Preclinical Evaluation of a Cabazitaxel Prodrug Using Nanoparticle Delivery for the Treatment of Taxane-Resistant Malignancies
Binbin Xie1,2, Jianqin Wan1, Xiaona Chen1, Weidong Han2, and Hangxiang Wang1
Taxane-based chemotherapeutics are clinically available as frontline treatment regimens for cervical cancer. However, drug resistance and life-threatening toxicity impair the clinical efﬁ- cacy of taxanes, so more effective and less toxic therapeutic modalities are urgently needed. Cabazitaxel has attracted increasing interest due to its potential to circumvent the drug resistance by taxanes. We previously showed that tethering docosahexaenoic acid (DHA) to cabazitaxel enabled the prodrug to self-assemble into nanoparticles in water. Despite this encour- aging ﬁnding, the DHA–cabazitaxel conjugate formulation requires further optimization to enhance nanoparticle retention and tumor delivery. We here integrated this conjugate into amphiphilic poly(ethylene glycol)-block-poly(D,L-lactic acid)
copolymers to assemble dCTX NPs. The nanoparticle abrogated P-glycoprotein–mediated resistance in cancer cells. In a doce- taxel-resistant cervical tumor xenograft-bearing mouse model, the efﬁcacy was augmented by the nanotherapy when compared with solution-based free drugs (i.e., docetaxel and cabazitaxel). Dose intensiﬁcation of dCTX NPs markedly suppressed the tumor growth in this model. Detailed studies revealed that systemic toxicity was alleviated, and MTD of dCTX NPs was at least 3 times higher than that of free cabazitaxel in animals, which may enable dose increases for clinical studies. In con- clusion, the new formulation addresses essential requirements in terms of the stability, safety, and translational capacity for initiating early-phase clinical trials.
Cervical cancer is one of the most prevalent gynecologic malig- nancies. Each year, cervical cancer accounts for more than half a million new cases, and over 300,000 females die from the disease worldwide (1, 2). Persistent infection by high-risk human papilloma- virus (HPV) is closely associated with the development of cervical cancer. Although effective prophylactic vaccines against the most lethal oncogenic HPV types are clinically available, only limited numbers of patients can receive the vaccine treatment (3, 4). In North America, approximately 85% of patients are diagnosed at early stages; however, in many less developed countries, most cervical cancers are diagnosed in the advanced stages because these patients are either lack of awareness or unaffordable for HPV vaccine (5–7).
Chemotherapy continues to be a mainstay treatment regimen for advanced and metastatic cervical cancer. Taxane-based chemother- apeutics [e.g., paclitaxel and docetaxel (DTX), Supplementary Fig. S1], which are the frontline treatments for cervical cancer, are clinically
1The First Afﬁliated Hospital, Collaborative Innovation Center for Diagnosis and
Treatment of Infectious Diseases, Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, School of Medicine, Zhejiang Univer- sity, Hangzhou, Zhejiang Province, PR China. 2Department of Medical Oncology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, PR China.
Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Authors: Hangxiang Wang, Zhejiang University, 79, Qingchun Road, Hangzhou 310003, PR China. Phone: 86-571-88208173; Fax: 86-571- 88208173; E-mail: [email protected]; and Weidong Han, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, PR China. E-mail: [email protected]
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available alone or in combination with other agents and have a signiﬁcant therapeutic beneﬁt (8–10). Taxanes induce the apoptosis of rapidly proliferating cancer cells by binding to tubulin and stabi- lizing microtubules (11). Unfortunately, the clinical efﬁcacy of taxane- based treatment regimens remains unsatisfactory because of the induction of drug resistance and dose-limiting toxicity (12). These limitations prevent cancer therapies from achieving stable and com- plete responses, and thus need for alternative modality.
Generally, only limited amounts (less than 1%) of small-molecule drugs reach tumors when they are systemically administered in a traditional solution-based dosage form (13). Nanoparticle-mediated drug delivery has been proposed as an alternative approach to deliver increased amounts of drugs to tumor lesions (14). Taking advantage of the unique pathologies observed in solid tumors, including defective leaky tumor vasculature and ineffective lymphatic drainage, nanopar- ticles with small sizes can passively accumulate in target tumors through the enhanced permeability and retention (EPR) effect (15–22). This ﬁnding has motivated many investigators to explore nanoparticle strategies used as a viable solution to achieve the magic bullet concept for cancer therapy proposed by Paul Ehrlich (23–26). Free chemother- apeutics must be stably entrapped in nanoparticles in the systemic circulation to decrease indiscriminatory distribution in healthy tissues. After accumulating in tumors, drug molecules must be released at a high efﬁciency (27–29). More importantly, nanoparticle approaches have been suggested to have the ability to partially overcome drug resistance by sustained drug release or by reducing the interaction of drug molecules with multidrug resistance (MDR) proteins (30, 31). Consistent with these approaches, some nanoparticle-based therapeu- tics have been approved for clinical use, and many others are under- going clinical trials (32, 33). Several taxane nanoformulations, includ- ing albumin-bound paclitaxel (e.g., Abraxane) and amphiphilic block copolymer-encapsulated paclitaxel (e.g., Genexol-PM), have been approved for clinical use (34–36).
Cabazitaxel (Jevtana; Sanoﬁ-Aventis), which is a potent mitotic inhibitor, has attracted rapidly increasing interest in preclinical studies
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Nanoparticle Enhances the Safety and Efﬁcacy of Cabazitaxel and clinical trials due to its potential to circumvent drug resistance induced by paclitaxel and DTX. The structure of cabazitaxel pro- vides this molecule with low afﬁnity for the MDR1 [or P-glyco- protein (P-gp), which is encoded by the ABCB1 gene] protein, thereby avoiding drug efﬂux (37, 38). Despite this advantage, high systemic toxicity of cabazitaxel has been observed in phase I studies, and its MTD has been established as only 25 mg/m2 in a 1-hour infusion (i.v. injection) every 3 weeks (39). Previously, we showed that tethering a polyunsaturated fatty acid (PUFA) such as doc- osahexaenoic acid (DHA) to cabazitaxel enables the spontaneous self-assembly of a DHA–cabazitaxel prodrug conjugate to create organic solvent-free supramolecular nanotherapeutics (40). “PUFAylation” of cabazitaxel not only enables this conjugate to self-assemble in water without any exogenous excipients but also enhances drug tolerability in animals. Thus, this conjugate was shown to be extremely effective with low toxicity, warranting further investigation.
To further optimize the delivery matrices, and to assess the ther- apeutic potential in drug-resistant cervical cancer, we formulated a DHA–cabazitaxel conjugate within the clinically approved copolymer poly(ethylene glycol)-block-poly(D,L-lactic acid) (PEG-b-PLA) to assemble dCTX NPs. Interestingly, the DHA–cabazitaxel conjugate showed higher compatibility with the PEG-b-PLA matrix compared with parent cabazitaxel, and this conjugate can be stably encapsulated in PEG-b-PLA–composed nanoparticles. The optimized formulation addresses essential requirements in terms of the stability, safety, and translational capacity for future evaluation in clinical trials. We examined the cytotoxicities of this new nanotherapy in DTX- resistant HeLa human cervical cancer cells. Furthermore, in a pre- clinical DTX-resistant HeLa tumor xenograft-bearing mouse model, compared with solution-based free drugs (i.e., DTX and cabazitaxel), the nanoparticles showed a signiﬁcantly improved antitumor efﬁcacy. Finally, detailed studies suggested that the systemic toxicity was substantially reduced in animals, enabling increased doses for clinical studies.
Materials and Methods
Materials and compounds
DTX and cabazitaxel were purchased from Knowshine Pharma- chemicals Inc. DHA was purchased from Tokyo Chemical Industry. The copolymer PEG-b-PLA was purchased from Advanced Polymer Materials Inc.
Preparation of DHA–cabazitaxel prodrug-loaded polymeric nanoparticles
The DHA–cabazitaxel conjugate was synthesized as described previously (40). The conjugate was obtained through one-step esteriﬁcation with an 80% yield from cabazitaxel and DHA. DHA– cabazitaxel prodrug-loaded nanoparticles were prepared using a nanoprecipitation method. Brieﬂy, cabazitaxel prodrugs (1 mg cabazitaxel equivalent) and PEG5k-b-PLA8k (19 mg) were dissolved in acetone (1 mL), and the solution was then added dropwise into deionized (DI) water (9 mL) while stirring. After stirring for 10 minutes, the remaining organic solvent was removed in a rotary evaporator under reduced pressure. Finally, the nanoparticle solution was concentrated with centrifugal ﬁlter devices (Amicon Ultra4, 10k molecular weight cut-off, Millipore Corp.) and washed with DI water. The particle size was measured by dynamic light scattering (DLS), and the drug concentration was determined by analytical high- performance liquid chromatography (HPLC).
Cell lines and cell culture
HeLa human cervical cancer cells and A549 human lung cancer cells were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 units/mL) and strepto- mycin (100 mg/mL). DTX-resistant HeLa (HeLa/DTX) cells and DTX- resistant A549 (A549/DTX) cells were maintained in RPMI 1640 medium. All cells were identiﬁed by short tandem repeat DNA ﬁngerprinting and cultured in a humidiﬁed incubator with 5% CO2 at 37◦C.
Cell apoptosis assay
FITC Annexin V Apoptosis Detection Kit (556547, BD Bios- ciences) was used to assess the apoptotic rate of cancer cells. Brieﬂy,
1 106 cells per well were seeded in 6-well plates overnight and exposed to drugs for 48 hours. Then, detached and attached cells were collected by trypsinization, washed twice with cold phosphate buffer saline (PBS, 0.01 mol/L phosphate, pH ¼ 7.4, 135 mmol/L NaCl, 2.7 mmol/L KCl, 1.5 mmol/L KH2PO4, and 8 mmol/L K2HPO4), and resuspended in 500 mL 1 binding buffer. Next, the cells were mixed with 5 mL FITC Annexin V and 5 mL propidium iodide (PI) and incubated for 15 minutes in the dark at room temperature. After incubation, the levels of apoptosis were analyzed with ﬂow cytometry (BD LSRFortessa). Untreated cells suspended in PBS were used as the control.
Cell-cycle distribution and DNA content were determined using PI staining and ﬂow cytometry. Cells (1 106 cells/well) were adhered to 6-well plates and treated with drugs for 24 hours. Next, the cells were harvested, washed with cold PBS, and ﬁxed in 75% ethanol at 20◦C overnight. Following incubation, the cells were washed with PBS twice and stained in 500 mL staining buffer containing 5 mL PI solution for 30 minutes in the dark at room temperature. Cell-cycle proﬁles were analyzed with ﬂow cytometry (BD LSRFortessa). Untreated cells suspended in PBS were used as the control.
Total cell lysates were prepared and fractionated on 10% SDS- PAGE gels and transferred to polyvinylidene diﬂuoride membranes. The membranes were blocked with 5% milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated overnight at 4◦C in the presence of anti-Bcl2, anti-survivin, anti-cleaved caspase 9, anti- cleaved PARP, anti-Cdc25c, anti-cyclin B1, anti-Cdc2, anti–p-Cdc2, and anti–b-actin primary antibodies (Cell Signaling Technology). After washing with TBST, the membranes were incubated with secondary antibodies for 1 hour at room temperature. Protein bands were detected using enhanced chemiluminescence (Fude Biological Technology). b-Actin was used for normalization of protein loading.
The experimental protocols were approved by the Ethics Commit- tee of the Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. All of the animal studies were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
In vivo pharmacokinetic study with dCTX NPs
Sprague–Dawley rats ( 250 g, n 6 in rats each group) were administered a single i.v. injection of free cabazitaxel (formulated in 1:1
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v/v polysorbate 80/ethanol) or dCTX NPs at a dose of 15 mg/kg (cabazitaxel equivalent). Blood samples were collected at 5, 15, and 30 minutes and at 1, 2, 4, 7, 24, and 48 hours in 1.5 mL centrifugal tubes containing sodium heparin. Plasma was separated from the blood by centrifuging for 10 minutes at 3,000 g and stored at 80◦C until analysis. To analyze the drug concentrations, the plasma samples (50 mL) were added to acetonitrile (300 mL) and ultrasonicated to extract cabazitaxel and the DHA–cabazitaxel conjugate. The drug concentrations were determined by reverse-phase HPLC (RP-HPLC). The pharmacokinetic parameters were obtained by ﬁtting the drug concentrations versus each time point to a noncompartmental pharmacokinetic model using PKSolver, an add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel (41).
In vivo biodistribution of dCTX NPs using near-infrared
Balb/c nude mice bearing HeLa/DTX tumor xenografts were used to evaluate the tissue biodistribution of dCTX NPs. We randomly divided the mice into two groups (n 6 mice in each group) when their tumor volumes reached approximately 100 mm3. The mice were administered a single injection of dCy5.5-labeled dCTX NPs (termed dCy5.5@dCTX NPs, 1 mg/kg Cy5.5-equivalent dose and 20 mg/kg of cabazitaxel equivalent dose) via the tail vein. Free Cy5.5 (1 mg/kg) was also injected as a reference. Whole-body and ex vivo NIR ﬂuorescence imaging were performed using a Xenogen IVIS Lumina imaging system (PerkinElmer). At time points of 24 and 48 hours post injection, the mice were sacriﬁced and the major organs and tumors were excised. Prior to ex vivo imaging, the tissues were washed with PBS 3 times to rule out the nanoparticle residue in the blood vessels. Finally, the tumor tissues were ﬁxed, processed into frozen sections, and stained with 4,6-diamidino-2-phenylindole. We observed the distri- bution of dCy5.5@dCTX NPs under a confocal ﬂuorescence micro- scope (ZEISS 880, Germany).
Antitumor activity in a preclinical DTX-resistant tumor xenograft-bearing mouse model
To evaluate the antitumor activity of the nanoparticles, 5-week-old Balb/c nude mice were subcutaneously implanted with cell suspen- sions containing 7 105 HeLa/DTX cells in the right ﬂank. The mice bearing HeLa/DTX tumor xenografts were established approximately 10 days after implantation. When the tumor volume reached approx- imately 100 mm3, the mice were randomly divided into six groups (n 6 mice in each group). The mice were i.v. injected with saline, free DTX (10 mg/kg), free cabazitaxel (5 mg/kg), and dCTX NPs (5, 10, and
15 mg/kg cabazitaxel equivalent) on days 0, 3, and 6. The tumor volume and body weight were logged until the tumors reached 2,500 mm3. The tumor volume (V) was evaluated by measuring the length (L) and width (W) and calculated using the following formula: V (L W2)/2, W being smaller than L. Finally, the mice were sacriﬁced by CO2 inhalation, and the tumor weights were obtained.
Evaluation of the drug toxicity in healthy ICR mice
The in vivo toxicity of free cabazitaxel and dCTX NPs was evaluated in healthy ICR mice (4–5 weeks old). The mice were i.v. injected with saline, free cabazitaxel, and dCTX NPs via the tail vein 3 times on days 0, 3, and 6. We obtained the body weight and peripheral blood cell counts, as well as the hepatorenal toxicity using biochemistry of mouse plasma. After the injection regimen, the major organs were excised and
subjected to hematoxylin and eosin (H&E) staining to analyze the damage induced by the drugs.
All quantitative data are presented as the mean SD. The statistical signiﬁcance between different groups was analyzed using an unpaired Student t test. A P value less than 0.05 was considered statistically signiﬁcant, and a P value less than 0.01 was considered highly signiﬁcant.
Preparation and characterization of DHA–cabazitaxel prodrug- loaded polymeric nanoparticles
We ﬁrst assessed whether the DHA–cabazitaxel conjugate can be formulated in clinically improved amphiphilic PEG-b-PLA copoly- mers. To test this, we followed a nanoprecipitation protocol by adding a mixture of DHA–cabazitaxel and PEG5k-b-PLA8k in acetone into DI water under stirring (Fig. 1A). Further removal of the organic solvent enabled to obtain a solution of dCTX NPs, which is expected to be systemically injectable for preclinical studies. Representative trans- mission electron microscope and scanning electron microscope images were shown in Fig. 1B, revealing the formation of spherical nanoparticles with a diameter of approximately 50 nm in water. Further DLS measurements suggested that the hydrodynamic dia- meters (DH) of dCTX NPs were approximately 73 nm (Fig. 1C). To exert the cytotoxic activity, free cabazitaxel must to be hydrolyzed from the DHA–cabazitaxel conjugate. Many forms of esterase play critical roles in the metabolism of lipids and bond cleavage of organic species. Speciﬁcally, previous studies suggest that various types of cancer cells highly express esterase, which could be used for selective conversion of ester prodrugs into therapeutically active drugs. Thus, we assessed the hydrolysis of the DHA–cabazitaxel conjugate analyzed by HPLC. Clearly, esterase activation substantially accelerated the conversion of the prodrug into active cabazitaxel, whereas the hydrolysis was negligibly observed in the absence of esterase (Fig. 1D). The results evidenced that once uptake by cancerous cells, intracellular esterase could trigger the release of active cabazitaxel. In addition, inhibition of premature hydrolysis and release of free drugs in the blood is a prerequisite to accomplish in vivo activity and to reduce side effects. We therefore evaluated the hydrolysis of the prodrug when dCTX NPs were incubated with rat whole blood or plasma (Fig. 1E). After 72-hour incubation, approximately 64% of the prodrugs remained intact. Considering that the circulation time of nanoparticles com- posed of PEG-b-PLA is usually less than 24 hours, we thus expect that this scaffold could constrain the drug payloads in nanoparticles during systemic circulation. Furthermore, under the current formulation, we determined the encapsulation efﬁciency and drug loading for dCTX NPs to be 99.8% and 4.7%, respectively.
Establishment of DTX-resistant cell lines
In this study, the DTX-resistant HeLa/DTX cancer cell line was established using chemosensitive HeLa human cervical cancer cells according to a previously established protocol. Pulsed exposure of HeLa cells to DTX with stepwise increments of time was performed (42). In the adaptation stage, the parent HeLa cells were exposed to DTX at a 15 nmol/L concentration for different times ranging from 0.5 to 48 hours (i.e., 0.5, 1, 2, 4, 12, 24, and 48 hours). Each treatment was repeated in triplicate. After each treatment, the surviving cells were harvested and expanded in DTX-free medium. In the following consolidation stage, previously surviving cells were
824 Mol Cancer Ther; 19(3) March 2020 MOLECULAR CANCER THERAPEUTICS
Nanoparticle Enhances the Safety and Efﬁcacy of Cabazitaxel
Preparation and characterization of DHA–cabazitaxel conjugate-formulated polymeric nanoparticles (dCTX NPs). A, Chemical structure of the DHA–cabazitaxel conjugate and schematic illustration of the nanoprecipitation protocol. B, Transmission electron microscope (TEM) image of dCTX NPs. Inset: scanning electron microscope (SEM) image of nanoparticles. C, The size distribution of dCTX NPs measured by DLS analysis. D, Hydrolytic release of free cabazitaxel from the DHA– cabazitaxel conjugate. The conjugate was incubated in the mixture solution of DMSO and HEPES buffer (1:3, v/v), and the hydrolytic event was monitored by analytical HPLC. E, Release of free cabazitaxel from the DHA–cabazitaxel conjugate incubated in whole blood or in plasma. The data are presented as the mean SD (n ¼ 3).
cultured in medium containing 15 nmol/L DTX for 72 hours until they expanded normally in this medium (42).
Eventually, we successfully established the DTX-resistant HeLa/DTX cells, as evidenced by the increased IC50. For instance,
the IC50 value of HeLa/DTX cells after 72 hours of DTX exposure was
63.5 6.0 nmol/L, which was 7.7-fold greater than that in the parent HeLa cells (Table 1). To further test the efﬁcacy of cabazitaxel, we established DTX-resistant A549 human lung cancer cell (A549/DTX)
Table 1. In vitro cytotoxicity of cabazitaxel dissolved in DMSO and dCTX NPs compared with DTX dissolved in DMSO after 72 hours of incubation (presented as IC50 SD in nmol/L)a.
Cabazitaxel 6.5 2.2 8.5 2.4 1.3 1.7 1.1 14.4 2.1 8.5
dCTX NPs 7.5 1.8 12.8 3.1 1.7 2.2 0.9 17.7 2.8 8.0
aDetermined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
bResistance fold (RF) was determined by dividing IC50 values of HeLa/DTX cells by those of HeLa cells.
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The cytotoxicity of dCTX NPs compared with cabazitaxel against DTX-resistant cell lines in vitro. A, Overexpression of P-gp protein was observed in A549/DTX and HeLa cells as determined by Western blot. B, HeLa/DTX cells were treated with varying concentrations (from 3 to 24 nmol/L) of cabazitaxel and dCTX NPs (cabazitaxel equivalent concentration) for 24, 48, 72, and 96 hours. Analysis of apoptosis (C and D) and cell cycle (E and F) of dCTX NPs of HeLa/DTX and A549/DTX cells. G and H, Quantiﬁcation of cell apoptosis– and cell-cycle–related proteins in A549/DTX and HeLa/DTX cells after treatment with drugs for 48 hours. The concentrations of drugs in HeLa/DTX and A549/DTX cells were 24 nmol/L for apoptosis and Western blot analysis and 12 nmol/L for cell-cycle analysis, respectively. The data are presented as the mean SD (n ¼ 3); ω, P < 0.05 and ωω, P < 0.01.
using the same protocol. As expected, the IC50 values in A549/DTX cells increased to 205.2 38.2 nmol/L after exposure to free DTX for 72 hours, indicating a 93.3-fold enhancement relative to DTX-sensitive A549 cells. To investigate the mechanism of drug resistance, we examined the expression of taxane resistance–associated P-gp in these cell lines by Western blot. A signiﬁcantly increased expression level of P-gp was observed in HeLa/DTX and A549/DTX cells compared with DTX-sensitive cancer cells (Fig. 2A).
In vitro cytotoxicity of cabazitaxel prodrug nanoparticles in DTX-resistant cells
Because of the low afﬁnity for P-gp, cabazitaxel has the potential to overcome drug resistance induced by paclitaxel and DTX. We thus assessed the activity of dCTX NPs compared with free DTX and free
cabazitaxel in DTX-sensitive and the established DTX-resistant cell lines. As shown in Table 1, the IC50 values in HeLa/DTX and A549/DTX cells after 72 hours of dCTX NP treatment were 12.8 3.1 and 17.7 2.8 nmol/L, respectively, suggesting that dCTX NPs possessed potent activity in DTX-resistant cells. Notably, the potency of dCTX NPs was in the same range as that of free cabazitaxel (i.e., the IC50 values of cabazitaxel were 14.4 2.1 and 8.5 5.4 in A549/DTX and HeLa/DTX cells, respectively). Furthermore, the growth inhibi- tion assay showed that dCTX NPs had a potent inhibitory effect in HeLa/DTX cells, and the effectiveness increased as the concentration increased when the treatment time ranged from 24 to 72 hours (Fig. 2B). Unexpectedly, compared with treatment with free cabazi- taxel, treatment with dCTX NPs for 96 hours showed a statistically greater inhibitory effect. This result likely indicated the sustained
826 Mol Cancer Ther; 19(3) March 2020 MOLECULAR CANCER THERAPEUTICS
Nanoparticle Enhances the Safety and Efﬁcacy of Cabazitaxel
release of therapeutically active cabazitaxel from the nanoparticles over time.
Efﬁcient apoptosis induction and G2–M cell-cycle arrest by cabazitaxel prodrug nanoparticles
Similar to other taxanes, cabazitaxel exerts cytotoxic effects through mitotic arrest by stabilizing microtubules, leading to cell death. To elucidate the mechanism of action, the cell apoptosis and cell-cycle progression induced by dCTX NPs were examined. DTX treatment induced a high level of apoptosis and effectively arrested the G2–M cell cycle in both sensitive A549 and HeLa cells, but showed negligible activities in HeLa/DTX and A549/DTX cells (Fig. 2C–F; Supplemen- tary Figs. S2 and S3). Compared with DTX, dCTX NPs increased the apoptotic rates of cells and promoted the accumulation of cells in the G2–M phase. For instance, in the HeLa/DTX cell line, the apoptotic rates for the DTX, free cabazitaxel, and dCTX NP treatments were 23.4%, 52.6%, and 49.6%, respectively. Moreover, the proportions of cells in the G2–M phase for the DTX, free cabazitaxel, and dCTX NP treatments were 11.1%, 69.1%, and 67.8%, respectively (Fig. 2E and F; Supplementary Fig. S2). Collectively, these cell-based results demon- strated that following the ligation of DHA and the nanoparticle formulation, the potency in inducing apoptosis and cell-cycle arrest by dCTX NPs compared with free cabazitaxel was not statistically decreased.
Furthermore, we conducted a detailed analysis of several regulatory proteins associated with drug-induced apoptosis and G2–M cell-cycle arrest. As shown in Fig. 2G and H, the exposure of cells to cabazitaxel and dCTX NPs led to highly efﬁcient cleavage of PARP, caspase 9. In addition, the downregulation of the antiapoptotic protein Bcl2, sur- viving, and the cell-cycle–related proteins Cdc25c, Cdc2, and cyclin B1 were conﬁrmed. Overall, our data demonstrated that cabazitaxel and dCTX NPs remained highly potent in DTX-resistant cancer cells by promoting apoptosis and G2–M phase arrest via the Bcl2/PARP and Cdc25c/Cdc2/cyclin B1 pathways.
We further studied the cellular uptake of dCTX NPs in HeLa/DTX cells. To visualize dCTX NPs under observation of ﬂuorescence microscope, a lipophilic ﬂuorescent probe, DiI, was loaded into dCTX NPs to form DiI-labeled dCTX NPs (termed DiI@dCTX NPs). HeLa/DTX cells were treated with free DiI and DiI@dCTX NPs and subjected to confocal laser ﬂuorescence microscopy (CLSM) obser- vation for cellular uptake as well as the intracellular distribution. As shown in Supplementary Fig. S4, we found that the ﬂuorescence signal distributed in the lysosomes as evidenced by the colocalization with the lysotracker after the treatment of DiI@dCTX NPs. By contrast, free DiI rapidly diffused into cells and randomly distributed in the cytosol and nucleus with no obvious colocalization with the lysotracker. These results indicated that dCTX NPs internalized into the cells through the endocytosis/lysosome pathway, whereas free DiI entered the cells by diffusion.
Pharmacokinetic studies of dCTX NPs
Premature release of drugs from nanocarriers during systemic circulation, which reduces drug delivery to tumor tissues and enhances systemic exposure of toxic drugs, remains a major obstacle for achieving favorable therapeutic efﬁcacy. Through the encapsulation of the DHA–cabazitaxel prodrug in long circulating PEG-b-PLA nanoparticles, the pharmacokinetic properties of drugs can be ratio- nally improved. To explore the role of PEG-b-PLA matrices as nanocarriers, we conducted pharmacokinetic studies by analyzing drug concentrations in the blood of Sprague–Dawley rats. Blood was obtained from the Sprague–Dawley rats at predetermined timepoints after single i.v. injection of dCTX NPs (at 15 mg/kg cabazitaxel equivalence) via the tail vein. The cabazitaxel concentrations in serum were determined by HPLC analysis. The concentration–time proﬁles of total cabazitaxel in plasma and related pharmacokinetic parameters are shown in Fig. 3A and B. Indeed, compared with the clinical formulation of free cabazitaxel, the PEG-b-PLA nanoparticles signif- icantly prolonged the drug retention and circulation in rats. Free cabazitaxel exhibited rapid clearance from the blood, making this agent untraceable in our HPLC system at 1 hour after injection. The area as extrapolated from the concentration versus time curve (AUC0-t) for dCTX NPs was 984.2 217.4 mg·h/mL, which was 55.9- fold greater than the plasma AUC0-t of the cabazitaxel formulation (Fig. 3B). On the basis of this substantial increase in AUC0-t, these results indicated that compared with free cabazitaxel, dCTX NPs showed extremely high drug accumulation in solid tumors.
Furthermore, we separately quantiﬁed the concentrations of
released cabazitaxel and DHA–cabazitaxel conjugate in blood samples. Unexpectedly, only a small portion ( 11%) of free cabazitaxel was hydrolyzed from dCTX NPs (Fig. 3C and D). The results suggested that the dCTX NP scaffold had excellent stability and showed neg- ligible hydrolysis and premature release in the blood after systemic administration. Limited drug exposure in the circulation likely sug- gests a low systemic toxicity.
Tumor-speciﬁc nanoparticle accumulation
Next, we investigated the retention and tissue distribution of our nanotherapy by conducting near infrared (NIR) ﬂuorescence imaging in HeLa/DTX xenograft-bearing mice. To mimic the tethering of PUFA to drugs, the NIR dye Cy5.5 was ligated to DHA, and this conjugate was coassembled with DHA–cabazitaxel in PEG-b-PLA matrices (termed dCy5.5@dCTX NPs). For comparison, free Cy5.5 dissolved in the clinical formulation of cabazitaxel was analyzed. Following a single i.v. injection, whole-body in vivo ﬂuorescence imaging was conducted. The NIR signal decayed rapidly in mice that received free Cy5.5, but the NIR signal did not decay rapidly in the dCy5.5@dCTX NP–treated mice (Fig. 4A). Moreover, in dCy5.5@dCTX NP–treated mice, strong NIR ﬂuorescence was observed in the tumor regions. Furthermore, we determined the quantity of Cy5.5 in the organs to indicate the distribution proﬁles of dCy5.5@dCTX NPs. At 24 and 48 hours post injection, we excised the major organs and tumors from the mice for ex vivo imaging and quantiﬁed the ﬂuorescence intensities in each organ (Fig. 4C and D). As expected, free Cy5.5 showed negligible tumor accumulation, but high accumulation in the liver and kidney was observed. Noticeably, dCy5.5@dCTX NPs showed higher accumulation than free Cy5.5 in tumors.
Furthermore, we examined the tumor cross-section under CLSM. The CLSM results clearly revealed that strong signals derived from Cy5.5 were distributed throughout the entire tumor tissues in the mice administered dCy5.5@dCTX NPs (Fig. 4E and F). However, the tumor tissues excised from the free Cy5.5-injected mice exhibited negligible Cy5.5 signals.
In addition to these ﬂuorescence imaging studies, we further comparatively analyzed cabazitaxel accumulation in tumors. After a single injection of cabazitaxel and dCTX NPs, the tumor tissues were excised and the concentration of drugs was determined by analytical HPLC. Noticeably, at both time points post administration, the tumors of mice receiving dCTX NPs demonstrated statistically higher caba- zitaxel concentration when compared with free cabazitaxel injected in Jevtana-mimicking formulation (Fig. 4G). Moreover, we separately quantiﬁed the amounts of released free cabazitaxel and nonhydrolytic
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In vivo pharmacokinetic studies of dCTX NPs. A and B, Plasma concentration–time proﬁles and pharmacokinetic parameters of dCTX NPs compared with free cabazitaxel following a single i.v. injection in Sprague–Dawley rats. The total amount of cabazitaxel molecules, including released cabazitaxel and intact DHA– cabazitaxel conjugate, in the plasma was extracted and analyzed by HPLC. Drugs were injected at a dose of 15 mg/kg (cabazitaxel-equivalent dose). C and D, A comparison of released free cabazitaxel and intact DHA-conjugate in the plasma of Sprague–Dawley rats after dCTX NPs were injected. In vivo plasma concentration– time proﬁles (C) and pharmacokinetic parameters (D) of free cabazitaxel and the respective intact DHA-conjugate after a single i.v. injection of dCTX NPs in rats. t1/2 indicates the half-life of the distribution phase; Cmax indicates maximum concentration; and AUC indicates the area under the concentration versus time curve. The data are presented as the mean SD (n ¼ 6).intact DHA–cabazitaxel conjugates in tumors (Fig. 4H). We con- ﬁrmed the presence of both drug forms, suggesting that the prodrug can be readily converted into the therapeutically active cabazitaxel in target tumors.
Collectively, these results were well correlated with the pharmaco- kinetic data, suggesting that our dCTX NP scaffold has the potential to enhance drug accumulation in tumors, with lower toxicity to normal tissues, making it a promising candidate for further investigation.
In vivo therapeutic efﬁcacy in a preclinical DTX-resistant tumor- bearing mouse model
Encouraged by the potent in vitro cytotoxicity and superior tumor accumulation, we thus explored the therapeutic efﬁcacy in Balb/c nude mice bearing subcutaneous DTX-resistant HeLa tumor xenografts. When the tumors grew to 100 mm3, we initiated the treatment by i.v. injecting of DTX, cabazitaxel, and dCTX NPs. Following three injec- tions, the tumor size and body weight were obtained (Fig. 5D). Unfortunately, the DTX treatment at a dose of 10 mg/kg resulted in rapid tumor growth, which was correlated with the in vitro cytotoxicity results showing that DTX lost its activity in DTX-resistant HeLa cancer cells. As shown in Fig. 5A–C, the administration of free cabazitaxel at a 5 mg/kg dose suppressed the tumor growth but additionally caused substantial body weight loss (approximately 22%) on day 12 post administration, suggesting severe systemic toxicity in Balb/c nude mice. A comparable therapeutic effect was achieved by administering dCTX NPs at a dose of 5 mg/kg (cabazitaxel equivalence); however, the
mice that were injected at this dose showed negligible body weight loss (Fig. 5D). The enhanced drug tolerability in animals inspired us to examine whether increasing the dose yields better therapeutic out- comes. Encouragingly, higher doses of dCTX NPs markedly delayed the growth of drug-resistant tumors. Speciﬁcally, treatment with dCTX NPs at 15 mg/kg was associated with the superior activity in this model. Administration of dCTX NPs continued to show durable tumor repression that persisted for at least 2 weeks after the last injection. At the end point of the therapeutic study, the mice were sacriﬁced, and the average weight of tumors excised from the mice that received 15 mg/kg (cabazitaxel-equivalent dose) of nanotherapy was
0.17 g, which was signiﬁcantly less than that of the tumors in mice that received cabazitaxel treatment (0.48 g, cabazitaxel vs. dCTX NPs, P < 0.01). More notably, increasing the dose of the nanotherapies compared with cabazitaxel substantially alleviated toxicity in animals, as indicated by no noticeable reduction in the body weight of the mice during the treatment with dCTX NPs. As shown in Fig. 5D, admin- istration of free cabazitaxel at 5 mg/kg led to approximately 22% body weight loss. However, dCTX NPs at 15 mg/kg (cabazitaxel equivalent) only produced a drop of approximately 11% in mouse body weight. We thus can safely conclude that the MTD of dCTX NPs was at least 3 times higher than that of free cabazitaxel in animals.
Histologic analysis of the excised HeLa/DTX tumors further val- idated the antitumor activity of dCTX NPs. H&E staining (Fig. 5E) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Fig. 5F) revealed that efﬁcient and extensive
828 Mol Cancer Ther; 19(3) March 2020 MOLECULAR CANCER THERAPEUTICS
Nanoparticle Enhances the Safety and Efﬁcacy of Cabazitaxel
In vivo biodistribution of dCy5.5@dCTX NPs in HeLa/DTX xenograft-bearing mice. A, In vivo whole-body NIR ﬂuorescence imaging of mice at 24 and 48 hours after i.
v. injection of free Cy5.5 dye and dCy5.5@dCTX NPs via the tail vein. The white dotted circles indicate the tumor regions. Ex vivo NIR ﬂuorescence imaging of the major organs (B) and tumors (C) excised from the mice. D, Quantitative analysis of ﬂuorescence intensities in the tumor region and major organs at 24 and 48 hours post administration. Representative ﬂuorescence images (E) and quantiﬁcation of ﬂuorescence intensities (F) of tumors obtained from the mice that were i.v. injected with free Cy5.5 and dCy5.5@dCTX NPs. The ﬂuorescence images were observed under a confocal laser scanning microscope. The scale bars represent 100 mm. The data are presented as the mean SD (n ¼ 4); ω, P < 0.05 and ωω, P < 0.01.
intratumoral apoptosis in the tumor tissues was elicited by the dCTX NP treatment (10 mg/kg, cabazitaxel equivalent), consistent with the observed reduction in the tumor volume. The percentage of TUNEL- positive cells in dCTX NP–treated mouse tumors increased compared with that of other treatments (Fig. 5G). IHC staining using the proliferation marker Ki67 further conﬁrmed the reduced intratumoral cell proliferation in the sections from the dCTX NP group (Fig. 5H), which was also veriﬁed by the quantiﬁcation of Ki67-positive cells (Fig. 5I). Therefore, these histologic analyses provided the evidence that compared with the cabazitaxel treatment (5 mg/kg), dCTX NPs at 10 mg/kg (cabazitaxel equivalent) exhibited superior cytotoxic effects.
In vivo safety of dCTX NPs
Finally, the safety proﬁles of the nanoparticle formulations were carefully examined in healthy mice. Clinically, cabazitaxel causes high systemic toxicity in patients with cancer. Neutropenia is one of the most severe side effects, which prevents increasing the dose of caba- zitaxel. We anticipate that the nanoparticle approach presented in this study could partially alleviate the in vivo toxicity of cabazitaxel when used in vivo. To validate this assumption, we performed a toxicity study in healthy ICR mice (n 10 mice in each group) using dCTX NPs compared with the clinical formulation of cabazitaxel. Figure 6A–C and Supplementary Table S1 show variations in the hematologic parameters in mice that received i.v. injections of dCTX NPs (10 and
AACRJournals.org Mol Cancer Ther; 19(3) March 2020 829Xie et al.
The therapeutic efﬁcacy of dCTX NPs was evaluated in a HeLa/DTX xenograft-bearing Balb/c nude mouse model. A, Changes in the tumor volume following treatment with saline (n 6), free DTX in its clinical formulation (10 mg/kg, n 6), cabazitaxel (5 mg/kg, n 6), dCTX NPs (5, 10, and 15 mg/kg cabazitaxel equivalent, n 6) for three successive injections on days 0, 3, and 6. B and C, Tumor weights and representative images of the excised tumors from each group. D, Changes in the body weights of the mice after the treatment. The data are presented as the mean SD (n 6); ω, P < 0.05 and ωω, P < 0.01. E, Representative images from H&E staining (E), TUNEL analysis (F), and Ki67 staining (H) of the excised tumors on day 21. The scale bars represent 100 mm in length. Quantiﬁcation of TUNEL-positive (G) and Ki-67–positive cells (I). Potent induction of apoptosis and inhibition of proliferation were observed in the dCTX NP–treated group, consistent with the H&E staining results.
830 Mol Cancer Ther; 19(3) March 2020 MOLECULAR CANCER THERAPEUTICS
Nanoparticle Enhances the Safety and Efﬁcacy of Cabazitaxel
Systemic toxicity of the nanotherapy com- pared with the clinical formulation of caba- zitaxel in ICR mice. A–C, WBC, NE, and LY counts in mice on days 0, 6, 9, and 15. The mice were administrated with dCTX NPs (10 and 20 mg/kg, cabazitaxel equivalent dose) 3 successive times via the tail vein. Saline and cabazitaxel were also injected as controls. Analysis of AST (D), ALT (E), total bilirubin (TBIL; F), ALP (G), creatinine (CR; H), and BUN (I) in the ICR mice on day 9 after three injections. The data are presented as the mean SD (n 6); ω, P < 0.05; ωω, P < 0.01; and ωωω, P < 0.001.
20 mg/kg, cabazitaxel equivalent) compared with those that received the same dosage of cabazitaxel. After the injection of cabazitaxel, the mice spontaneously exhibited a substantial reduction in the white blood cell (WBC) counts ( 70%), neutrophil leukocyte (NE) counts ( 95%), and lymphocyte (LY) counts ( 50%) on day 6 compared with these blood counts on day 0. As a result of dCTX NP administration, myelosuppression and a reduction in blood cells were observed, but the differences between day 6 and day 0 were not statistically signiﬁcant. In addition, after stopping the dosage, these hematologic parameters recovered to the normal range.
Furthermore, we analyzed a series of serological markers to indicate the presence of lesions in the liver and kidneys induced by the drugs. As shown in Fig. 6D–I, increased expression levels of aspartate amino- transferase (AST), alanine aminotransferase (ALT), alkaline phospha- tase (ALP), and blood urea nitrogen (BUN) were observed in the cabazitaxel-treated mice, indicating the acute hepatorenal toxicity. Notably, dCTX NPs show these types of changes for these indicators. The in vivo safety of dCTX NPs was further supported by the stable body weight of the ICR mice after receiving the treatment. Admin- istration of dCTX NPs at 10 mg/kg (cabazitaxel equivalence) exhibited stable body weight gain during the observation period. However, after injecting mice with cabazitaxel at a 10 mg/kg dose, a signiﬁcant body weight loss ( 16%) was observed (Fig. 7A). Finally, to assess drug toxicity, we performed histologic analyses of organs excised from the mice. As shown in Fig. 7B, the absence of signs of necrosis and cell
death in all organs indicated that the mice can tolerate dCTX NPs therapy at high doses. Unfortunately, renal damage was observed in the cabazitaxel-treated mice. Collectively, these results indicated that we successfully converted toxic cabazitaxel into a safe nanotherapy that is extremely well tolerated in animals by encapsulation of cabazitaxel prodrug in nanoparticles.
Microtubules are components of the cytoskeleton that are involved in many crucial cellular interphase functions, including maintenance of cell shape, intracellular transport, mitosis, motility, and cell signal transmission. They are composed of a-tubulin and b-tubulin hetero- dimers in dynamic equilibrium with tubulin polymers in cells. During cell division, the principal function of microtubules is the formation of the mitotic spindle (43). Therefore, the dynamic equilibrium is an attractive drug target. Taxane drugs bind to tubulin, thereby stabilizing microtubules and promoting tubulin polymerization (11). Eventually, these agents arrest quickly proliferating cells at the G2–M phase and lead to efﬁcient tumor cell apoptosis. Currently, FDA-approved taxane drugs that target the taxane-binding site include paclitaxel, DTX, and cabazitaxel. However, the clinical efﬁcacy of taxanes (e.g., pacli- taxel and DTX) is typically limited by the development of drug resistance. Repeated drug exposure causes cancer cells to evolve to overexpress the ATP-dependent drug efﬂux pump P-gp (44, 45).
AACRJournals.org Mol Cancer Ther; 19(3) March 2020 831Xie et al.
Toxicity evaluation of the nanotherapy compared with the clinical formulation of cabazitaxel in healthy ICR mice. A, Changes in the body weights were observed for 18 days after the three administrations of the drugs. The mice were i.v. injected with cabazitaxel and dCTX NPs (5 and 10 mg/kg, cabazitaxel equivalent dose). The data are presented as the mean SD (n 6). B, Representative images of H&E staining of the major organs.
Hence, overexpression of P-gp by tumor cells is responsible for constitutive and acquired resistance to taxanes. Accordingly, many efforts have been dedicated to developing new taxane derivatives that are not strong substrates for P-gp.
In addressing the issue of taxane resistance, cabazitaxel has emerged as a second-generation taxane. Compared with paclitaxel and DTX, cabazitaxel has been suggested to have a low afﬁnity for P-gp drug efﬂux, circumventing the resistance of current taxanes (37, 46). Many previous reports have suggested that cabazitaxel has high potency in a broad spectrum of taxane-resistant cancer cells according to in vitro cell-based assays (37, 47). Clinically, cabazitaxel has been approved by the FDA for treating the patients with metastatic castration-resistant prostate cancer (mCRPC; ref. 38). Compared with previous standard chemotherapeutic treatments, chemotherapy with DTX treatment provides a survival beneﬁt of approximately 2 to 3 months. Unfortu- nately, patients with mCRPC who had previously been treated with DTX become taxane resistance (48). Clinical data have shown that the patients receiving cabazitaxel have signiﬁcantly longer overall survival than those receiving other therapeutic modalities (49). Despite these favorable clinical outcomes, the success of using cabazitaxel has been limited in clinical practice due to the high toxicity of the drug. In phase
I studies, the MTD of cabazitaxel was established as only 25 mg/m2 in a 1-hour infusion every 3 weeks (39). This MTD is substantially lower than that of paclitaxel and DTX (175 and 60–100 mg/m2 for paclitaxel and DTX administration every 3 weeks, respectively; refs. 50, 51). However, similar to other taxanes, cabazitaxel is not soluble in aqueous solutions. To make cabazitaxel applicable for i.v. injections, surfactants and/or organic solvents are required. Currently, only one formulation of cabazitaxel in polysorbate 80 and ethanol (under the trademark Jevtana) has been approved for the clinical use. Although polysorbate 80 has relatively low toxicity, the use of this excipient inevitably causes severe hypersensitivity reactions (52, 53).
To mitigate these obstacles, we previously tethered the DHA motif to this potent agent through an ester linkage to create a DHA–cabazitaxel conjugate (40). Unexpectedly, despite the substan- tial hydrophobicity of the overall molecule, this prodrug is capable of self-assembling into nanoparticles without the use of excipients. After uptake by cells where a high concentration of esterases is presented, the prodrug undergoes cleavage of the ester bond, releasing therapeutically active cabazitaxel and a DHA moiety. The latter composite is abundant in the humans and plays an essential role in biological functions, obviating concerns over modiﬁcation-associated side effects.
832 Mol Cancer Ther; 19(3) March 2020 MOLECULAR CANCER THERAPEUTICS
Nanoparticle Enhances the Safety and Efﬁcacy of Cabazitaxel
Furthermore, our previous results demonstrated the effectiveness and low toxicity of this conjugate in animal models, making it a promising new drug candidate for further preclinical evaluation in taxane- resistant cancer. Previous studies have suggested that the formulation matrices have a substantial impact on the in vivo performance of nanoparticle therapeutics. Hence, with the aim of optimizing the delivery matrices and assessing the efﬁcacy in taxane-resistant tumors, a clinically approved copolymer matrix (i.e., PEG-b-PLA) with high stability in the blood and a low critical micelle concentration was used to formulate the DHA–cabazitaxel prodrug.
In DTX-resistant HeLa and A549 cells, dCTX NPs potently induced the cell apoptosis, arrested the cell cycle at the G2–M phase, and showed potency that was comparable with that of free cabazitaxel. Interestingly, compared with free cabazitaxel, treatment with dCTX NPs for 96 hours led to a statistically higher inhibitory activity (Fig. 2A). In general, prodrug constructs require additional cleavage and release steps to yield their active drug forms, decreasing the potency of prodrug formulations. In this study, we did not observe delayed in vitro potency. This lack of a ﬁnding could be attributed to the rapid release of active cabazitaxel under esterase-rich intracellular conditions because cabazitaxel is conjugated to DHA by a hydrolytic ester bond.
Furthermore, we explored the potential of using dCTX NPs in a preclinical mouse model bearing DTX-resistant cervical cancer xeno- grafts. Administration of DTX at 10 mg/kg showed limited antitumor efﬁcacy, indicating the maintenance of drug resistance when the tumor cells were grown in mice (Fig. 5). Interestingly, compared with cabazitaxel, dCTX NPs slightly improved the efﬁcacy (not statistically signiﬁcant) but markedly decreased drug toxicity in nude mice. Notably, we safely increased the dosage of dCTX NPs to 10 and 15 mg/kg, which enhanced the durability of tumor reduction. Finally, we administered dCTX NPs to healthy ICR mice via the tail vein to examine the tolerability of the nanoparticles. Compared with cabazi- taxel (formulated in 1:1 v/v polysorbate 80/ethanol), dCTX NPs were proven to have a better safety proﬁle, as indicated by a stable body weight and negligible changes in hematologic parameters (Figs. 6 and 7). In addition, the absence of liver and kidney dysfunction and the absence of signs of necrosis and cell death in the major organs (H&E staining) were conﬁrmed in the nanoparticle-treated mice, suggesting the marked safety of the nanoparticles. These results were well correlated with the in vivo pharmacokinetic analysis, strongly supporting the hypothesis that individual conjugates are stably encap- sulated within the hydrophobic core of the nanoparticles. Moreover, the long-circulation characteristics of PEG-b-PLA nanoparticles could extend the drug persistence in the blood, thus making the
nanoparticles preferentially accumulate in tumor lesions via the EPR effect. Detailed pharmacokinetic studies showed that the nanoparticle reservoir can impair the premature release of free cabazitaxel during circulation, supporting the markedly alleviated drug toxicity in ani- mals. Nonetheless, alleviation of the toxicity of the anticancer drug cabazitaxel facilitated the dose increases and thereby improved the efﬁcacy.
In conclusion, the data presented in this study provide compelling evidence that the integration of DHA–cabazitaxel into nanoparticles reduces relevant side effects by limiting the systemic exposure of free toxic drugs while enhancing the therapeutic efﬁcacy in a preclinical mouse model bearing a taxane-resistant cervical malignancy. The overall nanosystems (i.e., the formulation consisting of polymer matrices and encapsulated prodrug entities) are composed of FDA- approved materials without introducing additional uncertiﬁed mole- cules. Following further investigation, we aim to initiate early-phase clinical trials to assess the safety and efﬁcacy of this nanotherapy in patients with taxane-resistant cancer.
Disclosure of Potential Conﬂicts of Interest
No potential conﬂicts of interest were disclosed.
Conception and design: B. Xie, W. Han, H. Wang
Development of methodology: B. Xie, J. Wan, X. Chen, H. Wang Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): B. Xie, J. Wan, X. Chen
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): B. Xie, J. Wan, X. Chen, W. Han, H. Wang
Writing, review, and/or revision of the manuscript: B. Xie, J. Wan, W. Han,
H. Wang Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): B. Xie, J. Wan, X. Chen
Study supervision: W. Han, H. Wang
This work was supported by grants from the National Natural Science Foundation of China (grants 81773193 and 81571799 to H. Wang, and 81972745 and 81572361 to W. Han), the Zhejiang Province Preeminence Youth Fund (grant LR19H160002 to H. Wang), and the Ten Thousand Plan Youth Talent Support Program of Zhejiang Province (grant ZJWR0108009 to W. Han).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1. Cancer Genome Atlas Research Network, Albert Einstein College of Med- icine, Analytical Biological Services, Barretos Cancer Hospital, Baylor Col- lege of Medicine, Beckman Research Institute of City of Hope, et al. Integrated genomic and molecular characterization of cervical cancer. Nature 2017;543:378–84.
2. Cohen PA, Jhingran A, Oaknin A, Denny L. Cervical cancer. Lancet 2019;393: 169–82.
3. de Martel C, Plummer M, Vignat J, Franceschi S. Worldwide burden of cancer attributable to HPV by site, country and HPV type. Int J Cancer 2017;141:664–70.
4. Schiffman M, Doorbar J, Wentzensen N, de Sanjose S, Fakhry C, Monk BJ, et al. Carcinogenic human papillomavirus infection. Nat Rev Dis Primers 2016;2: 16086.
5. Goodman A. HPV testing as a screen for cervical cancer. BMJ 2015;350:h2372.
6. Elfstrom KM, Arnheim-Dahlstrom L, von Karsa L, Dillner J. Cervical cancer screening in Europe: quality assurance and organisation of programmes. Eur J Cancer 2015;51:950–68.
7. Mezei AK, Armstrong HL, Pedersen HN, Campos NG, Mitchell SM, Seki- kubo M, et al. Cost-effectiveness of cervical cancer screening methods in low- and middle-income countries: a systematic review. Int J Cancer 2017;141: 437–46.
8. Rein DT, Kurbacher CM, Breidenbach M, Schondorf T, Schmidt T, Konig E, et al. Weekly carboplatin and docetaxel for locally advanced primary and recurrent cervical cancer: a phase I study. Gynecol Oncol 2002;87:98–103.
9. Lee M-Y, Wu H-G, Kim K, Whan Ha S, Sung Kim J, Ah Kim I, et al. Concurrent radiotherapy with paclitaxel/carboplatin chemotherapy as a deﬁnitive treatment for squamous cell carcinoma of the uterine cervix. Gynecol Oncol 2007;104: 95–9.AACRJournals.org Mol Cancer Ther; 19(3) March 2020 833Xie et al.
10. Rowinsky EK. The development and clinical utility of the taxane class of antimicrotubule chemotherapy agents. Annu Rev Med 1997;48:353–74.
11. Schiff PB, Fant J, Horwitz SB. Promotion of microtubule assembly in vitro by taxol. Nature 1979;277:665–7.
12. Wang S, Qiu J, Shi Z, Wang Y, Chen M. Nanoscale drug delivery for taxanes based on the mechanism of multidrug resistance of cancer. Biotechnol Adv 2015; 33:224–41.
13. Wilhelm S, Tavares AJ, Dai Q, Ohta S, Audet J, Dvorak HF, et al. Analysis of nanoparticle delivery to tumours. Nat Rev Mater 2016;1:16014.
14. Zhang L, Gu FX, Chan JM, Wang AZ, Langer RS, Farokhzad OC. Nanoparticles in medicine: therapeutic applications and developments. Clin Pharmacol Ther 2008;83:761–9.
15. Maeda H, Tsukigawa K, Fang J. A retrospective 30 years after discovery of the enhanced permeability and retention effect of solid tumors: nextgeneration chemotherapeutics and photodynamic therapy—problems, solutions, and pro- spects. Microcirculation 2016;23:173–82.
16. Nakamura H, Etrych T, Chytil P, Ohkubo M, Fang J, Ulbrich K, et al. Two step mechanisms of tumor selective delivery of N-(2-hydroxypropyl) methacryla- mide copolymer conjugated with pirarubicin via an acid-cleavable linkage. J Control Release 2014;174:81–7.
17. Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res 1986;46:6387–92.
18. Saatchi K, Soema P, Gelder N, Misri R, McPhee K, Baker JH, et al. Hyper- branched polyglycerols as trimodal imaging agents: design, biocompatibility, and tumor uptake. Bioconjug Chem 2012;23:372–81.
19. Maeda H, Khatami M. Analyses of repeated failures in cancer therapy for solid tumors: poor tumor-selective drug delivery, low therapeutic efﬁcacy and unsus- tainable costs. Clin Transl Med 2018;7:11.
20. Maeda H. Polymer therapeutics and the EPR effect. J Drug Target 2017;25:781–5.
21. Nakamura H, Koziolová E, Chytil P, Tsukigawa K, Fang J, Haratake M, et al. Pronounced cellular uptake of pirarubicin versus that of other anthracyclines: comparison of HPMA copolymer conjugates of pirarubicin and doxorubicin. Mol Pharm 2016;13:4106–15.
22. Islam W, Fang J, Imamura T, Etrych T, Subr V, Ulbrich K, et al. Augmentation of the enhanced permeability and retention effect with nitric oxide–generating agents improves the therapeutic effects of nanomedicines. Mol Cancer Ther 2018;17:2643–53.
23. Strebhardt K, Ullrich A. Paul Ehrlich’s magic bullet concept: 100 years of progress. Nat Rev Cancer 2008;8:473–80.
24. Wang H, Xie H, Wang J, Wu J, Ma X, Li L, et al. Self assembling prodrugs by precise programming of molecular structures that contribute distinct stability, pharmacokinetics, and antitumor efﬁcacy. Adv Funct Mater 2015;25:4956–65.
25. Wan J, Qiao Y, Chen X, Wu J, Zhou L, Zhang J, et al. Structure-guided engineering of cytotoxic cabazitaxel for an adaptive nanoparticle formulation: enhancing the drug safety and therapeutic efﬁcacy. Adv Funct Mater 2018;28: 1804229.
26. Wang H, Xie H, Wu J, Wei X, Zhou L, Xu X, et al. Structure-based rational design of prodrugs to enable their combination with polymeric nanoparticle delivery platforms for enhanced antitumor efﬁcacy. Angew Chem Int Ed Engl 2014;53: 11532–7.
27. Mukalel AJ, Riley RS, Zhang R, Mitchell MJ. Nanoparticles for nucleic acid delivery: applications in cancer immunotherapy. Cancer Lett 2019;458:102–12.
28. Yu M, Zhang C, Tang Z, Tang X, Xu H. Intratumoral injection of gels containing losartan microspheres and (PLG-g-mPEG)-cisplatin nanoparticles improves drug penetration, retention and anti-tumor activity. Cancer Lett 2019;442:396–408.
29. Hoang B, Ernsting MJ, Tang WS, Bteich J, Undzys E, Kiyota T, et al. Cabazitaxel- conjugated nanoparticles for docetaxel-resistant and bone metastatic prostate cancer. Cancer Lett 2017;410:169–79.
30. Minko T, Kopeˇckov´a P, Pozharov V, Kopeˇcek J. HPMA copolymer bound adriamycin overcomes MDR1 gene encoded resistance in a human ovarian carcinoma cell line. J Control Release 1998;54:223–33.
31. Fang J, Sawa T, Maeda H. Factors and mechanism of “EPR” effect and the enhanced antitumor effects of macromolecular drugs including SMANCS. Adv Exp Med Biol 2003;519:29–49.
32. Caster JM, Patel AN, Zhang T, Wang A. Investigational nanomedicines in 2016: a review of nanotherapeutics currently undergoing clinical trials. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2017;9.
33. Wicki A, Witzigmann D, Balasubramanian V, Huwyler J. Nanomedicine in cancer therapy: challenges, opportunities, and clinical applications. J Control Release 2015;200:138–57.
34. Kinoshita R, Ishima Y, Chuang VTG, Nakamura H, Fang J, Watanabe H, et al. Improved anticancer effects of albumin-bound paclitaxel nano- particle via augmentation of EPR effect and albumin-protein interactions using S-nitrosated human serum albumin dimer. Biomaterials 2017;140: 162–9.
35. Kim TY, Kim DW, Chung JY, Shin SG, Kim SC, Heo DS, et al. Phase I and pharmacokinetic study of Genexol-PM, a cremophor-free, polymeric micelle- formulated paclitaxel, in patients with advanced malignancies. Clin Cancer Res 2004;10:3708–16.
36. Wolfram J, Ferrari M. Clinical cancer nanomedicine. Nano Today 2019;25: 85–98.
37. Galsky MD, Dritselis A, Kirkpatrick P, Oh WK. Cabazitaxel. Nat Rev Drug Discov 2010;9:677–8.
38. Mita AC, Figlin R, Mita MM. Cabazitaxel: more than a new taxane for metastatic castrate-resistant prostate cancer? Clin Cancer Res 2012;18:6574–9.
39. Mita AC, Denis LJ, Rowinsky EK, Debono JS, Goetz AD, Ochoa L, et al. Phase I and pharmacokinetic study of XRP6258 (RPR 116258A), a novel taxane, administered as a 1-hour infusion every 3 weeks in patients with advanced solid tumors. Clin Cancer Res 2009;15:723–30.
40. Wang H, Lu Z, Wang L, Guo T, Wu J, Wan J, et al. New generation nanome- dicines constructed from self-assembling small-molecule prodrugs alleviate cancer drug toxicity. Cancer Res 2017;77:6963–74.
41. Zhang Y, Huo M, Zhou J, Xie S. PKSolver: an add-in program for pharmaco- kinetic and pharmacodynamic data analysis in Microsoft Excel. Comput Methods Programs Biomed 2010;99:306–14.
42. Jiang D, Sui M, Zhong W, Huang Y, Fan W. Different administration strategies with paclitaxel induce distinct phenotypes of multidrug resistance in breast cancer cells. Cancer Lett 2013;335:404–11.
43. Nogales E. Structural insights into microtubule function. Annu Rev Biochem 2000;69:277–302.
44. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP- dependent transporters. Nat Rev Cancer 2002;2:48–58.
45. Fojo AT, Menefee M. Microtubule targeting agents: basic mechanisms of multidrug resistance (MDR). Semin Oncol 2005;32:S3–8.
46. Yared JA, Tkaczuk KH. Update on taxane development: new analogs and new formulations. Drug Des Devel Ther 2012;6:371–84.
47. Vrignaud P, Semiond D, Lejeune P, Bouchard H, Calvet L, Combeau C, et al. Preclinical antitumor activity of cabazitaxel, a semisynthetic taxane active in taxane-resistant tumors. Clin Cancer Res 2013;19:2973–83.
48. Gyawali B, Koomulli-Parambil S, Iddawela M. Continuous versus intermittent docetaxel for metastatic castration resistant prostate cancer. Crit Rev Oncol Hematol 2016;102:118–24.
49. de Bono JS, Oudard S, Ozguroglu M, Hansen S, Machiels JP, Kocak I, et al. Prednisone plus cabazitaxel or mitoxantrone for metastatic castration-resistant prostate cancer progressing after docetaxel treatment: a randomised open-label trial. Lancet 2010;376:1147–54.
50. Oudard S, Fizazi K, Sengelov L, Daugaard G, Saad F, Hansen S, et al. Cabazitaxel versus docetaxel as ﬁrst-line therapy for patients with metastatic castration- resistant prostate cancer: a randomized phase III trial-FIRSTANA. J Clin Oncol 2017;35:3189–97.
51. Eniu A, Palmieri FM, Perez EA. Weekly administration of docetaxel and paclitaxel in metastatic or advanced breast cancer. Oncologist 2005;10: 665–85.
52. Weiszhar Z, Czucz J, Revesz C, Rosivall L, Szebeni J, Rozsnyay Z. Complement activation by polyethoxylated pharmaceutical surfactants: Cremophor-EL, Tween-80 and Tween-20. Eur J Pharm Sci 2012;45:492–8.
53. Coors EA, Seybold H, Merk HF, Mahler V. Polysorbate 80 in medical products and nonimmunologic anaphylactoid reactions. Ann Allergy Asthma Immunol 2005;95:593–9834 Mol Cancer Ther; 19(3) March 2020 MOLECULAR CANCER THERAPEUTICS
Preclinical Evaluation of a Cabazitaxel Prodrug Using Nanoparticle Delivery for the Treatment of Taxane-Resistant Malignancies
Binbin Xie, Jianqin Wan, Xiaona Chen, et al.
Mol Cancer Ther 2020;19:822-834. Published OnlineFirst December 17, 2019.
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