Efficacy and Safety Profiles of Oral Atorvastatin-Loaded

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Efficacy and Safety Profiles of Oral Atorvastatin-Loaded Nanoparticles: Effect of Size Modulation on Biodistribution Iman S. Ahmed, Rania EL Hosary, Mariame A. Hassan, Mohamed Haider, and Marwa M. Abd-Rabo Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00856 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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Molecular Pharmaceutics

Efficacy and Safety Profiles of Oral Atorvastatin-Loaded Nanoparticles: Effect of Size Modulation on Biodistribution

1

Iman S. Ahmeda*, Rania EL Hosaryb, Mariame A. Hassana,c, Mohamed Haidera,c

2 3 4

Marwa M. Abd-Rabob

5

a

Department of Pharmaceutics & Pharmaceutical Technology, College of Pharmacy, University of Sharjah, Sharjah 27272, United Arab Emirates b National Organization for Drug Control and Research, Cairo, Egypt c Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo11562, Egypt

*Corresponding author: Iman Saad Ahmed. College of Pharmacy, University of Sharjah, UAE. Tel: +971503794374; Fax: +97165585812 E-mail address: [email protected]; [email protected]

1 ACS Paragon Plus Environment

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

18

Atorvastatin calcium (AC)-loaded nanoparticles (NPs) of mean particle diameter < 100 nm and

19

narrow distribution were prepared and characterized. Their in-vivo PK as well as PD measures

20

following oral administration in different dosage regimens in hyperlipidemic rats were evaluated.

21

The results revealed that the oral bioavailability of two selected AC-NPs formulations was 235%

22

®

and 169% relative to Lipitor . However, the treatment regimens were not superior in reducing

23

serum total cholesterol (TC), low-density lipoproteins (LDL) and triglycerides (TG) levels

24

compared to Lipitor®. Moreover, the AC-NPs treatments were associated with significant

25

adverse effects observed biochemically and histologically. These results were contradictory with

26

those obtained from a previous study in which similarly-formulated AC-NPs of mean particle

27

diameter > 200 nm were found to be more safe and effective in reducing TC, LDL and TG levels

28

when administered to hyperlipiemic rats at reduced dosing frequency compared to daily-dose of

29

Lipitor® despite their lower oral bioavailability. The discrepant correlation between

30

pharmacokinetics (PK) and pharmacodynamics (PD) results was suggested to pertain to the

31

different bio-distribution profiles of AC-NPs depending on their sizes. Hereby, we provide a

32

simple approach of particle size modulation to enhance the efficacy and safety of atorvastatin.

33 34

Keywords: Atorvastatin calcium (AC); LDL; TC; Nanoparticles (NPs); PK/PD correlation


35

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1. Introduction

36

Polymeric nanoparticles (NPs) have been widely studied as promising controlled-release drug

37

delivery systems that can increase the oral bioavailability and reduce toxicity of many important

38

1

drugs including proteins and genes . Many studies revealed that physico-chemical properties of

39

NPs, including particle size, play a crucial role in the interaction between particles and biological

40

barriers 2. It is acknowledged that the smaller the particle size is, the greater the extent of uptake

41

will be. By “uptake” two biological events are usually referred to; the uptake through a barrier

42

membrane, e.g. intestinal epithelium, through which particles move from administration site to

43

blood stream, and cellular internalization through cell membrane on single cell level 3. However,

44

the correlation between particle size and in-vivo uptake, and efficacy is not always

45

straightforward nor can be easily predicted for several reasons. For instance, the particle size

46

measured in-vitro may differ from the actual size in-vivo depending on how the particles interact

47

with biological fluids 4. This in-situ alteration of particle size may affect significantly the

48

permeation and internalization 5. Also, the use of different polymeric formulations of NPs, as

49

well as the use of different cell lines to simulate the in-vivo internalization, contribute largely to

50

the inconsistency in the reported cutoff sizes able to cross the biological membranes

5, 6

. In

51

addition to physico-chemical properties of carrier NPs, drug moieties of inherent poor PK/PD

52

correlation, e.g. statins 7, 8, further complicate the interpretation of the role of particle size on in-

53

vivo responses. Therefore, studies on nano-sized formulations should be carefully designed to

54

assess both PK and PD patterns in order to obtain reliable data and ultimately attain clinical

55

applicability.

56

Statins are associated with serious adverse effects, in particular; myopathy, which can progress

57

to rhabdomyolysis. Cerivastatin, for example, was withdrawn from the market following 52

58

incidences of drug-related fatalities in 2001. Despite the clinical hazard, statins cholesterol-

59

lowering effects are not only associated with reduced risk of cardiovascular events, but also

60

found to be associated with a range of “pleiotropic effects” including anti-inflammatory,

61

antioxidant, immunomodulatory and anti-thrombotic actions

9-11

. This wide therapeutic range

62

renders statins indispensable agents. In the near future, statins use may expand to manage other

63

diseases such as cancer, rheumatoid arthritis, COPD and neurodegenerative disorders. Therefore,

64

statins formulation and evaluation should receive special attention to improve their

65



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bioavailability, efficacy, and consequently reduce production costs on one side, and to minimize

66

their toxicity on the other 12.

67

In a previous study by Ahmed et al. 13, lyophilized Atorvastatin Calcium-loaded NPs (AC-NPs)

68

of average particle size diameter of 200 nm and span value 1.2 were found to be superior in

69

reducing low density lipoproteins (LDL) and triglycerides (TG) levels in albino hyperlipidemic

70

rats, demonstrating no measurable side effects. The enhanced efficacy and safety profiles were

71

attained at one-third the bioavailability of Lipitor®. From a translational point of view, these

72

results were significant and sparked our motivation to report on the effect of particle size along

73

with polydispersity for this class of drugs after removing formulation effects and manipulating

74

particle size with the same set of excipients. For our knowledge, this is the first study that

75

succeeds to eliminate formulation and experimental variables while studying particle size effect

76

for statins.

77 78

2. Materials and methods

79

2.1. Materials

80

Atorvastatin calcium (AC) was supplied by EPICO, Egypt. Poly-ε-caprolactone (P-ε-CL; M.W.

81

14,000) was purchased from Aldrich, Japan. Tween 60, Span 80 and Pluronic F-68 (PF-68) were

82

obtained from Sigma-Aldrich, Germany. Acetone, acetonitrile, methanol and glacial acetic acid

83

(HPLC grade) were from BHD, England. Water used was distilled deionized water.

84 85

2.2. Preparation of AC-NPs

86

Previously optimized formulations of AC-NPs had their composition modified through a

87

factorial design to obtain NPs with desired characteristics suitable for the objective of this study

88

14

. These characteristics were: (1) mean particle size (< 100 nm), (2) span value (0.1-0.2), (3)

89

entrapment efficiency (≥ 50%), (4) drug release (≤ 50% release during the first 4 h in-vitro) (5)

90

No visible particle aggregation

13

. Two formulations, designated as FP-NPs and FT-NPs, were

91

found to fulfill all of the above criteria. FP-NPs and FT-NPs contained Pluronic-68 and Tween

92

60, respectively, as stabilizers in the aqueous phase. For the preparation of NPs, 25 mL of

93

acetone containing 0.1% (w/v) P-ε-CL, 0.01% (w/v) AC and 0.05% or 0.14% (w/v) Span 80

94

were injected at controlled flow rate of 2 ml/min into 100 mL of aqueous solution containing

95

0.0125% (w/v) Pluronic F-68 (FP-NPs) or 0.015% (w/v) Tween 60 (FT-NPs) under constant

96


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sonication for 60 min to aid size reduction

15

. After NPs were formed, acetone and a large

97

proportion of water were removed using a rotary evaporator (Buchi, Switzerland) at 45o to 55o C

98

for 45 min. The solidified NPs were then reconstituted in distilled water. The composition and

99

the physicochemical characterization of the two formulations are summarized in Table 1. All

100

batches were freshly prepared and used on the same day or stored for no more than 5 days at 4°C.

101 102

2.3. Characterization of AC-NPs

103

2.3.1. Determination of particle size, polydispersity and zeta potential

104

The particle size, polydispersity (span values) and size distribution profiles were determined

105

using laser diffraction particle size analyzer (Master seizer Hydro MU 2000, Malvern MU

106

instruments, UK).

107

The d0.9 was used to assess the particle size. The small span values are indicative of narrow

108

particle size distribution. Zeta potential (ZP) of the samples was measured using laser light

109

scattering technique (Malvern Zetasizer ZS, Malvern, UK). The mean value ± SD for three

110

replicates was calculated.

111 112

2.3.2. Morphological characterization

113

The morphology of NPs was determined using transmission electron microscopy (TEM). A drop

114

of the diluted sample was placed on a copper grid coated with a carbon film, stained with 2%

115

phosphotungistic acid solution and then dried at room temperature. Images were taken using

116

TEM instrument (JEOL-2100, Jeol Ltd., Japan) via inverse contrast imaging.

117 118

2.3.3. Solid state characterization

119

The degree of crystallinity of AC in NPs formulations was determined using X-ray diffraction

120

(XRD) technique. Each sample was exposed to Copper (Cu) Kα radiation with a nickel filter, a

121

voltage of 45 kV, and a current of 30 mA. Diffraction patterns of pure AC, P-ε-CL, physical

122

mixture of AC and P-ε-CL and AC in NPs formulations were obtained using an XPERT-PRO

123

PANalytical X-ray diffractometer (USA).

124 125

2.3.4. Determination of entrapment efficiency


126


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The entrapment efficiency (EE) of AC in NPs was determined by dissolving one mL of NPs

127

suspension in 25 mL of acetonitrile. The solution was ﬁltered through 0.2 µm syringe ﬁlter and

128

the total AC was determined. Free AC was quantified in the collected aqueous supernatant

129

following centrifugation of a similar volume (1 mL) at 30,000 rpm for 1 h to precipitate the AC-

130

NPs. All amounts were determined by HPLC

16

and then expressed as a percentage of the total

drug in the preparation. The EE % was calculated from Eq (1). EE % =

Actual AC in NPs × 100 Theoretical AC

……………….. (1)

131 132

133

where the Actual AC entrapped in NPs = Total AC − Free AC, and the Theoretical AC is the

134

amount of AC used in the formulation.

135 136

The in-vitro release of AC from AC-NPs was determined in phosphate buffer solution (PBS; pH

137 138 139

7.4) over 24 h. A volume of AC-NPs suspension corresponding to 10 mg AC was placed in a

140

2.4. In-vitro release studies

. All vessels were kept at 37o C ± 0.5˚C with

141

paddle rotation speed of 100 rpm (USP Apparatus 2). Three mL samples were collected at 0.5,

142

1, 2, 3, 4, 5, 6, 8, 10, 12 and 24 h and immediately replaced with fresh media. Samples were

143

assayed for AC concentration using HPLC. Experiments were carried out in three replicates and

144

the average cumulative percentages of AC released were calculated using the calibration

145

equation after correction. Lipitor (10 mg; Pfizer) was used as a reference tablet for in-vitro

146

release studies.

147

dialysis bag (molecular cut off 12,000-14,000)

17

148 2.5. Short-term stability study

149

A short-term stability study was performed on FP-NPs and FT-NPs. The study was carried out at

150

room temperature and at 4°C for 5 days. The vials containing AC-NPs suspensions were sealed

151

and wrapped in aluminum foil and subdivided into two groups. One group is stored in

152

refrigerator at 4°C and the other group is stored at room temperature 25°C for 5 days. At the

153

predetermined time intervals, aliquots were taken and subjected to particle size analysis and %

154

drug entrapment studies as described above. The change in appearance (presence of aggregates),

155


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particle size, and % drug entrapped were recorded and compared to results obtained from freshly

156

prepared NPs.

157 158

2.6. Pharmacokinetics studies

159

The PK characteristics of AC in FP-NPs, FT-NPs and Lipitor, following oral administration of

160

a single dose equivalent to 10 mg/kg AC each, were determined. Male albino rats weighing from

161

0.21-0.25 kg were randomly divided into three treatment groups of seven rats each. The animals

162

were deprived from food 12 h prior to dosing with free access to water, but they were fed 4 h

163

post-dosing. Blood samples (0.2 mL) were withdrawn through the tail vein at predetermined time

164

intervals: 0 (predose), 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h. AC in blood samples was analyzed by

165

HPLC following the same procedures described previously 13, 18. All animal experiments were

166

approved by the Research Ethics Committee for Animal Subject Research at the National

167

Hepatology & Tropical Medicine Research Institute (NHTMRI), Cairo, Egypt, operating

168

according to the CIOMS and ICLAS international guiding principles for biomedical research

169

involving animals 2012. Also, all animal experiments comply with Directive 2010/63/EU.

170

Data were processed by WinNonlin® (version 1.5, Scientific consulting, Inc., NC) using non-

171

compartmental analytical model. Pharmacokinetic variables including Cmax (observed maximal

172

drug concentration; ng/mL) and Tmax (observed time to reach maximal drug concentration; h)

173

were calculated. The area under the curve (AUC0-t; ng h/mL) was determined as the area under

174

the plasma concentration-time curve from time zero up to the last measured time point. Apparent

175

terminal elimination half-life (t1/2) was calculated as 0.693/k where k is the terminal elimination

176

rate constant estimated by log-linear regression analysis of data visually assessed to be a terminal

177

log-linear phase. Mean transit time (MTT) was calculated from AUMC/AUC where AUMC is

178

the area under the first moment curve. The relative bioavailability (frel) was calculated as

179

(AUCAC-NPs/AUCLipitor)×100.

180 181

2.7. Pharmacodynamic studies

182

Forty-two male albino rats (0.21 - 0.25 kg each) were used in this study. The animals were

183

housed in plastic cages at the Animal Care Facility under controlled conditions of temperature

184

and humidity and exposed to a 12-h light/dark cycles. All rats were fed with commercially

185

available normal pellet diet (NPD) and had access to water ad libitum prior to dietary

186



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manipulation. For experiments, the animals were randomly divided into six-treatment groups

187

(seven animals each).

188 189

2.7.1. Induction of hyperlipidemia in rats

190

Except for Group 1 (negative control), all animals (groups 2-6) were fed with high fat diet (HFD)

191

19

. The HFD consisted of 58% fats, 25% proteins and

192

17% carbohydrates of the total kcal content. After six weeks, each group received the assigned

193

treatment for two consecutive weeks. The six groups were as follows: Group 1, negative control

194

(NPD- fed); Group 2, positive control (HFD-fed); Group 3, Lipitor; Group 4, FP-R1; Group 5,

195

FT-R1, Group 6, FP-R2. R1 and R2 stands for Regimen 1 and Regimen 2, respectively.

196

instead of NPD till the end of the study

197 2.7.2. Drug administration and dosing

198

Two different dosage regimens were followed in this study. In Regimen 1 (R1), Group 4 (FP-R1)

199

and Group 5 (FT-R1) received daily dose of FP-NPs and FT-NPs suspensions, respectively. In

200

Regimen 2 (R2), Group 6 (FP-R2) was given FP-NPs once every 3 days. A volume of NPs

201

suspension equivalent to 3 mg/kg of AC was administered through the oral route using an oral

202

gavage needle. Dosing of Lipitor was once daily at a dose equivalent to 3 mg/kg of AC. The

203

tablets were crushed and dispersed in water by sonication immediately before administration.

204

Prior to a treatment, the animals were examined for any abnormal behavior, morbidity or

205

mortality.

206 207

2.7.3. Blood sampling

208

Blood sampling was carried out on two occasions; following the six-week of HFD immediately

209

before dosing and then by the end of a 24-h fasting period at the end of the treatment period.

210

Blood was collected from retro-orbital veins into centrifuge tubes and left to clot. Clotted blood

211

samples were then centrifuged and fractionated at 10,000 rpm at -4°C for 10 min. Serum was

212

separated and kept at -20°C until analysis.

213 214

2.7.4. Biochemical tests

215

The pharmacological/toxic effects of AC were monitored by measuring selected biochemical

216

parameters in the serum. These were: total cholesterol (TC), triglycerides (TG), low density

217


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lipoproteins (LDL), high density lipoproteins (HDL), aspartate transaminase (AST), alanine

218

transaminase (ALT), creatinine kinase-MB (CK-MB), serum creatinine (CR), serum lactate

219

dehydrogenase (LD) and serum urea (U). All parameters were analyzed using commercial assay

220

kits purchased from QCA, Spain; Stanbio, USA; and Bio-diagnostic, Egypt.

221 222

2.7.5. Histological examination

223

At the end of the treatment period, liver tissue of sacrificed animals from different treatment

224

groups were isolated and homogenized in ice-cold 1.15% potassium chloride solution using a

225

glass homogenizer to yield 10% (w/v) liver tissue homogenates. Tissue sections were stained

226

and examined microscopically.

227 228

2.8. Statistical analysis

229

All in-vitro measurements were carried out in independent triplicates and values are presented as

230

mean ± SD unless otherwise noted. Statistics were carried out using Minitab 16 (UK). For

231

comparisons between two groups, two-tailed unpaired Student’s t-test was employed. For

232

multiple comparisons, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc

233

test was utilized. For PK studies, ANOVA was performed on untransformed data for Cmax,

234

AUC0-24 and t1/2. The non-parametric Signed Rank Test (Mann-Whitney's test) was used to

235

compare Tmax among the groups. A p-value of ≤ 0.05 was considered statistically significant.

236 237

3. Results and discussion

238

3.1. Properties of AC-NPs

239

Table 1 summarizes the composition and characteristics of the two selected formulations,

240

namely; FP-NPs and FT-NPs. The main difference between them is in the type of surfactant

241

added to the aqueous phase being polymeric or non-polymeric in FP-NPs and FT-NPs,

242

respectively. Non-polymeric surfactants are reported to show higher adsorption potential than

243

equal chain length polymers, and thus it was expected that FT-NPs would be smaller in size 20.

244

However, the particle size of FT-NPs (82±16 nm) was statistically no different compared to FP-

245

NPs (73±13 nm). Both formulations yielded suspensions of monodisperse particles of average

246

diameter < 100 nm. The particle size distribution of the two formulations showed unimodal

247

narrow distribution curves (Figure 1A). TEM micrographs of FP-NPs and FT-NPs showed that

248



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both formulations consisted of spherical particles and further confirmed the particle diameter to

249

be as obtained by the size analyzer. FP-NPs tended to be more uniform in shape and size (Figure

250

1B). The zeta potential, indicative of the magnitude of electric charges on NPs, was -18.5±1.1

251

mV and -20.4±1.4 mV for FP-NPs and FT-NPs, respectively.

252

Results from short-term stability studies showed an insignificant increase in the particle size of

253

FP-NPs. A significant increase in the particle size of FT-NPs was however observed after 5 days

254

storage at room temperature. No significant change in the particle size of both formulations was

255

observed at 4°C (Table 2). To eliminate possible changes in the particle size upon standing all

256

batches were freshly prepared and used on the same day or stored for no more than 5 days at 4°C.

257

There was no significant change in % EE for both formulations after 5 days at room temperature

258

or 4°C.

259

In order to identify any change in drug physical state upon formulation, crystallinity of AC in

260

AC-NPs was determined. Pure AC exhibited strong and characteristic XRD pattern dominated

261

by intense scattering peaks located between 10° and 30° 2θ, indicative of the crystalline nature of

262

the drug powder. The diffraction patterns of AC in NPs formulations retained these characteristic

263

peaks indicating that the formulation ingredients and procedures did not change the crystalline

264

nature of the drug (Figure 2).

265

The percentage of AC entrapped was ≈ 50% in the two formulations. Higher EE% (≈ 80%) was

266

previously attained when higher concentrations of Span 80, PF-68 and Tween 60 were used at a

267

ratio of 2:1 (Span 80 : PF-68 or Span 80 : Tween 60, respectively). The increased entrapment

268

efficiency might have been attributed either to the increased viscosity of the aqueous phase

269

which resulted in the reduction of counter diffusion rate of solvents

21

, or to the increased

270

solubility of drug/polymer in the aqueous phase. These concentrations however were associated

271

with significantly larger particles

13

. In this study, all surfactants were used at much lower

272

concentrations and the ratios of Span 80:PF-68 and Span 80:Tween 60 were 1:1 and 2.3:1,

273

respectively, in order to decrease the particle size diameter to lesser than 100 nm, and thus; EE%

274

decreased.

275 276

3.2. In-vitro release studies

277

The release profiles from FP-NPs, FT-NPs and Lipitor® are illustrated in Figure 3. The two AC-

278

NPs formulations were able to control the release of AC with no observed burst effect. More

279


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than 50% AC was released from Lipitor® in the first hour compared to less than 20% from the

280

NPs. At 4 h drug release from FP-NPs and FT-NPs was ≤ 50% which fulfilled one of the desired

281

NPs characteristic required for this study. Drug release from FT-NPs seemed to be slightly faster

282

compared to FP-NPs, however, there was no significant difference at all time points between the

283

two formulations. The in-vitro drug release mechanism was analyzed by calculating the

284

diffusional exponent (n) and was found to exhibit anomalous release (n>0.5) characterized by

285

diffusion and matrix erosion for both formulations

22, 23

. It has to be mentioned that release

286

studies in gastric fluid (pH=1.2) for 2 h were also performed and the two tested formulations

287

showed less than 3% drug release for 2 h. These results show that the NPs were able to protect

288

AC know to undergo acidic degradation 24.

289 290 291

3.3. In-vivo PK studies

292 The mean plasma concentration-time curves following oral administration of FP-NPs, FT-NPs

293

and Lipitor® to albino rats are shown in Figure 4. The mean PK parameters (Cmax, Tmax, AUC0-

294

24,

t1/2, MTT) for the three groups are summarized in Table 3. The data revealed significant

295

improvement in the rate and extent of drug absorption from the NPs compared to Lipitor. The

296

mean Cmax estimates from FP-NPs (950±160 ng/mL) and FT-NPs (487±128 ng/mL) were

297

significantly higher compared to the mean Cmax estimate from Lipitor® (259± 73 ng/mL). The

298

mean AUC0-24 estimates of FP-NPs (2438±438 ng h/mL) and FT-NPs (1753±395 ng h/mL)

299

represented 235% and 169%, respectively, of that of Lipitor® (1036±206 ng h/mL). The

300

statistically significant higher relative bioavailability of AC from NPs highlights the role of

301

particle size reduction in nano-formulations in the enhancement of drug absorption. On the other

302

hand, the mean Cmax and AUC0-24 estimates of FP-NPs were statistically significantly higher (p
200 nm in size) showed lower blood bioavailability compared to Lipitor® but was associated

331

with strikingly higher effectiveness in counteracting hyperlipidemia. This discrepancy suggests

332

that larger particles may behave differently in-vivo compared to smaller ones and emphasizes

333

that particle size plays an important role in AC treatment outcomes. This means that the small

334

NPs (< 100 nm) contribute to the higher bioavailability while the large NPs (> 200 nm)

335

contribute to the higher efficacy. Therefore, it is quite possible that higher percentage

336

distribution of large NPs in the liver results in apparent lower bioavailability in blood but

337

improved PD response due to liver targeting. On the other hand, smaller NPs distribute mainly in

338

the plasma resulting in higher bioavailability. In Regimen 2, AC in FP-NPs was dosed once

339

every 3 days to FP-R2 group and results showed no improvement in the levels of TC and TG

340

compared to the hyperlipidemic rats, yet, significant reduction in LDL level (almost 50%) was

341


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observed. The differential effects on the selected biochemical indicators could be attributed in part to the reported poor PK-PD correlation of statins

7, 8

. For HDL, there was no significant

difference among all groups in support to the reported poor effect of AC on HDL

26

342 343

however,

344

these results are not in accord with our previous findings in which a decrease in HDL levels was

345

observed following the administration of the large NPs

13

. The HDL results further suggest that

346

particle size may affect the PD profile of AC-NPs in-vivo.

347

The increase in TG levels observed in FP-R1 and FP-R2 groups compared to the positive control

348

might be due to the poloxamer (PF-68) used in the formulation of FP-NPs which is reported to

349

elevate plasma TG when administered to rats

13, 27

. In support, similar results were obtained in

13

350

our previous studies .

351

The measurement of biochemical parameters indicative of the adverse effects of oral statins

352

revealed that the administration of FP-R1 and FT-R1 was associated with the occurrence of

353

skeletal muscle damage manifested by the increase in CR and CK-MB (Figure 5A)

28

. CR and

354

CK-MB were not increased in FP-R2 group. The manifestation of AC adverse effects might be

355

thus correlated to bioavailability which was high in regimen R1 but low in R2. This can be taken

356

as a merit of drug encapsulation in nanoparticles that allows for dose reduction in terms of

357

amount and/or frequency of administration

13

. LD level indicative of non-specific severe tissue

358

damage was significantly increased in all treated groups compared to the positive control group

359

and was the highest in Lipitor group (Table 5) 29. AST, ALT and U levels indicative of major

360

liver or kidney problems did not change significantly in all groups. In support, histological

361

examination of liver tissue homogenates of HFD group revealed intra-cytoplasmic vacuolization

362

of fat in most of the hepatocytes as well as inflammatory cell infiltration surrounding the central

363

vein. Histomicrographs of Lipitor, FP-R1 and FT-R1 groups showed prominent decrease in fat

364

vacuoles count whereas significant inflammatory cell infiltration surrounding the dilated central

365

vein was still perceivable with FP-R1 and FT-R1 treated groups (Figure 5B). On the other hand,

366

micro-fatty vacuoles were observed in hepatocytes of FP-R2 group in association with lesser

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extent of inflammation.

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Taken together, it can be concluded that both treatment regimens with

Efficacy and Safety Profiles of Oral Atorvastatin-Loaded

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