Preclinical Antitumor Activity of Cabazitaxel, a Semi

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Author Manuscript Published OnlineFirst on April 15, 2013; DOI: 10.1158/1078-0432.CCR-12-3146 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Preclinical antitumor activity of cabazitaxel

1 Preclinical Antitumor Activity of Cabazitaxel, a Semi-

2 Synthetic Taxane Active in Taxane-Resistant Tumors

4 Patricia Vrignaud, Dorothée Sémiond, Pascale Lejeune, Hervé Bouchard, Loreley

5 Calvet, Cecile Combeau, Jean-François Riou, Alain Commerçon, François Lavelle, and

6 Marie-Christine Bissery

8 Authors’ Affiliation: Sanofi, Vitry-sur-Seine, France

10 Running title: Preclinical antitumor activity of cabazitaxel

12 Keywords: antiproliferative; taxane; docetaxel resistance; solid tumors; microtubule

14 Financial support: None

16 Corresponding author: Patricia Vrignaud, Sanofi Oncology, 13 quai Jules Guesde,

17 Vitry-sur-Seine, 94403 Cedex, France. Phone: +33 1 58 93 36 29; Fax: +33 1 58 93 34

18 71; Email: [email protected]

20 Potential conflicts of interest: All authors are employees of Sanofi or were employed

21 by companies subsequently acquired by Sanofi.

Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 15, 2013; DOI: 10.1158/1078-0432.CCR-12-3146 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Preclinical antitumor activity of cabazitaxel

1 Abstract

2 Purpose: Taxanes are important chemotherapeutic agents with proven efficacy in

3 human cancers, but their use is limited by resistance development. We report here the

4 preclinical characteristics of cabazitaxel (XRP6258), a semisynthetic taxane developed

5 to overcome taxane resistance.

6 Experimental Design: Cabazitaxel effects on purified tubulin and on taxane-

7 sensitive or chemotherapy-resistant tumor cells were evaluated in vitro. Antitumor

8 activity and pharmacokinetics of intravenously administered cabazitaxel were assessed

9 in tumor-bearing mice.

10 Results: In vitro, cabazitaxel stabilized microtubules as effectively as docetaxel,

11 but was ten-fold more potent than docetaxel in chemotherapy-resistant tumor cells (IC50

12 ranges: cabazitaxel, 0.013–0.414 µM; docetaxel, 0.17–4.01 µM). The active

13 concentrations of cabazitaxel in these cell lines were achieved easily and maintained for

14 up to 96 hours in the tumors of mice bearing MA16/C tumors treated with cabazitaxel at

15 40 mg/kg. Cabazitaxel exhibited antitumor efficacy in a broad spectrum of murine and

16 human tumors (melanoma B16, colon C51, C38, HCT 116 and HT-29, mammary

17 MA17/A and MA16/C, pancreas P03 and MIA PaCa-2, prostate DU145, lung A549 and

18 NCI-H460, gastric N87, head and neck SR475, and kidney Caki-1). Of particular note,

19 cabazitaxel was active in tumors poorly sensitive or innately resistant to docetaxel

20 (Lewis lung, pancreas P02, colon HCT-8, gastric GXF-209, mammary UISO BCA-1) or

21 with acquired docetaxel resistance (melanoma B16/TXT).

22 Conclusions: Cabazitaxel is as active as docetaxel in docetaxel-sensitive tumor

23 models but is more potent than docetaxel in tumor models with innate or acquired

Preclinical antitumor activity of cabazitaxel

1 resistance to taxanes and other chemotherapies. These studies were the basis for

2 subsequent clinical evaluation.

Preclinical antitumor activity of cabazitaxel

1 Translational Relevance

2 Mechanisms of resistance to taxanes in patients have not been fully elucidated. In

3 cell lines, overexpression of ATP-binding transporters, particularly P-glycoprotein, and

4 alteration of microtubule dynamics are the most common mechanisms of taxane

5 resistance. However, clinical data suggest that other mechanisms, including

6 dysfunctional regulation of apoptotic and intracellular signaling, may operate in tumors

7 escaping taxane therapy. To identify a docetaxel derivative with activity after taxane

8 failure, we developed a clinically relevant docetaxel-resistant tumor model, mimicking

9 tumor resistance development in patients who initially respond to docetaxel, but develop

10 resistance over time. Cabazitaxel was selected from 450 derivatives based on activity in

11 this model. Clinical proof of principle was achieved in a Phase II study in patients with

12 taxane-resistant metastatic breast cancer and a Phase III study in metastatic hormone-

13 refractory prostate cancer post-docetaxel therapy. The current study extends the

14 characterization of cabazitaxel, demonstrating wide-ranging in vitro and in vivo antitumor

15 activity.

Preclinical antitumor activity of cabazitaxel

1 Introduction

2 Microtubules are highly dynamic cytoskeletal fibers composed of two tubulin

3 subunits (α and β). The polymerization and depolymerization of these molecules are

4 crucial processes, not only to mitosis but also to intracellular trafficking. Microtubules are

5 the main target of taxanes, which bind to a specific binding site on the tubulin β-subunit

6 (1, 2). The taxanes paclitaxel and docetaxel suppress microtubule dynamics by

7 promoting tubulin assembly and stabilizing microtubules (3), blocking mitosis at the

8 metaphase/anaphase transition, which results in cell death (4) (Supplementary Fig.

9 S1[a] and [b]). By stabilizing microtubules, taxanes also impact intracellular trafficking.

10 This was recently reported as one of the main mechanisms of taxane action in prostate

11 cancer, where taxanes were shown to inhibit nuclear translocation of the androgen

12 receptor, thereby preventing androgen receptor transcriptional activity and leading to

13 prostate cancer cell death (5).

14 Paclitaxel and docetaxel form the backbone of both first-line and salvage

15 chemotherapy regimens for patients with a wide variety of tumor types. Paclitaxel is

16 indicated for first-line treatment of ovarian, breast and lung cancer, and for second-line

17 treatment of AIDS-related Kaposi’s sarcoma (6). Docetaxel is indicated for first-line

18 treatment of breast, head and neck, gastric, lung and prostate cancer, and for second-

19 line treatment of breast cancer (7). However, the utility of both paclitaxel and docetaxel

20 is limited by the development of tumor resistance (8–10). During the last two decades,

21 considerable efforts have been made to understand, and develop new agents to

22 overcome, taxane resistance.

23 Cabazitaxel (RPR116258; XRP6258; TXD258; Jevtana®) is a new semisynthetic

24 taxane derived from 10-deacetylbaccatin III, which is extracted from European yew

25 needles (11) (Supplementary Fig. S1[c]). Cabazitaxel was identified using a three-step

Preclinical antitumor activity of cabazitaxel

1 screening process, assessing activity against microtubule stabilization, in vitro activity in

2 resistant cell lines and in vivo activity in a tumor model in which docetaxel resistance had

3 been induced in vivo. This article describes the development and characterization of the

4 in vivo-induced docetaxel-resistant tumor model, the mechanism of action of cabazitaxel

5 on microtubules, and its preclinical evaluation in a wide range of taxane-sensitive and

6 taxane-resistant cell lines, both in vitro and in vivo.

Preclinical antitumor activity of cabazitaxel

1 Materials and Methods

2 Tubulin polymerization 3 The effects of cabazitaxel on tubulin polymerization and cold-induced microtubule

4 depolymerization were evaluated using tubulin purified from porcine brain (12, 13).

5 Tubulin was used at a concentration of 6 µM for polymerization assays (at 37°C) and

6 9 µM for depolymerization assays (at 8°C). Rates of polymerization/depolymerization

7 were measured by optical density (OD) at 350 nm and were expressed in ΔOD/min.

8 Upper and lower limits for drug concentrations reducing polymerization lag time by 50%

9 (LT50) and inhibiting cold-induced disassembly by 50% (dIC50) were determined.

10 Microtubule and enzymatic parameters in tumors 11 Microtubule parameters in B16 and B16/TXT tumors were characterized using rt-

12 PCR analysis of at least two samples per tumor. PCR values in arbitrary units were

13 obtained for the following genes: total α tubulin (TUBA), total β tubulin (TUBB), TUBB2,

14 TUBB3, TUBB4A, TUBB4B and TUBB7P.

15 Glutathione S-transferase (GST) activity was assayed as previously described

16 using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate (14). Formation of the CDNB

17 glutathione (GSH) conjugate by cytosols was measured continuously in a

18 spectrophotometer at 340 nm. The results were expressed as the quantity of CDNB

19 conjugated per minute per milligram of cytosolic protein (nmol/min/mg).

20 Total GSH concentration was determined as the sum of the reduced (GSH) and

21 oxidized (GSSG) forms of glutathione (15). In this assay, the sum of the reduced and

22 oxidized forms of glutathione is determined using a kinetic assay in which catalytic

23 amounts of GSH or GSSG and glutathione reductase bring about the continuous

24 reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by NADPH. The reaction rate is

Preclinical antitumor activity of cabazitaxel

1 proportional to the concentration of GSH below 2 µM. The formation of 5-thio-2-

2 nitrobenzoate was analyzed using a spectrophotometer at 412 nm. The results were

3 expressed as concentration per milligram of protein (nmol/mg).

4 Cytochrome P450 3A (CYP3A) levels were determined using the Amersham

5 ELISA system (code RPN 271, Amersham, Les Ulis, France). This assay uses a rabbit

6 primary antibody specific for rat CYP3A, a secondary conjugate of anti-rabbit Ig and

7 horseradish peroxidase antibody, and tetramethylbenzidine substrate. The horseradish

8 peroxidase color that develops is proportional to CYP3A levels. This assay was

9 validated against mouse CYP3A by the manufacturer. Protein concentrations of

10 microsomes, cytosols and homogenates were determined by the bicinchoninic acid

11 assay (16) using a commercial preparation (Pierce BCA Protein Assay Reagent).

13 In vitro antiproliferative activity 14 The HL60/TAX cell line (17) was a kind gift from Dr K. Bhalla (Medical University of

15 South Carolina, Charleston, CS, USA). Calc18/TXT and P388/TXT were developed

16 internally from P388 or Calc18 parental cell lines. The P388/TXT cell line was selected

17 by mutagenesis with ethyl methane sulfonate and soft agar cloning in the presence of

18 0.06 µM docetaxel. The Calc18/TXT cell line was established by 6-month exposure to

19 increasing concentrations of docetaxel (up to 0.019 µM). The cross-resistance pattern of

20 these two cell lines is shown in Supplementary Table S1. The other tumor cell lines were

21 obtained from the National Cancer Institute (NCI).

22 Parental and resistant tumor cells were incubated with different drug

23 concentrations for 96 hours at 37°C; cell viability was measured in quadruplicate using

24 neutral red uptake (18). The resistance factor for each drug was calculated by dividing

25 the mean half-maximal inhibitory concentration (IC50) in resistant cells by the mean IC50

Preclinical antitumor activity of cabazitaxel

1 in sensitive/parental cells, using data from at least three independent experiments.

2 Relative expression of ABCB1 mRNA was determined by northern blotting using a

3 human ABCB1 gene probe.

4 Antitumor activity in tumor-bearing mice 5 All experimental procedures were approved by Sanofi, Southern Research Institute

6 (SRI; Birmingham, AL, USA), and Molecular Imaging Research (MIR) Preclinical

7 Services (Ann Arbor, MI, USA) Laboratory Animal Care and Use committees. Protocol

8 design, chemotherapy techniques and methods of data analysis have been described

9 previously (19–21). Briefly, tumors were implanted subcutaneously (SC) and bilaterally

10 on Day 0. Animals were randomly assigned to treatment (T) or control (C) groups.

11 Tumors were measured using a caliper two to five times weekly (according to tumor

12 growth rate) until the tumor reached 2000 mm3. Tumor volumes were estimated from

13 two-dimensional measurements using the formula: tumor volume (mm3) = [length (mm) ×

14 width² (mm²)]/2. The day of death was recorded, and thoracic and abdominal cavities

15 were examined macroscopically to assess probable cause of death.

16 Mice: C57BL/6, B6D2F1 and Swiss nude mice were bred at Iffa Credo (Les Oncins,

17 France); C3H/HeN, BALB/c, BALB/c nude and SCID mice were bred at Charles River

18 (Les Oncins, France); and ICR and NCR nude mice were bred at Taconic (Hudson, NY,

19 USA). All mice weighed > 18 g at the start of treatment and had free access to food and

20 water.

21 Drugs: Cabazitaxel (RPR116258; XRP6258; TXD258; Jevtana®) and docetaxel

22 (RP 56976) were prepared by mixing one volume of ethanol stock solution, one volume

23 of polysorbate 80, and 18 volumes of 5% glucose in sterile water. Solutions were

24 administered intravenously (IV) as a slow bolus (0.4 mL/mouse). Drug doses were

25 adjusted based on body weight at start of treatment. For cytotoxic compounds, such as

Preclinical antitumor activity of cabazitaxel

1 docetaxel and cabazitaxel, a dose–response evaluation was performed in each trial to

2 determine the highest non-toxic dose (HNTD), defined as the highest drug dose inducing

3 < 20% body weight loss with no drug-related deaths. Animal body weights included the

4 tumor weights.

5 Tumor models: Murine tumors were obtained from Dr Corbett (Wayne State University,

6 Detroit, MI, USA) and included colon C51 and C38 (19), pancreas P02 and P03 (22),

7 mammary MA17/A and MA16/C (23), Lewis lung (24) and melanoma B16 (20). Tumors

8 were maintained by serial passage in the mouse strain of origin. B16/TXT was isolated

9 by treating C57BL/6 mice bearing docetaxel-sensitive B16 melanoma at the HNTD of

10 docetaxel (60 mg/kg) for 27 passages, until the B16 tumor acquired full resistance to

11 docetaxel. Human tumor cell lines were obtained from ATCC (Rockville, MD, USA) and

12 included prostate Du145 (25), lung NCI-H460 (26) and A549 (27), pancreas MIA PaCa2

13 (28), and colon HT-29 (29), HCT 116 (30) and HCT-8 (31). Mammary UISO-BCA-1 (32)

14 and gastric GXF-209 (33) tumors were obtained from SRI and gastric N87 (34) tumors

15 from MIR Preclinical Services. Murine tumors were grafted into syngenic mice and

16 human tumors were xenografted into immunocompromised mice.

17 Plasma pharmacokinetics and tumor distribution: Cabazitaxel concentrations in

18 plasma and tumor tissue were evaluated in mice bearing advanced-stage (400 mm3)

19 murine mammary adenocarcinoma MA16/C after administration of the HNTD of

20 cabazitaxel (40 mg/kg). Mice were treated on Day 8 after SC tumor implantation with a

21 single 45-second IV infusion of cabazitaxel in a polysorbate 80/ethanol/5% glucose

22 solution, with a dosing volume of 25 mL/kg and a rate of infusion of 1 mL/min. Blood and

23 tumor samples were collected from three animals per sampling time at 2, 5 and 15

24 minutes, and 2, 4, 8, 12, 24, 48, 96 and 168 hours after cabazitaxel treatment.

25 Cabazitaxel concentrations were analyzed by liquid chromatography–tandem mass

Preclinical antitumor activity of cabazitaxel

1 spectrometry, with limits of quantification of 2.5 ng/mL in plasma and 25 ng/g in tumor

2 tissue. Pharmacokinetic parameters were determined using WinNonLin software,

3 Version 1.0 (Scientific Consulting Inc., Apex, NC, USA), using a non-compartmental

4 infusion model.

5 Assessments of antitumor activity: Several endpoints were used. Tumor growth delay

6 (T−C) was defined as the difference between tumors in the T and C groups in the

7 median time (days) to reach a predetermined volume (750–1000 mm3). Tumor doubling

8 time (Td) in days was estimated from log linear tumor growth during the exponential

9 phase (range 100–1000 mm3). Log cell kill was calculated using the formula (T−C)/(3.32

10 x Td), with antitumor activity defined as a log cell kill value ≥ 0.7 (21). SRI score was

11 used to categorize antitumor activity based on log cell kill values, as follows: < 0.7 = −

12 (inactive); 0.7–1.2 = +; 1.3–1.9 = ++; 2.0–2.8 = +++; > 2.8 = ++++ (highly active).

13 Complete tumor regression (CR) was defined as tumor regression below the limit of

14 palpation (62 mm3). Animals without palpable tumors at the end of the study were

15 declared tumor-free survivors (TFS) and were excluded from the T-C value calculation.

16 Statistical analysis was performed using either a pairwise Wilcoxon rank sum test, with

17 p-value adjustment by the Holm method (N87 study), or by log rank multiple

18 comparisons test versus control (with Bonferroni–Holm correction for multiplicity) on

19 individual values for time to reach a prespecified tumor size for treated and control

20 groups (UISO BCA-1 study). A probability value of less than 5% (p < 0.05) was

21 considered significant.

Preclinical antitumor activity of cabazitaxel

1 Results

2 Isolation and characterization of B16/TXT, a docetaxel-resistant 3 melanoma 4 To identify taxane derivatives with activity following taxane failure, a docetaxel-

5 resistant tumor model (B16/TXT) was developed to mimic the gradual development of

6 resistance to docetaxel observed in some patients following an initial tumor response to

7 the agent. Mice bearing the sensitive murine B16 melanoma were treated with docetaxel

8 at the HNTD (60 mg/kg per passage; log cell kill 1.7; Table 1). Resistance occurred very

9 slowly, with 27 passages over 17 months needed to obtain a fully docetaxel-resistant

10 tumor (log cell kill < 0.7). B16/TXT was found to have similar Td (1.3–2 days) and

11 histologic characteristics to the parental B16 tumor. Cross-resistance (no antitumor

12 activity) was observed to the tubulin-binding drugs paclitaxel, vincristine and vinblastine,

13 but not to cyclophosphamide (log cell kill 2.9 in B16 versus 3.0 in B16/TXT), CCNU (log

14 cell kill 3.7 in B16 versus 4.7 in B16/TXT) and etoposide (log cell kill 1.2 in both).

15 B16/TXT was partially cross-resistant to doxorubicin (log cell kill 2.4 in B16 versus 0.9 in

16 B16/TXT). There was no difference between the docetaxel-sensitive and docetaxel-

17 resistant B16 tumors either in factors involved in drug resistance, such as GST activity

18 (B16: 0.42 ± 0.03 µmol/min/mg protein; B16/TXT: 0.39 ± 0.04 µmol/min/mg protein) and

19 glutathione content (GSH; B16: 21.7 ± 8.1 µmol/mg protein; B16/TXT: 21.2 ± 2.5

20 µmol/mg protein), or in activity of CYP3A, involved in TXT metabolism (B16: 2.4 ± 0.8

21 µg/mg protein; B16/TXT: 2.9 ± 0.06 µg/mg protein). Moreover, no overexpression of P-

22 glycoprotein was found in B16/TXT, either by flow cytometry or western blot analyses

23 (data not shown). Analyses by rt-PCR of microtubule components revealed that

24 B16/TXT expressed 3.13-fold higher levels of TUBB3 than the docetaxel-sensitive

25 parental B16 tumor, whereas levels of other microtubule parameters were similar

Preclinical antitumor activity of cabazitaxel

1 (Supplementary Table S2). As noted above, this model was pivotal in the selection of

2 cabazitaxel, the characteristics of which are described hereafter.

3 Microtubule stabilization 4 Cabazitaxel had similar efficiency compared with docetaxel for reducing the lag

5 time for tubulin assembly (LT50 = 0–0.1 µM for both) and the rate of cold-induced

6 microtubule depolymerization (dIC50 = 0.1–0.25 µM for both) in vitro (Table 2).

7 In vitro antiproliferative activity in chemotherapy-sensitive and 8 chemotherapy-resistant cell lines 9 Cabazitaxel demonstrated similar antiproliferative activity compared with docetaxel

10 in cell lines sensitive to chemotherapy (murine leukemia P388, human tumor HL60 and

11 KB, and breast Calc18), as shown by the similar IC50 ranges across different cell types

12 (cabazitaxel, 0.004–0.041 μM; docetaxel, 0.008–0.079 µM; Table 3). In P-glycoprotein-

13 expressing cell lines with in vitro acquired resistance to taxanes (P388/TXT, Calc18/TXT

14 and HL60/TAX) or to other chemotherapy agents (P388/DOX, P388/VCR and KBV1),

15 cabazitaxel was found to be more active than docetaxel (IC50 ranges: cabazitaxel,

16 0.013–0.414 µM; docetaxel, 0.17–4.01 µM). Resistance factors (an indication of the

17 difference in drug concentrations needed to inhibit resistant versus sensitive/parental cell

18 lines) were 2–10 for cabazitaxel and 5–59 for docetaxel. Cell lines expressing moderate

19 levels of P-glycoprotein (P388/TXT, P388/VCR, HL60/TAX and Calc18/TXT), which may

20 be more clinically representative, had minimal cross-resistance to cabazitaxel

21 (resistance factors = 2–4).

22 Plasma pharmacokinetics and drug distribution in tumors 23 The pharmacokinetic profile of cabazitaxel was evaluated in mice bearing docetaxel-

24 sensitive murine mammary MA16/C adenocarcinoma tumors. Cabazitaxel was highly

25 active in this tumor model, inducing CRs in 80% of mice and having a log cell kill of 3.7

26 at the HNTD of 40 mg/kg (Table 1). This antitumor activity was consistent with drug

Preclinical antitumor activity of cabazitaxel

1 uptake into the tumor, which was both rapid (maximum drug concentrations were

2 reached 15 minutes post-dosing) and sustained (at 48 hours post-dose, cabazitaxel

3 concentrations were 40-fold higher in the tumor versus plasma; Fig. 1). Ratios of

4 cabazitaxel exposure in tumors versus plasma were 1.6 from 0 to 48 hours and 2.9 over

5 the entire experimental period. Cabazitaxel concentrations were maintained above the

6 range of cellular antiproliferative IC50 values (0.004–0.041 μM [see Table 3],

7 corresponding to 3–29 ng/mL, 4-day exposure) for 24 hours in plasma and 96 hours in

8 the tumor.

9 Schedule of administration 10 The optimal schedule of cabazitaxel administration in vivo was initially determined

11 by assessing the total dose that could be injected without undue toxicity for different

12 schedules in non-tumor-bearing B6D2F1 female mice (Supplementary Table S3). Three

13 schedules of IV cabazitaxel were administered: intermittent (Days 1 and 5 [A1]), daily

14 (Days 1–5 [A2]) and split dose (Days 1–5, three times daily [A3]). HNTDs were 58 mg/kg

15 (A1), 29 mg/kg (A2) and 12 mg/kg (A3), suggesting a trend for schedule dependency.

16 These results indicate that, compared with intermittent treatment (A1) of the same

17 duration, the daily (A2) and split-dose (A3) schedules require 2-fold and 4.8-fold dose

18 reductions, respectively. In addition, host recovery time (time from last treatment to

19 recovery of initial body weight) was shorter with the intermittent schedule (A1; 8 days)

20 compared with the daily (A2; 12 days) and split-dose (A3; 15 days) schedules. Results of

21 these dose-scheduling studies were consistent with further evaluations performed in

22 C3H/HeN mice bearing mammary adenocarcinoma MA17/A tumors, which showed a

23 4.5-fold lower HNTD for a split-dose schedule (Days 3–7, three-times daily; HNTD 42

24 mg/kg) compared with an intermittent schedule (Days 3 and 7; HNTD 9.3 mg/kg)

25 (Supplementary Table S4). Cabazitaxel antitumor activity against MA17/A tumors was

26 also lower with the split-dose schedule compared with the intermittent schedule (log cell

Preclinical antitumor activity of cabazitaxel

1 kill 1.3 versus 4.6, respectively). These differences between split-dose and intermittent

2 schedules were confirmed in a second tumor model, murine mammary adenocarcinoma

3 MA16/C, which showed a 4.2-fold reduction in HNTD (11 versus 46 mg/kg, respectively)

4 and decreased antitumor activity (log cell kill 1.6 versus 5.4, respectively). As the

5 intermittent schedule allowed the highest drug dose to be administered, with the best

6 host recovery, and had the greatest antitumor activity of the schedules tested,

7 intermittent dosing was selected for further evaluation.

8 Antitumor activity in docetaxel-sensitive tumors 9 The antitumor properties of cabazitaxel in vivo were evaluated using murine

10 tumors grafted in syngenic mice and human tumors xenografted in immunocompromised

11 mice (Tables 1 and 4). Cabazitaxel was found to be very active against murine B16

12 melanoma (log cell kill 2.1 at HNTD of 20 mg/kg injection on days 3, 5 and 7), murine

13 colon adenocarcinoma C51 (log cell kill 2.6 at HNTD of 9.3 mg/kg injection on days 4, 6,

14 and 8), and mammary adenocarcinomas MA16/C and MA17/A (reported above).

15 Cabazitaxel was highly active, with complete regressions observed against the

16 advanced-stage murine tumors colon C38 (5/5 CR; 5/5 TFS) and pancreas P03 (5/5 CR;

17 4/5 TFS) as well as human tumor xenografts including: prostate DU145 (6/6 CR; 5/6

18 TFS); colon HCT116 (7/7 CR; 2/7 TFS) and HT29 (6/6 CR); pancreas MIAPaCa-2 (6/6

19 CR; 6/6 TFS); breast Calc18 (5/8 TFS); lung NCI-H460 (2/6 CR) and A549 (2/6 CR);

20 head and neck SR475 (6/6 CR; 6/6 TFS); and kidney Caki-1 (5/6 CRs). In most of the

21 above models, cabazitaxel and docetaxel exhibited similar antitumor activity.

22 Dose–response effects were examined in the advanced human gastric carcinoma

23 N87 model, a tumor expressing HER2 (34) (Fig. 2a). At the HNTD (24.4 mg/kg on days

24 27, 31 and 35), cabazitaxel was highly active and delayed tumor growth by 101 days

25 (log cell kill > 6, 1/8 TFS; p < 0.0001). The two dose levels below the HNTD (15 and 9.3

Preclinical antitumor activity of cabazitaxel

1 mg/kg per injection) also had a high level of antitumor activity (4.5 and 2.5 log cell kill; p

2 < 0.0001 and 0.091, respectively). In comparison, docetaxel was also highly active at the

3 HNTD, but to a lesser extent (T-C = 67 days; log cell kill 4.5; 1/8 TFS; p < 0.0001), and

4 the activity was observed only at one dose below the HNTD. Thus cabazitaxel had a

5 greater therapeutic index (three active dose levels) than docetaxel (two active dose

6 levels) in this gastric tumor model.

7 Antitumor activity in tumor models poorly or not sensitive to 8 docetaxel 9 Cabazitaxel showed antitumor activity against the fully docetaxel-resistant

10 B16/TXT tumor model (log cell kill 2.1 in B16 versus 1.3 in B16/TXT), but no antitumor

11 activity was obtained against the P-glycoprotein-overexpressing tumor Calc18/TXT in

12 which docetaxel resistance was induced in vitro (log cell kill 3.4 in Calc18 versus 0.5 in

13 Calc18/TXT). Cabazitaxel was also found to be active against two aggressive murine

14 tumors: Lewis lung carcinoma (which has innate resistance to vincristine and 5-

15 fluorouracil, and modest sensitivity to docetaxel; 35), and pancreatic adenocarcinoma

16 P02 (which has innate resistance to a broad spectrum of chemotherapeutic agents; 22)

17 (1.2 and 0.8 log cell kill, respectively). In addition, cabazitaxel was active against

18 three human tumors that are poorly or not sensitive to docetaxel, namely colon HCT-8,

19 gastric GXF-209, and breast UISO BCA-1 (log cell kill values for cabazitaxel versus

20 docetaxel of 1.9 versus 0.8, 1.4 versus 0.5, and > 6 versus 0.6, respectively). In UISO

21 BCA-1, docetaxel at the HNTD of 15 mg/kg per injection (IV on days 13, 16 and 19 after

22 tumor implantation) did not delay tumor growth (log cell kill 0.6 , p > 0.5), whereas

23 cabazitaxel was highly active, both at the HNTD of 15 mg/kg per injection (log cell kill >

24 6, p = 0.0016) and also at the dose level below the HNTD (log cell kill 4.4 at 9.3 mg/kg

25 per injection, p = 0.0016) (Fig. 2b).

Preclinical antitumor activity of cabazitaxel

1 Across different models, the HNTD of cabazitaxel ranged from 22.2–73.2 mg/kg,

2 and was influenced by the mouse strain in which the tumor was grafted and by tumor

3 aggressiveness.

Preclinical antitumor activity of cabazitaxel

1 Discussion

2 During the clinical development of docetaxel, we initiated a program with the

3 objective of selecting a taxane derivative as potent as docetaxel that had activity in

4 tumors unresponsive to docetaxel therapy. Cabazitaxel was selected for further

5 development based on positive results in a number of preclinical studies in which

6 cabazitaxel showed excellent antitumor activity in both docetaxel-sensitive and

7 chemotherapy-resistant tumors in vitro and in vivo.

8 The majority of patients receiving docetaxel therapy for advanced prostate cancer

9 eventually experience disease progression due to innate or acquired drug resistance

10 (36). Despite substantial efforts over many years, mechanisms of resistance to taxanes

11 in patients have not been fully elucidated, and resistance appears not to be mediated by

12 a single mechanism. In tumor cell lines in which taxane resistance is induced in vitro, the

13 two mechanisms most commonly associated with resistance are overexpression of

14 members of the ATP-binding cassette family of transporters, of which P-glycoprotein is

15 the best known, and mutations in tubulin, the cellular target of taxanes (9, 37, 38).

16 Clinical data suggest that additional mechanisms may operate in patient tumors, such as

17 altered expression of tubulin isotypes and expression or binding of microtubule-

18 regulatory proteins (39). Regulators of mitotic checkpoints, including Aurora A, BUBR1,

19 MAD2 and synuclein-γ, and specific checkpoint proteins, such as BRCA1, have all

20 emerged as potential predictive markers of taxane resistance (40). However, conflicting

21 results with these markers have been reported in patient samples. Loss of functional p53

22 may facilitate the development of resistance, potentially by providing a clonal advantage

23 (41). Dysfunctional regulation of apoptotic and intracellular signaling (such as by HER2

24 overexpression) may also contribute to taxane resistance (42). Finally, resistance may

25 be caused by decreased tumor cell permeability, limiting the passive influx of taxanes.

Preclinical antitumor activity of cabazitaxel

1 Based on the multiplicity of factors potentially involved in taxane resistance, it was

2 important when screening taxane derivatives to use a clinically relevant tumor model

3 rather than a cell line in which resistance was induced by continuous exposure to

4 taxanes. For this purpose, we developed a docetaxel-resistant tumor model with

5 behavior similar to that observed in the clinic, in which tumors initially respond to

6 docetaxel before developing resistance over time. The acquisition of full resistance to

7 docetaxel was slow (17 months of repeated docetaxel exposure) when compared with

8 the development of resistance to other chemotherapeutic agents, such as anthracycline,

9 against which resistance took 6 months to emerge under similar in vivo conditions. In

10 addition, the resistance factor for docetaxel was less than 4, which is potentially more

11 clinically relevant. Cross-resistance was mainly observed with tubulin-binding agents.

12 The B16/TXT tumor did not overexpress P-glycoprotein but changes in β-tubulin

13 isotypes suggested that the resistance may be due to changes in microtubule dynamics.

14 Approximately 450 taxane derivatives were screened in the in vivo B16/TXT model, with

15 cabazitaxel emerging as a promising candidate based on its similar activity against fully

16 docetaxel-resistant B16/TXT and taxane-sensitive B16 tumors (log cell kill 1.3 versus

17 2.1, respectively). In a preliminary target assay, cabazitaxel demonstrated equivalent

18 potency to docetaxel for stabilizing microtubules, suggesting a cytotoxic mechanism of

19 action similar to that of docetaxel. In addition, antiproliferative IC50 values in vitro were

20 similar for cabazitaxel and docetaxel in a range of chemotherapy-sensitive tumor cell

21 lines. One of the most striking observations was the improved antiproliferative activity of

22 cabazitaxel versus docetaxel in P-glycoprotein-expressing docetaxel-resistant cell lines

23 (18, 43). Resistance ratios were lower for cabazitaxel than for docetaxel in cell lines with

24 acquired P-glycoprotein-mediated resistance to doxorubicin, vincristine, vinblastine and

25 paclitaxel. Furthermore, in murine and human cell lines with resistance mechanisms

Preclinical antitumor activity of cabazitaxel

1 other than P-glycoprotein overexpression (as shown by similar cytotoxicity of doxorubicin

2 1 µM in both parental and resistant cell lines), there was no cross-resistance to

3 cabazitaxel (Supplementary Table S5).

4 These in vitro data were supported by subsequent studies in in vivo tumors. In

5 mice bearing very advanced-stage MA16/C tumors, drug concentrations were higher

6 than the cellular antiproliferative IC50 for up to 24 hours in plasma and up to 96 hours in

7 the tumor. This rapid and sustained drug uptake into the tumor was consistent with the

8 high antitumor activity of cabazitaxel in this model, which included induction of CRs. A

9 trend toward schedule dependency was observed: maximum tolerated doses were

10 4.8-fold higher with an intermittent schedule than with a split-dose schedule (2 versus

11 15 administrations over 5 days). Optimum antitumor activity and therapeutic index were

12 obtained with schedules allowing administration of the highest doses of cabazitaxel.

13 Using the intermittent IV schedule, cabazitaxel exhibited a broad spectrum of antitumor

14 activity in murine tumors, including activity against advanced-stage disease. Cabazitaxel

15 also showed a high level of antitumor activity in human tumor models, including not only

16 prostate but other tumor types, such as colon, lung, pancreatic, gastric, head and neck,

17 and renal tumors. Most importantly, cabazitaxel was also found to be active in vivo in

18 tumor models poorly or not sensitive to docetaxel, not only in a model with acquired

19 resistance to docetaxel (B16/TXT), but also in models innately resistant to docetaxel:

20 two aggressive murine tumors (Lewis lung and pancreas P02); and three human tumors

21 (colon HCT-8, gastric GXF-209 and mammary UISO BCA-1). Although UISO BCA-1 was

22 obtained from a patient never treated with a taxane, the HNTD of docetaxel was inactive

23 in this model, whereas cabazitaxel was highly active. UISO BCA-1 does not express P-

24 glycoprotein but does express HER2 (32), which might explain its docetaxel-refractory

25 properties (42). Overall, these preclinical data show that cabazitaxel has the potential to

Preclinical antitumor activity of cabazitaxel

1 be active not only in patients with acquired resistance to taxanes but also in patients

2 innately resistant (refractory) to taxanes.

3 Clinical proof of concept was first achieved for cabazitaxel in a Phase II study that

4 showed responses to cabazitaxel in patients with metastatic breast cancer resistant to

5 previous docetaxel therapy (44, 45). As suggested by the in vivo preclinical studies, the

6 responders included both patients with tumors refractory to taxane therapy and

7 patients with relapse following taxane therapy. This was further validated in the pivotal

8 Phase III study in metastatic hormone-refractory prostate cancer (mHRPC) previously

9 treated with docetaxel, in which cabazitaxel in combination with prednisone/prednisolone

10 provided a statistically significant overall survival benefit compared with mitoxantrone

11 plus prednisone/prednisolone (46), leading to regulatory approval in various countries

12 and regions worldwide in this indication.

13 Overall, the data presented here provide a comprehensive overview of preclinical

14 studies with the new taxane cabazitaxel, supporting its current use in patients with

15 mHRPC experiencing disease progression following docetaxel therapy. These results

16 also offer an insight into a successful development process for a new anticancer agent,

17 highlighting the importance of conducting relevant, rationally designed studies to

18 accelerate progress in drug discovery.

Preclinical antitumor activity of cabazitaxel

1 Acknowledgments

2 The authors gratefully acknowledge S. d’Agostino, L. Blot, V. Do Vale, S. d’Heilly,

3 D. Huet, C. Janvier, O. Petitgenet, Y. Ruffin, E. Serres, A. Thomas; Drs Y. Archimbaud,

4 J.P. Sarsat, T. Granda, C. Zuany-Amorim; Drs A.W. Aller and M. Stackhouse from

5 Southern Research Institute, and Dr W.R. Leopold from MIR Preclinical Services for

6 their contribution to the preclinical development of cabazitaxel. They also thank Dr C.

7 Dumontet and Dr GG Chabot for providing a summary of their unpublished results on

8 docetaxel-resistant B16/TXT melanoma. Editorial assistance for preparing this article

9 was supported by Sanofi.

10 Grant Support

11 Not applicable.

Preclinical antitumor activity of cabazitaxel

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Table 1. Dose–response antitumor activity of cabazitaxel and docetaxel in mice bearing murine tumors. Murine tumors were grafted Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. Author ManuscriptPublishedOnlineFirstonApril15,2013;DOI:10.1158/1078-0432.CCR-12-3146 in the syngenic strain of mice of origin of the tumor for MA17/A (C3H/HeN) and C51 (BABL/C) and in B6D2F1 mice for the C57BL/6 syngenic tumors (B16, B16/TXT, C38, P03, P02, 3LL).

clincancerres.aacrjournals.org Dose, mg/kg per Cabazitaxel Docetaxel Tumor type injection Total T-C, Log cell CR TFS Total HNTD, T-C, Log cell CR TFS (schedule days) HNTD, days killb mg/kg days killb mg/kg B16 melanoma 32.2, 20, 12.4, 7.7 60 8.5 2.1 NA 0/5 60 6.6 1.7 N/A 0/5 (Td : 1.2 days) (3, 5, 7) B16/TXT melanoma 32.2, 20, 12.4, 7.7 60 5.5 1.3 N/A 0/5 60 2.8 0.6 N/A 0/5 (Td : 1.3 days) (3, 5, 7) Colon C38 32.2, 20, 12.4, 7.7 a 60 – – 5/5 5/5 60 29.1 3.1 0/5 0/5 (Td : 2.8 days) (14 , 17, 20) Colon C51 24.2, 15, 9.3, 5.8 45 25.8 2.6 N/A 0/5 45 31.1 3.1 N/A 0/5

on September 30, 2021. © 2013American Association for Cancer (Td : 3 days) (4, 6, 8) Pancreas P03 32.2, 20, 12.4, 7.7 Research. a 60 – – 5/5 4/5 ND ND ND ND ND (Td : 3.5 days) (17 , 19, 21) Pancreas P02 32.2, 20, 12.4, 7.7 60 6.6 0.8 N/A 0/5 ND ND ND ND ND (Td : 2.5 days) (3, 5, 7) Mammary MA16/C 64.5, 40, 24.8, 15.4 a 40 13.4 3.7 4/5 0/5 ND ND ND ND ND (Td : 1.1 days) (8 ) Mammary MA17/A 19.4, 12, 7.4, 4.6 36 15.7 3.9 N/A 0/5 ND ND ND ND ND (Td : 1.2 days) (3, 5, 7) Lung 3LL 31.5, 19.5, 12.1, 7.5 58.5 4.6 1.2 N/A 0/5 ND ND ND ND ND (Td : 1.2 days) (3, 5, 7) a Median tumor burden at start of therapy: 290 mm3, 310 mm3 and 400 mm3 for C38, P03 and MA16/C studies, respectively. b Definition of antitumor activity: log cell kill total < 0.7 = inactive; > 2.8 = highly active. HNTD = highest non-toxic dose; T-C = tumor growth delay; CR = complete regression; TFS = long-term tumor-free survival; N/A = not available as treatment performed on early-stage disease; ND = not determined in the same study.

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Preclinical antitumor activity of cabazitaxel

Table 2. Effects of taxoids on the assembly–disassembly process of pure tubulin. Tubulin was used at a concentration of 6 µM for polymerization (at 37°C) and 9 µM for depolymerization (at 8°C). OD was measured at 350 nm. Rates of depolymerization were expressed in ΔOD/min. Ratios between depolymerization rates were calculated for each drug concentration. Boundaries of drug concentrations for dIC50 and LT50 are given.

Drug Rate of cold-induced microtubule Ratio Lag time, min concentration, disassembly, ΔOD/min µM Cabazitaxel Docetaxel Cabazitaxel Docetaxel Control 7.42 x 10-2 (n=5) 40 0.1 5.36 x 10-2 (n=4) 6 x 10-2 (n=3) 0.89 4.18 9.16 0.25 2.82 x 10-2 (n=5) 3.05 x 10-2 (n=5) 0.92 1.57 2.61 0.5 1.8 x 10-2 (n=6) 1.95 x 10-2 (n=5) 0.92 1.0 1.04 1 1.2 x 10-2 (n=5) 1.25 x 10-2 (n=4) 0.96 0.5 0.65 2.5 0.61 x 10-2 (n=4) 0.5 x 10-2 (n=6) 1.22 5 0.56 x 10-2 (n=3) 0.38 x 10-2 (n=3) 1.47 dIC : dIC : Mean: 50 50 LT : 0–0.1 µM LT : 0–0.1 µM 0.1–0.25 µM 0.1–0.25 µM 1.06 50 50

dIC50: concentration of drug that inhibits the cold-induced disassembly by 50%. LT50: concentration of drug that reduces the lag time by 50%.

Preclinical antitumor activity of cabazitaxel

Table 3. In vitro antiproliferative effects of cabazitaxel and docetaxel against sensitive and P-glycoprotein-expressing resistant cell lines. Cells were incubated for 96 h at 37°C in liquid medium with drugs at different concentrations. Viability was assessed by neutral red, with the mean of at least three results obtained.

a Mean IC50, µM, ± SD Resistance factor ABCB1 Cell line mRNA Docetaxel Cabazitaxel Docetaxel Cabazitaxel levelb P388 murine leukemia 0.079 ± 0.004 0.041 ± 0.017 – – – P388/DOX 4.01 ± 0.28 0.414 ± 0.036 51 10 +++ P388 murine leukemia 0.039 ± 0.012 0.013 ± 0.005 – – – P388/TXT 0.188 ± 0.022 0.024 ± 0.015 5 2 ++ P388 murine leukemia 0.039 ± 0.012 0.013 ± 0.005 – – – P388/VCR 0.227 ± 0.038 0.024 ± 0.003 6 2 ++ HL60 human leukemia 0.031 ± 0.004 0.022 ± 0.010 – – – HL60/TAX 0.25 ± 0.11 0.060 ± 0.029 8 3 ++ Calc18 human breast 0.008 ± 0.002 0.004 ± 0.002 – – – adenocarcinoma Calc18/TXT 0.17 ± 0.04 0.016 ± 0.004 21 4 ++ KB human epidermoid carcinoma 0.042 ± 0.0212 0.035 ± 0.026 – – – KB V1 2.48 ± 0.12 0.27 ± 0.013 59 8 ++++ a Resistance factor = IC50 (resistant)/IC50 (parental) from the same experiment. b Relative expression obtained from northern blot experiments using the human ABCB1 gene as probe. IC50 = concentration required to reduce cell survival by 50%; ABCB1 = ATP-binding cassette, sub-family B, member 1; P388/DOX = P388 murine leukemia resistant to doxorubicin; P388/TXT = P388 murine leukemia resistant to docetaxel; P388/VCR = P388 murine leukemia resistant to vincristine; HL60/TAX = HL60 human leukemia resistant to paclitaxel; Calc18/TXT = Calc18 human breast adenocarcinoma resistant to docetaxel; KB V1 = KB human epidermoid carcinoma resistant to vinblastine.

Table 4. Dose–response antitumor activity of cabazitaxel and docetaxel in mice bearing human tumors. Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. Median tumor Cabazitaxel Docetaxel Author ManuscriptPublishedOnlineFirstonApril15,2013;DOI:10.1158/1078-0432.CCR-12-3146 Dose per injection, burden at start Total Log Total Tumor type mg/kg T-C, T-C, Log cell of therapy, HNTD, cell CR TFS HNTD, a CR TFS (Schedule in days) 3 days days kill mm mg/kg killa mg/kg clincancerres.aacrjournals.org Prostate Du145 19.4, 12, 7.4, 4.6 210 48.0 – – 6/6 5/6 ND ND ND ND ND (Td: 4.5 days) (24, 30, 36, 42) Colon HCT 116 19.4, 12, 7.5, 4.6 220 36.0 48.2 3.4 7/7 2/7 ND ND ND ND ND (Td: 4.3 days) (16, 19, 22) Colon HT-29 19.4, 12, 7.5, 4.6 140 22.2 27.2 2.0 6/6 0/6 96.6b 45.8 3.4 6/6 0/6 (Td: 4 days) (8 12, 16) Colon HCT-8 22.4, 14, 8.6, 5.4 250 28.0 21.5 1.9 0/5 0/5 50b 8.8 0.8 0/5 0/5 (Td: 3.5 days) (13, 17) Pancreas MIA PaCa-2 19.4, 12, 7.5, 4.6 b c 310 48.0 – – 6/6 6/6 75 – – 6/6 6/6 (Td: 3 days) (15, 19, 23, 27 ) Breast Calc18 33, 20.5, 12.7, 7.9 N/A 61.5 50.2 3.4 N/A 5/8 ND ND ND ND ND

Research. Breast Calc18/TXT 33, 20.5, 12.7, 7.9 N/A 38.1 7.3 0.5 N/A 0/8 ND ND ND ND ND (Td: 4 days) (5, 7, 9) Breast UISO BCA-1 24.2, 15, 9.3, 5.8 70 45.0 75.0 > 6 N/A 0/5 45 4.1 0.6 N/A 0/5 (Td: 2.1 days) (13, 16, 19) Lung NCI-H460 19.4, 12, 7.4, 4.6 130 24.0 17.8 2.7 2/6 0/6 ND ND ND ND ND (Td: 2 days) (10, 13) Lung A549 19.4, 12, 7.4, 4.6 130 36.0 46.0 2.2 2/6 0/6 96.6b 40 1.9 0/5 0/5 (Td: 6.4 days) (21, 27, 33) Gastric N87 39.4, 24.4, 15, 9.3 140 73.2 100.9 > 6 N/A 1/8 73.2 66.8 4.5 N/A 1/8 (Td: 4.5 days) (27, 31, 35) Gastric GXF-209 32.3, 20, 12.4, 7.7 130 37.2 18.0 1.4 0/8 0/8 23.1 6.8 0.5 0/8 0/8 (Td: 4 days) (14, 17, 20) Head and neck SR475 22.5, 14, 8.6, 5.4 250 42.0 – – 6/6 6/6 45b 40.5 2.5 1/6 0/6 (Td: 4.9 days) (16, 20, 24) Kidney Caki-1 31.2, 19.4, 12, 7.5 140 24.0 25.8 1.7 5/6 0/6 64.4b 21.3 1.4 2/5 0/5 (Td: 4.6 days) (10, 14) a Definition of antitumor activity: log cell kill total < 0.7 = inactive; > 2.8 = highly active. b The dose–response pattern for docetaxel was different from that of cabazitaxel in the following studies: HT-29, A549 and Caki-1 studies: 51.9, 32.2, 20 and 12.4 mg/kg per injection; HCT-8, MIA PaCa-2 and SR475 studies: 41.7, 25 and 15 mg/kg per injection. c Docetaxel groups were not treated on day 27. HNTD = highest non-toxic dose; T-C = tumor growth delay; CR = complete regression; TFS = long-term tumor-free survival; N/A = not available as treatment performed on early-stage disease; ND = not determined in the same study.

Author Manuscript Published OnlineFirst on April 15, 2013; DOI: 10.1158/1078-0432.CCR-12-3146 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Preclinical antitumor activity of cabazitaxel

Figure Legends

Fig. 1. Pharmacokinetics of cabazitaxel in plasma and tumor tissue in mice.

Cabazitaxel concentrations in plasma and tumor tissue were measured after a single

intravenous dose of cabazitaxel at its highest non-toxic dose of 40 mg/kg in C3H/HeN

female mice bearing advanced-stage (≥ 400 mm3) mammary adenocarcinoma MA16/C.

Mean concentration ± SD from three animals was determined at each sampling time.

Fig. 2. Head-to-head comparison of the antitumor activity of cabazitaxel and docetaxel in

human tumor xenografts.

Antitumor activity was measured in human tumors xenografted subcutaneously into

female nude mice and reported as median tumor volumes ± interquartile ranges. (a)

Human gastric N87. Both drugs were administered intravenously at 5.8 (asterisk), 9.3

(triangle), 15.0 (circle) and 24.4 mg/kg/inj (highest non-toxic dose [HNTD]; no symbol) on

Days 25, 31 and 35 to mice bearing 140 mm3 tumors implanted SC monolaterally at start

of therapy on Day 25. Interquartile ranges for medians and numbers of animals per

group are provided in Supplementary Table S6. (b) Human breast UISO BCA-1. Drugs

were administered intravenously on Days 13, 16 and 19 to mice bearing palpable tumors

implanted SC bilaterally at start of therapy on Day 13. Doses were 5.8 (triangle), 9.3

(circle) and 15.0 mg/kg/inj (HNTD; no symbol) for cabazitaxel and 15.0 mg/kg/inj (HNTD;

cross) for docetaxel. Interquartile ranges for medians and numbers of animals per group

are provided in Supplementary Table S7.

IV, intravenously; HNTD, highest non-toxic dose.

Figure 1

5 10 Plasma Tumor

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103

102

101 Cabazitaxel concentration (ng/mL or ng/g) or (ng/mL Cabazitaxel concentration 100 0 5040302010 908 07060 100 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2013 American Association for Cancer Time (h)Research. Author Manuscript Published OnlineFirst on April 15, 2013; DOI: 10.1158/1078-0432.CCR-12-3146 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 2a

1400

1200 ) 3 1000

800

600

400 Tumor volume (mm volume Tumor

200

0 0 20 40 60 80 100 120 140 Days post implantation

Docetaxel (24.4 mg/kg/inj) (HNTD) Docetaxel (15.0 mg/kg/inj) Docetaxel (9.3 mg/kg/inj) Docetaxel (5.8 mg/kg/inj) Cabazitaxel (24.4 mg/kg/inj) (HNTD) Cabazitaxel (15.0 mg/kg/inj) Cabazitaxel (9.3 mg/kg/inj) Cabazitaxel (5.8 mg/kg/inj) Control Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2013 American Association for Cancer IV treatment Research. Tumor volume (mm3) 2b Figure 1600 1800 2000 1000 1200 1400 2 400 600 800 00 0 0 1 03 05 07 09 100 90 80 70 60 50 40 30 20 0 IV treatments treatments IV Control (HNTD)(15.0 mg/kg/inj) Docetaxel (5.8mg/kg/inj) Cabazitaxel (9.3 mg/kg/inj) Cabazitaxel (HNTD)(15.0 mg/kg/inj) Cabazitaxel Downloaded from Days post implantation post Days Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. Author ManuscriptPublishedOnlineFirstonApril15,2013;DOI:10.1158/1078-0432.CCR-12-3146 clincancerres.aacrjournals.org on September 30, 2021. © 2013American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 15, 2013; DOI: 10.1158/1078-0432.CCR-12-3146 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Preclinical Antitumor Activity of Cabazitaxel, a Semi-Synthetic Taxane Active in Taxane-Resistant Tumors

Patricia Vrignaud, Dorothée Sémiond, Pascale Lejeune, et al.

Clin Cancer Res Published OnlineFirst April 15, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-12-3146

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2013/04/17/1078-0432.CCR-12-3146.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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