- Email: [email protected]
Amphiphilic drugs as surfactants to fabricate excipient-free stable nanodispersions of hydrophobic drugs for cancer chemotherapy
Journal of Controlled Release 220 (2015) 175–179
Contents lists available at ScienceDirect
Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel
Amphiphilic drugs as surfactants to fabricate excipient-free stable nanodispersions of hydrophobic drugs for cancer chemotherapy Shiqi Hu a, Eunhye Lee b, Chi Wang c, Jinqiang Wang a, Zhuxian Zhou a, Yixian Li b, Xiaoyi Li c, Jianbin Tang a, Don Haeng Lee b, Xiangrui Liu a, Youqing Shen a,⁎ a b c
Center for Bio-nanoengineering, Key Laboratory of Biomass Chemical Engineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China Utah-Inha DDA & Advanced Therapeutics Research Center, Incheon 406840, South Korea College of Materials Science and Opto-Electronic Technology, University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e
i n f o
Article history: Received 29 July 2015 Received in revised form 13 October 2015 Accepted 15 October 2015 Available online 19 October 2015 Keywords: Nanomedicine Nanodispersion Excipient-free 7-Ethyl-10-hydroxycamptothecin (SN38) Water-insoluble drugs
a b s t r a c t Nanoformulations have been extensively explored to deliver water-insoluble drugs, but they generally use exotic new materials, for instance, amphiphilic block copolymers, which must first go through extensively clinical trials and be approved as drug excipients before any clinical uses. We hypothesize that using clinical amphiphilic drugs as surfactants to self-assemble with and thus solubilize hydrophobic drugs will lead to readily translational nanoformulations as they contain no new excipients. Herein, we show the first example of such excipient-free nanodispersions using an amphiphilic anti-tumor drug, irinotecan hydrochloride (CPT11). CPT11 selfassembles with its insoluble active parent drug, 7-ethyl-10-hydroxy camptothecin (SN38), into stable and water-dispersible nanoparticles, increasing SN38's water solubility by thousands of times up to 25 mg/mL with a loading efficiency close to 100%. The versatility of this approach is also demonstrated by fabricating nanodispersions of CPT11 with other water-insoluble drugs including paclitaxel (PTX) and camptothecin (CPT). These nanodispersions have much increased bioavailability and thereby improved anti-cancer activities. Thus, this strategy, using clinically proven amphiphilic drugs as excipients to fabricate nanodispersions, avoids new materials and makes readily translational nanoformulations of hydrophobic drugs. © 2015 Elsevier B.V. All rights reserved.
1. Concept and hypothesis Small molecular cytotoxin based anti-cancer drugs are still the main arsenal in fighting against cancer. However, most drugs are poorly water-soluble and the number of insoluble drug candidates in drug discovery is still increasing with almost 70% of new drug candidates [1]. Furthermore, their off-targeted intoxication to healthy cells causes severe adverse effects [2–4]. Hence, great efforts have been made to formulate these insoluble drugs for easy administration [5] using nanocarriers including polymers, liposomes and dendrimers [6,7]. These nanocarriers are also expected to improve drugs' pharmacokinetics via the enhanced permeability and retention effect [8]. Many such nanomedicines indeed show substantially reduced adverse effects, and several are already in clinical use and many more are in clinical trials [7]. For instance, 7-ethyl-10-hydroxycamptothecin (SN38) is a very potent inhibitor of topoisomerase I inhibiting growth of various tumor cells [9], but is poorly soluble in aqueous solution with a water solubility of about 11 μg/mL [10]. It is also insoluble in most pharmaceutically acceptable solvents and excipients including ethanol, polysorbate 80 and
⁎ Corresponding author. Tel.: +86 571 87953993. E-mail address: [email protected] (Y. Shen).
http://dx.doi.org/10.1016/j.jconrel.2015.10.031 0168-3659/© 2015 Elsevier B.V. All rights reserved.
cremophor, making it difficult to prepare suitable formulations for clinical use. So nanoparticles (e.g. SN38-loaded poly-lactide-co-glycolic acid [11]), polymer–drug conjugates (e.g. EZN-2208 [12]), and liposomal formulations (e.g. LE-SN38 [10]) of SN38 have been attempted to solve this challenge. Among them, EZN-2208 and NK012 (amphiphilic prodrugs) have entered the Phase II clinical trial, while some including SN2310 [13] (a lipophilic prodrug), DTS-108 [14] (a peptidic prodrug) and IMMU-130 [15] (an antibody–drug conjugate) are in the Phase I trial [16]. For example, EZN-2208 is a multi-arm 40 kDa PEG conjugate of SN38 via a glycine linker with an SN38 loading of 3.7% (wt/wt) and water solubility of 6.7 mg/mL. After extensive clinical trials, though well tolerated, it did not show expected therapeutic efficacy but unanticipated side effects, like neutropenia [17]. Up to date there is still no commercial SN38 formulations in clinics. Given a large number of nanomedicine systems reported to date, only few have entered clinical use [18]. One major bottleneck to clinical translation is that current nanomedicines often use new carrier materials or chemical modifications to drug molecules. These new materials or new molecules themselves need intensive clinical trials and final FDA approvals to be used as new excipients; particularly, the carriers may go through a more difficult path through the clinic [18]. Furthermore, the fabrications of the nanomedicines are often too complicated to establish proper manufacture process for scaling up [19].
176
S. Hu et al. / Journal of Controlled Release 220 (2015) 175–179
Motivated by our previous work that amphiphilic prodrugs could self-assemble into nanoparticles/nanocapsules for drug delivery [20], we hypothesized that directly using clinically proven amphiphilic drugs as surfactants to fabricate nanomedicines would avoid using non-clinically proven materials and thus facilitate their clinical translation. Herein, we demonstrated this concept that using CPT11 as the sole excipient to solubilize SN38 or other highly hydrophobic drugs. CPT11 is a clinically used water-soluble drug with a protonated tertiary amine head. Its hydrophobic moiety may interact with hydrophobic drugs such as SN38 like an amphiphilic surfactant to form stable nanodispersions (Supporting information, Fig. S1). 2. Experimental methods SN38/CPT11 nanodispersions were prepared using an anti-solvent method. A typical procedure is as follows: SN38 (10 mg) and CPT11 (10 mg) were dissolved in 150 μL of dimethyl sulfoxide (DMSO), and then DI water (10 mL) was added into the solution with vigorous stirring or ultrasonication for 5 min (ultrasonic cleaner, FRQ-1004HT, 200 W). The bluish solution was then loaded into a dialysis bag (MWCO 3500) and dialyzed against water for 10 h or freeze-dried directly to remove the organic solvent. This formulation is referred to as S1C1, which stands for the weight ratio of SN38 (S) to CPT11 (C) at 1 to 1. Other formulations at different ratios were prepared similarly (Supporting information). For in vivo experiments, the nanodispersions were prepared in 5% glucose or 0.9% sodium chloride injection medium. 3. Discovery and interpretation CPT11 or irinotecan hydrochloride, a prodrug of SN38 and widely used as a first-line drug for treatments of many types of cancer, is insoluble in water at pH 5.7 (2 mg/mL), and no particles were detected by
DLS in the solution, but some large and undefined nanostructures formed as the solution pH increased to neutral (Fig. S2). SN38 is insoluble in water. Once water was added to its organic solution (e.g. DMSO solution), it quickly precipitated out (Fig. S3) and could not form any stable dispersion even in the presence of various surfactants, such as Tween 80 and Cremophor. However, when the same amounts of SN38 and CPT11 were dissolved in DMSO together and then water was added, unexpectedly, they formed stable nanodispersion (S1C1) without any precipitation even after removing DMSO by dialysis (Fig. S3) and no free SN38 was detected in the dialysate. The nanodispersion remained well dispersed for several months in 4 °C storage. Lyophilization of the solution produced S1C1 powders that were re-dispersed in water, but the particle size became much larger to 500 nm. Adding 6% D-trehalose as a cryoprotectant to the solution resolved the problem. Lyophilization of the nanodispersion solution with 6% D-trehalose produced a powder well re-dispersible in water, or 5% glucose, or 0.9% sodium chloride injection medium used in clinics, and the nanoparticle sizes remained unchanged (Fig. S4). The Z-average size of S1C1 nanodispersion was 79.7 nm with a PDI of 0.176 (Fig. 1A) and a zetapotential of +31.3 mV in water, resulting from the protonated tertiary amine of CPT11. TEM and AFM images further proved the formation of uniform nanoparticles (Fig. 1B). The nanodispersion could form even in the presence of a very small amount of CPT11; for instance, as low as 1 wt.% of CPT11 was sufficient to make a stable dispersion (S99C1). Thus, the nanodispersions could form in a very broad weight ratio range of SN38 to CPT11, from 1:0.01 to 1:5. Thanks to the nanodispersion, the concentration of SN38 could reach 25 mg/mL in water, increased by thousands of times to free SN38, which had not yet been achieved by other SN38 formulations so far. This simultaneous formation of the SC nanoparticles can be ascribed to the strong interaction force between SN38 and its prodrug, CPT11 (Fig. S1). Though hydrophobic, SN38 is generally difficult to form
Fig. 1. Characterizations of nanodispersions. A) Particle size distribution of SN38/CPT11 nanodispersions (S1C1) measured by dynamic light scattering. B) Typical TEM (up) and AFM (down) images of S1C1 nanodispersion. Scale bar: 100 nm. C) Modeling of the self-assembly of S1C1, where CPT11 molecules are shown in red and SN38 molecules are in green. D) Xray diffraction (XRD) diagrams of SN38, CPT11 and S1C1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
S. Hu et al. / Journal of Controlled Release 220 (2015) 175–179
formulations with other hydrophobic segments because of its strong tendency to crystallization induced by the strong π–π interaction among the molecules. CPT11 has the same parent aromatic segment as SN38 and thus their aromatic parts have the same strong π–π stacking force to form the hydrophobic core, while the hydrophilic piperidine heads of CPT11 stick out and make the particle dispersible. This is confirmed by molecular simulation of SN38/CPT11 assembly (Fig. 1C and Supporting information), in which CPT11 molecules randomly mixed with SN38 molecules. The inhibited crystallization of SN38 or CPT11 in the nanodispersion further verified the conclusion. As shown in Fig. 1D, the strong Bragg peaks in the diffraction patterns of SN38 (A: 10.82°, B: 13.08°, C: 17.66°, D: 23.76°, and E: 25.94°) indicate its strong crystallization, while in the S1C1 nanoparticles, these typical SN38 peaks disappear, which indicates that SN38 molecules either distributed
177
in an amorphous state or as crystals with very small sizes in CPT11. Such an amorphous state or small crystals may be advantageous for a higher dissolution rate and better bioavailability [21]. The cytotoxicity of S1C1 was estimated in four tumor cell lines (human cancer cell lines BCap37, HeLa, SW620 and HepG2) using MTT assay (Fig. 2A). CPT11 was known less cytotoxic than its active component SN38 with an IC50 of 16.50 μg/mL to BCap37 cells, while the IC50 of S1C1 was 0.12 μg/mL, which was close to that of free SN38. In addition, Table 1 showed that the CPT11-assisted nanodispersion showed cytotoxicity close to free SN38 and could be 50–100 times more cytotoxic than CPT11. The pharmacokinetics of i.v. injected CPT11 and S1C1 were preliminarily studied and shown in Fig. 2B and summarized in Table 2. Because CPT11 can be quickly excreted by kidneys and its conversion to SN38 is
Fig. 2. Cytotoxicity and pharmacokinetics (A, B), in vivo chemotherapy-induced toxicity (C, D) and in vivo anti-tumor efficacy (E) of the nanodispersions. A) MTT assays of S1C1, SN38, and CPT11 against human breast cancer BCap37 cells for 48 h. Error bars represent standard deviation of means (n = 3). B) The SN38 concentration in plasma as a function of time in mice after i.v. administration of S1C1 or CPT11 at an SN38-eq. dose of 10 mg/kg body weight; Error bars represent standard deviation of means (n = 3). C) The body weight and D) diarrhea scoring after BALB/c mice treated with S1C1 nanodispersion or the controls at an SN38-eq. dose of 9.4 mg/kg (q1d × 5). Diarrhea in animals was graded as follows: Grade 1, small amount of watery diarrhea that lasted for 3 to 5 days followed by full recovery; Grade 2, moderate to severe diarrhea that lasted N5 days without blood or mucus and could be followed by recovery; Grade 3, severe diarrhea with mucus but without blood and no recovery; and Grade 4, severe diarrhea with blood and mucus and no recovery; nanodispersion or controls were intraperitoneal injected as indicated by the arrows (q1d × 5); Error bars represent standard error of means (n = 12). E) BCap37 xenografted tumor volume as a function of time after the nude mice treated with different doses of S1C1 nanodispersion or CPT11 via intravenous injection as indicated by the arrows (q3d × 5); tumors were excised from mice at the end of the treatment shown in the right panel. Error bars represent standard deviation of means (n = 9); Statistical significance: P values were obtained using the Student's t-test. *p b 0.05, **p b 0.005 and ***p b 0.0005.
178
S. Hu et al. / Journal of Controlled Release 220 (2015) 175–179
Table 1 In vitro cytotoxicity data of CPT11, SN38⁎ and S1C1 nanodispersions toward four cancer cell lines. The cells were treated with drugs for 48 h before analysis by MTT assay. (mean ± SD, n = 3). IC50 (μg/mL) Cancer type
Cell line
CPT11
SN38⁎
S1C1
Oral Cervical Breast Liver
KB HeLa BCap37 HepG2
17.23 ± 1.46 18.07 ± 1.54 16.50 ± 1.16 10.38 ± 1.24
0.35 ± 0.05 0.20 ± 0.06 0.12 ± 0.01 0.20 ± 0.03
0.39 ± 0.06 0.12 ± 0.03 0.13 ± 0.02 0.10 ± 0.02
⁎ Dissolved in DMSO at 10 mg/mL and then diluted by culture medium.
slow, the injected CPT11 resulted in very low plasma concentrations of SN38, well below 0.1 μg/mL at most times. On the other hand, the highest plasma SN38 concentration after the administration of S1C1 was 36.45 μg/mL, and then underwent a slower clearance. The resulting AUC value of SN38 was 15 times that of CPT11. Equally important, the nanodispersion showed even lower side effects than CPT11 (Fig. 2C and D). The nanodispersion did not significantly decrease the body weights of the mice compared with day 0, but the body weights of the CPT11 group decreased by 20% of their initial weights. Diarrhea is the main dose-limiting side effect of SN38-based drugs [22,23]. CPT11 at SN38-eq. 9 mg/kg (q1d × 5) caused severe life-threatening Grade 3 diarrhea on day 9, but the nanodispersion showed less severe diarrhea (scoring to Grade 2), which was agreed with the body weight loss of the mice. The dissected large intestines of S1C1 treated mice showed no obvious organ damage or lesion similar to the control, while localized ulcer, necrosis and erosion were observed in the CPT11-treated group (Fig. S5). In the nanodispersion, SN38 was buried inside and thus protected from glucuronidation by the enzyme UGT1A1. Thus, the reduced production of glucuronidated SN38 (SN38G) and the presence of less bispiperidine group of CPT11 [24] accounted for the substantially reduced diarrhea of S1C1. The hydrophobic microenvironment of the nanodispersion also favored the preservation of the active lactone form of both CPT11 and SN38 [25]. The therapeutic efficacy of S1C1 nanodispersion and CPT11 against different tumor models in mice was evaluated. The five time treatments of mice with CPT11 at a dose of 5 or 15 mg/kg (eq. SN38) every 3 days (Fig. 2E) led to an inhibition of BCap37 breast tumor growth by 36% or 47%, respectively, on 29th day posttreatment. In contrast, the mice treated with S1C1 at the same SN38-eq. doses gave 43% or 84% tumor growth inhibition rates, indicating a much improved efficacy. A striking anti-tumor response was observed in the high dose group (p b 0.0005). The mouse body weights of the treated groups were not significantly different from the control group (Fig. S6). As for the SW620 colon tumor model, since CPT11 is particularly active against human colon tumors, all tumors regressed and remained in stable remission for 16 days, but tumors treated with CPT11 rebounded earlier and grew faster than those treated with S1C1 (p b 0.05 on 28th day and p b 0.0005 on 31st day) (Fig. S7). The nanodispersions at different SN38 to CPT11 weight ratios (S1C2 and S2C1) showed a similar anti-cancer activity
Table 2 Pharmacokinetic parameters of SN38 after intravenous administration of S1C1 or CPT11 (equivalent to 10 mg/kg SN38 on ICR mice, mean, n = 3). Parameters
CPT11
S1C1
T1/2αa (h) T1/2βb (h) AUCINFc (h∗μg/ml) CLd (L/h/kg)
0.13 3.04 0.38 25.89
0.08 3.97 5.76 1.73
a b c d
T1/2α represents distribution phase half-life time. T1/2β represents elimination phase half-life time. AUCINF represents area under the curve. CL represents the clearance.
to S1C1. These results suggested that, in general, CPT11-assisted nanodispersions showed higher and more persistent therapeutic potency than free CPT11. The increased anti-tumor efficiency of the nanodispersion compared with CPT11 may result from the improved pharmacokinetics, bioavailability, and tumor accumulation of SN38 as it directly delivered active SN38 without need of enzymatic conversion like CPT11 (Fig. S8). More importantly, the nanodispersions are expected to produce even higher therapeutic efficacy than CPT11 in humans because the conversion of CPT11 into active SN38 is much less inefficient than in mice, which was estimated to be only 2% to 8% of the administered CPT11 converted to SN38 in 24 h in patients, due to the much lower level of the needed carboxylesterase enzymes in humans [26]. Following the same concept, other drug nanodispersions were also fabricated, e.g., from camptothecin (CPT/CPT11) and paclitaxel (PTX/ CPT11) using CPT11 as the amphiphilic drug or using toptecan (TPT) as the amphiphilic drug to make SN38 nanodispersion (SN38/TPT). Since CPT, SN38, CPT11 and TPT share the same parent structure, which leads to relative strong interaction force between molecules, their nanodispersions could be prepared similarly as described above. The preparation of PTX nanodispersions using CPT11 or TPT as the amphiphilic drug was more challenging. Since the inter-molecular force between PTX to camptothecins, was not as strong as SN38, the preparation process need more energy input and more amphiphilic drug, such as higher ultrasonic power and higher CPT11 to PTX ratio. The morphologies of the nanodispersions were different, as observed by AFM (Fig. S9). CPT/CPT11 nanodispersions were spherical nanoparticles, while PTX/CPT11 and SN38/TPT particles were nanorods. The cytotoxicity data of different formulations toward BCap37 breast cancer cells are summarized in Table S1. All the nanodispersions had cytotoxicities close to those of the loaded free drugs. Further, in vivo tests showed that PTX/ CPT11 had the anti-tumor efficiency against BCap37 breast cancer comparable to commercially paclitaxel formulation Taxol (Fig. S10). 4. Conclusions This study shows a novel concept using clinical drugs as surfactants to avoid usage of new materials and fabricate excipient-free readily translational nanomedicines. As an example, amphiphilic drug CPT11, which is an FDA approved first-line drug in clinics, can formulate SN38, CPT and PTX into stable nanodispersions. These nanodispersions are characterized by carrier materials free and improved pharmacokinetics, bioavailability and therapeutic efficacy but much reduced side effects. Furthermore, these nanodispersions may contain two types of drugs and thus be useful for combinational therapy. Further studies will be optimizing the systems for better stability, synergistic actions and clinical translation. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (51390481, 21174128 and 21274125), National Basic Research Program. Grant No: 2014CB931900 and the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20110101130007). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2015.10.031. References [1] Y. Kawabata, K. Wada, M. Nakatani, S. Yamada, S. Onoue, Formulation design for poorly water-soluble drugs based on biopharmaceutics classification system: basic approaches and practical applications, Int. J. Pharm. 420 (2011) 1–10.
S. Hu et al. / Journal of Controlled Release 220 (2015) 175–179 [2] S.E. Lipshultz, T.R. Cochran, V.I. Franco, T.L. Miller, Treatment-related cardiotoxicity in survivors of childhood cancer, Nat. Rev. Clin. Oncol. 10 (2013) 697–710. [3] V. Sahni, D. Choudhury, Z. Ahmed, Chemotherapy-associated renal dysfunction, Nat. Rev. Nephrol. 5 (2009) 450–462. [4] D. Schiff, P.Y. Wen, M.J. van den Bent, Neurological adverse effects caused by cytotoxic and targeted therapies, Nat. Rev. Clin. Oncol. 6 (2009) 596–603. [5] J.H. Hu, K.P. Johnston, R.O. Williams, Nanoparticle engineering processes for enhancing the dissolution rates of poorly water soluble drugs, Drug. Dev. Ind. Pharm. 30 (2004) 233–245. [6] L.H. Reddy, D. Bazile, Drug delivery design for intravenous route with integrated physicochemistry, pharmacokinetics and pharmacodynamics: illustration with the case of taxane therapeutics, Adv. Drug Deliv. Rev. 71 (2014) 34–57. [7] D. Peer, J.M. Karp, S. Hong, O.C. Farokhzad, R. Margalit, R. Langer, Nanocarriers as an emerging platform for cancer therapy, Nat. Nanotechnol. 2 (2007) 751–760. [8] H. Maeda, H. Nakamura, J. Fang, The EPR effect for macromolecular drug delivery to solid tumors: improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo, Adv. Drug Deliv. Rev. 65 (2013) 71–79. [9] H. Yoshizawa, S. Nishino, K. Shiomori, S. Natsugoe, T. Aiko, Y. Kitamura, Surface morphology control of polylactide microspheres enclosing irinotecan hydrochloride, Int. J. Pharm. 296 (2005) 112–116. [10] J.A. Zhang, T. Xuan, M. Parmar, L. Ma, S. Ugwu, S. Ali, I. Ahmad, Development and characterization of a novel liposome-based formulation of SN-38, Int. J. Pharm. 270 (2004) 93–107. [11] P. Ebrahimnejad, R. Dinarvand, A. Sajadi, M.R. Jaafari, A.R. Nomani, E. Azizi, M. RadMalekshahi, F. Atyabi, Preparation and in vitro evaluation of actively targetable nanoparticles for SN-38 delivery against HT-29 cell lines, Nanomedicine 6 (2010) 478–485. [12] P. Sapra, H. Zhao, M. Mehlig, J. Malaby, P. Kraft, C. Longley, L.M. Greenberger, I.D. Horak, Novel delivery of SN38 markedly inhibits tumor growth in xenografts, including a camptothecin-11-refractory model, Clin. Cancer Res. 14 (2008) 1888–1896. [13] J.F. Marier, L. Pheng, M.M. Trinh, H.A. Burris, S. Jones, K. Anderson, S. Warner, D. Porubek, Pharmacokinetics of SN2310, an injectable emulsion that incorporates a new derivative of SN-38 in patients with advanced solid tumors, J. Pharm. Sci. 100 (2011) 4536–4545. [14] F. Meyer-Losic, C. Nicolazzi, J. Quinonero, F. Ribes, M. Michel, V. Dubois, C. de Coupade, M. Boukaissi, A.S. Chene, I. Tranchant, V. Arranz, I. Zoubaa, J.S. Fruchart, D. Ravel, J. Kearsey, DTS-108, a novel peptidic prodrug of SN38: in vivo efficacy and toxicokinetic studies, Clin. Cancer Res. 14 (2008) 2145–2153.
179
[15] E. Dotan, J. Berlin, A. Starodub, M.J. Guarino, S.J. Cohen, P.P. Maliakal, S.V. Govindan, W.A. Wegener, R.M. Sharkey, D.M. Goldenberg, Activity of IMMU-130 antiCEACAM5-SN-38 antibody-drug conjugate (ADC) on metastatic colorectal cancer (mCRC) having relapsed after CPT-11: phase I study, J. Clin. Oncol. 32 (2014). [16] V. Bala, S.S. Rao, B.J. Boyd, C.A. Prestidge, Prodrug and nanomedicine approaches for the delivery of the camptothecin analogue SN38, J. Control. Release 172 (2013) 48–61. [17] C.R. Garrett, T.S. Bekaii-Saab, T. Ryan, G.A. Fisher, S. Clive, P. Kavan, E. ShachamShmueli, A. Buchbinder, R.M. Goldberg, Randomized phase 2 study of pegylated SN-38 (EZN-2208) or irinotecan plus cetuximab in patients with advanced colorectal cancer, Cancer 119 (2013) 4223–4230. [18] V.J. Venditto, F.C. Szoka Jr., Cancer nanomedicines: so many papers and so few drugs! Adv. Drug Deliv. Rev. 65 (2013) 80–88. [19] Q.H. Sun, M. Radosz, Y.Q. Shen, Challenges in design of translational nanocarriers, J. Control. Release 164 (2012) 156–169. [20] Y.Q. Shen, E.L. Jin, B. Zhang, C.J. Murphy, M.H. Sui, J. Zhao, J.Q. Wang, J.B. Tang, M.H. Fan, E. Van Kirk, W.J. Murdoch, Prodrugs forming high drug loading multifunctional nanocapsules for intracellular cancer drug delivery, J. Am. Chem. Soc. 132 (2010) 4259–4265. [21] D. Douroumis, A. Fahr, Drug Delivery Strategies for Poorly Water-soluble Drugs, John Wiley & Sons, Chichester, West Sussex, 2012. [22] A. Stein, W. Voigt, K. Jordan, Chemotherapy-induced diarrhea: pathophysiology, frequency and guideline-based management, Ther. Adv. Med. Oncol. 2 (2010) 51–63. [23] L.B. Saltz, Understanding and managing chemotherapy-induced diarrhea, J. Support. Oncol. 1 (2003) 35–46. [24] Y. Pommier, Topoisomerase I inhibitors: camptothecins and beyond, Nat. Rev. Cancer 6 (2006) 789–802. [25] I. Nabiev, F. Fleury, I. Kudelina, Y. Pommier, F. Charton, J.F. Riou, A.J. Alix, M. Manfait, Spectroscopic and biochemical characterisation of self-aggregates formed by antitumor drugs of the camptothecin family: their possible role in the unique mode of drug action, Biochem. Pharmacol. 55 (1998) 1163–1174. [26] J.G. Slatter, L.J. Schaaf, J.P. Sams, K.L. Feenstra, M.G. Johnson, P.A. Bombardt, K.S. Cathcart, M.T. Verburg, L.K. Pearson, L.D. Compton, L.L. Miller, D.S. Baker, C.V. Pesheck, R.S. Lord, Pharmacokinetics, metabolism, and excretion of irinotecan (CPT-11) following i.v. infusion of [C-14] CPT-11 in cancer patients, Drug Metab. Dispos. 28 (2000) 423–433.
Contents lists available at ScienceDirect
Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel
Amphiphilic drugs as surfactants to fabricate excipient-free stable nanodispersions of hydrophobic drugs for cancer chemotherapy Shiqi Hu a, Eunhye Lee b, Chi Wang c, Jinqiang Wang a, Zhuxian Zhou a, Yixian Li b, Xiaoyi Li c, Jianbin Tang a, Don Haeng Lee b, Xiangrui Liu a, Youqing Shen a,⁎ a b c
Center for Bio-nanoengineering, Key Laboratory of Biomass Chemical Engineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China Utah-Inha DDA & Advanced Therapeutics Research Center, Incheon 406840, South Korea College of Materials Science and Opto-Electronic Technology, University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e
i n f o
Article history: Received 29 July 2015 Received in revised form 13 October 2015 Accepted 15 October 2015 Available online 19 October 2015 Keywords: Nanomedicine Nanodispersion Excipient-free 7-Ethyl-10-hydroxycamptothecin (SN38) Water-insoluble drugs
a b s t r a c t Nanoformulations have been extensively explored to deliver water-insoluble drugs, but they generally use exotic new materials, for instance, amphiphilic block copolymers, which must first go through extensively clinical trials and be approved as drug excipients before any clinical uses. We hypothesize that using clinical amphiphilic drugs as surfactants to self-assemble with and thus solubilize hydrophobic drugs will lead to readily translational nanoformulations as they contain no new excipients. Herein, we show the first example of such excipient-free nanodispersions using an amphiphilic anti-tumor drug, irinotecan hydrochloride (CPT11). CPT11 selfassembles with its insoluble active parent drug, 7-ethyl-10-hydroxy camptothecin (SN38), into stable and water-dispersible nanoparticles, increasing SN38's water solubility by thousands of times up to 25 mg/mL with a loading efficiency close to 100%. The versatility of this approach is also demonstrated by fabricating nanodispersions of CPT11 with other water-insoluble drugs including paclitaxel (PTX) and camptothecin (CPT). These nanodispersions have much increased bioavailability and thereby improved anti-cancer activities. Thus, this strategy, using clinically proven amphiphilic drugs as excipients to fabricate nanodispersions, avoids new materials and makes readily translational nanoformulations of hydrophobic drugs. © 2015 Elsevier B.V. All rights reserved.
1. Concept and hypothesis Small molecular cytotoxin based anti-cancer drugs are still the main arsenal in fighting against cancer. However, most drugs are poorly water-soluble and the number of insoluble drug candidates in drug discovery is still increasing with almost 70% of new drug candidates [1]. Furthermore, their off-targeted intoxication to healthy cells causes severe adverse effects [2–4]. Hence, great efforts have been made to formulate these insoluble drugs for easy administration [5] using nanocarriers including polymers, liposomes and dendrimers [6,7]. These nanocarriers are also expected to improve drugs' pharmacokinetics via the enhanced permeability and retention effect [8]. Many such nanomedicines indeed show substantially reduced adverse effects, and several are already in clinical use and many more are in clinical trials [7]. For instance, 7-ethyl-10-hydroxycamptothecin (SN38) is a very potent inhibitor of topoisomerase I inhibiting growth of various tumor cells [9], but is poorly soluble in aqueous solution with a water solubility of about 11 μg/mL [10]. It is also insoluble in most pharmaceutically acceptable solvents and excipients including ethanol, polysorbate 80 and
⁎ Corresponding author. Tel.: +86 571 87953993. E-mail address: [email protected] (Y. Shen).
http://dx.doi.org/10.1016/j.jconrel.2015.10.031 0168-3659/© 2015 Elsevier B.V. All rights reserved.
cremophor, making it difficult to prepare suitable formulations for clinical use. So nanoparticles (e.g. SN38-loaded poly-lactide-co-glycolic acid [11]), polymer–drug conjugates (e.g. EZN-2208 [12]), and liposomal formulations (e.g. LE-SN38 [10]) of SN38 have been attempted to solve this challenge. Among them, EZN-2208 and NK012 (amphiphilic prodrugs) have entered the Phase II clinical trial, while some including SN2310 [13] (a lipophilic prodrug), DTS-108 [14] (a peptidic prodrug) and IMMU-130 [15] (an antibody–drug conjugate) are in the Phase I trial [16]. For example, EZN-2208 is a multi-arm 40 kDa PEG conjugate of SN38 via a glycine linker with an SN38 loading of 3.7% (wt/wt) and water solubility of 6.7 mg/mL. After extensive clinical trials, though well tolerated, it did not show expected therapeutic efficacy but unanticipated side effects, like neutropenia [17]. Up to date there is still no commercial SN38 formulations in clinics. Given a large number of nanomedicine systems reported to date, only few have entered clinical use [18]. One major bottleneck to clinical translation is that current nanomedicines often use new carrier materials or chemical modifications to drug molecules. These new materials or new molecules themselves need intensive clinical trials and final FDA approvals to be used as new excipients; particularly, the carriers may go through a more difficult path through the clinic [18]. Furthermore, the fabrications of the nanomedicines are often too complicated to establish proper manufacture process for scaling up [19].
176
S. Hu et al. / Journal of Controlled Release 220 (2015) 175–179
Motivated by our previous work that amphiphilic prodrugs could self-assemble into nanoparticles/nanocapsules for drug delivery [20], we hypothesized that directly using clinically proven amphiphilic drugs as surfactants to fabricate nanomedicines would avoid using non-clinically proven materials and thus facilitate their clinical translation. Herein, we demonstrated this concept that using CPT11 as the sole excipient to solubilize SN38 or other highly hydrophobic drugs. CPT11 is a clinically used water-soluble drug with a protonated tertiary amine head. Its hydrophobic moiety may interact with hydrophobic drugs such as SN38 like an amphiphilic surfactant to form stable nanodispersions (Supporting information, Fig. S1). 2. Experimental methods SN38/CPT11 nanodispersions were prepared using an anti-solvent method. A typical procedure is as follows: SN38 (10 mg) and CPT11 (10 mg) were dissolved in 150 μL of dimethyl sulfoxide (DMSO), and then DI water (10 mL) was added into the solution with vigorous stirring or ultrasonication for 5 min (ultrasonic cleaner, FRQ-1004HT, 200 W). The bluish solution was then loaded into a dialysis bag (MWCO 3500) and dialyzed against water for 10 h or freeze-dried directly to remove the organic solvent. This formulation is referred to as S1C1, which stands for the weight ratio of SN38 (S) to CPT11 (C) at 1 to 1. Other formulations at different ratios were prepared similarly (Supporting information). For in vivo experiments, the nanodispersions were prepared in 5% glucose or 0.9% sodium chloride injection medium. 3. Discovery and interpretation CPT11 or irinotecan hydrochloride, a prodrug of SN38 and widely used as a first-line drug for treatments of many types of cancer, is insoluble in water at pH 5.7 (2 mg/mL), and no particles were detected by
DLS in the solution, but some large and undefined nanostructures formed as the solution pH increased to neutral (Fig. S2). SN38 is insoluble in water. Once water was added to its organic solution (e.g. DMSO solution), it quickly precipitated out (Fig. S3) and could not form any stable dispersion even in the presence of various surfactants, such as Tween 80 and Cremophor. However, when the same amounts of SN38 and CPT11 were dissolved in DMSO together and then water was added, unexpectedly, they formed stable nanodispersion (S1C1) without any precipitation even after removing DMSO by dialysis (Fig. S3) and no free SN38 was detected in the dialysate. The nanodispersion remained well dispersed for several months in 4 °C storage. Lyophilization of the solution produced S1C1 powders that were re-dispersed in water, but the particle size became much larger to 500 nm. Adding 6% D-trehalose as a cryoprotectant to the solution resolved the problem. Lyophilization of the nanodispersion solution with 6% D-trehalose produced a powder well re-dispersible in water, or 5% glucose, or 0.9% sodium chloride injection medium used in clinics, and the nanoparticle sizes remained unchanged (Fig. S4). The Z-average size of S1C1 nanodispersion was 79.7 nm with a PDI of 0.176 (Fig. 1A) and a zetapotential of +31.3 mV in water, resulting from the protonated tertiary amine of CPT11. TEM and AFM images further proved the formation of uniform nanoparticles (Fig. 1B). The nanodispersion could form even in the presence of a very small amount of CPT11; for instance, as low as 1 wt.% of CPT11 was sufficient to make a stable dispersion (S99C1). Thus, the nanodispersions could form in a very broad weight ratio range of SN38 to CPT11, from 1:0.01 to 1:5. Thanks to the nanodispersion, the concentration of SN38 could reach 25 mg/mL in water, increased by thousands of times to free SN38, which had not yet been achieved by other SN38 formulations so far. This simultaneous formation of the SC nanoparticles can be ascribed to the strong interaction force between SN38 and its prodrug, CPT11 (Fig. S1). Though hydrophobic, SN38 is generally difficult to form
Fig. 1. Characterizations of nanodispersions. A) Particle size distribution of SN38/CPT11 nanodispersions (S1C1) measured by dynamic light scattering. B) Typical TEM (up) and AFM (down) images of S1C1 nanodispersion. Scale bar: 100 nm. C) Modeling of the self-assembly of S1C1, where CPT11 molecules are shown in red and SN38 molecules are in green. D) Xray diffraction (XRD) diagrams of SN38, CPT11 and S1C1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
S. Hu et al. / Journal of Controlled Release 220 (2015) 175–179
formulations with other hydrophobic segments because of its strong tendency to crystallization induced by the strong π–π interaction among the molecules. CPT11 has the same parent aromatic segment as SN38 and thus their aromatic parts have the same strong π–π stacking force to form the hydrophobic core, while the hydrophilic piperidine heads of CPT11 stick out and make the particle dispersible. This is confirmed by molecular simulation of SN38/CPT11 assembly (Fig. 1C and Supporting information), in which CPT11 molecules randomly mixed with SN38 molecules. The inhibited crystallization of SN38 or CPT11 in the nanodispersion further verified the conclusion. As shown in Fig. 1D, the strong Bragg peaks in the diffraction patterns of SN38 (A: 10.82°, B: 13.08°, C: 17.66°, D: 23.76°, and E: 25.94°) indicate its strong crystallization, while in the S1C1 nanoparticles, these typical SN38 peaks disappear, which indicates that SN38 molecules either distributed
177
in an amorphous state or as crystals with very small sizes in CPT11. Such an amorphous state or small crystals may be advantageous for a higher dissolution rate and better bioavailability [21]. The cytotoxicity of S1C1 was estimated in four tumor cell lines (human cancer cell lines BCap37, HeLa, SW620 and HepG2) using MTT assay (Fig. 2A). CPT11 was known less cytotoxic than its active component SN38 with an IC50 of 16.50 μg/mL to BCap37 cells, while the IC50 of S1C1 was 0.12 μg/mL, which was close to that of free SN38. In addition, Table 1 showed that the CPT11-assisted nanodispersion showed cytotoxicity close to free SN38 and could be 50–100 times more cytotoxic than CPT11. The pharmacokinetics of i.v. injected CPT11 and S1C1 were preliminarily studied and shown in Fig. 2B and summarized in Table 2. Because CPT11 can be quickly excreted by kidneys and its conversion to SN38 is
Fig. 2. Cytotoxicity and pharmacokinetics (A, B), in vivo chemotherapy-induced toxicity (C, D) and in vivo anti-tumor efficacy (E) of the nanodispersions. A) MTT assays of S1C1, SN38, and CPT11 against human breast cancer BCap37 cells for 48 h. Error bars represent standard deviation of means (n = 3). B) The SN38 concentration in plasma as a function of time in mice after i.v. administration of S1C1 or CPT11 at an SN38-eq. dose of 10 mg/kg body weight; Error bars represent standard deviation of means (n = 3). C) The body weight and D) diarrhea scoring after BALB/c mice treated with S1C1 nanodispersion or the controls at an SN38-eq. dose of 9.4 mg/kg (q1d × 5). Diarrhea in animals was graded as follows: Grade 1, small amount of watery diarrhea that lasted for 3 to 5 days followed by full recovery; Grade 2, moderate to severe diarrhea that lasted N5 days without blood or mucus and could be followed by recovery; Grade 3, severe diarrhea with mucus but without blood and no recovery; and Grade 4, severe diarrhea with blood and mucus and no recovery; nanodispersion or controls were intraperitoneal injected as indicated by the arrows (q1d × 5); Error bars represent standard error of means (n = 12). E) BCap37 xenografted tumor volume as a function of time after the nude mice treated with different doses of S1C1 nanodispersion or CPT11 via intravenous injection as indicated by the arrows (q3d × 5); tumors were excised from mice at the end of the treatment shown in the right panel. Error bars represent standard deviation of means (n = 9); Statistical significance: P values were obtained using the Student's t-test. *p b 0.05, **p b 0.005 and ***p b 0.0005.
178
S. Hu et al. / Journal of Controlled Release 220 (2015) 175–179
Table 1 In vitro cytotoxicity data of CPT11, SN38⁎ and S1C1 nanodispersions toward four cancer cell lines. The cells were treated with drugs for 48 h before analysis by MTT assay. (mean ± SD, n = 3). IC50 (μg/mL) Cancer type
Cell line
CPT11
SN38⁎
S1C1
Oral Cervical Breast Liver
KB HeLa BCap37 HepG2
17.23 ± 1.46 18.07 ± 1.54 16.50 ± 1.16 10.38 ± 1.24
0.35 ± 0.05 0.20 ± 0.06 0.12 ± 0.01 0.20 ± 0.03
0.39 ± 0.06 0.12 ± 0.03 0.13 ± 0.02 0.10 ± 0.02
⁎ Dissolved in DMSO at 10 mg/mL and then diluted by culture medium.
slow, the injected CPT11 resulted in very low plasma concentrations of SN38, well below 0.1 μg/mL at most times. On the other hand, the highest plasma SN38 concentration after the administration of S1C1 was 36.45 μg/mL, and then underwent a slower clearance. The resulting AUC value of SN38 was 15 times that of CPT11. Equally important, the nanodispersion showed even lower side effects than CPT11 (Fig. 2C and D). The nanodispersion did not significantly decrease the body weights of the mice compared with day 0, but the body weights of the CPT11 group decreased by 20% of their initial weights. Diarrhea is the main dose-limiting side effect of SN38-based drugs [22,23]. CPT11 at SN38-eq. 9 mg/kg (q1d × 5) caused severe life-threatening Grade 3 diarrhea on day 9, but the nanodispersion showed less severe diarrhea (scoring to Grade 2), which was agreed with the body weight loss of the mice. The dissected large intestines of S1C1 treated mice showed no obvious organ damage or lesion similar to the control, while localized ulcer, necrosis and erosion were observed in the CPT11-treated group (Fig. S5). In the nanodispersion, SN38 was buried inside and thus protected from glucuronidation by the enzyme UGT1A1. Thus, the reduced production of glucuronidated SN38 (SN38G) and the presence of less bispiperidine group of CPT11 [24] accounted for the substantially reduced diarrhea of S1C1. The hydrophobic microenvironment of the nanodispersion also favored the preservation of the active lactone form of both CPT11 and SN38 [25]. The therapeutic efficacy of S1C1 nanodispersion and CPT11 against different tumor models in mice was evaluated. The five time treatments of mice with CPT11 at a dose of 5 or 15 mg/kg (eq. SN38) every 3 days (Fig. 2E) led to an inhibition of BCap37 breast tumor growth by 36% or 47%, respectively, on 29th day posttreatment. In contrast, the mice treated with S1C1 at the same SN38-eq. doses gave 43% or 84% tumor growth inhibition rates, indicating a much improved efficacy. A striking anti-tumor response was observed in the high dose group (p b 0.0005). The mouse body weights of the treated groups were not significantly different from the control group (Fig. S6). As for the SW620 colon tumor model, since CPT11 is particularly active against human colon tumors, all tumors regressed and remained in stable remission for 16 days, but tumors treated with CPT11 rebounded earlier and grew faster than those treated with S1C1 (p b 0.05 on 28th day and p b 0.0005 on 31st day) (Fig. S7). The nanodispersions at different SN38 to CPT11 weight ratios (S1C2 and S2C1) showed a similar anti-cancer activity
Table 2 Pharmacokinetic parameters of SN38 after intravenous administration of S1C1 or CPT11 (equivalent to 10 mg/kg SN38 on ICR mice, mean, n = 3). Parameters
CPT11
S1C1
T1/2αa (h) T1/2βb (h) AUCINFc (h∗μg/ml) CLd (L/h/kg)
0.13 3.04 0.38 25.89
0.08 3.97 5.76 1.73
a b c d
T1/2α represents distribution phase half-life time. T1/2β represents elimination phase half-life time. AUCINF represents area under the curve. CL represents the clearance.
to S1C1. These results suggested that, in general, CPT11-assisted nanodispersions showed higher and more persistent therapeutic potency than free CPT11. The increased anti-tumor efficiency of the nanodispersion compared with CPT11 may result from the improved pharmacokinetics, bioavailability, and tumor accumulation of SN38 as it directly delivered active SN38 without need of enzymatic conversion like CPT11 (Fig. S8). More importantly, the nanodispersions are expected to produce even higher therapeutic efficacy than CPT11 in humans because the conversion of CPT11 into active SN38 is much less inefficient than in mice, which was estimated to be only 2% to 8% of the administered CPT11 converted to SN38 in 24 h in patients, due to the much lower level of the needed carboxylesterase enzymes in humans [26]. Following the same concept, other drug nanodispersions were also fabricated, e.g., from camptothecin (CPT/CPT11) and paclitaxel (PTX/ CPT11) using CPT11 as the amphiphilic drug or using toptecan (TPT) as the amphiphilic drug to make SN38 nanodispersion (SN38/TPT). Since CPT, SN38, CPT11 and TPT share the same parent structure, which leads to relative strong interaction force between molecules, their nanodispersions could be prepared similarly as described above. The preparation of PTX nanodispersions using CPT11 or TPT as the amphiphilic drug was more challenging. Since the inter-molecular force between PTX to camptothecins, was not as strong as SN38, the preparation process need more energy input and more amphiphilic drug, such as higher ultrasonic power and higher CPT11 to PTX ratio. The morphologies of the nanodispersions were different, as observed by AFM (Fig. S9). CPT/CPT11 nanodispersions were spherical nanoparticles, while PTX/CPT11 and SN38/TPT particles were nanorods. The cytotoxicity data of different formulations toward BCap37 breast cancer cells are summarized in Table S1. All the nanodispersions had cytotoxicities close to those of the loaded free drugs. Further, in vivo tests showed that PTX/ CPT11 had the anti-tumor efficiency against BCap37 breast cancer comparable to commercially paclitaxel formulation Taxol (Fig. S10). 4. Conclusions This study shows a novel concept using clinical drugs as surfactants to avoid usage of new materials and fabricate excipient-free readily translational nanomedicines. As an example, amphiphilic drug CPT11, which is an FDA approved first-line drug in clinics, can formulate SN38, CPT and PTX into stable nanodispersions. These nanodispersions are characterized by carrier materials free and improved pharmacokinetics, bioavailability and therapeutic efficacy but much reduced side effects. Furthermore, these nanodispersions may contain two types of drugs and thus be useful for combinational therapy. Further studies will be optimizing the systems for better stability, synergistic actions and clinical translation. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (51390481, 21174128 and 21274125), National Basic Research Program. Grant No: 2014CB931900 and the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20110101130007). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2015.10.031. References [1] Y. Kawabata, K. Wada, M. Nakatani, S. Yamada, S. Onoue, Formulation design for poorly water-soluble drugs based on biopharmaceutics classification system: basic approaches and practical applications, Int. J. Pharm. 420 (2011) 1–10.
S. Hu et al. / Journal of Controlled Release 220 (2015) 175–179 [2] S.E. Lipshultz, T.R. Cochran, V.I. Franco, T.L. Miller, Treatment-related cardiotoxicity in survivors of childhood cancer, Nat. Rev. Clin. Oncol. 10 (2013) 697–710. [3] V. Sahni, D. Choudhury, Z. Ahmed, Chemotherapy-associated renal dysfunction, Nat. Rev. Nephrol. 5 (2009) 450–462. [4] D. Schiff, P.Y. Wen, M.J. van den Bent, Neurological adverse effects caused by cytotoxic and targeted therapies, Nat. Rev. Clin. Oncol. 6 (2009) 596–603. [5] J.H. Hu, K.P. Johnston, R.O. Williams, Nanoparticle engineering processes for enhancing the dissolution rates of poorly water soluble drugs, Drug. Dev. Ind. Pharm. 30 (2004) 233–245. [6] L.H. Reddy, D. Bazile, Drug delivery design for intravenous route with integrated physicochemistry, pharmacokinetics and pharmacodynamics: illustration with the case of taxane therapeutics, Adv. Drug Deliv. Rev. 71 (2014) 34–57. [7] D. Peer, J.M. Karp, S. Hong, O.C. Farokhzad, R. Margalit, R. Langer, Nanocarriers as an emerging platform for cancer therapy, Nat. Nanotechnol. 2 (2007) 751–760. [8] H. Maeda, H. Nakamura, J. Fang, The EPR effect for macromolecular drug delivery to solid tumors: improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo, Adv. Drug Deliv. Rev. 65 (2013) 71–79. [9] H. Yoshizawa, S. Nishino, K. Shiomori, S. Natsugoe, T. Aiko, Y. Kitamura, Surface morphology control of polylactide microspheres enclosing irinotecan hydrochloride, Int. J. Pharm. 296 (2005) 112–116. [10] J.A. Zhang, T. Xuan, M. Parmar, L. Ma, S. Ugwu, S. Ali, I. Ahmad, Development and characterization of a novel liposome-based formulation of SN-38, Int. J. Pharm. 270 (2004) 93–107. [11] P. Ebrahimnejad, R. Dinarvand, A. Sajadi, M.R. Jaafari, A.R. Nomani, E. Azizi, M. RadMalekshahi, F. Atyabi, Preparation and in vitro evaluation of actively targetable nanoparticles for SN-38 delivery against HT-29 cell lines, Nanomedicine 6 (2010) 478–485. [12] P. Sapra, H. Zhao, M. Mehlig, J. Malaby, P. Kraft, C. Longley, L.M. Greenberger, I.D. Horak, Novel delivery of SN38 markedly inhibits tumor growth in xenografts, including a camptothecin-11-refractory model, Clin. Cancer Res. 14 (2008) 1888–1896. [13] J.F. Marier, L. Pheng, M.M. Trinh, H.A. Burris, S. Jones, K. Anderson, S. Warner, D. Porubek, Pharmacokinetics of SN2310, an injectable emulsion that incorporates a new derivative of SN-38 in patients with advanced solid tumors, J. Pharm. Sci. 100 (2011) 4536–4545. [14] F. Meyer-Losic, C. Nicolazzi, J. Quinonero, F. Ribes, M. Michel, V. Dubois, C. de Coupade, M. Boukaissi, A.S. Chene, I. Tranchant, V. Arranz, I. Zoubaa, J.S. Fruchart, D. Ravel, J. Kearsey, DTS-108, a novel peptidic prodrug of SN38: in vivo efficacy and toxicokinetic studies, Clin. Cancer Res. 14 (2008) 2145–2153.
179
[15] E. Dotan, J. Berlin, A. Starodub, M.J. Guarino, S.J. Cohen, P.P. Maliakal, S.V. Govindan, W.A. Wegener, R.M. Sharkey, D.M. Goldenberg, Activity of IMMU-130 antiCEACAM5-SN-38 antibody-drug conjugate (ADC) on metastatic colorectal cancer (mCRC) having relapsed after CPT-11: phase I study, J. Clin. Oncol. 32 (2014). [16] V. Bala, S.S. Rao, B.J. Boyd, C.A. Prestidge, Prodrug and nanomedicine approaches for the delivery of the camptothecin analogue SN38, J. Control. Release 172 (2013) 48–61. [17] C.R. Garrett, T.S. Bekaii-Saab, T. Ryan, G.A. Fisher, S. Clive, P. Kavan, E. ShachamShmueli, A. Buchbinder, R.M. Goldberg, Randomized phase 2 study of pegylated SN-38 (EZN-2208) or irinotecan plus cetuximab in patients with advanced colorectal cancer, Cancer 119 (2013) 4223–4230. [18] V.J. Venditto, F.C. Szoka Jr., Cancer nanomedicines: so many papers and so few drugs! Adv. Drug Deliv. Rev. 65 (2013) 80–88. [19] Q.H. Sun, M. Radosz, Y.Q. Shen, Challenges in design of translational nanocarriers, J. Control. Release 164 (2012) 156–169. [20] Y.Q. Shen, E.L. Jin, B. Zhang, C.J. Murphy, M.H. Sui, J. Zhao, J.Q. Wang, J.B. Tang, M.H. Fan, E. Van Kirk, W.J. Murdoch, Prodrugs forming high drug loading multifunctional nanocapsules for intracellular cancer drug delivery, J. Am. Chem. Soc. 132 (2010) 4259–4265. [21] D. Douroumis, A. Fahr, Drug Delivery Strategies for Poorly Water-soluble Drugs, John Wiley & Sons, Chichester, West Sussex, 2012. [22] A. Stein, W. Voigt, K. Jordan, Chemotherapy-induced diarrhea: pathophysiology, frequency and guideline-based management, Ther. Adv. Med. Oncol. 2 (2010) 51–63. [23] L.B. Saltz, Understanding and managing chemotherapy-induced diarrhea, J. Support. Oncol. 1 (2003) 35–46. [24] Y. Pommier, Topoisomerase I inhibitors: camptothecins and beyond, Nat. Rev. Cancer 6 (2006) 789–802. [25] I. Nabiev, F. Fleury, I. Kudelina, Y. Pommier, F. Charton, J.F. Riou, A.J. Alix, M. Manfait, Spectroscopic and biochemical characterisation of self-aggregates formed by antitumor drugs of the camptothecin family: their possible role in the unique mode of drug action, Biochem. Pharmacol. 55 (1998) 1163–1174. [26] J.G. Slatter, L.J. Schaaf, J.P. Sams, K.L. Feenstra, M.G. Johnson, P.A. Bombardt, K.S. Cathcart, M.T. Verburg, L.K. Pearson, L.D. Compton, L.L. Miller, D.S. Baker, C.V. Pesheck, R.S. Lord, Pharmacokinetics, metabolism, and excretion of irinotecan (CPT-11) following i.v. infusion of [C-14] CPT-11 in cancer patients, Drug Metab. Dispos. 28 (2000) 423–433.