formula- tions (Rapamune® (Wyeth), Emend® (Merck), Tricor® (Abbott Laboratories) and Megace® ES (Bristol-Myers Squibb11)).

There are many literature reports about drug deliv- ery systems of SIM, such as inclusion complex12, lipid nanoparticles13,14, self-microemulsifying drug delivery systems15 and so on. However, these drug delivery sys- tems have their disadvantages. As the chemical structure of lactone, SIM is chemical unstability. However, these drug delivery systems lack stability study of SIM. They did not demonstrate the stability of SIM. Besides, they are difficult to produce on large scale, and the clinical use is also not easy to realize in a short time. So, it is neces- sary to produce a stable SIM preparation which can both improve the bioavailability of SIM and be easy to produce on a large scale.

In this study, pellet-layered SIM nanosuspensions was studied. This preparation improved the dissolution and bioavailability of SIM, and most of all the pellets were stable. First of all, SIM nanosuspensions were prepared with hydroxypropyl methyl cellulose (HPMC) by low tem- perature grinding. SIM is not stable, it is susceptible to slow oxidative degradation. And the SIM solution was less stable than the solid SIM. So, the nanosuspensions were then layered on the surface of sugar pellets in a fluid-bed. This method was verified by the pilot-scale production. The characterization and stability of the SIM pellets was studied in detail. Compared with commercial tablets (Zocor®, SIM 20 mg), the dissolution and bioavailability of pellet-layered SIM nanosuspensions were both improved and the bioavailability of SIMA was improved signifi- cantly (p < 0.05). The results indicated that pellets layered SIM nanosuspensions were stable and can improve the dissolution and bioavailability of SIM to a great degree.

Materials and methods

Materials

The raw SIM (purity 99.8%) was purchased from Henan Tianfang Pharmaceutical Corporation (Henan, China). Zocor® tablets were purchased from Merck Sharp & Dohme (Hangzhou, China). The SIMA ammo- nium salt (purity 98%) was purchased from Toronto Research Chemicals Inc (Toronto, Canada). Lovastatin (LOV) (internal standard, IS) (purity 99.9%) was pur- chased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HPMC E5 was provided by Colorcon (Shanghai, China). Sugar pellets, which were produced by sucrose, were provided by Gaocheng Biotech & Health Co. LTD (Hangzhou, China). Butylated hydroxyanisole (BHA), citric acid and sodium dodecyl sulfate (SDS) were pur- chased from Bodi Chemical Company (Tianjin, China). All the excipients were either of analytical or chromato- graphic grade.

Methods

Preparation of SIM pellets

Preparation of SIM nanosuspensions

To achieve this, 30 g SIM was dispersed in 200 mL HPMC solution at a concentration of 15% (w/v). The concentra- tion of the HPMC solution was 5%, 7% and 10% (w/v). Then, 0.03 % BHA (m/v) and 0.2 % citric acid (m/v) were added to the drug suspensions and then the suspensions were stirred on the magnetic stirring apparatus (Gongyi Yuhua Instrument Factory, Zhengzhou, China) for 30 min to obtain pre-milled suspensions.The pre-milled suspensions were ground in a MiniEasy Nano-grinder (Retsch Topway Technology CO. LTD, Beijing, China) at 3000 r/min for 45 min and the grinding temperature was kept below 20°C using cold water. This allowed the SIM nanosuspensions to be obtained.

Preparation of SIM pellets

After grinding, SDS was evenly dispersed in the SIM nanosuspensions whose concentration of the HPMC was 7%, and the ratio of SDS to SIM was 1:5 (m/m). Then, the nanosuspensions were diluted with water in a ratio of 1:2 (v/v) and these diluted nanosuspensions were layered on the surface of sugar pellets in a fluid-bed with a bottom spray (FD-MP-01, Powrex, Japan). The weight of SIM layered on the surface of pellets was 7% of the sugar pellets (w/w). The process parameters were as follows: inlet temperature 25°C, outlet temperature 20°C, spray rate 2.5 mL/min, atomization pressure 0.3 MPa, and rate of air flow 80 m3/h. After the layering process, SIM pellets were dried at 40°C for 3 h.

Characterization

Particle size distribution

The particle size distribution (PSD) was determined using a Laser material Path Analyzer LS230 (Coulter Instruments, Brea, CA, USA) and data evaluation was carried out using Coulter LS software (version 3.19, Coulter Instruments, Brea, CA, USA). The samples were pre-milled suspensions, nanosuspensions and suspen- sions re-dispersed by the pellets. Before determination, samples were diluted with the saturated aqueous solu- tion of SIM. The particles of SIM were fully dispersed in the suspension during the determination process.

Scanning electron microscopy

A scanning electron microscope (SEM) (SUPRA35, LEO, German) was used to examine the dried nanosuspen- sions, the surface and cross-section of the layered pellets, and the pellets which was stored at room temperature for 12 months.

Optical microscopy

Optical micrographs were obtained using a XSZ-G type light microscope (Chongqing Optical Instrument Co., Chongqing, China). Micrographs with the same magnify- ing power of 400 were recorded to study the morphologi- cal characteristics of the suspensions.

Differential scanning calorimetry

The SIM pre-milled suspensions and nanosuspen- sions were dried and then milled in mortars for further study. The pellets-layered nanosuspensions was also milled in mortars. The SIM, dried pre-milled suspen- sions, nanosuspensions and powder of pellets-layered nanosuspensions were determined by Differential scanning calorimetry (DSC). DSC analysis was carried out using a Modulated temperature DSC (MTDSC), DSC 1 (Mettler Toledo, Zurich, Switzerland) and apply- ing TOPEMR with a modulation amplitude of ±0.5°C. Nitrogen was used as the purge gas at a flow rate of 40 mL/min. Samples were crimped in aluminum pans with a pinhole. For this, 2 mg samples were weighed in aluminum pans and analyzed at a heating rate of 2°C/ min in an atmosphere of nitrogen. As the melting point of SIM was 135~138°C3, the temperature range was 100–170°C.

X-ray powder diffraction

X-ray powder diffraction (XRPD) was performed using a D/Max-2400 X-ray Fluorescence Spectrometer (Rigaku, Osaka, Japan) with a CuKa line as the source of radiation. Standard runs were carried out using a voltage of 56 kV, a current of 182 mA and a scanning rate of 2°min−1 over a 2θ range of 5 to 60°. The samples were powdered SIM, dried pre-milled suspensions, nanosuspensions and the powder of pellets-layered nanosuspensions.

Chemical degradation

As SIM is susceptible to slow oxidative degradation, the chemical degradation during grinding was assessed by high pressure liquid chromatography (HPLC). To deter- mine the degradation, the samples were examined by HPLC using methods prescribed by BP and USP. Compared with the raw SIM, the increase in related substances was considered as degradation. A Hitachi series 2100 liquid chromatograph (Hitachi, Tokyo, Japan) comprising of a PU-2130 pump, an autosampler and a UV-2400 UV detec- tor were used for determination. Chromatographic sepa- ration was achieved using a Venusil ASB C18 analytical column (4.6 mm × 33 mm, particle size 3 μm).

Dissolution study

The dissolution of the SIM formulations was investigated using a ZRS-8G dissolution apparatus (Tianjin, China) with USP31-NF26 Apparatus II (paddle method) in 900 mL medium. The medium was 0.01M sodium dihy- drogen orthophosphate containing 0.1% (w/v) SDS, and it was adjusted to pH 7.0 with 1M sodium hydroxide. The paddle was set at 50 revolutions per minute at 37 ± 0.5°C. Then, 4 mL samples were withdrawn and replaced by fresh medium at 5, 10, 15, 20, 30, 45 and 60 min. Each sample was passed through a 0.22 μm cellulose acetate type mem- brane filter. According to the Ch.P, samples were analyzed by HPLC (Hitachi Organizer, PU-2130 pump and UV-2400 UV detector, Tokyo, Japan) at 238 nm. The dissolution specification was that each sample contained 20 mg SIM.

Stability study

The SIM pellets enclosed in a foil were kept in a Drug Stability Test Chamber [LRH-150(250)-Y, Guangdong, China] at 40°C and 75% relative humidity (RH) for 3 months and kept at room temperature for 12 months. In addition, the SIM pellets stored openly exposed to sun- light light intensity of which was 4500 ± 500 lx for 30 days. The dissolution behavior and the related substances of the pellets were both evaluated.

Bioavailability study

Administration program

The study protocol was approved by the Ethics Committee of Shenyang Pharmaceutical University. The study involved six beagle dogs, raised in the Experimental Animal Center of Shenyang Pharmaceutical University, weighing 10 ± 1 kg. The dogs were divided into two groups and a single-dose, randomized, crossover study was carried out with a washout period of 7 days. The two groups were the test given pellet-layered nanosuspensions and the reference group given commercial tablets. After fasting overnight, one tablet or one capsule which both contain 20 mg SIM were given to the beagle dogs with 200 mL water. The dogs were provided with standard food 8 h after dosing. On each dosing day, blood samples were taken before and 0.17, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12 and 24 h after dosing. Plasma was separated from blood samples by centrifugation at 4000 rpm for 10 min and then stored at −20°C until analysis within 1 month.

Plasma sample preparation

For this, 200 µL plasma samples were spiked with 20 µL internal standard solution (LOV, 250 µg/mL dissolved in methanol) and the samples were then vortexed for 1 min, followed by the addition of 3 mL ethyl acetate. After being vortexed for 10 min, the samples were then cen- trifuged at 4000 rpm/min for 10 min. The organic layers were transferred to other tubes and evaporated at 40°C using a Centrifugal Concentrator (CentriVap®78120-03, Labconco Corp., Kansas City, MO, USA). The residues were dissolved in 200 μL mobile phase and centrifuged at 12000 rpm for 10 min then 5 μL samples of the superna- tant were subjected to UPLC-MS/MS analysis.

Analysis conditions and method validation

The analysis was carried out on an ACQUITYTM UPLC system (Waters Corp., Milford, MA, USA) with a cooling autosampler and column oven. An ACQUITY UPLCTM BEH C18 column (50 mm × 2.1 mm, 1.7 µm; Waters Corp., Milford, MA, USA) was employed for separation and the column temperature was maintained at 40°C. The chromatographic separation was achieved with gradient elution using a mobile phase composed of 0.01 M ammo- nium acetate water and acetonitrile. The gradient elution started at 20% acetonitrile, increased linearly to 80% acetonitrile over 0.8 min, then maintained at 80% aceto- nitrile for 1.9 min, then returned to the initial percentage over 0.3 min and maintained there for another 0.5 min. The flow rate was set at 0.25 mL/min. The autosampler temperature was kept at 4°C.

A Waters ACQUITYTM TQD triple quadrupole tandem mass spectrometer (Waters Corp., Manchester, UK) with an electrospray ionization (ESI) interface was employed for mass analysis. The ESI source was operated in posi- tive ionization model with optimal operation parameters as follows: capillary voltage 0.2 kV, cone voltage 30 V, extractor 20V and RF 0.1V for SIM and SIMA, capillary voltage 0.2 kV, cone voltage 25 V, extractor 10V and RF 0.1V for LOV with a source temperature of 100°C and a desolvation temperature of 400°C. Nitrogen was used as the desolvation and cone gas at a flow rate of 450 L/h and 50 L/h, respectively. For collision-induced dissocia- tion (CID), argon was used as the collision gas at a flow rate of 0.17 mL/min. Quantification was performed using multiple reaction monitoring (MRM) of the transitions of m/z 405.24→285.19 for SIM, m/z 419.20→285.11 for SIMA and m/z 437.29→303.20 for LOV (IS), respectively. All data were collected in centroid mode and processed using MassLynx TM NT 4.1 software with a QuanLynx TM program (Waters Corp., Milford, MA, USA16–19).

The UPLC-MS/MS method had been validated previ- ously. The linear range for SIM and SIMA was 1–1000 ng/ mL with an R (correlation coefficient) value of not less than 0.99. The LLOQ values of SIM and SIMA were both 1 ng/mL. The RSD. values, which reflected the intra-day and the inter-day precision of the QC samples, were both not more than 10.0%. The extraction recoveries of SIM from the three different QC plasma samples were 72.2%, 79.8% and 72.0%. Also, the extraction recoveries of SIMA from the three different QC plasma samples were 54.2%, 52.3% and 51.4% and the mean extraction recovery of LOV was 73.3%.

Results and discussion

Preparation of pellet-layered SIM nanosuspensions During the grinding process, hydrophilic polymer HPMC was added as a stabilizer. HPMC substantially reduced the rate of recrystallization20. At the surface of the drug crystals, the concentration of drug was decided by a bal- ance of the dissolution of the solid drug and deposition of crystals from the surrounding solution. The presence of a polymer at the surface slowed down the diffusion of the drug molecules to the solid surface and reduced the surface area available for nucleation on the particle sur- face so that the process of crystallization was also slowed down21. In addition, HPMC, which was hydrophilic, increased the wettability and hydrophilicity of SIM.
After grinding, SIM nanosuspensions were layered on the surface of the sugar pellets. Before layering, SDS was added to the nanosuspensions. The nanosuspensions aggregated into larger particles on the surface of the pel- lets, and SDS improved the dispersion of the SIM pellets. It also allowed the pellets convert into nanoparticles much more quickly.

Characterization

PSD

SIM nanosuspensions resulted in a significant reduction in particle size (Table 1). Compared with the particle size of pre-milled SIM, the particles in the nanosuspensions were reduced markedly. Moreover, when HPMC was present at a concentration of 7%, 80.6% of the particles were below 1 µm in size compared with 63.7% and 55.8% at a concentration of 5% and 10%. These results showed that hydrophilic polymer HPMC in different concentrations had different effects on the particle size reduction of SIM. With an increase in the concentration of HPMC, the particle size of SIM decreased. This was because HPMC slowed down the diffusion of the drug molecules to the solid surface. Also it reduced the surface area available for nucleation on the particle surface21. Consequently, the process of crystallization was slowed down. However, compared with 7% HPMC, the particle size of SIM in 10% HPMC increased. This may be due to the fact that the viscosity of the suspensions increased with an increase in the concentration of HPMC. This would reduce the mechanical efficiency. So, during the same time period, the particle size reduction of SIM in a high concentration of HPMC was slower than that in a low concentration of HPMC. In addition, the HPMC molecules would produce an inclusion effect on the SIM particles and the presence of a high concentration of polymer at the dissolving surface would restrict access of water molecules to the crystal surface22–24. All the above findings indicated that HPMC should be at an optimal concentration. Also for this experiment, 7% HPMC was the best choice for the grinding of SIM.
The re-dispersibility of the pellet-layered nanosus- pensions, the HPMC concentration of which was 7%, was studied in a saturated solution of SIM under the same conditions as the dissolution study. Pellets began re-dispersing as soon as they came into contact with the solution, and the pellets dispersed completely after about 10 min. The solution was then withdrawn after 30 min to determine the particle size of SIM and this is shown in Table 1. The result obtained showed that the d90 was slightly bigger than the nanosuspensions, but the d10 and d50 were similar to the nanosuspensions. The increase in d90 may be because the particles aggregated during their layering on the surface of the pellets while compared with the data for the pre-milled suspensions, aggregation was so minor that it could be ignored. So, it was concluded that SIM pellets could re-disperse to the original nanoparticle state.

SEM

Figure 2 shows the raw SEM images of SIM, dried nano- suspensions and layered pellets. Raw drug consisted of a mixture of some large crystals (most about 20 µm) with microparticles, which might have been generated by micronization or any other size reduction process at the time of manufacturing. Dried nanosuspensions revealed significant changes in particle shape and surface topog- raphy due to the effect of grinding. It can be seen from Figure 2a and Figure 2b that the particle size of raw SIM was large and non-uniform, while the particle size of nanosuspensions was reduced and highly uniform.

The surface of the pellets was even and smooth indi- cating that the nanosuspensions were layered evenly on the surface of the pellets. The pellets exhibited a clear nanosuspenisons layer, and the thickness was almost equal to each other. This indicated that nanosuspen- sions were layered on the pellets layer by layer. Also, the state of the layer was similar to that of the dried nano- suspensions, except that the nanosuspensions layer was denser than that of the dried nanosuspensions. This also explained why the dissolution rate of the dried nanosuspensions was a little faster than the pellet-lay- ered nanosuspensions.

As shown in the Figure 2b and Figure 2e, nanosus- pensions has no significant change after storing at room temperature for 12 months. And the morphology of pel- lets also changed seldom by comparing Figure 2c, 2d and Figure 2f, 2g. This results indicated that the pellets was stable.

Optical microscopy

The optical micrographs are shown in Figure 3. These show that the particle size of pre-milled suspensions is large and non-uniform, while the particle size of nano- suspensions was reduced and uniform. From the optical micrographs, the particle size of the suspensions re-dis- persed by the pellet-layered nanosuspensions was no difference from that of the nansuspensions. The optical micrographs show that nanosuspensions layered on the surface of pellets could re-disperse to the original state before layering.

DSC

Figure 4 shows the DSC thermograms of raw SIM (a), nanosuspensions (b), pre-milled suspensions (c) with concentrations of HPMC 7% and the powder of pellets-layered nanosuspensions (d). The SIM exhib- ited a sharp endothermic peak at 139.41°C indicating the melting point of SIM. In the thermograms of the dried pre-milled suspensions and nanosuspensions, the endothermic peaks were at 138.75°C and 134.97°C indicating that the SIM was still in crystalline form after grinding. The drift of endothermic peak was due to the size reduction of the crystals25,26. It has been reported that small particles have a lower melting point than raw drug. With the reduction of the size of the particles, the melting temperature of the drugs decreased. This also indicated that the particles of nanosuspensions reduced by grinding. The endothermic peak of powder of pellets indicated that SIM was still crystal in the pel- lets, the process of layering did not change the physical state of nanocrystals.

XRPD

Figure 5 shows the XRPD patterns of raw SIM (a), pre- milled suspensions (b) and nanosuspensions (c) with concentrations of HPMC 7% (c), and the powder of pellets- layered nanosuspensions (d). The results of the XRPD investigations were similar to those of the DSC analysis. The XRPD pattern of the SIM showed characteristic high- energy diffraction peaks at 2θ values between 5°and 60°, indicating the crystalline structure of SIM. The dried nanosuspensions showed similar XRPD patterns to that of SIM, indicating that the initial crystalline state had been maintained during the grinding process. However, there was a significant reduction in the intensity of some major SIM crystalline peaks (28.8, 22.8, 19.6, 18, 9.6) in the diffractogram of the nanosuspensions. In general, this partial loss of crystallinity may be due to the physical presence of amorphous excipients HPMC27. The DSC and XRPD results both indicated that grinding did not destroy the crystal form of SIM, and SIM was still crystals state. Besides the results of DSC, the crystalline peaks of powder of pellets indicated again that SIM was still crystal in the pellets, the process of layering did not change the physical state of nanocrystals.

Chemical degradation

HPLC analysis of SIM nanosuspensions indicated no deg- radation after grinding. Except for the peaks produced by raw SIM, no extra peaks were observed in the chromato- grams. This proved that SIM was stable during grinding. This may because SIM was ground at a low temperature, and the conditions of grinding were suitable for SIM.

Dissolution study

The in vitro dissolution profiles are shown in Figure 6 and Figure 7. The dissolution of the dried nanosuspensions, layered pellets, physical mixture of SIM and HPMC, and commercial tablets was studied in pH 7.0 phosphate solutions which did not contain surfactant SDS. In addi- tion, in order to achieve sink condition, 0.1% SDS was added to the medium, and dissolution was also studied under this condition.

Nanosuspensions which were layered on the surface of the pellets began dispersing as soon as they were put into the dissolution medium. Also it took about 10 min for SIM pellets to disperse completely in pH 7.0 phos- phate buffer solutions. From the dissolution profiles, it can be seen that the nanosuspensions and pellets has no significant difference in dissolution under sink condi- tion. The PSD and optical micrographs of suspensions re- dispersed by the pellets indicated that nanosuspensions layered on pellets could re-disperse to a nanoparticle state after 30 min. This was also showed by the dissolu- tion profiles. Compared with the physical mixture and commercial tablets in Figure 6, the dissolution rate of SIM nanosuspensions and SIM pellets was markedly faster. The nanosuspensions significantly improved the dissolution rate of SIM. Under sink conditions, nearly 100% of the drug dissolved from the pellets within 5 mins, as opposed to only 7% and 27% for the physical mixture and commercial tablets. The fact that the simple mixture of the components did not improve the dissolution of SIM suggested that the enhancement in the nanosuspen- sions was not correlated with solubilization or a wetting effect of the HPMC additives on the drug. In addition, this proved that dissolution of dried nanosupensions and layered pellets was rarely different under sink conditions. According to the Nernst–Brunner/Noyes–Whitney equation28, the dissolution rate is proportional to the surface area available for dissolution. A reduction in the particle size will increase the effective particle surface area, thus increasing the dissolution rate. Furthermore, the diffusion layer thickness will also be reduced with a reduction in particle size, thus resulting in an even faster dissolution rate29. In addition, an increase in the satura- tion solubility of nanosuspensions is also expected30, which would lead to a further increase in the dissolution rate. The results obtained indicated that pellet-layered nanosuspensions are useful solid dosage forms for
improving the dissolution rate of SIM.

Stability study

Because of the special chemical structure, SIM is eas- ily degradated. So, the stability study of SIM solid dos- age forms was of prime importance. The layered pellets under the conditions of 40°C, 75% RH for 3 months, room temperature for 12 months, and 4500 lx ± 500 lx light for 30 days were all studied. Compared with the initial solid dosage form, the dissolution did not change markedly and the related substances were still below 2%, which was within the limit of 3%31.

The results of PSD showed that the particle size of nano- suspensions was reduced significantly. Consequently, the dissolution rate of SIM was improved. In the stability study, the dissolution of SIM rarely changed. This may be because the SIM particles were fixed inside the HPMC, and the particles were kept at a distance from each other, thus preventing particle aggregation. For SIM layered pellets, SDS not only played a role of solubilizer, but also improved the re-dispersibility of the nanosuspensions.

Because of its special chemical structure, SIM was eas- ily oxidized and degraded by oxygen, and the degrada- tion products were generated by a series of degradation reactions9. With the reduction in particle size, the surface area increased. This would cause the SIM to be more susceptible to slow oxidative degradation than the raw SIM. So, BHA was added as an antioxidant to prevent this oxidation of SIM. As the content of BHA in drugs should be carefully controlled, citric acid was added as a syner- gist, which could improve the antioxidant effect of BHA dramatically32.

In addition to the presence of antioxidant, the stability of the pellets could also be explained by several effects: (i) the oxygen diffusion rate is significantly lower in the crystalline region than in the amorphous region10 and both the DSC and XRPD results proved that SIM remains crystalline form in the nanosuspensions; (ii) the molecu- lar mobility of SIM nanosuspensions layered on the pel- lets was very slow and the low molecular mobility could slow down the chemical reactivity during storage33; (iii) hydrogen bonding between the SIM and the polymer HPMC increased the stability of SIM34.
The lactone ring of SIM was easily opened by acid hydrolysis. The presence of citric acid may accelerate the reaction. However, from the chromatograms of the pellets at the starting time (Figure 8b), SIMA was not produced in a high concentration. This may be due to the inclusion of HPMC and hydrogen bonding between the SIM and the polymer HPMC34, which would protect the drug during the grinding and layering processes.All the studies indicated that SIM nanosuspen- sion-layered pellets exhibited good physical and chemical stability.

Bioavailability study

After oral administration, SIM is converted to the corre- sponding SIMA, which is a potent inhibitor of HMG-CoA reductase. So, in the bioavailability study, SIM and SIMA were both determined. The bioavailability of SIM and SIMA in layered pellets was studied and compared with that of commercial tablets (Zocor®, SIM 20 mg, Merck Sharp & Dohme). The curves of the plasma concentration of SIM and SIMA versus time for the two preparations are shown in Figure 9 and Figure 10, and the correspond- ing bioavailability parameters are given in Table 2 and Table 3.

The AUC0→24 of SIM in layered pellets and commer- cial tablets was 260.1 ± 162.8 and 222.3 ± 132.3 ng h/mL, respectively. Also the AUC0→24 of SIMA in layered pellets and commercial tablets was 159.2 ± 58.8 and 92.3 ± 35.5 ng h/mL, respectively. For SIM and SIMA, the relative bio- availability of the nanosuspension-layered pellets to the this is also confirmed by the Tmax of SIM and SIMA. The time until SIM and SIMA achieved their maximum con- centration was very similar.

SIM is a BCS Class II drug (poorly soluble and highly permeable), which means that dissolution is the rate- limiting factor for its absorption35. The increased oral bio- availability of the pellet-layered nanosuspensions could be explained by the improved dissolution rate, which would keep the drug in soluble form during the gastroin- testinal dilution and permeation processes.

Conclusion

In this paper, pellet-layered SIM nanosuspensions were investigated. The nanosuspensions prepared by grind- ing were layered on the surface of pellets, and charac- terization of these pellets showed that the pellets could re-disperse to the nanoparticles state. The nanosuspen- sions remained in the crystalline state after grinding, and it also was crystals after layering on the pellets. During the stability studies, SIM nanosuspension-layered pel- lets exhibited good physical and chemical stability. In the in vitro study, the dissolution of SIM increased to a great degree. Moreover, in the in vivo study, the bioavail- ability of SIM was improved markedly (p < 0.05). Thus, the present study demonstrates that the pellet-layered nanosuspensions improved both the dissolution and bioavailability of SIM.