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A new approach to produce drug nanosuspensions CO2-assisted effervescence to produce drug nanosuspensions
Colloids and Surfaces B: Biointerfaces 143 (2016) 107–110
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A new approach to produce drug nanosuspensions CO2 -assisted effervescence to produce drug nanosuspensions Xiangfei Han, Menglin Wang, Zhihui Ma, Peng Xue, Yongjun Wang ∗ School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, 110016, China
a r t i c l e
i n f o
Article history: Received 12 January 2016 Received in revised form 4 March 2016 Accepted 5 March 2016 Available online 7 March 2016 Keywords: Nanomedicine Nanoamorphization CO2 Itraconazole Cabazitaxel
a b s t r a c t The exploration of a simple and robust approach to produce nanosuspensions is a meaningful attempt for clinical translation. CO2 -assisted effervescence was firstly developed to prepare nanosuspensions and was found to be easy for scale-up. Drug nanosuspensions were easily obtained by adding aqueous carbonate to the pre-treated mixture of drug, stabilizer and organic acid. The burst of CO2 bubbles resulted from the acid-base reaction insert a micro gas bubble smashing and mixing effect to the formation of nanosuspensions, leading to smaller sizes and a refined size distribution. We successfully prepared nanosuspensions with twelve structurally diverse drugs. Alternatively, solid carbonate blended with the mixture, allowing for later addition of water, also facilitates the formation of amorphous nanosuspensions. We defined this approach as in situ nanoamorphization (ISN). Intensive in vitro and in vivo investigations for itraconazole and cabazitaxel nanosuspensions validate the availability for administration. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Nanosuspensions are liquid sub-micron colloidal dispersions of nanosized pure drug particles stabilized by polymer and/or surfactant. A nanosuspension platform is a very efficient drug delivery system for water-insoluble drugs because these platforms provide increased dissolution velocity and high mass per volume loading. [1] To date, existing techniques to produce nanosuspensions are divided into “bottom-up”, “top-down” techniques, or a combination of both. The top-down technique utilizes mechanical attrition, high-pressure homogenization or media milling to invert coarse drugs into nanosized particles. When the “top-down” technique is used, the drug often exists in a crystalline state in the dispersion medium [2]. In contrast, the “bottom-up” process involves dissolving the drug in a solvent and precipitating it in a controlled manner to achieve nanosized particles at the addition of an antisolvent (socalled antisolvent precipitation technique). With this method, the drug can exist in either a needle-shaped crystalline state or amorphous state, depending on the applied technique [3]. The top-down technique is more widely employed in the pharmaceutical industry due to its straightforward process features. Six products based on
∗ Corresponding author at: Mailbox 59#, Department of Biopharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, 110016, China. E-mail address: [email protected] (Y. Wang). http://dx.doi.org/10.1016/j.colsurfb.2016.03.017 0927-7765/© 2016 Elsevier B.V. All rights reserved.
the top-down technique have been successfully marketed in recent years [4]. However, many challenges remain as outlined below: i) Nanosuspensions are thermodynamically unstable colloid dispersion systems with a high surface energy that inevitably induces growth of particle size, known as Ostwald ripening. In addition, nanosized systems tend to reduce the Gibbs free energy causing nanosuspensions to aggregate during the preparation process and storage. During the bottom-up process, stability issues are more severe because the drug nanoparticles are usually generated in a mestastable polymorphic form, which can cause transformation to occur. Therefore, although the amorphous form is more soluble and has a higher dissolution rate than the crystalline state, no products based on the bottom-up technique have yet appeared on the market. ii) In comparison with conventional approaches that attempt to solubilize insoluble drugs using agents such as co-solvents and inclusion complexes, a high-pressure homogenization or media milling process consumes high energy and requires special equipment such as a homogenizer or ball grinder [5]. Furthermore, a solidification process using spray drying or freeze-drying method is typically required due to the instability of aqueous nanosuspensions [3]. This results in higher production costs and concerns over the ability to re-disperse the dried powder are also needed. iii) The media milling process generates considerable heat, which may cause crystal defects or degradation of heat-sensitive drugs. Moreover, the erosion of milling pearls may contaminate the nanosuspension [6]. Because of these limitations, alternative
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Fig. 1. (A) The dissolution profiles of ITC formulation 1 and Sporanox® in pH 1.2 with 0.6% SDS (mean ± SD, n = 3). (B) Plasma concentration profiles of ITC after oral administration of ITC formulation 1, ITC formulation 2 and Sporanox® (mean ± SD, n = 6). (C) XRD and (D) DSC images for ITC, blank excipient, the mixture of ITC and excipients and ITC/soluplus (1:3, w/w) nanosuspension.
nanosuspension production processes are attractive to the pharmaceutical industry. In this study, we explored a simple but robust approach to prepare nanosuspensions (Scheme). The process utilizes an organic solvent to dissolve water-insoluble drugs along with organic acids and stabilizers. Then, the organic solvent is evaporated—leaving an organic acid-phase material. Finally, drug nanosuspensions are obtained following the addition of aqueous carbonate, which rapidly generates CO2 bubbles by acid-base reaction. Alternatively, the solid acid-phase material can be blended with dry carbonate and prepared into final solid dosage forms at a later time. Of importance, the nanosuspensions could not be produced in the absence of the organic acid or carbonate; rather, the drugs gradually aggregated together and precipitated over time. In this new approach, CO2 bubbles exert a rapid micro-mixing effect to suppress crystal growth while stabilizers simultaneously absorb onto the hydrophobic surface of the drugs and prevent aggregation and agglomeration. Unlike the top-down and bottom-up techniques, it was demonstrated that the drugs of final nanosuspensions existed as amorphous form. Ideally, formulated nanosuspensions occur in the amorphous form to improve solubility and the dissolution rate. However, limited commercialization of nanosuspensions in the amorphous state has occurred due to stability issues. The method described here bypasses concerns over stability because the final products can be produced in a sub-packaging form consisting of the organic-acid phase with drug and stabilizer separated from the aqueous carbonate. Consequently, the nanosuspensions could be achieved by combining the aqueous carbonate with the
organic acid-phase. In this new approach, the nanosuspension is prepared only at the time of usage. We define this new technique as in situ nanoamorphization (ISN). ISN may not only solve the stability issues associated with current manufacturing techniques, but is also a low-energy consumption process.
2. Results and discussion Here, we show that this approach is suitable for the preparation of nanosuspensions of structurally diverse drugs with different physicochemical properties (Molucular weight, MW, from 206.28 to 1202.62; logP, from 3.07 to 9.05; melting point, from 75 to 213 ◦ C) (Table S1). As shown in Table 1, the particle size of the nanosuspended drugs range from 92.21 nm (cabazitaxel) to 653.9 nm (loratadine) (Table 1). Due to the different steric stabilization effects, the use of different stabilizers resulted in varying particle sizes with the nanosuspensions. For example, the stabilizers Soluplus (a polyvinyl caprolactam − polyvinyl acetate − polyethylene glycol graft copolymer), TPGS (D-alpha tocopheryl polyethylene glycol 1000 succinate), and HPMC E5 (a hydroxypropyl methyl cellulose) resulted in cyclosporin A particle sizes of 128.9 nm (polydispersity index, PDI, 0.234), 290.3 nm (PDI, 0.191) and 510.9 nm (PDI, 0.214), respectively. In short, nanosuspensions prepared by ISN present favorable small particle sizes and narrow particle size distributions. In comparison, the bottom-up technique is usually followed by an ultrasonication or homogenization step due to the difficulty in controlling particle growth, which is the
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Fig. 2. (A) The particle size and morphology of CTX/TPGS nanosuspension with a ratio of 1:2.5(w/w) (B) XRD and (C) DSC images CTX/TPGS nanosuspension with a ratio of 1:2.5 (w/w) (D) Stability of CTX/TPGS nanosuspensions stored at room temperature. (E) TEM image of CTX/TPGS nanosuspension with a ratio of 1:2.5(w/w) prepared using an antisolvent precipitation coupled ultrasonication technique.
Table 1 Particle sizes and size distributions of nanosuspensions using different drug stabilizers (Ratio = drug: stabilizer) prepared with the ISN technique. (mean ± SD, n = 3). Drug
Stabilizer
Ratio Particle size(nm) PDI
Cabazitaxel Cabazitaxel Cabazitaxel Paclitaxel Paclitaxel Docetaxel Itraconazole Itraconazole Itraconazole Tacrolimus Abamectin Fenofibrate Fenofibrate Loratadine Loratadine Loratadine Cyclosporin A Cyclosporin A Cyclosporin A Ibuprofen paliperidone palmitate Spironolactone Spironolactone
TPGS Tween 80 Cremophor RH TPGS Tween 80 TPGS TPGS HPMC E5 Soluplus TPGS TPGS TPGS Soluplus TPGS Soluplus HPMC E5 TPGS Soluplus HPMC E5 TPGS TPGS TPGS HPMC E5
1:2.5 1:5 1:5 1:5 1:5 1:5 1:5 1:5 1:5 1:2.5 1:1.5 1:5 1;5 1:3 1:3 1:3 1:4 1:4 1:4 1:1.5 1:5 1:5 1:5
92.21 ± 2.51 212.8 ± 2.29 103.1 ± 2.10 118.4 ± 1.86 267.3 ± 2.96 312.6 ± 3.02 135.4 ± 2.35 338.6 ± 3.02 109.2 ± 1.69 212.3 ± 0.53 177.1 ± 0.83 361.9 ± 2.91 224.4 ± 1.52 397.2 ± 2.13 176.4 ± 0.18 653.9 ± 2.52 290.3 ± 0.09 128.9 ± 0.53 510.9 ± 1.35 244.1 ± 1.38 105.5 ± 2.63 249.7 ± 2.51 106.1 ± 1.36
0.228 ± 0.006 0.185 ± 0.056 0.251 ± 0.029 0.172 ± 0.078 0.194 ± 0.022 0.222 ± 0.035 0.140 ± 0.036 0.079 ± 0.018 0.156 ± 0.015 0.178 ± 0.009 0.193 ± 0.019 0.338 ± 0.009 0.101 ± 0.021 0.215 ± 0.016 0.172 ± 0.018 0.152 ± 0.019 0.191 ± 0.005 0.234 ± 0.017 0.214 ± 0.021 0.324 ± 0.008 0.320 ± 0.009 0.301 ± 0.012 0.289 ± 0.026
primary reason that the bottom-up techniques have not become a standard approach in the pharmaceutical industry. To further demonstrate the efficiency of ISN for producing nanosuspensions, itraconazole (ITC) nanosuspensions based on the ISN technique were developed for oral administration. ITC, a broad-spectrum antifungal agent, is a BCS II drug with poor water solubility (∼1 ng/mL at neutral pH and ∼4 g/mL at pH 1) and high permeability [7]. Many approaches have been developed to improve the dissolution and oral absorption (eg self-emulsion, solid dispersions and nanosuspensions). [8,9] Sarnes et al. demonstrated that the relative bioavailability of an ITC nanocrystal that was prepared using a wet milling technique was only 39.9% compared to Sporanox® (ITC commercial product) [10]. Herein, we prepared two kinds of ITC formulations. Briefly, a sodium carbonate solution was added to the organic-acid phase and then the formed nanosuspensions were solidified using a lyophilization method. The lyophilized powders were encapsuled into gastric-soluable capsules (ITC formulation 1). Alternatively, the solid organic-acid phase was mixed with sodium carbonate powder, and then the mixture was embeded into enteric-coated capsules (ITC formulation 2). Fig. 1B shows the mean plasma concentration-time curves of ITC after administration of Sporanox® , ITC formulation 1 and formulation 2 to beagle dogs by oral gavage. The pharmacokinetic parameters are listed in Table S2. The AUC (0–48h) of ITC formulation 1 and formulation 2 were about 1.74-fold (p < 0.05) and 1.58-fold (p < 0.05)
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that of Sporanox® . Interestingly, this was not consistent with the dissolution profile showing that the release rate of Sporanox® was faster than that of ITC nanosuspensions (Fig. 1A), although X-ray diffraction (XRD) and differential scanning calorimetry (DSC) characterization of the freeze-dried nanosuspensions confirmed the amorphous state of ITC (Fig. 1C and D). However, it is difficult to predict the in vivo behavior on the basis of in vitro dissolution profiles; superior in vitro dissolution does not always lead to enhancement of in vivo drug absorption [11,12]. Cabazitaxel (CTX) is a semi-synthetic derivative of a natural toxoid that is used for the treatment of patients with hormonerefractory metastatic prostate cancer and has poor water solubility. Therefore, we investigated the efficiency of ISN for producing CTX nanosuspensions for intravenous administration. Due to its poor water solubility, CTX is clinically formulated using Tween 80 and ethanol (JEVTANA® ), which may cause severe side effects such as hypersensitivity, neurotoxicity, allergic reaction, etc [13]. The pharmaceutical form of JEVTANA® includes a concentrate vial and solvent vial. The concentrate is diluted with the supplied solvent before adding to the infusion solution and needs to be used within 1 h; otherwise CTX may crystallize and precipitate over time. Therefore, the use of JEVTANA® clinically requires special disposal considerations and must be administered by personnel trained in handling cytotoxic agents. By virtue of ISN, the pharmaceutical form of CTX nanosuspensions could also be divided in two parts: a solid organic-acid phase and sodium carbonate solution. In contrast to JEVTANA® , the preparation of CTX nanosuspensions is simple and only involves the addition of the sodium carbonate solution into the vial of solid organic-acid phase and then manually mixed. Transmission electron micrograph images indicate that CTX exists as homogeneous spheres with a mean diameter of approximately 92.21 nm with no visible drug crystals (Fig. 2A). XRD and DSC assays also demonstrate that CTX exists in the amorphous state (Fig. 2B and C). Additionally, the CTX nanosuspension prepared using ISN results in a stable product at room temperature for 24 h (Fig. 2D), which is adequate for in-use storage time. In contrast, CTX occurs in a rod-shape crystalline state when antisolvent precipitation coupled with an ultrasonication technique is applied to prepare the nanosuspension (Fig. 2E). 3. Conclusion The search for a simple and easy-scale approach to producing nanosuspensions remains of major clinical importance in nanomedicine. The major obstacle of the conventional bottomup technique is aggregation of the molecular drug. By virtue of a micro-mixing effect due to CO2 bubbles and the inhibition effect of stabilizers, we successfully prevented aggregation in the nanosuspensions tested. Our study presents an ISN technique with huge potential for the field of nanosuspension preparations that fulfills important properties, including: 1) a low energy-consumption process, 2) no specialized equipment needed, 3) no stability issues for the final products, and 4) good reproducibility. The results in this study set the stage for a thorough evaluation of in vivo efficacy. In fact, experiments using nanosuspensions based on the ISN technique for intramuscular and ophthalmic applications are currently ongoing.
Acknowledgements This work was financially supported by the National Basic Research Program of China (973 Program, No. 2015CB932100), National Nature Science Foundation of China (No. 81273450), Nature Science Foundation of Liaoning Province (No. 2014020079), and the General Project in Education Department of Liaoning Province (No. L2014396). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.03. 017. References [1] B.E. Rabinow, Nanosuspensions in drug delivery, Nat. Rev. Drug Discov. 3 (2004) 785–796. [2] Y. Liu, P. Xie, D. Zhang, Q. Zhang, A mini review of nanosuspensions development, J. Drug Target. 20 (2012) 209–223. [3] Y. Wang, Y. Zheng, L. Zhang, Q. Wang, D. Zhang, Stability of nanosuspensions in drug delivery, J. Controlled Release 172 (2013) 1126–1141. [4] W.W.L. Chin, J. Parmentier, M. Widzinski, E.H. Tan, R. Gokhale, A Brief literature and patent review of nanosuspensions to a final drug product, J. Pharm. Sci. 103 (2014) 2980–2999. [5] J.P. Moeschwitzer, Drug nanocrystals in the commercial pharmaceutical development process, Int. J. Pharm. 453 (2013) 142–156. [6] K. Moribe, K. Ueda, W. Limwikrant, K. Higashi, K. Yamamoto, Nano-Sized crystalline drug production by milling technology, Curr. Pharm. Des. 19 (2013) 6246–6258. [7] K. Zhang, H. Yu, Q. Luo, S. Yang, X. Lin, Y. Zhang, B. Tian, X. Tang, Increased dissolution and oral absorption of itraconazole/Soluplus extrudate compared with itraconazole nanosuspension, Eur. J. Pharm. Biopharm. 85 (2013) 1285–1292. [8] F. Le Devedec, S. Strandman, P. Hildgen, G. Leclair, X.X. Zhu, PEGylated bile acids for use in drug delivery systems: enhanced solubility and bioavailability of itraconazole, Mol. Pharm. 10 (2013) 3057–3066. [9] A.L. Sarode, P. Wang, S. Obara, D.R. Worthen, Supersaturation, nucleation, and crystal growth during single- and biphasic dissolution of amorphous solid dispersions: polymer effects and implications for oral bioavailability enhancement of poorly water soluble drugs, Eur. J. Pharm. Biopharm. 86 (2014) 351–360. [10] A. Sarnes, M. Kovalainen, M.R. Hakkinen, T. Laaksonen, J. Laru, J. Kiesvaara, J. Ilkka, O. Oksala, S. Ronkko, K. Jarvinen, J. Hirvonen, L. Peltonen, Nanocrystal-based per-oral itraconazole delivery: superior in vitro dissolution enhancement versus Sporanox (R) is not realized in vivo drug absorption, J. Controlled Release 180 (2014) 109–116. [11] Q. Fu, J. Sun, D. Zhang, M. Li, Y. Wang, G. Ling, X. Liu, Y. Sun, X. Sui, C. Luo, L. Sun, X. Han, H. Lian, M. Zhu, S. Wang, Z. He, Nimodipine nanocrystals for oral bioavailability improvement: preparation, characterization and pharmacokinetic studies, Colloids Surf. B 109 (2013) 161–166. [12] J.E. Polli, In vitro studies are sometimes better than conventional human pharmacokinetic in vivo studies in assessing bioequivalence of immediate-release solid oral dosage forms, Aaps Journal 10 (2008) 289–299. [13] F.A. Schutz, A.C. Buzaid, O. Sartor, Taxanes in the management of metastatic castration-resistant prostate cancer: efficacy and management of toxicity, Crit. Rev. Oncol. Hematol. 91 (2014) 248–256.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A new approach to produce drug nanosuspensions CO2 -assisted effervescence to produce drug nanosuspensions Xiangfei Han, Menglin Wang, Zhihui Ma, Peng Xue, Yongjun Wang ∗ School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, 110016, China
a r t i c l e
i n f o
Article history: Received 12 January 2016 Received in revised form 4 March 2016 Accepted 5 March 2016 Available online 7 March 2016 Keywords: Nanomedicine Nanoamorphization CO2 Itraconazole Cabazitaxel
a b s t r a c t The exploration of a simple and robust approach to produce nanosuspensions is a meaningful attempt for clinical translation. CO2 -assisted effervescence was firstly developed to prepare nanosuspensions and was found to be easy for scale-up. Drug nanosuspensions were easily obtained by adding aqueous carbonate to the pre-treated mixture of drug, stabilizer and organic acid. The burst of CO2 bubbles resulted from the acid-base reaction insert a micro gas bubble smashing and mixing effect to the formation of nanosuspensions, leading to smaller sizes and a refined size distribution. We successfully prepared nanosuspensions with twelve structurally diverse drugs. Alternatively, solid carbonate blended with the mixture, allowing for later addition of water, also facilitates the formation of amorphous nanosuspensions. We defined this approach as in situ nanoamorphization (ISN). Intensive in vitro and in vivo investigations for itraconazole and cabazitaxel nanosuspensions validate the availability for administration. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Nanosuspensions are liquid sub-micron colloidal dispersions of nanosized pure drug particles stabilized by polymer and/or surfactant. A nanosuspension platform is a very efficient drug delivery system for water-insoluble drugs because these platforms provide increased dissolution velocity and high mass per volume loading. [1] To date, existing techniques to produce nanosuspensions are divided into “bottom-up”, “top-down” techniques, or a combination of both. The top-down technique utilizes mechanical attrition, high-pressure homogenization or media milling to invert coarse drugs into nanosized particles. When the “top-down” technique is used, the drug often exists in a crystalline state in the dispersion medium [2]. In contrast, the “bottom-up” process involves dissolving the drug in a solvent and precipitating it in a controlled manner to achieve nanosized particles at the addition of an antisolvent (socalled antisolvent precipitation technique). With this method, the drug can exist in either a needle-shaped crystalline state or amorphous state, depending on the applied technique [3]. The top-down technique is more widely employed in the pharmaceutical industry due to its straightforward process features. Six products based on
∗ Corresponding author at: Mailbox 59#, Department of Biopharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, 110016, China. E-mail address: [email protected] (Y. Wang). http://dx.doi.org/10.1016/j.colsurfb.2016.03.017 0927-7765/© 2016 Elsevier B.V. All rights reserved.
the top-down technique have been successfully marketed in recent years [4]. However, many challenges remain as outlined below: i) Nanosuspensions are thermodynamically unstable colloid dispersion systems with a high surface energy that inevitably induces growth of particle size, known as Ostwald ripening. In addition, nanosized systems tend to reduce the Gibbs free energy causing nanosuspensions to aggregate during the preparation process and storage. During the bottom-up process, stability issues are more severe because the drug nanoparticles are usually generated in a mestastable polymorphic form, which can cause transformation to occur. Therefore, although the amorphous form is more soluble and has a higher dissolution rate than the crystalline state, no products based on the bottom-up technique have yet appeared on the market. ii) In comparison with conventional approaches that attempt to solubilize insoluble drugs using agents such as co-solvents and inclusion complexes, a high-pressure homogenization or media milling process consumes high energy and requires special equipment such as a homogenizer or ball grinder [5]. Furthermore, a solidification process using spray drying or freeze-drying method is typically required due to the instability of aqueous nanosuspensions [3]. This results in higher production costs and concerns over the ability to re-disperse the dried powder are also needed. iii) The media milling process generates considerable heat, which may cause crystal defects or degradation of heat-sensitive drugs. Moreover, the erosion of milling pearls may contaminate the nanosuspension [6]. Because of these limitations, alternative
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Fig. 1. (A) The dissolution profiles of ITC formulation 1 and Sporanox® in pH 1.2 with 0.6% SDS (mean ± SD, n = 3). (B) Plasma concentration profiles of ITC after oral administration of ITC formulation 1, ITC formulation 2 and Sporanox® (mean ± SD, n = 6). (C) XRD and (D) DSC images for ITC, blank excipient, the mixture of ITC and excipients and ITC/soluplus (1:3, w/w) nanosuspension.
nanosuspension production processes are attractive to the pharmaceutical industry. In this study, we explored a simple but robust approach to prepare nanosuspensions (Scheme). The process utilizes an organic solvent to dissolve water-insoluble drugs along with organic acids and stabilizers. Then, the organic solvent is evaporated—leaving an organic acid-phase material. Finally, drug nanosuspensions are obtained following the addition of aqueous carbonate, which rapidly generates CO2 bubbles by acid-base reaction. Alternatively, the solid acid-phase material can be blended with dry carbonate and prepared into final solid dosage forms at a later time. Of importance, the nanosuspensions could not be produced in the absence of the organic acid or carbonate; rather, the drugs gradually aggregated together and precipitated over time. In this new approach, CO2 bubbles exert a rapid micro-mixing effect to suppress crystal growth while stabilizers simultaneously absorb onto the hydrophobic surface of the drugs and prevent aggregation and agglomeration. Unlike the top-down and bottom-up techniques, it was demonstrated that the drugs of final nanosuspensions existed as amorphous form. Ideally, formulated nanosuspensions occur in the amorphous form to improve solubility and the dissolution rate. However, limited commercialization of nanosuspensions in the amorphous state has occurred due to stability issues. The method described here bypasses concerns over stability because the final products can be produced in a sub-packaging form consisting of the organic-acid phase with drug and stabilizer separated from the aqueous carbonate. Consequently, the nanosuspensions could be achieved by combining the aqueous carbonate with the
organic acid-phase. In this new approach, the nanosuspension is prepared only at the time of usage. We define this new technique as in situ nanoamorphization (ISN). ISN may not only solve the stability issues associated with current manufacturing techniques, but is also a low-energy consumption process.
2. Results and discussion Here, we show that this approach is suitable for the preparation of nanosuspensions of structurally diverse drugs with different physicochemical properties (Molucular weight, MW, from 206.28 to 1202.62; logP, from 3.07 to 9.05; melting point, from 75 to 213 ◦ C) (Table S1). As shown in Table 1, the particle size of the nanosuspended drugs range from 92.21 nm (cabazitaxel) to 653.9 nm (loratadine) (Table 1). Due to the different steric stabilization effects, the use of different stabilizers resulted in varying particle sizes with the nanosuspensions. For example, the stabilizers Soluplus (a polyvinyl caprolactam − polyvinyl acetate − polyethylene glycol graft copolymer), TPGS (D-alpha tocopheryl polyethylene glycol 1000 succinate), and HPMC E5 (a hydroxypropyl methyl cellulose) resulted in cyclosporin A particle sizes of 128.9 nm (polydispersity index, PDI, 0.234), 290.3 nm (PDI, 0.191) and 510.9 nm (PDI, 0.214), respectively. In short, nanosuspensions prepared by ISN present favorable small particle sizes and narrow particle size distributions. In comparison, the bottom-up technique is usually followed by an ultrasonication or homogenization step due to the difficulty in controlling particle growth, which is the
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Fig. 2. (A) The particle size and morphology of CTX/TPGS nanosuspension with a ratio of 1:2.5(w/w) (B) XRD and (C) DSC images CTX/TPGS nanosuspension with a ratio of 1:2.5 (w/w) (D) Stability of CTX/TPGS nanosuspensions stored at room temperature. (E) TEM image of CTX/TPGS nanosuspension with a ratio of 1:2.5(w/w) prepared using an antisolvent precipitation coupled ultrasonication technique.
Table 1 Particle sizes and size distributions of nanosuspensions using different drug stabilizers (Ratio = drug: stabilizer) prepared with the ISN technique. (mean ± SD, n = 3). Drug
Stabilizer
Ratio Particle size(nm) PDI
Cabazitaxel Cabazitaxel Cabazitaxel Paclitaxel Paclitaxel Docetaxel Itraconazole Itraconazole Itraconazole Tacrolimus Abamectin Fenofibrate Fenofibrate Loratadine Loratadine Loratadine Cyclosporin A Cyclosporin A Cyclosporin A Ibuprofen paliperidone palmitate Spironolactone Spironolactone
TPGS Tween 80 Cremophor RH TPGS Tween 80 TPGS TPGS HPMC E5 Soluplus TPGS TPGS TPGS Soluplus TPGS Soluplus HPMC E5 TPGS Soluplus HPMC E5 TPGS TPGS TPGS HPMC E5
1:2.5 1:5 1:5 1:5 1:5 1:5 1:5 1:5 1:5 1:2.5 1:1.5 1:5 1;5 1:3 1:3 1:3 1:4 1:4 1:4 1:1.5 1:5 1:5 1:5
92.21 ± 2.51 212.8 ± 2.29 103.1 ± 2.10 118.4 ± 1.86 267.3 ± 2.96 312.6 ± 3.02 135.4 ± 2.35 338.6 ± 3.02 109.2 ± 1.69 212.3 ± 0.53 177.1 ± 0.83 361.9 ± 2.91 224.4 ± 1.52 397.2 ± 2.13 176.4 ± 0.18 653.9 ± 2.52 290.3 ± 0.09 128.9 ± 0.53 510.9 ± 1.35 244.1 ± 1.38 105.5 ± 2.63 249.7 ± 2.51 106.1 ± 1.36
0.228 ± 0.006 0.185 ± 0.056 0.251 ± 0.029 0.172 ± 0.078 0.194 ± 0.022 0.222 ± 0.035 0.140 ± 0.036 0.079 ± 0.018 0.156 ± 0.015 0.178 ± 0.009 0.193 ± 0.019 0.338 ± 0.009 0.101 ± 0.021 0.215 ± 0.016 0.172 ± 0.018 0.152 ± 0.019 0.191 ± 0.005 0.234 ± 0.017 0.214 ± 0.021 0.324 ± 0.008 0.320 ± 0.009 0.301 ± 0.012 0.289 ± 0.026
primary reason that the bottom-up techniques have not become a standard approach in the pharmaceutical industry. To further demonstrate the efficiency of ISN for producing nanosuspensions, itraconazole (ITC) nanosuspensions based on the ISN technique were developed for oral administration. ITC, a broad-spectrum antifungal agent, is a BCS II drug with poor water solubility (∼1 ng/mL at neutral pH and ∼4 g/mL at pH 1) and high permeability [7]. Many approaches have been developed to improve the dissolution and oral absorption (eg self-emulsion, solid dispersions and nanosuspensions). [8,9] Sarnes et al. demonstrated that the relative bioavailability of an ITC nanocrystal that was prepared using a wet milling technique was only 39.9% compared to Sporanox® (ITC commercial product) [10]. Herein, we prepared two kinds of ITC formulations. Briefly, a sodium carbonate solution was added to the organic-acid phase and then the formed nanosuspensions were solidified using a lyophilization method. The lyophilized powders were encapsuled into gastric-soluable capsules (ITC formulation 1). Alternatively, the solid organic-acid phase was mixed with sodium carbonate powder, and then the mixture was embeded into enteric-coated capsules (ITC formulation 2). Fig. 1B shows the mean plasma concentration-time curves of ITC after administration of Sporanox® , ITC formulation 1 and formulation 2 to beagle dogs by oral gavage. The pharmacokinetic parameters are listed in Table S2. The AUC (0–48h) of ITC formulation 1 and formulation 2 were about 1.74-fold (p < 0.05) and 1.58-fold (p < 0.05)
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that of Sporanox® . Interestingly, this was not consistent with the dissolution profile showing that the release rate of Sporanox® was faster than that of ITC nanosuspensions (Fig. 1A), although X-ray diffraction (XRD) and differential scanning calorimetry (DSC) characterization of the freeze-dried nanosuspensions confirmed the amorphous state of ITC (Fig. 1C and D). However, it is difficult to predict the in vivo behavior on the basis of in vitro dissolution profiles; superior in vitro dissolution does not always lead to enhancement of in vivo drug absorption [11,12]. Cabazitaxel (CTX) is a semi-synthetic derivative of a natural toxoid that is used for the treatment of patients with hormonerefractory metastatic prostate cancer and has poor water solubility. Therefore, we investigated the efficiency of ISN for producing CTX nanosuspensions for intravenous administration. Due to its poor water solubility, CTX is clinically formulated using Tween 80 and ethanol (JEVTANA® ), which may cause severe side effects such as hypersensitivity, neurotoxicity, allergic reaction, etc [13]. The pharmaceutical form of JEVTANA® includes a concentrate vial and solvent vial. The concentrate is diluted with the supplied solvent before adding to the infusion solution and needs to be used within 1 h; otherwise CTX may crystallize and precipitate over time. Therefore, the use of JEVTANA® clinically requires special disposal considerations and must be administered by personnel trained in handling cytotoxic agents. By virtue of ISN, the pharmaceutical form of CTX nanosuspensions could also be divided in two parts: a solid organic-acid phase and sodium carbonate solution. In contrast to JEVTANA® , the preparation of CTX nanosuspensions is simple and only involves the addition of the sodium carbonate solution into the vial of solid organic-acid phase and then manually mixed. Transmission electron micrograph images indicate that CTX exists as homogeneous spheres with a mean diameter of approximately 92.21 nm with no visible drug crystals (Fig. 2A). XRD and DSC assays also demonstrate that CTX exists in the amorphous state (Fig. 2B and C). Additionally, the CTX nanosuspension prepared using ISN results in a stable product at room temperature for 24 h (Fig. 2D), which is adequate for in-use storage time. In contrast, CTX occurs in a rod-shape crystalline state when antisolvent precipitation coupled with an ultrasonication technique is applied to prepare the nanosuspension (Fig. 2E). 3. Conclusion The search for a simple and easy-scale approach to producing nanosuspensions remains of major clinical importance in nanomedicine. The major obstacle of the conventional bottomup technique is aggregation of the molecular drug. By virtue of a micro-mixing effect due to CO2 bubbles and the inhibition effect of stabilizers, we successfully prevented aggregation in the nanosuspensions tested. Our study presents an ISN technique with huge potential for the field of nanosuspension preparations that fulfills important properties, including: 1) a low energy-consumption process, 2) no specialized equipment needed, 3) no stability issues for the final products, and 4) good reproducibility. The results in this study set the stage for a thorough evaluation of in vivo efficacy. In fact, experiments using nanosuspensions based on the ISN technique for intramuscular and ophthalmic applications are currently ongoing.
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