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Pharmacological and histological examination of atorvastatin-PVP K30 solid dispersions
Powder Technology 286 (2015) 538–545
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Pharmacological and histological examination of atorvastatin-PVP K30 solid dispersions Azin Jahangiri a,b, Mohammad Barzegar-Jalali a, Alireza Garjani d, Yousef Javadzadeh c, Hamed Hamishehkar a, Arash Afroozian a,d, Khosro Adibkia a,⁎ a
Drug Applied Research Center and Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran Students Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran Biotechnology Research Center and Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran b c
a r t i c l e
i n f o
Article history: Received 29 June 2015 Received in revised form 26 August 2015 Accepted 29 August 2015 Available online 3 September 2015 Keywords: Atorvastatin Solid dispersions Physicochemical characterization Histological examination Pharmacological efficiency
a b s t r a c t The objective of the present study was to investigate the pharmacological efficiency of orally administered atorvastatin calcium (ATV) solid dispersions (SDs). SDs of ATV were prepared using polyvinylpyrrolidone K30 (PVP) through the solvent evaporation method. Physicochemical characteristics of the prepared formulations were assessed benefitting scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and powder X-ray diffractometry (PXRD). The drug dissolution rates (under sink and non-sink conditions) as well as solubility studies were also examined. Serum lipid levels, liver index and histological analysis of the liver tissue in hyperlipidemic rats were considered to evaluate the pharmacological efficiency of prepared SDs. According to our findings, the drug crystallinity was reduced and the drug dissolution characteristics were improved in the prepared SDs. In vivo studies revealed that oral administration of ATV (3 mg/kg/day) in the SD form (ASDT) for 14 days along with high fat diet (HFD) to the hypercholesterolemic rats led to a significant decline (P b 0.05) in serum level of total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C). Moreover, ASDT exhibited more beneficial effects on the liver steatosis compared to ATV physical mixture (APMT) and hyperlipidemic control (HC) groups. In the present study, it was concluded that the SDs of ATV with improved physicochemical characteristics provided an increased therapeutic potential for management of hyperlipidemia compared to the corresponding physical mixtures (PMs). © 2015 Elsevier B.V. All rights reserved.
1. Introduction Atorvastatin calcium (ATV), a lipid-lowering agent, acts by the reversible inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [1]. ATV is administered for treatment of hypercholesterolemia and other dyslipidemic disorders alone or in conjunction with other anti-hyperlipidemic drugs. It is also helpful in primary prevention of cardiovascular disease and reducing the risk of myocardial infarction [2,3]. ATV belongs to class ІІ of biopharmaceutical classification system (BCS) [4] and tends to exhibit solubility or dissolution rate limited absorption. The marketed products of ATV suffer from poor oral bioavailability (~ 12%) [5]. Poor aqueous solubility, pre-systemic clearance in the gastrointestinal mucosa and/or hepatic first-pass metabolism are responsible for the low oral bioavailability of ATV [6]. Achieving appropriate therapeutic effects is possible by using higher doses of drug which results in dose related undesirable adverse effects. ⁎ Corresponding author at: Faculty of Pharmacy, Tabriz University of Medical Sciences, Golgasht Street, Daneshgah Ave., Tabriz, Iran. E-mail address: [email protected] (K. Adibkia).
http://dx.doi.org/10.1016/j.powtec.2015.08.047 0032-5910/© 2015 Elsevier B.V. All rights reserved.
Moreover, development of ATV formulation due to its poor aqueous solubility and insufficient bioavailability after oral administration is challenging [5]. Therefore, developing effective approaches to minimize the associated problems could be highly advantageous. Several techniques have been applied to enhance the solubility and dissolution rate of ATV, including nanosuspension formulation [7], preparation of the self-emulsifying drug delivery systems (SEDDS) [8,9], complexation with hydroxypropyl-β-cyclodextrin [6], preparation of the solid dispersions (SDs) [10–12], applying supercritical antisolvent (SAS) process [13,14] and spray drying technique [15]. SDs are used as one of the most practical and effective strategies in order to increase the dissolution behavior of poorly water-soluble drugs. The advantages of SDs include particle size reduction (possibly to molecular level), increase in wettability and porosity, decrease of drug crystallinity and sometimes conversion into amorphous state [16]. Accordingly, the drug substance could disperse as separate molecules, amorphous particles or crystalline particles, whereas, the carrier might present in the crystalline or amorphous state. Improvement in drug solubility and dissolution rate through SD technique has been shown by numerous studies [16]. Oral bioavailability of some poorly water-soluble drugs [17]
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such as ibuprofen [18], carbamazepine [19], naproxen [20] and ritonavir [21] has also been enhanced using SD technique. According to the conducted researches, polyvinylpyrrolidone (PVP)-based SDs of ATV exhibited noticeable increase in ATV absorption following oral administration [10]. Therefore, in this study, SDs of ATV were prepared using PVP K30 as an amorphous carrier, via the solvent evaporation method. Regarding literature review, evaluation of the therapeutic efficiency of ATV SDs (especially in liver tissue) via in vivo study has not been considered by other researchers. Although, the majority of researches are focused on effects of hypercholesterolemia on atherosclerosis and heart disease; however, toxic effects of cholesterol on the liver are also of particular importance [22]. Thus, in this work, after preparation and physicochemical characterization of ATV SDs, in order to investigate the therapeutic efficiency of prepared formulations in the diet induced hyperlipidemic rats, serum lipid levels, liver index and histopathological examination of the rat livers were evaluated. 2. Materials and methods 2.1. Materials ATV was purchased from Abidi pharmaceutical company (Tehran, Iran). PVP K30 and sodium hydroxide were supplied from Merck (Germany). Methanol was of high-performance liquid chromatography (HPLC) grade (Caledon Labs, Ontario, Canada). All other chemicals were of analytical grade.
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2.6. Fourier transformed infrared spectroscopy (FTIR) A Shimadzu 43000 FTIR spectrophotometer was employed for FTIR analysis. Samples were examined in the transmission mode and prepared using the KBr disk method (1 mg sample in 100 mg KBr powder). A spectral range of 400–4000 cm−1 and a resolution of 2 cm−1 were used in order to obtain the spectra. 2.7. Solubility of ATV Solubility measurements were performed according to the Higuchi and Connors' method [23]. Briefly, an excess amount of ATV was added into the test flasks containing various concentrations of PVPK30 in the dissolution medium (10 mL, phosphate buffer, pH 6.8). The samples were sonicated (Metler Electronics, model ME5.5, USA) for 30 min at room temperature. Afterward, the sealed test flasks were shaken at 25 °C (room temperature) for 48 h on a rotary shaker incubator (Heidolph unimax 1010, Inkubator 1000). The suspensions were centrifuged at 10,000 rpm for 5 min (Eppendorf 5810R centrifuge) and filtered through a 0.45 μm membrane filter. After suitable dilution, the samples were analyzed spectrophotometrically at a wavelength of 241 nm using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The polymer did not interfere with the UV measurement and no significant adsorption of the drug to the filter membranes was detected. All solubility experiments were performed in triplicate and the obtained results were presented as mean ± SD. 2.8. Dissolution study under sink condition
2.2. Preparation of the solid dispersions SDs of ATV were prepared by PVP K30 in different drug to polymer ratios, using the common solvent evaporation method. Briefly, different weight ratios (1:1, 1:3, 1:5 w/w) of drug:polymer combinations were dissolved in minimum volume of methanol and stirred (Heidolph, MR Hei-Tec, Germany) at 600 rpm for 15 min at room temperature. After complete dissolving, the solvent was evaporated using rota-evaporator (Heidolph, Laborota 4010 digital, Germany) at room temperature, 50 rpm for 12 h. Then the residue was pulverized by mortar and pestle, sieved (mesh size 60) and then kept in a desiccator. Recrystallized ATV was also prepared through recrystallization of ATV from methanol.
Dissolution study was performed using USP apparatus II, paddle stirrer (Erweka dissolution tester). The pure drug, SDs and PMs, all equivalent to 20 mg ATV, were added to the 900 mL dissolution medium (phosphate buffer, pH 6.8) under stirring rate of 75 rpm at 37 ± 0.5 °C. At appropriate time intervals, 2 mL of sample was withdrawn and filtered through 0.45 μm membrane filter. The removed volume was refilled with the fresh medium at 37 °C. The filtrate was suitably diluted and analyzed spectrophotometrically at 241 nm using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The obtained results were deducted from a standard calibration curve of ATV and the percent of the dissolved drug was plotted against the time. The reported results were the average value of at least three replicates ± SD.
2.3. Scanning electron microscopy (SEM)
2.9. Dissolution study under non-sink condition
The morphology of prepared samples was observed using scanning electron microscopy (SEM; Tescan, Brno Czech) operating at 20 kV. The samples were directly dispersed on a double-sided tape and coated by gold in a vacuum condition, before the examination.
Non-sink condition represented an insightful dissolution situation to further evaluation of the dissolution phenomena [24]. In this condition, dissolution medium is not enough for dissolving the total amount of drug [25]. The apparent dissolution profiles of SDs were investigated under non-sink condition as well. An accurately weighed amount of the SDs, each equivalent to 10 mg drug, was added to test flasks containing 10 mL phosphate buffer and shacked at 37 °C, 100 rpm (Heidolph unimax 1010, Inkubator 1000). The experiment was performed on five time points with three replicates. The samples were centrifuged at 10,000 rpm for 3 min and then filtered using 0.45 μm membrane filter. The supernatant was then further diluted and analyzed spectrophotometrically at 241 nm.
2.4. Differential scanning calorimetry (DSC) Thermal behavior of the SDs, PMs as well as pure drug and polymer was evaluated using differential scanning calorimeter (DSC60 Shimadzu, Kyoto, Japan). Accurately weighed samples (3 mg) were placed into crimp sealed aluminum pans. The measurement was performed at a heating rate of 20 °C/min from 50 to 210 °C. The aluminum oxide and indium powders were applied as reference and standard, respectively. 2.5. Powder X-ray diffraction (PXRD) X-ray diffraction patterns of the pure drug, polymer, PMs as well as prepared SDs were obtained by the X-ray diffractometer (Siemens D5000, Munich, Germany). Samples were scanned over the range of 2θ from 2 to 40°, under Cu Kα radiation, at a voltage of 40 kV and a current of 30 mA.
2.10. Animal study 2.10.1. Animals Male Wistar rats were purchased from Pasteur Institute of Iran (Tehran). Following their arrival to the animal care facility center, they were housed for one week prior to examination. Afterward, rats weighing 250 ± 20 g were divided into four groups and employed in this research. Rats were kept in the Animal House of Tabriz University of Medical Sciences at a controlled ambient temperature of 25 ± 2 °C
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Table 1 Animal groups and respective treatments. Groups
1 2 3 4
Diet
Normal control group (NC) Hyperlipidemic control group (HC) PM treated group (APMT) SD treated group (ASDT)
Treatment
(6 week)
(2 week)
NCD HFD HFD HFD
NCD HFD + PVP HFD + APM HFD + ASD
NCD: normal chow diet, HFD: high fat diet, APMT: treatment with ATV physical mixture, ASDT: treatment with ATV solid dispersion.
with 50 ± 10% relative humidity and a 12 h light/dark cycle. They had free access to food and water as well. The present study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of Tabriz University of Medical Sciences, Tabriz-Iran (Nation al Institutes of Health Publication No 85-23, revised 1985). 2.10.2. High fat diet A high-fat diet (HFD) reported by Garjani and coworkers [26] was applied in this study with some little modifications. Our HFD contained standard rodent chow powder (Sahand Niroo, Tabriz, Iran) (62.75% w/w), melted sheep's tail fat (15% w/w), wheat flour (10% w/ w), sucrose (10% w/w), cholesterol (2% w/w) and cholic acid (0.25% w/w). 2.10.3. Experimental protocol In vivo studies were performed on four groups of animals; each group was consisted of six rats. The total duration of experiment was eight weeks for both experimental and control groups. At the end of the sixth week, hyperlipidemia was developed and the treatment was begun (Table. 1). At the end of trial, rats were fasted for 10–12 h, with free access to water, then anesthetized through intraperitoneal injection of sodium pentobarbital. Blood samples were collected from inferior vena cava and transferred into gel and clot activator tube based on the manufacturer's suggested protocol. The livers were removed immediately by dissection, washed in ice-cold saline and blotted between two filter papers to eliminate the surface water. The livers subsequently weighed for measurement of the liver index. Isolated livers were kept in 10% neutralbuffered formaldehyde for further histological examination. 2.10.4. Drug administration ATV SDs and PMs, equivalent to 3 mg/kg ATV, based on dose translation from animal to human studies [27], were immediately gavaged (orally intubated) after suspending in 1 mL of distilled water. The formulation with the drug:polymer ratio of 1:5 was selected for in vivo studies because of its enhanced performance. The treatment was administered once a day for two weeks.
2.10.5. Biochemical analysis Fasting blood samples were taken for measurement of the serum concentrations of total cholesterol (TC), high density lipoproteincholesterol (HDL-C), and triglycerides (TG). Measurements were carried out using enzymatic colorimetric methods by commercially available kits (Pars Azmoon Inc., Tehran, Iran). The assays were performed using Autoanalyzer (Abbott Alycon 300, USA). HDL-C concentration was determined after precipitation of VLDL-cholesterol (VLDL-C) and LDL-cholesterol (LDL-C) with phosphotungstic acid and magnesium chloride. The LDL-C concentration was deliberated from the Friedewald formula [28] in which, LDL-C = (TC) − (HDL-C + VLDL-C), and since VLDL-C = (TG) / 5, thus, the concentration of LDL-C was calculated through LDL-C = TC − (HDL-C + 0.2(TG)). 2.10.6. Liver index and liver histological examination Liver index was calculated from the ratio of liver weight to body weight. Liver index = (Liver weight / Body weight) × 100%. The removed livers were fixed in 10% neutral-buffered formaldehyde for histological study. The tissues were embedded in paraffin and cut into 5 μm thick sections and subsequently stained by hematoxylin and eosin (H&E) for histological examination (n = 3). Microscope study was performed using camera equipped Olympus (Tokyo, Japan) BX50 microscope with 20× or 40× objective lens. 2.10.7. Statistical analysis Statistical evaluations were determined using oneway ANOVA followed by LSD posthoc test using SPSS 16 software. Any differences between the groups were considered significant at P b 0.05 level. All data were displayed as the mean ± SEM of values obtained from six rats. 3. Results and discussion 3.1. SEM studies SEM images of the pure ATV, recrystallized ATV and corresponding SD in 1:1 ratio are shown in Fig. 1. ATV demonstrated a few needlelike crystallites within an irregular shaped particle (Fig. 1(A)). The morphological feature of recrystallized ATV was completely different from the pure drug so that the surface area was meaningfully decreased in the recrystallized drug (Fig. 1(B)). Emergence of the new morphological shape in SD (Fig. 1(C)) compared to the pure drug was indicative of the efficient formation of SD system [18,29]. Moreover, according to SEM image of the SD, there were no observable drug particles on the surface of prepared SD, representing homogeneous distribution of the drug molecules in the SD system [30]. 3.2. DSC studies Fig. 2 represents the DSC thermograms of the pure ATV, PVP, recrystallized ATV as well as ATV:PVP PMs and SDs in 1:1 and 1:5 ratios. The
Fig. 1. SEM images of pure ATV (A), recrystallized ATV (B) and SD of ATV:PVP in 1:1 ratio (C).
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21.36°, which are indicative of the low crystallinity of the pure ATV [36]. The semicrystalline nature of the pure ATV was also confirmed by DSC. However, no characteristic diffraction peaks related to the pure ATV were observed in the recrystallized ATV, representing decrease in the drug crystallinity [37]. Moreover, SDs of ATV did not show any diffraction peak in 2θ = 2–40°, indicating a transition of ATV from a semicrystalline to an amorphous state [38]. Similar findings have been previously reported for the amorphous nanoparticles and solid dispersions of ATV prepared by supercritical antisolvent (SAS) and spray drying techniques [13,39]. Taken together with DSC data, these results specify a formation of amorphous ATV in the SDs. 3.4. FTIR studies
Fig. 2. DSC thermograms of PVP, ATV, recrystallized ATV (RCT ATV), physical mixtures (PMs) and solid dispersions (SDs) of ATV:PVP in the 1:1 and 1:5 ratios.
DSC curve of PVP displayed a broad endothermic peak between 75 and 135 °C, demonstrating the loss of water from the hygroscopic PVP [31]. DSC thermogram of the pure ATV displayed two endotherms, a broad endotherm at 50–130 °C corresponding to water loss or dehydration (probably indicates the presence of trihydrate form), followed by another endotherm at 182 °C related to the pure ATV melting point. These thermal events have been confirmed by thermal gravimetric analysis (TGA) in the previous conducted studies [7,13,15,32]. No endotherm was observed around the melting point of pure ATV in the thermogram of recrystallized ATV signifying the reduced crystallinity of recrystallized ATV. Physical mixtures of ATV: PVP in the ratio of 1:5 showed a melting endotherm with very low intensity, which could be due to the polymer dilution effect [20]. No endotherm related to the pure ATV melting point was observed in the SDs, which could be because of the reduced crystallinity; and/or dispersion of the drug molecules in the polymer solution [33–35]. Disappearance of the melting endotherm in the recrystallized ATV and the SDs might be attributed to the drug amorphization. According to similar previous studies, alteration of the drug physical state from a crystalline form to an amorphous state during SD formation is most probable [20,29,33].
3.3. PXRD studies Fig. 3 represents the X-ray diffraction patterns of the pure and recrystallized ATV, PVP, the PMs and SDs of ATV:PVP in 1:1 and 1:5 ratios. The low intensity peaks were observed at 2θ = 8.24°, 10.09° 18.82° and
FTIR studies were conducted to estimate any possible interactions between the ATV and PVP in the SDs. The FTIR spectra of PVP, ATV, recrystallized ATV, drug:polymer PMs and SDs in the ratios of 1:1 and 1:5 are represented in Fig. 4. The spectrum of PVP showed important bands at 2916 cm− 1 (C–H stretch) and 1656 cm−1 (C_O). A broad band from 3200 to 3600 cm−1 was assigned to the presence of water in the polymer [40], which was in agreement with a broad endotherm detected in the DSC finding. The spectrum of pure ATV exhibited characteristic peaks at 1660 cm−1 (acid carboxylic C_O stretch), 2930 cm−1 (C–H stretch), between 3200–3500 cm− 1 (intermolecular hydrogen bond, O–H stretch), three peaks between 1400 and 1600 cm−1 (aromatic C_C stretch), 1215 cm− 1 (C–F stretch), 834 cm− 1 (ring vibration due to para-substituted benzene), 1431 cm−1 (C–N stretch) as well as 1500 cm−1 and 3390 cm− 1 (N–H stretch) [13]. As can be seen in Fig. 4, the characteristic peak of the carbonyl group at 1660 cm−1 in the pure ATV became broader in the recrystallized ATV, which might be due to the drug amorphization. A similar finding has been stated by other researchers for melt-quenched and milled ketoprofen [41]. Regarding the chemical structure of PVP, carbonyl group of the polymer tends to form hydrogen bond with the favorable groups of drugs [40]. Hydrogen bonding usually leads to bathochromic shifting or broadening the peaks of functional groups [42]. According to ATV structure, hydrogen bonding could be expected between the hydroxyl groups of ATV and the carbonyl group of PVP. Comparing the FTIR spectra of the SDs with those of the corresponding PMs showed that O–H stretch between 3200 and 3500 cm−1 and C_O stretch at 1660 cm−1 in ATV spectrum were replaced by a broader band in the SDs, indicating the possible hydrogen bonding between ATV and PVP. Similar outcomes were formerly reported for other PVP-containing SDs including indomethacin [43], carbamazepine [40] and piroxicam [44]. Hydrogen bonding formation between the drug and polymer could prevent the drug re-crystallization and increase the stability of
Fig. 3. Powder X-ray diffraction patterns of PVP, ATV, recrystallized ATV (RCT ATV), solid dispersions (SDs) of ATV: PVP in 1:1 and 1:5 ratios.
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A. Jahangiri et al. / Powder Technology 286 (2015) 538–545 Table 2 Apparent solubility of recrystallized ATV (RCT ATV) and SDs of ATV-PVP in the 1:1, 1:3 and 1:5 drug:polymer ratios (after 24 h).
Fig. 4. FTIR spectra of PVP, ATV, recrystallized ATV (RCT ATV), physical mixtures (PMs) and solid dispersions (SDs) of ATV:PVP in the 1:1 and 1:5 ratios.
SDs [44]. Therefore, physical stability as well as in vivo performance of the SDs could be enhanced by using appropriate polymers [16,25]. 3.5. Solubility studies Fig. 5 illustrates the effect of PVP concentration on the solubility of ATV at 25 °C in phosphate buffer. As can be seen, aqueous solution of PVP led to increase in ATV solubility. There was no remarkable increase in the ATV solubility at lower concentrations of the polymer (below 1%). By increasing the polymer concentration, solubility of ATV was enhanced linearly up to 4.3 fold in 15% w/v. Increase in the solubility of ATV through PVP could probably be due to the formation of soluble complex between PVP as a water-soluble carrier and ATV as a poorly water-soluble drug [40]. Solubility of the pure ATV and apparent solubility of the prepared SDs and recrystallized ATV are exhibited in Table 2. Recrystallized ATV, which was present in complete amorphous form, revealed greater solubility. Moreover, the SDs of ATV presented a substantial increase in the drug solubility. Although the solubility of amorphous materials is meaningfully higher than their crystalline state, determining the solubility of amorphous form under true equilibrium condition is faced with difficulties. As a result, their experimental solubility is typically less than their thermodynamic predicted solubility [45]. 3.6. Dissolution studies
Samples
Apparent solubility (mg/mL) ± SD pH 6.8
Pure ATV RCT ATV SD 1:1 SD 1:3 SD 1:5
0.432 ± 0.010 0.543 ± 0.0141 0.990 ± 0.002 1.129 ± 0.026 1.149 ± 0.008
bioavailability [40]. Based on the obtained dissolution outcomes (Fig. 6), application of PVP could significantly improve the dissolution characteristics of ATV in both PMs and SDs. Results revealed that the SDs caused a further increase in ATV dissolution rate compared to the pure drug and PMs. The complete drug was released from all SDs after 60 min (Q60min). However, almost 100% of the drug was released from the SD with drug:polymer ratio of 1:5 during first 5 min (Q5min). The resulted value (Q5min) for pure ATV was 65% and the complete drug release occurred approximately within 210 min (3.5 h). The improved dissolution rate of ATV in the PMs might be attributed to the hydrophilic nature of PVP, which can facilitate the dissolution rate by reduction in the interfacial tension between the drug and the release medium. The enhancement of dissolution properties could also be explained by the solubilizing and wetting effect of PVP [46]. In addition to the cited mechanisms, an improvement in the dissolution rate of SDs could also be ascribed to the drug particle size reduction and/or decrease in the drug crystallinity during the SD preparation process [47,48]. Because of the higher internal energy of amorphous materials, they dissolve more rapidly than crystalline forms [49]. It is important to highlight that, in spite of the higher solubility of recrystallized ATV, which was present in amorphous state, it showed lower dissolution rate at the initial time intervals (Fig. 6). This phenomenon could probably be related to formation of a sticky mass with the reduced surface area in the hygroscopic amorphous drugs [50]. So therefore, solvent evaporation alone (recrystallization) could just produce an amorphous drug with enhanced solubility, however, application of PVP in the SD preparation process led to a further improvement in both drug solubility and dissolution rate. Any increase in the drug dissolution rate might also be attributable to the supersaturation phenomenon. However, the drug precipitates from unstable supersaturated solution to reach an equilibrium state [51]. The ability of a selected polymer to maintain the supersaturation state in the aqueous solution is of particular importance in SD performance [25]. According to the non-sink dissolution study of SDs (Fig. 7), drug
Dissolution rate in the gastrointestinal tract is most likely the ratelimiting step of absorption in the case of poorly water-soluble drugs such as ATV. Thus, they commonly exhibit dissolution dependent
Fig. 5. The effect of PVP concentration on the ATV solubility.
Fig. 6. Dissolution profiles of ATV, recrystallized ATV (RCT ATV), physical mixtures (PMs) and solid dispersions (SDs) of ATV:PVP in the 1:1, 1:3 and 1:5 ratios under sink condition.
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of TC and LDL-C in the hyperlipidemic control (HC), ATV PM treated (APMT) and SD treated (ASDT) groups, all compared to normal control group (NC). Treatment with both PM and SD of ATV (APMT and ASDT) revealed an outstanding decrease in the serum level of TC and LDL-C in comparison with HC group (P b 0.001). As seen in Table 3, the ASDT was more effective than APMT in reducing TC and LDL-C serum levels (P b 0.01). Serum level of TG was meaningfully (P b 0.05) reduced, while there was no significant change (P N 0.05) in the serum level of HDL-C between treated and HC groups. Altogether, the biochemical analysis showed that ASDT provided greater reduction in the serum lipid profile compared to APMT. However, the administered dose of ATV could not completely improve the hyperlipidemic condition.
Fig. 7. Dissolution profiles of ATV soli dispersions (SDs) in the 1:1, 1:3 and 1:5 ratios under non-sink condition.
concentration at the initial time intervals reached to more than 10-folds of ATV saturated solubility. Subsequently, the drug molecules precipitated from the supersaturated solution and reached to a plateau value after about 5 h, which could be considered as the drug amorphous solubility. During the drug precipitation, the adsorbed polymer on the surface of particles could prevent crystal growth by blocking the active surfaces and providing steric stabilization [52]. Moreover, hydrogen bonding between the drugs and polymers may help the maintenance of supersaturation state [53]. At the highest polymer ratio (SD 1:5), supersaturated concentration of the ATV in the non-sink condition as well as dissolution characteristics under sink condition were at the uppermost level. Accordingly, this formulation was selected for further in vivo studies. 3.7. In vivo studies 3.7.1. Effects of the treatments on serum lipid levels Elevated levels of cholesterol and LDL along with decreased HDL play an important role in the development of atherosclerosis, while lipid lowering agents reduce the risk of atherosclerosis and coronary heart disease (CHD) [54]. The measured serum lipid profiles at the end of the sixth week just before the beginning of the treatments were found to be as follows: TC = 185.7 ± 0.1, LDL-C = 164.3 ± 0.2, HDL-C = 11.0 ± 0.3 and TG = 51.7 ± 0.7. Reported data are mean ± SEM values of six independent experiments. According to the results obtained from biochemical analysis (Table 3), HFD at the end of the eighth weeks could significantly (P b 0.001) increase the serum levels
3.7.2. Effects of the treatments on liver visual appearance and liver index Consumption of HFD for eight weeks led to enlargement and paleness of the liver (Table 3 and Fig. 8(B–D)). Regarding the obtained data, healthy livers of the NC group exhibited the lowest liver index value and the highest redness (Fig. 8(A)). As shown in Table 3, HFD induced a notable increase in the liver index in all groups comparing the NC group (P b 0.001). Treatment with both APMT and ASDT improved the visual appearance of liver tissue from pale red to a more reddish color (Fig. 8(C and D)). The liver enlargement and paleness in ASDT group were lower than APMT group. Reduction in the liver index by using high doses of ATV (30 mg/kg/day) has been reported by other researchers [55]. However, it seems that the administered dose of ATV (3 mg/kg/day) was not sufficient to cause any significant effect on the liver index in the treated groups. 3.7.3. Histological examination of the liver tissue In addition to the harmful effects of hypercholesterolemia on atherosclerosis and CHD, its toxic effects on the liver are also challenging [22]. In today's world, due to excessive consumption of high-cholesterol foods and also lack of sufficient physical activity, the prevalence of obesity, diabetes, and the metabolic syndrome is expanding every day. Thus, non-alcoholic fatty liver disease (NAFLD) accompanied with lipid accumulation in the liver (steatosis) is a growing public health concern in most countries. Conducted researches revealed that the overconsumption of HFD plays an important role in the etiology of hepatic steatosis [56]. Moreover, nonalcoholic steatohepatitis (NASH), the liver steatosis combined with inflammation or advanced form of NAFLD, may develop end stage liver diseases which associates with about twenty percent mortality [57]. NAFLD is concomitant with hepatomegaly, elevated alanine aminotransferase hepatic steatosis and in some cases, varying degrees of liver fibrosis and steatohepatitis [57]. Steatosis, lobular inflammation, hepatocellular ballooning and fibrosis are the number of contributing factors linked to the existence of NAFLD. Histological evaluation remains the only way to appropriately assess these disorders [58].
Table 3 Effects of the ATV treatments on serum lipid levels and liver index in the diet induced hyperlipidemic rats. Groups
TC (mg/dL)
LDL-C (mg/dL)
HDL-C (mg/dL)
TG (mg/dL)
LI
NC HC APMT ASDT
61.80 ± 3.75###,⁎⁎⁎ 342.25 ± 11.43†††,⁎⁎⁎ 181.8 ± 6.79†††,### 147.5 ± 7.72†††,###,⁎⁎
20.88 ± 3.23###,⁎⁎⁎ 304.15 ± 14.10†††,⁎⁎⁎ 151.76 ± 5.89†††,### 111.5 ± 4.80†††,###,⁎⁎
34.80 ± 0.73 27.75 ± 2.25 24.00 ± 4.35 29.25 ± 3.47
30.60 ± 7.07# 51.75 ± 6.67 30.20 ± 3.12# 33.75 ± 3.06#
3.18 ± 0.08###,⁎⁎⁎ 5.80 ± 0.22††† 5.74 ± 0.20††† 5.4 ± 0.21 †††
Values are presented as mean ± SEM, n = 6 per group. Abbreviations: NC, normal control; HC, hyperlipidemic control; APMT, ATV physical mixture treated; ASDT, ATV solid dispersion treated; TC, total cholesterol; LDL-C, LDL-cholesterol; HDL-C, HDL-cholesterol; TG, triglyceride; LI, liver index. ††† P b 0.001 vs. NC group. # P b 0.05 vs. HC group. ### P b 0.001 vs. HC group. ⁎⁎ P b 0.01 vs. APMT. ⁎⁎⁎ P b 0.001 vs. APMT.
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Fig. 8. Liver visual appearance (A–D) and histological staining using H&E (E–H) followed by examining under a light microscope (magnification 20×), A&E: normal control, B&F: hyperlipidemic control, C&G: PM treated (APMT), D&H: SD treated (ASDT).
Fig. 8(E–H) demonstrates the histological examination of the liver tissues. The histological appearance of normal liver tissue without any steatosis, inflammation or fibrosis is represented in Fig. 8(E). High degrees of steatosis, megamitochondria and ballooning degeneration as well as inflammatory cells were observed in HC group (Fig. 8(F)) indicating the presence of NAFLD or NASH. It should be noted that an accurate diagnosis between NAFLD cases with steatohepatitis from those with only steatosis and inflammation is demanding [58]. In the APMT group, some levels of ballooning injury, inflammation and megamitochondria are detectable, suggestive of the NAFLD (Fig. 8(G)). Interestingly, after treatment with ASDT, diet-induced pathological abnormalities such as megamitochondria were almost completely disappeared in ASDT group (Fig. 8(H)). As shown in this figure, only slight degree of ballooning injury and inflammation was observable in the liver histology of ASDT group. The obtained data revealed that HFD feeding resulted in the hepatic steatosis and increased the liver index, whereas feeding of ATV in SD form (ASDT) inhibited the progression of hepatic steatosis in NAFLD rats. Histological findings together with the previous outcomes in our study confirmed the superior performance of ASDT compared to APMT. These findings suggest that an improvement in physicochemical characteristics of ATV could enhance its therapeutic efficiency with reduced side effects. 4. Conclusion The prepared SDs of ATV were present in amorphous form and resulted in higher dissolution characteristics as compared to the PMs. The serum levels of TC and LDL-C were significantly (P b 0.01) decreased following the administration of ATV in SD form compared to the corresponding PM. Furthermore, treatment with ATV especially its SD form could outstandingly reduce the HFD toxic effects on the liver tissue. This study demonstrated the greater performance of the SDs in reduction of serum lipid levels and inhibition of NAFLD progression in HFDinduced hyperlipidemic rats compared to the pure drug and the related PMs. Declaration of interest The authors report no declaration of Interest. Acknowledgments The authors would like to thank the Drug Applied Research Center, Tabriz University of Medical Sciences (93/121) for the financial support
of this study. This article is a part of a thesis (no. 94) submitted for PhD degree in Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
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Powder Technology journal homepage: www.elsevier.com/locate/powtec
Pharmacological and histological examination of atorvastatin-PVP K30 solid dispersions Azin Jahangiri a,b, Mohammad Barzegar-Jalali a, Alireza Garjani d, Yousef Javadzadeh c, Hamed Hamishehkar a, Arash Afroozian a,d, Khosro Adibkia a,⁎ a
Drug Applied Research Center and Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran Students Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran Biotechnology Research Center and Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran b c
a r t i c l e
i n f o
Article history: Received 29 June 2015 Received in revised form 26 August 2015 Accepted 29 August 2015 Available online 3 September 2015 Keywords: Atorvastatin Solid dispersions Physicochemical characterization Histological examination Pharmacological efficiency
a b s t r a c t The objective of the present study was to investigate the pharmacological efficiency of orally administered atorvastatin calcium (ATV) solid dispersions (SDs). SDs of ATV were prepared using polyvinylpyrrolidone K30 (PVP) through the solvent evaporation method. Physicochemical characteristics of the prepared formulations were assessed benefitting scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and powder X-ray diffractometry (PXRD). The drug dissolution rates (under sink and non-sink conditions) as well as solubility studies were also examined. Serum lipid levels, liver index and histological analysis of the liver tissue in hyperlipidemic rats were considered to evaluate the pharmacological efficiency of prepared SDs. According to our findings, the drug crystallinity was reduced and the drug dissolution characteristics were improved in the prepared SDs. In vivo studies revealed that oral administration of ATV (3 mg/kg/day) in the SD form (ASDT) for 14 days along with high fat diet (HFD) to the hypercholesterolemic rats led to a significant decline (P b 0.05) in serum level of total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C). Moreover, ASDT exhibited more beneficial effects on the liver steatosis compared to ATV physical mixture (APMT) and hyperlipidemic control (HC) groups. In the present study, it was concluded that the SDs of ATV with improved physicochemical characteristics provided an increased therapeutic potential for management of hyperlipidemia compared to the corresponding physical mixtures (PMs). © 2015 Elsevier B.V. All rights reserved.
1. Introduction Atorvastatin calcium (ATV), a lipid-lowering agent, acts by the reversible inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [1]. ATV is administered for treatment of hypercholesterolemia and other dyslipidemic disorders alone or in conjunction with other anti-hyperlipidemic drugs. It is also helpful in primary prevention of cardiovascular disease and reducing the risk of myocardial infarction [2,3]. ATV belongs to class ІІ of biopharmaceutical classification system (BCS) [4] and tends to exhibit solubility or dissolution rate limited absorption. The marketed products of ATV suffer from poor oral bioavailability (~ 12%) [5]. Poor aqueous solubility, pre-systemic clearance in the gastrointestinal mucosa and/or hepatic first-pass metabolism are responsible for the low oral bioavailability of ATV [6]. Achieving appropriate therapeutic effects is possible by using higher doses of drug which results in dose related undesirable adverse effects. ⁎ Corresponding author at: Faculty of Pharmacy, Tabriz University of Medical Sciences, Golgasht Street, Daneshgah Ave., Tabriz, Iran. E-mail address: [email protected] (K. Adibkia).
http://dx.doi.org/10.1016/j.powtec.2015.08.047 0032-5910/© 2015 Elsevier B.V. All rights reserved.
Moreover, development of ATV formulation due to its poor aqueous solubility and insufficient bioavailability after oral administration is challenging [5]. Therefore, developing effective approaches to minimize the associated problems could be highly advantageous. Several techniques have been applied to enhance the solubility and dissolution rate of ATV, including nanosuspension formulation [7], preparation of the self-emulsifying drug delivery systems (SEDDS) [8,9], complexation with hydroxypropyl-β-cyclodextrin [6], preparation of the solid dispersions (SDs) [10–12], applying supercritical antisolvent (SAS) process [13,14] and spray drying technique [15]. SDs are used as one of the most practical and effective strategies in order to increase the dissolution behavior of poorly water-soluble drugs. The advantages of SDs include particle size reduction (possibly to molecular level), increase in wettability and porosity, decrease of drug crystallinity and sometimes conversion into amorphous state [16]. Accordingly, the drug substance could disperse as separate molecules, amorphous particles or crystalline particles, whereas, the carrier might present in the crystalline or amorphous state. Improvement in drug solubility and dissolution rate through SD technique has been shown by numerous studies [16]. Oral bioavailability of some poorly water-soluble drugs [17]
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such as ibuprofen [18], carbamazepine [19], naproxen [20] and ritonavir [21] has also been enhanced using SD technique. According to the conducted researches, polyvinylpyrrolidone (PVP)-based SDs of ATV exhibited noticeable increase in ATV absorption following oral administration [10]. Therefore, in this study, SDs of ATV were prepared using PVP K30 as an amorphous carrier, via the solvent evaporation method. Regarding literature review, evaluation of the therapeutic efficiency of ATV SDs (especially in liver tissue) via in vivo study has not been considered by other researchers. Although, the majority of researches are focused on effects of hypercholesterolemia on atherosclerosis and heart disease; however, toxic effects of cholesterol on the liver are also of particular importance [22]. Thus, in this work, after preparation and physicochemical characterization of ATV SDs, in order to investigate the therapeutic efficiency of prepared formulations in the diet induced hyperlipidemic rats, serum lipid levels, liver index and histopathological examination of the rat livers were evaluated. 2. Materials and methods 2.1. Materials ATV was purchased from Abidi pharmaceutical company (Tehran, Iran). PVP K30 and sodium hydroxide were supplied from Merck (Germany). Methanol was of high-performance liquid chromatography (HPLC) grade (Caledon Labs, Ontario, Canada). All other chemicals were of analytical grade.
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2.6. Fourier transformed infrared spectroscopy (FTIR) A Shimadzu 43000 FTIR spectrophotometer was employed for FTIR analysis. Samples were examined in the transmission mode and prepared using the KBr disk method (1 mg sample in 100 mg KBr powder). A spectral range of 400–4000 cm−1 and a resolution of 2 cm−1 were used in order to obtain the spectra. 2.7. Solubility of ATV Solubility measurements were performed according to the Higuchi and Connors' method [23]. Briefly, an excess amount of ATV was added into the test flasks containing various concentrations of PVPK30 in the dissolution medium (10 mL, phosphate buffer, pH 6.8). The samples were sonicated (Metler Electronics, model ME5.5, USA) for 30 min at room temperature. Afterward, the sealed test flasks were shaken at 25 °C (room temperature) for 48 h on a rotary shaker incubator (Heidolph unimax 1010, Inkubator 1000). The suspensions were centrifuged at 10,000 rpm for 5 min (Eppendorf 5810R centrifuge) and filtered through a 0.45 μm membrane filter. After suitable dilution, the samples were analyzed spectrophotometrically at a wavelength of 241 nm using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The polymer did not interfere with the UV measurement and no significant adsorption of the drug to the filter membranes was detected. All solubility experiments were performed in triplicate and the obtained results were presented as mean ± SD. 2.8. Dissolution study under sink condition
2.2. Preparation of the solid dispersions SDs of ATV were prepared by PVP K30 in different drug to polymer ratios, using the common solvent evaporation method. Briefly, different weight ratios (1:1, 1:3, 1:5 w/w) of drug:polymer combinations were dissolved in minimum volume of methanol and stirred (Heidolph, MR Hei-Tec, Germany) at 600 rpm for 15 min at room temperature. After complete dissolving, the solvent was evaporated using rota-evaporator (Heidolph, Laborota 4010 digital, Germany) at room temperature, 50 rpm for 12 h. Then the residue was pulverized by mortar and pestle, sieved (mesh size 60) and then kept in a desiccator. Recrystallized ATV was also prepared through recrystallization of ATV from methanol.
Dissolution study was performed using USP apparatus II, paddle stirrer (Erweka dissolution tester). The pure drug, SDs and PMs, all equivalent to 20 mg ATV, were added to the 900 mL dissolution medium (phosphate buffer, pH 6.8) under stirring rate of 75 rpm at 37 ± 0.5 °C. At appropriate time intervals, 2 mL of sample was withdrawn and filtered through 0.45 μm membrane filter. The removed volume was refilled with the fresh medium at 37 °C. The filtrate was suitably diluted and analyzed spectrophotometrically at 241 nm using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The obtained results were deducted from a standard calibration curve of ATV and the percent of the dissolved drug was plotted against the time. The reported results were the average value of at least three replicates ± SD.
2.3. Scanning electron microscopy (SEM)
2.9. Dissolution study under non-sink condition
The morphology of prepared samples was observed using scanning electron microscopy (SEM; Tescan, Brno Czech) operating at 20 kV. The samples were directly dispersed on a double-sided tape and coated by gold in a vacuum condition, before the examination.
Non-sink condition represented an insightful dissolution situation to further evaluation of the dissolution phenomena [24]. In this condition, dissolution medium is not enough for dissolving the total amount of drug [25]. The apparent dissolution profiles of SDs were investigated under non-sink condition as well. An accurately weighed amount of the SDs, each equivalent to 10 mg drug, was added to test flasks containing 10 mL phosphate buffer and shacked at 37 °C, 100 rpm (Heidolph unimax 1010, Inkubator 1000). The experiment was performed on five time points with three replicates. The samples were centrifuged at 10,000 rpm for 3 min and then filtered using 0.45 μm membrane filter. The supernatant was then further diluted and analyzed spectrophotometrically at 241 nm.
2.4. Differential scanning calorimetry (DSC) Thermal behavior of the SDs, PMs as well as pure drug and polymer was evaluated using differential scanning calorimeter (DSC60 Shimadzu, Kyoto, Japan). Accurately weighed samples (3 mg) were placed into crimp sealed aluminum pans. The measurement was performed at a heating rate of 20 °C/min from 50 to 210 °C. The aluminum oxide and indium powders were applied as reference and standard, respectively. 2.5. Powder X-ray diffraction (PXRD) X-ray diffraction patterns of the pure drug, polymer, PMs as well as prepared SDs were obtained by the X-ray diffractometer (Siemens D5000, Munich, Germany). Samples were scanned over the range of 2θ from 2 to 40°, under Cu Kα radiation, at a voltage of 40 kV and a current of 30 mA.
2.10. Animal study 2.10.1. Animals Male Wistar rats were purchased from Pasteur Institute of Iran (Tehran). Following their arrival to the animal care facility center, they were housed for one week prior to examination. Afterward, rats weighing 250 ± 20 g were divided into four groups and employed in this research. Rats were kept in the Animal House of Tabriz University of Medical Sciences at a controlled ambient temperature of 25 ± 2 °C
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Table 1 Animal groups and respective treatments. Groups
1 2 3 4
Diet
Normal control group (NC) Hyperlipidemic control group (HC) PM treated group (APMT) SD treated group (ASDT)
Treatment
(6 week)
(2 week)
NCD HFD HFD HFD
NCD HFD + PVP HFD + APM HFD + ASD
NCD: normal chow diet, HFD: high fat diet, APMT: treatment with ATV physical mixture, ASDT: treatment with ATV solid dispersion.
with 50 ± 10% relative humidity and a 12 h light/dark cycle. They had free access to food and water as well. The present study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of Tabriz University of Medical Sciences, Tabriz-Iran (Nation al Institutes of Health Publication No 85-23, revised 1985). 2.10.2. High fat diet A high-fat diet (HFD) reported by Garjani and coworkers [26] was applied in this study with some little modifications. Our HFD contained standard rodent chow powder (Sahand Niroo, Tabriz, Iran) (62.75% w/w), melted sheep's tail fat (15% w/w), wheat flour (10% w/ w), sucrose (10% w/w), cholesterol (2% w/w) and cholic acid (0.25% w/w). 2.10.3. Experimental protocol In vivo studies were performed on four groups of animals; each group was consisted of six rats. The total duration of experiment was eight weeks for both experimental and control groups. At the end of the sixth week, hyperlipidemia was developed and the treatment was begun (Table. 1). At the end of trial, rats were fasted for 10–12 h, with free access to water, then anesthetized through intraperitoneal injection of sodium pentobarbital. Blood samples were collected from inferior vena cava and transferred into gel and clot activator tube based on the manufacturer's suggested protocol. The livers were removed immediately by dissection, washed in ice-cold saline and blotted between two filter papers to eliminate the surface water. The livers subsequently weighed for measurement of the liver index. Isolated livers were kept in 10% neutralbuffered formaldehyde for further histological examination. 2.10.4. Drug administration ATV SDs and PMs, equivalent to 3 mg/kg ATV, based on dose translation from animal to human studies [27], were immediately gavaged (orally intubated) after suspending in 1 mL of distilled water. The formulation with the drug:polymer ratio of 1:5 was selected for in vivo studies because of its enhanced performance. The treatment was administered once a day for two weeks.
2.10.5. Biochemical analysis Fasting blood samples were taken for measurement of the serum concentrations of total cholesterol (TC), high density lipoproteincholesterol (HDL-C), and triglycerides (TG). Measurements were carried out using enzymatic colorimetric methods by commercially available kits (Pars Azmoon Inc., Tehran, Iran). The assays were performed using Autoanalyzer (Abbott Alycon 300, USA). HDL-C concentration was determined after precipitation of VLDL-cholesterol (VLDL-C) and LDL-cholesterol (LDL-C) with phosphotungstic acid and magnesium chloride. The LDL-C concentration was deliberated from the Friedewald formula [28] in which, LDL-C = (TC) − (HDL-C + VLDL-C), and since VLDL-C = (TG) / 5, thus, the concentration of LDL-C was calculated through LDL-C = TC − (HDL-C + 0.2(TG)). 2.10.6. Liver index and liver histological examination Liver index was calculated from the ratio of liver weight to body weight. Liver index = (Liver weight / Body weight) × 100%. The removed livers were fixed in 10% neutral-buffered formaldehyde for histological study. The tissues were embedded in paraffin and cut into 5 μm thick sections and subsequently stained by hematoxylin and eosin (H&E) for histological examination (n = 3). Microscope study was performed using camera equipped Olympus (Tokyo, Japan) BX50 microscope with 20× or 40× objective lens. 2.10.7. Statistical analysis Statistical evaluations were determined using oneway ANOVA followed by LSD posthoc test using SPSS 16 software. Any differences between the groups were considered significant at P b 0.05 level. All data were displayed as the mean ± SEM of values obtained from six rats. 3. Results and discussion 3.1. SEM studies SEM images of the pure ATV, recrystallized ATV and corresponding SD in 1:1 ratio are shown in Fig. 1. ATV demonstrated a few needlelike crystallites within an irregular shaped particle (Fig. 1(A)). The morphological feature of recrystallized ATV was completely different from the pure drug so that the surface area was meaningfully decreased in the recrystallized drug (Fig. 1(B)). Emergence of the new morphological shape in SD (Fig. 1(C)) compared to the pure drug was indicative of the efficient formation of SD system [18,29]. Moreover, according to SEM image of the SD, there were no observable drug particles on the surface of prepared SD, representing homogeneous distribution of the drug molecules in the SD system [30]. 3.2. DSC studies Fig. 2 represents the DSC thermograms of the pure ATV, PVP, recrystallized ATV as well as ATV:PVP PMs and SDs in 1:1 and 1:5 ratios. The
Fig. 1. SEM images of pure ATV (A), recrystallized ATV (B) and SD of ATV:PVP in 1:1 ratio (C).
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21.36°, which are indicative of the low crystallinity of the pure ATV [36]. The semicrystalline nature of the pure ATV was also confirmed by DSC. However, no characteristic diffraction peaks related to the pure ATV were observed in the recrystallized ATV, representing decrease in the drug crystallinity [37]. Moreover, SDs of ATV did not show any diffraction peak in 2θ = 2–40°, indicating a transition of ATV from a semicrystalline to an amorphous state [38]. Similar findings have been previously reported for the amorphous nanoparticles and solid dispersions of ATV prepared by supercritical antisolvent (SAS) and spray drying techniques [13,39]. Taken together with DSC data, these results specify a formation of amorphous ATV in the SDs. 3.4. FTIR studies
Fig. 2. DSC thermograms of PVP, ATV, recrystallized ATV (RCT ATV), physical mixtures (PMs) and solid dispersions (SDs) of ATV:PVP in the 1:1 and 1:5 ratios.
DSC curve of PVP displayed a broad endothermic peak between 75 and 135 °C, demonstrating the loss of water from the hygroscopic PVP [31]. DSC thermogram of the pure ATV displayed two endotherms, a broad endotherm at 50–130 °C corresponding to water loss or dehydration (probably indicates the presence of trihydrate form), followed by another endotherm at 182 °C related to the pure ATV melting point. These thermal events have been confirmed by thermal gravimetric analysis (TGA) in the previous conducted studies [7,13,15,32]. No endotherm was observed around the melting point of pure ATV in the thermogram of recrystallized ATV signifying the reduced crystallinity of recrystallized ATV. Physical mixtures of ATV: PVP in the ratio of 1:5 showed a melting endotherm with very low intensity, which could be due to the polymer dilution effect [20]. No endotherm related to the pure ATV melting point was observed in the SDs, which could be because of the reduced crystallinity; and/or dispersion of the drug molecules in the polymer solution [33–35]. Disappearance of the melting endotherm in the recrystallized ATV and the SDs might be attributed to the drug amorphization. According to similar previous studies, alteration of the drug physical state from a crystalline form to an amorphous state during SD formation is most probable [20,29,33].
3.3. PXRD studies Fig. 3 represents the X-ray diffraction patterns of the pure and recrystallized ATV, PVP, the PMs and SDs of ATV:PVP in 1:1 and 1:5 ratios. The low intensity peaks were observed at 2θ = 8.24°, 10.09° 18.82° and
FTIR studies were conducted to estimate any possible interactions between the ATV and PVP in the SDs. The FTIR spectra of PVP, ATV, recrystallized ATV, drug:polymer PMs and SDs in the ratios of 1:1 and 1:5 are represented in Fig. 4. The spectrum of PVP showed important bands at 2916 cm− 1 (C–H stretch) and 1656 cm−1 (C_O). A broad band from 3200 to 3600 cm−1 was assigned to the presence of water in the polymer [40], which was in agreement with a broad endotherm detected in the DSC finding. The spectrum of pure ATV exhibited characteristic peaks at 1660 cm−1 (acid carboxylic C_O stretch), 2930 cm−1 (C–H stretch), between 3200–3500 cm− 1 (intermolecular hydrogen bond, O–H stretch), three peaks between 1400 and 1600 cm−1 (aromatic C_C stretch), 1215 cm− 1 (C–F stretch), 834 cm− 1 (ring vibration due to para-substituted benzene), 1431 cm−1 (C–N stretch) as well as 1500 cm−1 and 3390 cm− 1 (N–H stretch) [13]. As can be seen in Fig. 4, the characteristic peak of the carbonyl group at 1660 cm−1 in the pure ATV became broader in the recrystallized ATV, which might be due to the drug amorphization. A similar finding has been stated by other researchers for melt-quenched and milled ketoprofen [41]. Regarding the chemical structure of PVP, carbonyl group of the polymer tends to form hydrogen bond with the favorable groups of drugs [40]. Hydrogen bonding usually leads to bathochromic shifting or broadening the peaks of functional groups [42]. According to ATV structure, hydrogen bonding could be expected between the hydroxyl groups of ATV and the carbonyl group of PVP. Comparing the FTIR spectra of the SDs with those of the corresponding PMs showed that O–H stretch between 3200 and 3500 cm−1 and C_O stretch at 1660 cm−1 in ATV spectrum were replaced by a broader band in the SDs, indicating the possible hydrogen bonding between ATV and PVP. Similar outcomes were formerly reported for other PVP-containing SDs including indomethacin [43], carbamazepine [40] and piroxicam [44]. Hydrogen bonding formation between the drug and polymer could prevent the drug re-crystallization and increase the stability of
Fig. 3. Powder X-ray diffraction patterns of PVP, ATV, recrystallized ATV (RCT ATV), solid dispersions (SDs) of ATV: PVP in 1:1 and 1:5 ratios.
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A. Jahangiri et al. / Powder Technology 286 (2015) 538–545 Table 2 Apparent solubility of recrystallized ATV (RCT ATV) and SDs of ATV-PVP in the 1:1, 1:3 and 1:5 drug:polymer ratios (after 24 h).
Fig. 4. FTIR spectra of PVP, ATV, recrystallized ATV (RCT ATV), physical mixtures (PMs) and solid dispersions (SDs) of ATV:PVP in the 1:1 and 1:5 ratios.
SDs [44]. Therefore, physical stability as well as in vivo performance of the SDs could be enhanced by using appropriate polymers [16,25]. 3.5. Solubility studies Fig. 5 illustrates the effect of PVP concentration on the solubility of ATV at 25 °C in phosphate buffer. As can be seen, aqueous solution of PVP led to increase in ATV solubility. There was no remarkable increase in the ATV solubility at lower concentrations of the polymer (below 1%). By increasing the polymer concentration, solubility of ATV was enhanced linearly up to 4.3 fold in 15% w/v. Increase in the solubility of ATV through PVP could probably be due to the formation of soluble complex between PVP as a water-soluble carrier and ATV as a poorly water-soluble drug [40]. Solubility of the pure ATV and apparent solubility of the prepared SDs and recrystallized ATV are exhibited in Table 2. Recrystallized ATV, which was present in complete amorphous form, revealed greater solubility. Moreover, the SDs of ATV presented a substantial increase in the drug solubility. Although the solubility of amorphous materials is meaningfully higher than their crystalline state, determining the solubility of amorphous form under true equilibrium condition is faced with difficulties. As a result, their experimental solubility is typically less than their thermodynamic predicted solubility [45]. 3.6. Dissolution studies
Samples
Apparent solubility (mg/mL) ± SD pH 6.8
Pure ATV RCT ATV SD 1:1 SD 1:3 SD 1:5
0.432 ± 0.010 0.543 ± 0.0141 0.990 ± 0.002 1.129 ± 0.026 1.149 ± 0.008
bioavailability [40]. Based on the obtained dissolution outcomes (Fig. 6), application of PVP could significantly improve the dissolution characteristics of ATV in both PMs and SDs. Results revealed that the SDs caused a further increase in ATV dissolution rate compared to the pure drug and PMs. The complete drug was released from all SDs after 60 min (Q60min). However, almost 100% of the drug was released from the SD with drug:polymer ratio of 1:5 during first 5 min (Q5min). The resulted value (Q5min) for pure ATV was 65% and the complete drug release occurred approximately within 210 min (3.5 h). The improved dissolution rate of ATV in the PMs might be attributed to the hydrophilic nature of PVP, which can facilitate the dissolution rate by reduction in the interfacial tension between the drug and the release medium. The enhancement of dissolution properties could also be explained by the solubilizing and wetting effect of PVP [46]. In addition to the cited mechanisms, an improvement in the dissolution rate of SDs could also be ascribed to the drug particle size reduction and/or decrease in the drug crystallinity during the SD preparation process [47,48]. Because of the higher internal energy of amorphous materials, they dissolve more rapidly than crystalline forms [49]. It is important to highlight that, in spite of the higher solubility of recrystallized ATV, which was present in amorphous state, it showed lower dissolution rate at the initial time intervals (Fig. 6). This phenomenon could probably be related to formation of a sticky mass with the reduced surface area in the hygroscopic amorphous drugs [50]. So therefore, solvent evaporation alone (recrystallization) could just produce an amorphous drug with enhanced solubility, however, application of PVP in the SD preparation process led to a further improvement in both drug solubility and dissolution rate. Any increase in the drug dissolution rate might also be attributable to the supersaturation phenomenon. However, the drug precipitates from unstable supersaturated solution to reach an equilibrium state [51]. The ability of a selected polymer to maintain the supersaturation state in the aqueous solution is of particular importance in SD performance [25]. According to the non-sink dissolution study of SDs (Fig. 7), drug
Dissolution rate in the gastrointestinal tract is most likely the ratelimiting step of absorption in the case of poorly water-soluble drugs such as ATV. Thus, they commonly exhibit dissolution dependent
Fig. 5. The effect of PVP concentration on the ATV solubility.
Fig. 6. Dissolution profiles of ATV, recrystallized ATV (RCT ATV), physical mixtures (PMs) and solid dispersions (SDs) of ATV:PVP in the 1:1, 1:3 and 1:5 ratios under sink condition.
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of TC and LDL-C in the hyperlipidemic control (HC), ATV PM treated (APMT) and SD treated (ASDT) groups, all compared to normal control group (NC). Treatment with both PM and SD of ATV (APMT and ASDT) revealed an outstanding decrease in the serum level of TC and LDL-C in comparison with HC group (P b 0.001). As seen in Table 3, the ASDT was more effective than APMT in reducing TC and LDL-C serum levels (P b 0.01). Serum level of TG was meaningfully (P b 0.05) reduced, while there was no significant change (P N 0.05) in the serum level of HDL-C between treated and HC groups. Altogether, the biochemical analysis showed that ASDT provided greater reduction in the serum lipid profile compared to APMT. However, the administered dose of ATV could not completely improve the hyperlipidemic condition.
Fig. 7. Dissolution profiles of ATV soli dispersions (SDs) in the 1:1, 1:3 and 1:5 ratios under non-sink condition.
concentration at the initial time intervals reached to more than 10-folds of ATV saturated solubility. Subsequently, the drug molecules precipitated from the supersaturated solution and reached to a plateau value after about 5 h, which could be considered as the drug amorphous solubility. During the drug precipitation, the adsorbed polymer on the surface of particles could prevent crystal growth by blocking the active surfaces and providing steric stabilization [52]. Moreover, hydrogen bonding between the drugs and polymers may help the maintenance of supersaturation state [53]. At the highest polymer ratio (SD 1:5), supersaturated concentration of the ATV in the non-sink condition as well as dissolution characteristics under sink condition were at the uppermost level. Accordingly, this formulation was selected for further in vivo studies. 3.7. In vivo studies 3.7.1. Effects of the treatments on serum lipid levels Elevated levels of cholesterol and LDL along with decreased HDL play an important role in the development of atherosclerosis, while lipid lowering agents reduce the risk of atherosclerosis and coronary heart disease (CHD) [54]. The measured serum lipid profiles at the end of the sixth week just before the beginning of the treatments were found to be as follows: TC = 185.7 ± 0.1, LDL-C = 164.3 ± 0.2, HDL-C = 11.0 ± 0.3 and TG = 51.7 ± 0.7. Reported data are mean ± SEM values of six independent experiments. According to the results obtained from biochemical analysis (Table 3), HFD at the end of the eighth weeks could significantly (P b 0.001) increase the serum levels
3.7.2. Effects of the treatments on liver visual appearance and liver index Consumption of HFD for eight weeks led to enlargement and paleness of the liver (Table 3 and Fig. 8(B–D)). Regarding the obtained data, healthy livers of the NC group exhibited the lowest liver index value and the highest redness (Fig. 8(A)). As shown in Table 3, HFD induced a notable increase in the liver index in all groups comparing the NC group (P b 0.001). Treatment with both APMT and ASDT improved the visual appearance of liver tissue from pale red to a more reddish color (Fig. 8(C and D)). The liver enlargement and paleness in ASDT group were lower than APMT group. Reduction in the liver index by using high doses of ATV (30 mg/kg/day) has been reported by other researchers [55]. However, it seems that the administered dose of ATV (3 mg/kg/day) was not sufficient to cause any significant effect on the liver index in the treated groups. 3.7.3. Histological examination of the liver tissue In addition to the harmful effects of hypercholesterolemia on atherosclerosis and CHD, its toxic effects on the liver are also challenging [22]. In today's world, due to excessive consumption of high-cholesterol foods and also lack of sufficient physical activity, the prevalence of obesity, diabetes, and the metabolic syndrome is expanding every day. Thus, non-alcoholic fatty liver disease (NAFLD) accompanied with lipid accumulation in the liver (steatosis) is a growing public health concern in most countries. Conducted researches revealed that the overconsumption of HFD plays an important role in the etiology of hepatic steatosis [56]. Moreover, nonalcoholic steatohepatitis (NASH), the liver steatosis combined with inflammation or advanced form of NAFLD, may develop end stage liver diseases which associates with about twenty percent mortality [57]. NAFLD is concomitant with hepatomegaly, elevated alanine aminotransferase hepatic steatosis and in some cases, varying degrees of liver fibrosis and steatohepatitis [57]. Steatosis, lobular inflammation, hepatocellular ballooning and fibrosis are the number of contributing factors linked to the existence of NAFLD. Histological evaluation remains the only way to appropriately assess these disorders [58].
Table 3 Effects of the ATV treatments on serum lipid levels and liver index in the diet induced hyperlipidemic rats. Groups
TC (mg/dL)
LDL-C (mg/dL)
HDL-C (mg/dL)
TG (mg/dL)
LI
NC HC APMT ASDT
61.80 ± 3.75###,⁎⁎⁎ 342.25 ± 11.43†††,⁎⁎⁎ 181.8 ± 6.79†††,### 147.5 ± 7.72†††,###,⁎⁎
20.88 ± 3.23###,⁎⁎⁎ 304.15 ± 14.10†††,⁎⁎⁎ 151.76 ± 5.89†††,### 111.5 ± 4.80†††,###,⁎⁎
34.80 ± 0.73 27.75 ± 2.25 24.00 ± 4.35 29.25 ± 3.47
30.60 ± 7.07# 51.75 ± 6.67 30.20 ± 3.12# 33.75 ± 3.06#
3.18 ± 0.08###,⁎⁎⁎ 5.80 ± 0.22††† 5.74 ± 0.20††† 5.4 ± 0.21 †††
Values are presented as mean ± SEM, n = 6 per group. Abbreviations: NC, normal control; HC, hyperlipidemic control; APMT, ATV physical mixture treated; ASDT, ATV solid dispersion treated; TC, total cholesterol; LDL-C, LDL-cholesterol; HDL-C, HDL-cholesterol; TG, triglyceride; LI, liver index. ††† P b 0.001 vs. NC group. # P b 0.05 vs. HC group. ### P b 0.001 vs. HC group. ⁎⁎ P b 0.01 vs. APMT. ⁎⁎⁎ P b 0.001 vs. APMT.
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Fig. 8. Liver visual appearance (A–D) and histological staining using H&E (E–H) followed by examining under a light microscope (magnification 20×), A&E: normal control, B&F: hyperlipidemic control, C&G: PM treated (APMT), D&H: SD treated (ASDT).
Fig. 8(E–H) demonstrates the histological examination of the liver tissues. The histological appearance of normal liver tissue without any steatosis, inflammation or fibrosis is represented in Fig. 8(E). High degrees of steatosis, megamitochondria and ballooning degeneration as well as inflammatory cells were observed in HC group (Fig. 8(F)) indicating the presence of NAFLD or NASH. It should be noted that an accurate diagnosis between NAFLD cases with steatohepatitis from those with only steatosis and inflammation is demanding [58]. In the APMT group, some levels of ballooning injury, inflammation and megamitochondria are detectable, suggestive of the NAFLD (Fig. 8(G)). Interestingly, after treatment with ASDT, diet-induced pathological abnormalities such as megamitochondria were almost completely disappeared in ASDT group (Fig. 8(H)). As shown in this figure, only slight degree of ballooning injury and inflammation was observable in the liver histology of ASDT group. The obtained data revealed that HFD feeding resulted in the hepatic steatosis and increased the liver index, whereas feeding of ATV in SD form (ASDT) inhibited the progression of hepatic steatosis in NAFLD rats. Histological findings together with the previous outcomes in our study confirmed the superior performance of ASDT compared to APMT. These findings suggest that an improvement in physicochemical characteristics of ATV could enhance its therapeutic efficiency with reduced side effects. 4. Conclusion The prepared SDs of ATV were present in amorphous form and resulted in higher dissolution characteristics as compared to the PMs. The serum levels of TC and LDL-C were significantly (P b 0.01) decreased following the administration of ATV in SD form compared to the corresponding PM. Furthermore, treatment with ATV especially its SD form could outstandingly reduce the HFD toxic effects on the liver tissue. This study demonstrated the greater performance of the SDs in reduction of serum lipid levels and inhibition of NAFLD progression in HFDinduced hyperlipidemic rats compared to the pure drug and the related PMs. Declaration of interest The authors report no declaration of Interest. Acknowledgments The authors would like to thank the Drug Applied Research Center, Tabriz University of Medical Sciences (93/121) for the financial support
of this study. This article is a part of a thesis (no. 94) submitted for PhD degree in Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
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