Nanosuspensions of a new compound, ER-β005, for enhanced oral bioavailability and improved analgesic efficacy

International Journal of Pharmaceutics 531 (2017) 246–256

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Research paper

Nanosuspensions of a new compound, ER-β005, for enhanced oral bioavailability and improved analgesic efficacy ⁎

Ling Yea,b,d,1, Mingxing Miaoa,c,1, Suning Lie, , Kun Haoa,

MARK

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a

Key Lab of Drug Metabolism & Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, PR China School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China c National Experimental Teaching Demonstration Center of Pharmacy, China Pharmaceutical University, Nanjing 210009, PR China d School of Traditional Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 51006, PR China e China National Center for Biotechnology Development, Beijing 100039, PR China b

A R T I C L E I N F O

A B S T R A C T

Chemical compounds studied in this article: ER-β005 (PubChem CID: 16450315) sulprostone (PubChem CID: 5312153) phenylephrine (PubChem CID: 6041) morphine (PubChem CID: 5288826) sodium dodecyl sulfate (PubChem CID: 3423265) acetone (PubChem CID: 180) capsaicin (PubChem CID: 1548943) isoliquiritigenin (PubChem CID: 638278) ethyl acetate (PubChem CID: 8857) dimethyl sulfoxide (PubChem CID: 679)

Estrogen receptor-β005 (ER-β005) is a novel compound developed by our group; however, its application has been greatly hindered due to its low solubility. A nanosuspension of insoluble drugs is a nanoscale colloidal dispersion that has extremely higher drug-loading compared with other nanomedicines. In this study, nanosuspensions of ER-β005 (Nano-ER-β005) stabilized by a food protein, β-casein (β-CN), were prepared via an antisolvent-precipitation method to improve oral absorption and thus promote therapeutic efficacy. Nano-ERβ005, which has a diameter of 110 nm and drug-loading of 50%, was developed. Analyses of fluorescence and circular dichroism (CD) spectra demonstrated a strong interaction between β-CN and drug particles in Nano-ERβ005, indicating that β-CN is a potent nanosuspension stabilizer. The oral bioavailability of Nano-ER-β005 was 1.6-fold greater than that of raw drug particles. Additionally, ER-β005 was confirmed to have a strong therapeutic effect against pain reactions in animal models, and inhibition of this effect was significantly increased with Nano-ER-β005 treatment. In conclusion, by using β-CN as a stabilizer, nanosuspensions of ER-β005 were developed and oral absorption was enhanced. Moreover, ER-β005 is a powerful drug that inhibits pain reactions, and its therapeutic efficacy was markedly increased in the Nano-ER-β005.

Keywords: Nanosuspensions Stabilizer Estrogen receptor agonist Interaction Bioavailability Analgesic efficacy

1. Introduction Neuropathic pain induced by damage or dysfunction of the nervous system is characterized in terms of allodynia, hyperalgesia and spontaneous pain (Sorge et al., 2012). It is estimated that 7%–8% of the adult population worldwide is impacted by this disease (van Hecke et al., 2014), which dramatically affects a person’s quality of life and social and economic wellbeing. Unfortunately, treatment of neuropathic pain is still a great challenge owing to the complicated pathophysiological mechanisms of this disease. Estrogen receptor (ER)-β005, an ER agonist with a molecular weight (MW) of 277.7 and log P value of 3.66, is a novel compound developed by our group, the chemical structure of which is shown in Fig. 1(A). By binding to ER-β and regulating ER-dependent transcription in reporter

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1

Corresponding authors. E-mail addresses: [email protected] (S. Li), [email protected] (K. Hao). Both authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ijpharm.2017.08.103 Received 12 May 2017; Received in revised form 31 July 2017; Accepted 21 August 2017 Available online 25 August 2017 0378-5173/ © 2017 Elsevier B.V. All rights reserved.

systems (Cordey and Pike, 2005), it is assumed that ER-β005 can be used to treat hyperalgesia or allodynia. However, ER-β005 is a biopharmaceutics classification system (BCS) II drug that is characterized by low solubility (approximately 5.3 μg/mL) and high permeability. Its oral absorption is poor, and thus, a high dose of the drug must be administered to patients, compromising the therapeutic efficacy. In addition, this low solubility results in gastrointestinal toxicity, enlarged individual variation, poor stability, and so on (Wang et al., 2017). As a result, development of a potent formulation to promote the delivery of ER-β005 is strongly desired. A nanosuspension of insoluble drugs with a diameter of 1 to 100 nm is a nanoscale colloidal dispersion that consists of pure drug particles having 100% drug and a small amount of stabilizer (Rabinow, 2004). Because of their theoretical drug-loading up to 100%, nanosuspensions

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Fig. 1. (A) Chemical structure of ER-β005. Effect of (B) the drug concentration in acetone and (C) pH of the protein solution on the average particle size of Nano-ER-β005. The protein concentration was 1 mg/mL (1%, w/v). (D) TEM image of Nano-ER-β005. The scale bar is 200 nm.

(Shapira et al., 2012, 2010). Furthermore, β-CN is a natural food protein that is inexpensive and is thus biocompatible, biodegradable, and able to be metabolized. Indeed, β-CN might induce potential inflammatory reactions in mucous membrane of patients with celiac disease (Miciński et al., 2013), limiting its use in this case. However, βCN is ready digested in gastrointestinal tract in healthy people (Monogioudi et al., 2011). Collectively, β-CN is a promising material for the development of an oral drug-delivery system. In the current study, by using β-CN as a stabilizer, nanosuspensions of ER-β005 (Nano-ER-β005) were prepared via an antisolvent-precipitation method to enhance oral bioavailability and promote therapeutic efficacy against neuropathic pain. The interplay between drug particles in Nano-ER-β005 and β-CN was studied by analyses of fluorescence and CD spectra. Studies of oral absorption and biodistribution were performed in rats, and the treatment efficacy was assessed in different models. This work is the first report to demonstrate that β-CN exhibits a stabilization effect on nanosuspensions. In addition, it was also demonstrated that ER-β005 has a profound anti-allodynia effect that is significantly promoted by Nano-ER-β005. This work confirms the analgesic effects of ER agonists and thus widens the clinical application of this type of drug.

have overwhelming advantages over other nanomedicines, such as liposomes, polymeric micelles, nanoemulsions, among others. (Li et al., 2015; Müller et al., 2011; Mishra et al., 2016). Moreover, the smaller drug particles in nanosuspensions enable increase in surface area and therefore enhancement in drug dissolution, according to the NoyesWhitney equation (Merisko-Liversidge and Liversidge, 2011). In addition, nanosuspensions possess other benefits, including ease of scale-up for manufacturing, low toxicity, protection of the drug against degradation in the gastrointestinal tract (He et al., 2017), relatively low cost of preparation, applicability to various administration routes (Bi et al., 2017; Gao et al., 2012), and so on. Currently, more than 20 nanosuspension products have been commercially marketed; typical products include Megace ES (Megestrol acetate), Zanaflex Capsules (Tizanidine), Invega (Paliperidone palmitate), among others (Moschwitzer, 2013; Shegokar and Müller, 2010). Despite the merits of nanosuspensions, they still have various drawbacks, such as toxicity and stability issues, with regard to sedimentation/creaming, agglomeration, crystal growth and changes in the crystallinity state (Wang et al., 2013; Wu et al., 2011). In particular, stability is an essential factor in guaranteeing the safety and efficacy of nanosuspension products (Wu et al., 2011). Therefore, a stabilizer must be incorporated into the formulation to coat the drug particles (Verma et al., 2011; Yuminoki et al., 2016). β-casein (β-CN), a milk protein, is a calcium-sensitive phosphoprotein with a MW of approximately 24 kDa, isoelectric point (pI) of 5, and diameter of 5 nm (Elzoghby et al., 2011). Unlike other globular proteins, β-CN has a definite sequence that consists of amino acids 1–52, 88–130 and 158–183 and is stable against heating (Horne, 2002). Importantly, distinct hydrophobic and hydrophilic domains are present in β-CN and, as a result, this protein is able to act like a surfactant and interact with hydrophobic materials

2. Materials and methods 2.1. Materials ER-β005 (batch No. EW5779-2-P1, 99.53% purity) was provided by WuXi AppTec Co., Ltd. (Wuhan, China). Bovine β-CN (SLBN8470V, 98% purity), capsaicin, sulprostone, phenylephrine and morphine were purchased from Sigma Aldrich (Shanghai, China). Sodium dodecyl 247

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were used: bandwidth, 1 nm; response, 1 s; wavelength range, 250–190 nm; scan rate, 100 nm/min; cell length,0.1 cm; temperature, 25 °C; and β-CN concentration,0.2 mg/mL (pH 7.0).

sulfate (SDS) was purchases from SCR Co., Ltd. (Shanghai, China). Methanol and other reagents were of chromatographic grade. 2.2. Animals

2.8. Powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC)

Male Sprague-Dawley rats (200–220 g) were obtained from Shanghai SIPPR-BK laboratory animal Co., Ltd. (Shanghai, China). Male Black6-C57 mice (20–25 g) were from Changzhou CAVENS laboratory animal Co., Ltd. (Changzhou, China). All animals were maintained in an air-conditioned animal facility at 25 ± 2 °C and a relative humidity of 50 ± 10%. Water and food were provided ad libitum. Animals were acclimatized to the facilities for one week prior to experiments. All animal experiments were in accordance with the guidelines set out by the Institutional Animal Care and Use Committee of China Pharmaceutical University.

The drug state in the optimized formulation was detected with a D8 Advance diffractometer equipped with Cu Kα radiation (λ = 0.154 nm) (Bruker AXS, Germany) and a TA-Q2000 DSC instrument (TA Instruments Ltd., New Castle, USA). To facilitate analysis, dried NanoER-β005 was prepared by centrifuging the sample at 50,000 g (Beckman Coulter Optima L-80XP) for 10 min at 4 °C, followed by drying at 60 °C. PRXD analysis was performed under the following conditions: 2θ range, 2–40°; scan rate, 1°/min; step size/time,0.02°/1 s; and voltage, 40 kV/40 mA. The parameters for DSC determination were as follows: heating range and rate, 30−300 °C and 10 K/min; nitrogen, 100 mL/min. A fixed weight of 5 mg sample was placed in an aluminum pan and sealed.

2.3. Preparation of Nano-ER-β005 Nano-ER-β005 was prepared via a precipitation–ultrasonication method as described in previous reports (He et al., 2013b; Li et al., 2015). In brief, 2 mL of acetone that contained a fixed amount of ERβ005 was added to 10 mL of a β-CN solution (1 mg/mL); after that, the mixture was subjected to ultrasonic treatment (20–25 kHz, Scientz Biotechnology Co., Ltd, Ningbo, China) for 8 min at 400 W/4 °C. Before mixing, the acetone solution and protein solution were cooled to 4 °C in an ice-water bath. The ultrasound conditions were as follows: probe diameter, 8 mm; ultrasound burst and pause, 3 s; and depth, 1 cm. Finally, the sample was centrifuged at 50,000g (Beckman Coulter Optima L-80XP) for 10 min at 4 °C to remove free protein. The physical mixture (PM) of ER-β005 and β-CN employed as control in drug state assay and dissolution test was prepared by mixing the two in a mortar at a mass ratio of 1:2 ER-β005 and β-CN.

2.9. In vitro dissolution

The particle size and polydispersion index (PI) of Nano-ER-β005 were detected with a 90Plus Particle Size Analyzer (Brookhaven Instruments, Holtsville, NY, USA) at room temperature, according to the dynamic light scattering (DLS) principle. The sample was diluted by approximately 10-fold before testing. Data were collected at 5 min and 90 °C, and the particle size was indicated as a volume-weighted distribution.

In vitro dissolution was evaluated using a Chinese Pharmacopoeia type III Apparatus in a ZRS-8G release tester (Tianjin, China). The following parameters were used: rotation speed, 100 rpm; temperature, 37 ± 0.5 °C; medium volume: 250 mL PBS (pH 6.8) with 2% SDS (w/ v); and sampling volume, 5 mL. The sample was centrifuged for 5 min at 100, 000g to collect the supernatant for high performance liquid chromatography (HPLC) assay. Determination of drug content in supernatant was performed on a Shimadzu LC-2010 HPLC system (Japan). Separation was performed via a Shimadzu VP-ODS C18 column (150 mm × 4.6 mm, Japan) at 40 °C. The mobile phase was composed of a 0.1% formic acid aqueous solution and methanol (45/55, v/v) with a total running time of 9.5 min at a flow rate of 1.0 mL/min. The injection volume was 10 μL. The ultraviolet wavelength was 260 nm. A good liner relationship between the drug concentration and the peak area was displayed within the range of 1.0–50.0 μg/mL. The regression equation was Peak area = 719.888C + 178.464, r = 0.9999, where C is the drug concentration and r is correction coefficient. The average recovery and RSD were 101.3% and 0.83, respectively.

2.5. Transmission electron microscopy (TEM)

2.10. Oral bioavailability and biodistribution in rats

The shape of Nano-ER-β005 was observed on a JEM-1230 TEM (Tokyo, Japan) at an acceleration voltage of 200 kV. Briefly, one drop of sample was added to a carbon mesh, followed by absorption of excess sample with filter paper. The sample was dried at room temperature for 10 min, one drop of 2% uranyl acetate (w/v) was used to stain the sample for 5 min, and finally, the sample was dried at room temperature for 10 min.

Thirty-six rats were randomly divided into 6 groups (6 rats per group), followed by administration of ER-β005 suspensions that were fabricated by dispersing raw drug particles (4–6 μm) in water that contained 2.5% (w/w) hydroxypropyl methylcellulose E5 (HPMC) or in Nano-ER-β005 via intragastric gavage (i.g.) at doses of 25 mg/kg, 50 mg/kg, and 100 mg/kg. At predetermined time points, serial blood samples (0.5 mL) were collected via retro-orbital venous plexus puncture with the aid of glass heparinized capillaries in EDTA centrifuge tubes. Samples were centrifuged at 10,000g for 5 min; plasma was obtained and then stored at −80 °C for further analysis. To study the biodistribution, ER-β005 suspensions or Nano-ER-β005 was administered to rats via i.g. at a dose of 50 mg/kg (n = 6). At 0.5 h, 2 h and 8 h, the rats were killed and the main organs, including the heart, liver, spleen, lung, brain and kidney, were harvested. Tissue homogenates were prepared with PBS (w/v = 1/4) and frozen at −80 °C until analysis.

2.4. Particle size measurement

2.6. Fluorescence spectra The fluorescence assay was conducted on a SHIMADZU RF-5301PC Spectrofluorometer (Japan) at 25 °C. The conditions for analysis were as follows: emission wavelength, 300–500 nm; excitation wavelength, 295 nm; emission and excitation wavelengths, 5 nm and 15 nm; and protein concentration, 0.2 mg/mL. 2.7. Circular dichroism (CD) spectra

2.11. Plasma and tissue sample processing and analytical assay A J-810 spectrometer (Tokyo, Japan) with a temperature-controlling unit and a quartz cuvette were used to record the CD spectra, and the ellipticity is provided in millidegrees. The following conditions

Plasma samples and tissue homogenates were extracted via liquid–liquid extraction. A 100-μL aliquot of plasma sample or tissue 248

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homogenate was spiked with 10 μL of internal standard solution (isoliquiritigenin) and mixed for 30 s. Then, 1 mL of ethyl acetate was added to each tube for drug extraction. The tubes were vortexed for 5 min and then centrifuged at 10,000g for 5 min. The supernatants (900 μL) were then transferred to clean plastic tubes, followed by evaporation to dryness under a gentle stream of nitrogen at 37 °C. The extracted residues were reconstituted in 100 μL of methanol-water (50: 50, v/v) with vortex-mixing for 3 min. After centrifugation at 18,000g for 5 min, the supernatant was collected for analysis. Analytes were analyzed on an HPLC–MS/MS system that included an HPLC unit (Shimadzu) and AB Sciex triple quadrupole 4000+. Separation was performed on a Shimadzu VP-ODS C18 column (150 mm × 2.0 mm). The mobile phase was composed of a 0.1% formic acid aqueous solution (A) and methanol (B) with a total running time of 9.5 min at a flow rate of 0.2 mL/min. From 0 to 1.50 min, the A/B ratio was 90/10; from 1.50 to 2.20 min, the ratio was gradually changed from 90/10 to 10/90; from 2.20 to 5.00 min, the ratio was maintained at 10/90; from 5.00 to 8.50 min, the ratio was gradually altered from 10/90 to 90/10; and from 8.50 to 9.50 min, the ratio was kept at 90/10. The temperature of the column oven was set at 40 °C, and the injection volume was 10 μL. The mass spectrometer was operated in positive mode for the analytes and internal standard. Quantification was performed in the multiple reactions monitoring mode of the transitions of m/z 278.1 → 125.1for the analytes and m/z 257.1 → 137.0 for the internal standard. Data acquisition and processing were performed with analyst software (AB SCIEX Analyst 1.5.2).

1980; Ho Kim and Mo Chung, 1992). In brief, after anesthesia by inhaling a mixture of isoflourane/oxygen, an incision was made from the XIII thoracic vertebra toward the sacrum, followed by isolating the muscle from the spinal vertebra at the levels of L4–S2 and removing the transverse process with a small rongeur to confirm the identity of the L4–L6 spinal nerve. After that, the L5 and L6 spinal nerves were separated, and then, the isolated nerves were ligated with silk thread. The mice were then sutured and placed at room temperature for recovery. One week later, allodynia was evaluated with a light tactile stimulus to the affected surgical paw. Eight filaments were used with logarithmically spaced increments from 4 to 150 mN; each filament was oriented vertically to the plantar surface of the ligated paw. The withdrawal threshold was defined as the change from increasing to reducing the stimulus strength; accordingly, a positive response was displayed as a sharp withdraw of the paw, and the 50% threshold was recorded as an up-down manner relying on the response. Allodynia reversal was calculated by the following equation: Allodynia reversal (%) = PoDT − PrDT/15 − PrDT × 100, where PoDT and PrDT are defined as the Post and Pre Drug Thresholds, respectively. To investigate the effect of different doses of ER-β005 on the inhibition of allodynia, three doses of Nano-ER-β005 (25, 50 and 100 mg/ kg) were administered to the animals via i.g., with ER-β005 suspensions (50 mg/kg) serving as the control. The standard pain reducer, morphine (15 mg/kg i.p.), was also employed for comparison purposes. Assessment of allodynia was performed at 1 h after administration. Moreover, three different regimens were studied: acute drug effect (regimen 1), prolonged drug effect (regimen 2) and continuous drug effect (regimen 3). ER-β005 suspensions or Nano-ER-β005 were administered to animals via i.g. at an ER-β005 dose of 50 mg/kg, followed by assessment of allodynia for regimen 1 and regimen 2 30 min and 6 h later, respectively. Regimen 3 included i.g. administration 3 times with a 3 h interval and evaluation of the inhibition of allodynia 30 min later.

2.12. Therapeutic efficacy 2.12.1. Capsaicin-induced mechanical hyperalgesia Mice (20–25 g) were used in this study. The model of mechanical hyperalgesia was prepared via a similar process described in a previous report (Piu et al., 2008). In brief, baseline determination was performed as follows: the plantar surface of the left paw was poked 10 consecutive times over 3 periods per day using 5.07 Von Frey Hairs with an interval of 10 min for each period. The mice that did not respond by withdrawing their paws or that responded more than 4 times each trial were not used. After the baseline determination, on the day of the study, ERβ005 suspensions (50 mg/kg), Nano-ER-β005 (25, 50 and 100 mg/kg, respectively) or other formulations were administered to mice via i.g. (n = 6 per group). One hour later, 10 μL of capsaicin (0.3%) was injected into the plantar surface of the left hind paw. The left paw was measured with the procedure described above at 30 and 60 min postcapsaicin treatment. Hyperalgesia over time was monitored by counting the number of flinches in response to the poking of the paw with a Von Frey Hair.

2.13. Data analysis and statistics All of the pharmacokinetic parameters were estimated with the Winnonlin software and non-compartmental model. The peak plasma concentration (Cmax) and time to reach Cmax (tmax) were obtained directly from the observed concentration versus time profiles. The area under the curve was calculated with the linear trapezoidal rule. Oneway analysis of variance used to evaluate the statistical significance of the differences between samples. The results are expressed as the mean ± standard deviation. The difference was significant at a P value of less than 0.05. 3. Results

2.12.2. Chemically induced tactile allodynia The model of chemically induced tactile allodynia was prepared via i.p. administration of sulprostone in dimethyl sulfoxide (DMSO) at a dose of 300 ng/kg or phenylephrine in water at a dose of 100 ng/kg (Yaksh and Harty, 1988). Briefly, one hour before injection of the allodynia-inducing agents, ER-β005 suspensions (50 mg/kg), Nano-ERβ005 (25, 50 and 100 mg/kg, respectively) or other control formulations were administered to the mice via i.g. (n = 6 per group). Following injection of the allodynic agent, animals were subjected to a 15–50 min allodynia evaluation with intervals of 5 min. Three levels of allodynia response were defined: 0, no response; 1, mild squeaking and trying to escape away from the paintbrush; and 2, strong squeaking with biting at the paintbrush and intense efforts to escape. Each mouse could have a maximum score of 16 in the overall period.

3.1. Preparation and characterization of Nano-ER-β005 Nano-ER-β005 was prepared via an antisolvent-precipitation method. To prevent aggregation, β-CN with an amphiphilic structure was used to coat the drug particles according to the interaction between the hydrophobic domains in β-CN and drug particles. To optimize the formulation of Nano-ER-β005, the impacts of drug-loading and the pH on the particle size were studied. As depicted in Fig. 1(B), the particle size rose as amount of drug in 2 mL of acetone increased from 0 to 0.5 mg; inversely, the diameter of Nano-ER-β005 decreased as the drug concentration changed from 0.5 to 5 mg/mL. However, the particle size increased markedly as the drug concentration rose to 10 mg/mL. The critical micellization concentration (CMC) of β-CN was approximately 0.2% in water; above the CMC, β-CN self-assembles into micelles (Turovsky et al., 2015). On the other hand, the addition of a small amount of hydrophobic compound breaks protein micelles and then induces co-assembly into new nanoaggregates (Chen et al., 2015). Nevertheless, increased drug-loading destroys the nanosystem. The protein coating on the nanoscaled drug particles prevents the

2.12.3. L5/L6 spinal nerve ligation model Male Sprague–Dawley rats (200–220 g, n = 6 per group) were used in this study, and tactile allodynia was established by surgical ligation of the L5 and L6 spinal nerves (Decosterd and Woolf, 2000; Dixon, 249

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to 359 nm. The fluorescence quenching was ascribed to the destruction of the hydrophobic local environment of Trp. Interestingly, in Nano-ERβ005, the fluorescence of β-CN rose when the drug-loading changed from 0.005 to 0.2 mg/mL; by contrast, the fluorescence decreased when the drug-loading changed from 0.5 to 5 mg/mL. At low drug-loading, the drug associated with protein to form drug-protein complexes that assembled into nanoparticles, thereby enabling the exposed Trp to reenter a relatively hydrophobic environment. At high drug-loading, the drug-protein complexes were inclined to coat the drug particles due to the stronger affinity of the complexes to the hydrophobic surface of the drug particles (He et al., 2017). The CD spectrum is a potent tool that can be used to study alterations of a protein’s secondary and tertiary structures. The CD spectrum of β-CN displays a fold typical of proteins that includes a β-sheet structure, as demonstrated by a radian of ellipticity, and an α-helix, as indicated by an ellipticity minimum located at 200 nm (Stroylova et al., 2011). The addition of ER-β005 led to a decrease in the minimum and radian of ellipticity, with a trend of reduction of the minimum ellipticity with increasing drug-loading from 0.005 to 5 mg/mL. These results demonstrated that the addition of ER-β005 reduced the α-helix content and increased the β-sheet content, therefore changing the protein’s secondary and tertiary structures. Collectively, the interaction between β-CN and the drug (particles), which was displayed as an alteration of the local-environment of Trp and changes in the secondary and tertiary structures, was confirmed. Generally, once the protein aqueous solution was blended with hydrophobic drug dissolved in organic phase, two events would occur: forming protein-drug complexes and drug particles stabilized by protein or complexes, depending on the drug-loading. At low drug-loading, the protein tended to bind with drug and form protein-drug complexes probably due to their robust binding ability (Zhang et al., 2017). At high drug-loading, the protein would associate with drug and form protein-drug complexes at first; and subsequently, the complexes rather

aggregation of drug particles; yet, a further increase in drug-loading led to insufficient surface-coating by the stabilizer, thereby generating larger drug particles (He et al., 2017). The pH of the medium had a significant influence on the particle size of Nano-ER-β005 (Fig. 1C). In general, a low pH close to the pI (∼5) of β-CN resulted in a reduction of the particle size. The repulsive force between the protein molecules decreased as the pH of the media approached the pI, facilitating protein coating on drug particles. Collectively, the conditions for the preparation of the optimized formulation were as follows: protein concentration, 1% (w/v)/10 mL; amount of drug, 5 mg in 2 mL acetone; and medium pH, 6. The average particle size of Nano-ER-β005 from an optimized formulation with a drug-loading of 50% (w/w) was approximately 110 nm with a PI value of 0.066, demonstrating a narrow size-distribution. TEM examination displays spherical particles with a diameter of approximately 100 nm (Fig. 1D), consistent with the DLS results. After storage for 30 days at 4 °C, the particle size of Nano-ER-β005 was approximately 100 nm, demonstrating that Nano-ER-β005 was stable against aggregation over a period of 1 month. 3.2. Interplay between β-CN and drug particles in Nano-ER-β005 To explore the interactions between β-CN and drug particles in Nano-ER-β005, both fluorescence and CD spectra were examined. Tryptophan (Trp), an amino acid residue located at position 143 in the protein, has a high radiative rate and fluorescence yield; however, its fluorescence is greatly impacted by its local environment owing to the presence of an indole chromophore in Trp (Liu and Guo, 2007). Therefore, the interaction between a protein and other materials induces fluorescence quenching of the protein, which is easy to study (He et al., 2013a). As shown in Fig. 2(A and B), the addition of ER-β005, irrespective of the difference in drug-loading, led marked fluorescencequenching of the protein, accompanied by a red shift of λmax from 339

Fig. 2. Effect of the drug concentration in acetone on (A/B) the intrinsic fluorescence and (C) UV-CD spectra of β-CN in Nano-ER-β005. The protein concentration was 1 mg/mL. The sample was prepared by adding 1 mL of acetone containing different amounts of the drug to 10 mL of protein solution.

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Fig. 3. PXRD diffractograms of raw drug particles (ER-β005), β-CN, PM containing drug and β-CN at 1:2 mass ratio, and dried Nano-ERβ005. Dried Nano-ER-β005 was prepared by centrifuging the sample at 15,000 g for 5 min at 37 °C, collecting the deposit and drying the sample at 60 °C.

amorphous material, and therefore, any endothermic peak was absent. The characteristic melting peak was also shown for Nano-ER-β005, confirming the absence of crystal state of ER-β005. Interestingly, an exothermic peak appeared at around 230 °C. Previous reports indicated that nanosuspensions with coexistence of amorphous and crystal drug would form at high drug-loading as a protein was used as a stabilizer, owing to that protein-drug complexes forming at initial stage preferred to coat the drug particles due to their stronger affinity to hydrophobic surface than protein (He et al., 2017; Zhang et al., 2017). Therefore, this peak at 230 °C probably stemmed from the decomposition of the protein-drug complexes coating the drug particles.

than the protein coated the drug particles because the complexes had increased hydrophobic regions resulted from the drug binding and thereby facilitated their association with drug particles (He et al., 2017). Therefore, it was speculated that the stabilized effect on the drug particles in Nano-ER-β005 was predominantly ascribed to β-CN-drug complexes. 3.3. Drug state in Nano-ER-β005 To study the drug state in an optimized formulation of Nano-ERβ005, PXRD and DSC analyses were performed. As depicted in Fig. 3, pure raw particles of ER-β005 have diffraction peaks at 2θ angles of 4.62, 18.77, 19.70, and 27.48, thus demonstrating that raw ER-β005 was present in a crystal state. β-CN did not exhibit diffraction peaks because of its amorphous state, while the main diffraction peaks of ERβ005 were observed from PM. The diffractogram of dried Nano-ERβ005 showed characteristic peaks of ER-β005 and thus indicated that the drug in Nano-ER-β005 was mainly in a crystal state. The drug state in Nano-ER-β005 was further studied by DSC analysis. Pure ER-β005 and PM had an endothermic melting peak at approximately 242 °C and an exothermic decomposing peak at around 253 °C, demonstrating that it was in a crystal state (Fig. 4). β-CN was an

The dissolution profiles of ER-β005 from the raw drug particles, PM of ER-β005 and β-CN at mass ratio 1:2 and dried Nano-ER-β005 are shown in Fig. 5. The cumulative dissolution of ER-β005 in Nano-ERβ005 was up to approximately 90% at 10 min; by contrast, only 37% and 45% of the drug from PM and raw particles, respectively, were dissolved at 10 min, and the dissolution of 90% occurred at 120 min. This enhancement stemmed from the increased surface area of the nanosized drug particles in Nano-ER-β005, according to the Noyes-

Fig. 4. DSC thermograms of raw drug particles (ER-β005), β-CN, PM containing drug and β-CN at 1:2 mass ratio, and dried Nano-ER-β005.

Fig. 5. In vitro cumulative dissolution profile of different formulations in pH 6.8 PBS containing 2% (w/w) SDS at 37 °C. PM of ER-β005 and β-CN was at mass ratio 1:2.

3.4. In vitro dissolution

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Fig. 6. Plasma drug concentration-time curves after i.g. administration of ER-β005 suspensions and Nano-ER-β005 to rats at doses of (A) 25 mg/kg, (B) 50 mg/kg, and (C) 100 mg/kg (n = 6). The control formulation, the ER-β005 suspension, was prepared by dispersing raw drug particles into a 2.5% (w/w) HPMC solution.

Whitney equation. Indeed, the drug dissolution from PM was slightly faster than raw particles, probably owing to the wetting effect from βCN.

drug accumulated in the heart, liver, spleen, lung, kidney, and brain; Nano-ER-β005 exhibited higher drug accumulation in these tissues due to its enhanced oral absorption.

3.5. Oral bioavailability and biodistribution in rats

3.6. Therapeutic efficacy

To investigate oral absorption, suspensions of ER-β005 or Nano-ERβ005 were orally administered to rats at doses of 25 mg/kg, 50 mg/kg, and 100 mg/kg. The plasma-concentration curves and pharmacokinetic parameters are shown in Fig. 6 and Table 1, respectively. Nano-ERβ005 led to in general markedly higher plasma levels of the drug compared with ER-β005 suspensions, and the plasma levels rose with the increase in dose from 25 to 100 mg/kg. Despite the difference in doses, the Cmax and area under the concentration (AUC0-∞) of Nano-ERβ005 were approximately 1.6-fold greater than ER-β005 suspensions. These results demonstrated that the bioavailability of the drug was significantly improved when the drug was orally administered with Nano-ER-β005. ER-β005 is a BCS II drug with low solubility and high permeability; thus, the enhancement in dissolution was ascribed to improved bioavailability. The distributions of ER-β005 at 0.5 h, 2 h and 8 h after oral administration of ER-β005 or Nano-ER-β005 at a dose of 50 mg/kg are displayed in Fig. 7. Upon administration of the two formulations, the

3.6.1. Capsaicin-induced mechanical hyperalgesia Injection of capsaicin (0.3%) triggered marked mechanical hyperalgesia within 15 min. This response was stable for over 1 h and reached a maximum of 4.8 ± 0.8 flinches at 30 min in the saline group and 4.7 ± 0.9 flinches for the blank formulation group (Fig. 8). One hour prior to capsaicin injection, the ER-β005 suspension at a dose of 50 mg/kg or Nano-ER-β005 at doses of 25, 50 and 100 mg/kg were orally administered to mice. Strikingly, fewer flinches were recorded at 30 min (Fig. 8A) or 60 min (Fig. 8B) after treatment with these two formulations compared to the two controls, irrespective of the differences in dose, demonstrating that ER-β005 was efficient at preventing mechanical hyperalgesia. On the other hand, at 30 min, approximately 2.8 flinches were recorded in animals treated with 50 mg/kg of ERβ005 and 2.6, 1.7 and 1.1 flinches were recorded in animals treated with 25, 50 and 100 mg/kg of Nano-ER-β005. Importantly, the reduction in the number of flinches from 25 mg/kg of Nano-ER-β005 was similar to that of 50 mg/kg of ER-β005. Similar results were also

Table 1 Pharmacokinetic parameters of ER-β005 in rats after i.g. administration of ER-β005 suspensions and Nano-ER-β005 at doses of 25 mg/kg, 50 mg/kg, or 100 mg/kg (n = 6). 25 mg/kg

Cmax (ng/ml) Tmax (h) t1/2 (h) AUC0-∞ (ng*h/mL)

50 mg/kg

100 mg/kg

ER-β005

Nano-ER-β005

ER-β005

Nano-ER-β005

ER-β005

Nano-ER-β005

3109.20 ± 480.70 2.00 6.43 ± 1.06 4724.10 ± 551.20

4908.40 ± 513.40* 2.00 6.89 ± 1.13 7638.30 ± 616.80*

6689.10 ± 797.40 2.00 6.50 ± 1.26 9032.30 ± 867.30

9438.60 ± 882.60* 2.00 6.65 ± 1.87 14137.30 ± 1123.50*

9512.30 ± 975.80 2.00 6.56 ± 0.97 14098.90 ± 1210.20

15438.50 ± 1023.40* 2.00 6.99 ± 1.21 24074.00 ± 2038.60*

Cmax, maximum plasma concentration; AUC, area under the concentration curve; tmax, time to reach Cmax; t1/2, biological half-life. *p < 0.01 and #p < 0.05 vs. control of ER-β005 suspensions.

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Fig. 7. Drug concentration in rat tissues after i.g. administration of ER-β005 suspensions or Nano-ER-β005 at an ER-β005 dose of 50 mg/kg at (A) 0.5 h, (B) 2 h, or (C) 8 h (n = 6). Significant differences were observed in all tissues at the three time-points. The control formulation, the ER-β005 suspension, was prepared by dispersing raw drug particles into 2.5% (w/ w) HPMC solution.

pain scores for the sulprostone-induced model treated with Nano-ERβ005 were 16, 10 and 7 for doses of 25, 50 and 100 mg/kg, respectively (Fig. 9A); the scores for the phenylephrine-induced model treated with Nano-ER-β005 were 16, 9 and 6 for doses of 25, 50 and 100 mg/kg, respectively (Fig. 9B). In particular, treatment with Nano-ER-β005 at a dose of 100 mg/kg restored the pain thresholds to levels that were comparable to those of the saline treatment. These results suggested that Nano-ER-β005 had profound anti-allodynia effects, which were further improved by increasing the dose from 25 to 100 mg/kg. Compared with ER-β005 suspensions, Nano-ER-β005 had enhanced antiallodynia effects: the pain score for Nano-ER-β005 (50 mg/kg) was significantly lower than that for the ER-β005 suspensions (50 mg/kg) in both phenylephrine- and sulprostone-induced tactile allodynia.

observed at 60 min. These results indicated that Nano-ER-β005 was more efficient in alleviating capsaicin-induced mechanical hyperalgesia than ER-β005 suspensions.

3.6.2. Chemically induced tactile allodynia Two pain sensitizers, sulprostone and phenylephrine, were used to evoke an allodynia response by activating different pathways. As shown in Fig. 9, treatment with the two agents produced marked and significant allodynia responses, with a pain score of up to approximately 20, which a baseline pain score of 4–5 was caused by saline or the blank formulation. After oral administration of ER-β005 suspensions at a dose of 50 mg/kg, the pain scores declined to approximately 15 for sulprostone and phenylephrine-induced tactile allodynia, demonstrating that ER-β005 could antagonize the effects of these pain sensitizers. The

Fig. 8. Suppression of capsaicin-induced mechanical hyperalgesia. Hyperalgesia was assessed by counting the number of flinches in response to the poking of a paw. ER-β005 suspensions (50 mg/kg) and Nano-ER-β005 at doses of 25, 50 and 100 mg/kg were administered i.g. to the model (A) at 30 min and (B) 60 min, respectively, before injecting capsaicin. n = 6, *p < 0.05, **p < 0.01 versus control. The blank formulation was a β-CN solution without drug.

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Fig. 9. Suppression of chemically induced allodynia. The model was prepared via injecting pain sensitizers, sulprostone (300 ng/kg i.p.) or phenylephrine (100 ng/kg i.p.), in mice. Influence of ER-β005 (50 mg/kg) and different doses of Nano-ER-β005 (25, 50 and 100 mg/kg, respectively) were studied in (A) sulprostone-induced allodynia or (B) phenylephrineinduced allodynia. n = 6, *p < 0.05, **p < 0.01 versus control. The blank formulation was a β-CN solution without the drug.

et al., 2017), β-CN is generally recognized as safe, inexpensive, readily available, non-toxic and highly stable (Elzoghby et al., 2011). In particular, β-CN is easily digested after oral administration compared with other food proteins (Katz et al., 2009), thereby enabling drug particles in nanosuspensions to be readily exposed in the gastrointestinal tract, facilitating drug dissolution. Thus, β-CN is a promising biomaterial for the development of oral nanosuspensions. ER-β005 is an efficient drug for anti-allodynia, and its therapeutic efficacy was significantly improved with Nano-ER-β005. Like other ER modulators, ER-β005 can selectively interact with ER-β, which is a member of the nuclear hormone receptor superfamily, and thus exert therapeutic effects on cancer, cardiovascular disease, multiple sclerosis and Alzheimer’s disease, among others. (Nilsson et al., 2011). Herein, oral administration of ER-β005 had profound anti-allodynia effects in models of capsaicin-induced mechanical hyperalgesia, chemically induced tactile allodynia or spinal nerve ligation. Few reports have indicated that ER agonists can alleviate or abolish allodynia; accordingly, our work demonstrates a new therapeutic effect of ER agonists. However, due to the low solubility of ER-β005, a high dose must be administered to the patient, resulting in an increased risk of reproductive tissue cancer, thus restricting clinic applications and long-term compliance issues (Henke et al., 2002). Importantly, the therapeutic efficacy of Nano-ER-β005 at a dose of 25 mg/k was similar to that the ERβ005 suspensions at 50 mg/kg (Figs. 8–10), which was beneficial for the reduction of side effects. Additionally, at other doses, Nano-ERβ005 also markedly promoted the inhibition of allodynia compared with ER-β005 suspensions. These results were ascribed to the enhanced oral absorption or higher plasma concentration of the drug from NanoER-β005 as well as the resultant improved accumulation in other organs. As a result, Nano-ER-β005 has potential for improving the therapeutic outcomes of ER-β005 treatment.

3.6.3. Spinal nerve ligation Inhibition of allodynia was further evaluated in the spinal nerve ligation model, which was established via surgical ligation of the L5–L6 spinal nerves in rats. First, the dose of ER-β005 on allodynia inhibition was assessed. As shown in Fig. 10(A), the percent of reversal allodynia increased from 20% to 60% with administration of Nano-ER-β005 to the model at doses ranging from 25 to 100 mg/kg. Nano-ER-β005 administered at a dose of 50 mg/kg exhibited greater reversal of allodynia (%) compared with ER-β005 suspensions (50 mg/kg), demonstrating enhancement in anti-allodynia effects. Additionally, the reversal of allodynia (%) from the dose of 100 mg/kg of Nano-ER-β005 was similar to that of the typical pain reducer morphine, further confirming the inhibition effect of Nano-ER-β005 on allodynia. Second, three therapeutic regimens, acute, prolonged and continuous responses of ERβ005 to pain suppression, were performed after administration of NanoER-β005 or ER-β005 suspensions at a dose of 50 mg/kg. The reversal allodynia (%) from ER-β005 suspensions was 7% for an acute drug response (regimen 1), 9% for a prolonged drug response (regimen 2), and 30% for a continuous drug response (regimen 3); the reversal from Nano-ER-β005 was 16% for an acute drug response (regimen 1), 18% for a prolonged drug response (regimen 2), and 57% for a continuous drug response (regimen 3) (Fig. 10B and C). Therefore, these results indicated that Nano-ER-β005 had a stronger inhibitory effect on allodynia in terms of acute, prolonged and continuous drug responses.

4. Discussion β-CN is a potent stabilizer that is used for preparation of nanosuspensions. In general, β-CN is able to self-assemble into micelles that are predominantly employed as vehicles for poorly water-soluble drugs (Bachar et al., 2012; Bar-Zeev et al., 2016; Shapira et al., 2010). However, the drug-loading of micelles is extreme low, not greater than 10% (Shen et al., 2010). To the best of our knowledge, this is the first report to indicate that β-CN can adsorb on nanosized drug particles and thus stabilize nanosuspensions of ER-β005 with drug-loading of up to 50%. The driving force for the adsorption of β-CN on drug particles resulted from the interactions between them; indeed, these interactions were confirmed by the examination of fluorescence and CD spectra, which indicated fluorescence quenching of β-CN and changes in its secondary and tertiary structures. Moreover, unlike other traditional surfactants, such as Tween-80, D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and SDS, which have potential toxicity (Wang

5. Conclusions In this study, by using β-CN as a stabilizer, Nano-ER-β005 with a diameter of 110 nm and drug-loading of up to 50% were developed. To the best of our knowledge, this study is the first to demonstrate that βCN is a potent stabilizer of nanosuspensions. Moreover, the dissolution and absorption of ER-β005 from Nano-ER-β005 were significantly increased compared with the suspension formulation, and Nano-ER-β005 had an AUC that was 1.6-fold higher than that of ER-β005. In addition, a profound inhibitory effect of ER-β005 (an ER agonist) on allodynia 254

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Fig. 10. Suppression of allodynia in the spinal nerve ligation model created via surgical ligation of the L5–L6 spinal nerves in rats. The allodynia assessment was performed by employing a light tactile stimulus to the paw. (A) The reversal effects of ER-β005 (50 mg/kg) and Nano-ER-β005 (25, 50 and 100 mg/kg) on allodynia were assessed at 30 min post i.g. administration. A pain inhibitor, morphine (15 mg/kg i.p.), was employed as a positive control. (B/C) Different regimens were studied after administration of ER-β005 or Nano-ER-β005 at a dose of 50 mg/kg. In regimen 1, the acute effects of a single dose were tested 30 min post administration. In regimen 2, the prolonged effect of a single dose was assessed 6 h post administration. In regimen 3, after the administration of 3 doses at an interval of 3 h, the continuous drug effects were determined at 30 min post administration. n = 6, *p < 0.05, **p < 0.01 versus control. The blank formulation was a β-CN solution without the drug. multidrug resistance in human gastric cancer cells. Oncotarget 7, 23322–23334. Bi, C., Miao, X.Q., Chow, S.F., Wu, W.J., Yan, R., Liao, Y.H., Chow, A.H.-L., Zheng, Y., 2017. Particle size effect of curcumin nanosuspensions on cytotoxicity, cellular internalization, in vivo pharmacokinetics and biodistribution. Nanomed.: Nanotechnol. Biol. Med. 13, 943–953. Chen, Q., Wang, X., Wang, C., Feng, L.Z., Li, Y.G., Liu, Z., 2015. Drug-induced self-assembly of modified albumins as nano-theranostics for tumor-targeted combination therapy. ACS Nano 9, 5223–5233. Cordey, M., Pike, C.J., 2005. Neuroprotective properties of selective estrogen receptor agonists in cultured neurons. Brain Res. 1045, 217–223. Decosterd, I., Woolf, C.J., 2000. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 87, 149–158. Dixon, W.J., 1980. Efficient analysis of experimental observations. Annu. Rev. Pharmacol. Toxicol. 20, 441–462. Elzoghby, A.O., El-Fotoh, W.S., Elgindy, N.A., 2011. Casein-based formulations as promising controlled release drug delivery systems. J. Controlled Release 153, 206–216. Gao, L., Liu, G., Ma, J., Wang, X., Zhou, L., Li, X., 2012. Drug nanocrystals: in vivo performances. J. Controlled Release 160, 418–430. He, W., Lu, Y., Qi, J., Chen, L., Hu, F., Wu, W.v., 2013a. Food proteins as novel nanosuspension stabilizers for poorly water-soluble drugs. Int. J. Pharm. 441, 269–278. He, W., Lu, Y., Qi, J., Chen, L., Yin, L., Wu, W., 2013b. Formulating food protein-

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