Nanoparticle Formulation Derived from Carboxymethyl Cellulose

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A Novel Nanoparticle Formulation Derived from Carboxymethyl-Cellulose, Polyethylene Glycol and Cabazitaxel for Chemotherapy Delivery to the Brain Joseph Bteich, Mark J Ernsting, Mohammed Z Mohammed, Taira Kiyota, Trevor McKee, Mohit Trikha, Henry Lowman, and Kenneth K. Sokoll Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 7, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

A Novel Nanoparticle Formulation Derived from Carboxymethyl-Cellulose, Polyethylene Glycol and Cabazitaxel for Chemotherapy Delivery to the Brain

AUTHORS Joseph Bteich1, Mark J. Ernsting1,2, Mohammed Mohammed1, Taira Kiyota1, Trevor McKee3, Mohit Trikha4, Henry B. Lowman4† and Kenneth K. Sokoll5† 1

Drug Delivery and Formulation, Drug Discovery Program, Ontario Institute for Cancer Research, 101 College Street, Suite 800, Toronto, Ontario, Canada, M5G 0A3

2

Faculty of Engineering and Architectural Science, Ryerson University, Toronto, Ontario, Canada, M5B 1Z2

3

STTARR Innovation Centre, Radiation Medicine Program, Princess Margaret Cancer Centre, University Health Network, Toronto ON, M5G 1L7

4

Triphase Accelerator, 3366 North Torrey Pines Court, Suite 210, La Jolla, California 92037 USA

5

Fight Against Cancer Innovation Trust, MaRs Centre, West Tower, 661 University Avenue, suite 510, Toronto, Ontario, Canada, M5G 0A3

ABSTRACT Nanoparticles provide a unique opportunity to explore the benefits of selective distribution and release of cancer therapeutics at sites of disease through varying particle sizes and compositions that exploit the enhanced permeability of tumor-associated blood vessels. Though delivery of

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larger as opposed to smaller and/or actively transported molecules to the brain is prime face a challenging endeavor, we wondered whether nanoparticles could improve the therapeutic index of existing drugs for use in treating brain tumors via these vascular effects. We therefore selected a family of nanoparticles composed of cabazitaxel-carboxymethylcellulose amphiphilic polymers to investigate the potential for delivering a brain-penetrant taxane to intracranial brain tumors in mice. Among a small set of nanoparticle formulations, we found evidence for nanoparticle accumulation in the brain, and one such formulation demonstrated activity in an orthotopic model of glioma, suggesting that such nanoparticles could be useful for the treatment of glioma and brain metastases of other tumor types.

INTRODUCTION CBZed-Nano is a novel polymer conjugate formulation consisting of the drug cabazitaxel (CBZ) covalently conjugated to a cellulose polymer modified with polyethylene glycol (termed a Cellax polymer) that assembles into well-defined nanoparticles, NPs (Figure 1).1,2,3

Figure 1. Cellax polymers can be engineered to self-assemble into nanoparticles within a biologically relevant range of sizes, while achieving long-term formulation stability.

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Bioconjugate Chemistry

Development of CBZed-Nano began as an alternative delivery approach for Jevtana® (Cabazitaxel), a taxane drug that was approved for prostate cancer, but was limited in use and effectiveness by significant toxicities leading to morbidity, treatment discontinuation, and sometimes death.4 Considering the activity and the liabilities of CBZ in treating prostate cancer, CBZed-Nano was initially evaluated for tolerability and efficacy in a subcutaneous xenograft prostate cancer model (PC3) in NCR nude mice.5 Early results showed that CBZed-Nano formulations provide durable cures in the PC3 model, and therefore evaluation of the platform for other indications was undertaken. We have postulated that CBZed-Nano formulations may mitigate off-target toxicity due to their small 20-70 nm (diameter) size range and narrow distribution as determined by the polydispersity index (0.07-0.18), particulate morphology and long circulation times providing invivo stability with respect to release of CBZ. Recent work has examined toxicity in mice and shown that CBZed-Nano formulations enable an increased maximum tolerated dose (MTD) relative to native CBZ.5 Particles of this size cannot cross the endothelial boundary of normaltissue vasculature, but do accumulate in or near tumors; we have found that CBZed-Nano particles accumulate to 4-5 fold higher concentrations in tumor tissue versus muscle tissue in mouse xenograft models when administered at a 20 mg/Kg dose.5 The CBZ drug can be released by various hydrolysis mechanisms which may include the action of carboxylesterases.6 The presence of carboxylesterases in brain extracts has been established and tumor cells typically have much higher concentrations than normal cells.7 Moreover, the potential for acquired drug resistance is reduced by the resistance of the Cellax conjugate to P-glycoprotein, Pgp.8 It is known that Pgp protein can be competed away from taxane by verapamil in vitro and that CBZ is

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a substrate for Pgp.9 Studies have shown (unpublished results) that the corresponding IC50 of CBZ is reduced for CBZ in the presence of verapamil (~280 fold reduction), whereas verapamil has been shown to have a modest effect on the IC50 of CBZed-Nano (10 fold reduction). These findings confirmed that CBZed-Nano particles are not good substrates for Pgp. These attributes may lead to improved efficacy and safety by overcoming limitations to the use of taxanes in the clinic. Accordingly, CBZed-Nano and the Cellax nanoparticle platform offer the potential for an improved therapeutic index and improved therapeutic outcome in a variety of solid-tumor indications. Of the numerous challenging targets, malignant gliomas are the most common and most dangerous primary CNS cancers in humans. Current treatments are temporarily effective, but unfortunately ultimately fail leading to neurological deficits and precipitous death. There is therefore an urgent need to develop new and more tolerable therapies for glioma. No taxanes have been approved in glioma despite promising preclinical data, because taxanes generally have poor brain penetration. However, preclinical evidence of CBZ activity in brain tumors, including intracranial xenograft models comes from two reports.10,11 Furthermore, cabazitaxel showed clinical activity in a limited number of patients with prostate cancer who had metastatic lesions in the brain.12 These findings suggest that CBZ itself can penetrate the blood-brain barrier (BBB) and could provide clinical benefit in glioma and other brain cancers. Despite these promising results, delivery of sufficient amounts of CBZ to the brain is potentially hindered by the systemic toxicity of CBZ. We hypothesized that an appropriate nanoparticle composition might provide for selective distribution of CBZ to the brain and local release of CBZ to produce cytotoxic effects on

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Bioconjugate Chemistry

intracranial tumors. The Cellax polymer can be modified by altering the conjugation chemistry to provide a wide range of drug- loaded nanoparticle compositions having tunable features, in particular, size and drug loading. Smaller particles might be expected to distribute more efficiently through tissue, yet could be less selective for tumor tissue or have different safety profiles as compared to larger particles. We therefore produced several different CBZed-Nano compositions derived from Cellax polymers identified as CLX-703, CLX-706 and CLX-861, based on our experience with the prototype composition known as CLX-661, previously reported,13 and tested them in glioma models. The CLX numbering system reflects the chronology of the manufacturing with the 600 series being protoype, 700 series lab scale and 800 series scalable conjugates providing formulations suitable for development. In the case of CLX861 this represents a scalable version of the Cellax prototype polymer CLX-661 which was prepared in continuous mode as opposed to single batch and accordingly only select in-vitro studies reported herein have both CLX-661 and CLX-861 tested concurrently.

Here we show that CBZed-Nano particles can accumulate in the brain with preferential accumulation in intracranial brain tumors, and that they show evidence of activity in an orthotopic mouse model of glioma. Combined with previous observations of taxane sensitivity in such models, this suggests the opportunity for a unique therapeutic approach to the treatment of malignant glioma and other tumors of the brain. More generally, the technology provides a platform for improving the therapeutic index of difficult-to-formulate hydrophobic drugs having known toxicity concerns.

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RESULTS Chemical and Physical Characterization of Cellax-CBZ Polymers and CBZed-Nano Formulations Studied in GBM Models. The different Cellax-CBZ polymer analogues of interest for the DiI encapsulation and GBM screening PK and efficacy studies were synthesized and characterized by 1H-NMR analysis, and MALS. CLX-661 and CLX-861conjugates were engineered to produce lower molecular weight polymers, with a higher weight percent composition of CBZ to PEG ratio when compared to conjugates derived from CLX-703 and CLX-706. Specific material properties and composition data are tabulated and shown in Table 1 for Cellax polymers and derived CBZed-Nano formulations examined in imaging studies DiI encapsulation).

Table 1. Summary of properties of Cellax-CBZ polymers and dose concentrations for the corresponding CBZed-Nano formulations encapsulated with DiI. Conc.

Conc.

CBZ

PEG

mg/mL

mg/mL

14.0

10.1

22.2

13.1

9.3

10.9

Wt%

Wt%

MW

CBZ

PEG

(kDa)

CLX-706

22.9

35.0

CLX-703

36.2

CLX-661

31.4

Polymer

DiI

Size

(µg/mL)

(nm)

13.91

722.9

25.2

10.0

5.91

443.1

46.6

9.4

2.18

466.6

57.7

In all three cases the concentration of conjugated CBZ was adjusted to be comparable for this study. Nanoparticles were formulated at 10 mg polymer/gram MeCN. Nanoparticle saline solutions exhibited colors of bright fluorescent fuschia.

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Bioconjugate Chemistry

Polymer conjugates were formulated with the aid of microfluidics (Precision Nanosystems Inc., Vancouver BC, Canada) to produce nanoparticles with encapsulated DiI for measuring uptake in orthotopic GBM tumors, and without encapsulated DiI for all other efficacy and PK studies. Representative CBZed-Nano formulations with and without encapsulated DiI were examined in IC50 studies in the PC3 cancer cell line. Prior to use CBZed-Nano formulations were dialyzed, sterile-filtered (0.22 um) and concentrated to approximately 10 mg CBZ/mL as indicated in the fifth column of Table 1 for DiI encapsulated formulations. The corresponding concentrations of encapsulated DiI are shown in the seventh column of Table 1. Specific material properties and composition data are shown in Table 2 for Cellax polymers and derived CBZed-Nano formulations used in the GBM PK and efficacy studies.

Table 2. Summary of properties of Cellax-CBZ polymers and dose concentrations for the corresponding CBZed-Nano formulations used for the GBM screening PK and efficacy studies.

Polymer

CLX-706

CLX-861

Wt%

Wt%

MW

CBZ

PEG

(kDa)

22.9

35.0

14.0

32.3

11.0

8.1

Dose

Conc.

Conc.

Level

CBZ

PEG

(mg/Kg)

mg/mL

mg/mL

150

19.52

25.26

24.2

100

12.97

16.98

24.1

25

3.68

4.32

24.1

150

20.97

5.99

67.7

100

14.58

3.97

68.5

25

3.37

1.02

68.0

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Size (nm)

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nanoparticles were formulated at 30 mg polymer/gram MeCN via microfluidics, concentrated and diluted in saline to obtain the targeted CBZ dosing levels used in the GBM PK and efficacy studies are shown in the sixth column of Table 2.

Tables 1 and 2 provide a list of the nanoparticle sizes, for each Cellax-CBZ polymer synthesized and the corresponding CBZed-Nano formulation used in the fluorescent BD (Table 1) and GBM screening PK and efficacy studies (Table 2). The range of nanoparticle sizes examined are all below the typical “pore cutoff size” reported by Jain and colleagues,15 of less than 200 nm for tumors grown in the cranial microenvironment, however we sought to obtain a more detailed understanding of potential differences in biodistribution and differential uptake by analyzing nanoparticles less than 100 nm and specifically ranging from 20-70 nm as provided by CellaxCBZ polymers CLX-661, CLX-703, CLX-706 and CLX-861. Concentrations of unbound free CBZ drug and PEG were uniformly found to be very low or undetectable. Unconjugated CBZ represented less than 0.12 % of total CBZ dose. It was noted that the introduction of DiI (Table 1) produces a more variable DLS reading due to the fluorescence interfering with the detector. TEM were thus run to verify that morphology and size were unchanged by the encapsulation of the DiI chromophore.

Nanoparticle Morphology. TEM was used to contrast DiI encapsulated CBZed nanoparticles with DiI free nanoparticles in terms of size and morphology as DLS measurements were not

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Bioconjugate Chemistry

reliable due to fluorescence. Figure 2 depicts typical morphology contrasted with DiI encapsulated CBZed-Nano particles. Morphology and size was shown to be similar.

Figure 2. TEM images contrasting similar Cellax-CBZ analogue polymers with (top row) and without (bottom row) DiI encapsulation. Magnification = 70,000x. Nanoparticle morphology and size appear unaltered when hydrophobic dye is encapsulated during nanoparticle selfassembly.

Accumulation of Fluorescently Labeled Nanoparticles in U87MG GBM Models. We examined normal brain and tumor tissue from an intracranial orthotopic model using the U87MG cell line at a time point 24h after i.v. injection of nanoparticles. As depicted in the following Figures 3-6 DiI intensity was quantified both as mean total intensity measured across the entire (tumor or normal brain) volume, or as the area fraction occupied by DiI signal within each

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region. Mean total intensity should relate more to overall uptake within each tissue; area fraction would relate more to how much of the tumor is occupied by significant regions of CBZed-Nano particles / dye in vessel periphery.

For this imaging study three CBZed-Nano formulations (derived from CLX-661, CLX-703 and CLX-706 conjugates) were available for evaluation. Nanoparticles prepared from CLX-861 are a scalable version of CLX-661 with very similar physical properties and thus CLX-861 formulations were evaluated in subsequent in-vivo screening PK and efficacy studies.

A

B

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Bioconjugate Chemistry

C

■ Tumor ■ Normal Brain

D

Figure 3. Image preparation; A) DiI Fluorescence Image, B) H&E Image, C) Registration and overlay, D) Region of interest – Tumor (orange) / Normal Brain (purple). Scale bar = 500µm

A

B

Intensity Thresholds ■ Low ■ Medium ■ High

■ DiI overlay

Figure 4. Image quantification example of a cross-section brain sample at 24h post inoculation (IV) of CLX-661. In A, NP containing DiI are depicted by a white overlay, representing their fluorescence intensity. In B, thresholds were chosen to quantify the area of Cellax / DiI marker

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present within each region (Tumor, normal brain), 3 thresholds corresponding to more permissive (low, intensity of 60) to more restrictive (high, intensity of 150), were chosen, and areas were calculated for each sample and region of interest (Tumor, normal brain). Scale bar = 100µm.

Cross-sections of tissue showed targeted accumulation of nanoparticles in cancerous regions. Figure 5 shows a close-up example for each formulation at 48h.

CLX-661

CLX-703

CLX-706

Figure 5. Magnified image quantification examples at 48 hs showing DiI fluorescence in CLX661 (left), CLX-703 (middle), and CLX-706 (right) formulations. Scale bar = 100µm

The corresponding CBZed-Nano / DiI intensity and area fractions were calculated at 24 and 48h and are shown in Figure 6. It is unlikely that DiI leakage could account for the current findings as the DiI signal observed from the tissue sections during the quantitative image analysis work appeared as primarily punctate in nature, indicating that it arose from dye encapsulated in nanoparticles or nanoparticle aggregates extravasated from vessels, rather than free dye which would appear as a more uniform distribution of fluorescence within the tissue.

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DiI intensity within whole ROI - 24h

DiI intensity within whole ROI - 48h LX Mean DiI Intensity within ROI -6 6 1 -6 Tu 61 m N or or m al C B LX ra C in -7 LX 0 3 -7 T 03 um N or or m al C B LX ra C in -7 LX 0 6 -7 Tu 06 m N or or m al B ra in

100

100

80

80

60

60

40

40

20

20

C

0

Treatment / Region of Interest

Treatment / Region of Interest

DiI Area Fraction - 24h

DiI Area Fraction - 48h

C

Tu m or al B ra in

LX

-7 06

N

or m

70 6

al B

or m

LX C C

m or Tu N

or m N

C

LX

LX

-7 03

Tu 66 1 -6 61

LX C

C

ra in

0

m or

0

ra in

10

10

70 3

20

20

al B

30

LX -

40

30

C

Percent DiI positive area

50

LX -6 61 Tu 61 m N or or m al C B LX ra C in -7 LX 0 3 -7 T 03 um N or or m al C B LX ra C in -7 LX 0 6 -7 Tu 06 m N or or m al B ra in

Percent DiI positive area

C

C LX

LX -6 6

C

0

C LX -6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

LX Mean DiI Intensity within ROI -6 61 Tu 1 m N or or m al C B LX ra C in -7 LX 0 3 -7 T 03 um N or or m al C B LX ra C in -7 LX 0 6 -7 Tu 06 m N or or m al B ra in

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Treatment / Region of Interest

Treatment / Region of Interest

Figure 6. CBZed-Nano/DiI intensity and area fractions calculated at 24 and 48h.

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An evaluation of the statistical significance of the mean DiI intensity in the region of interest (ROI) and area fractions at 24 and 48h for tumor versus normal brain was completed using the Independent-Samples T-Test. Results are highlighted in Table 3.

Table 3. Statistical summary for CBZed-Nano/DiI formulations. Polymer

24h ROI mean DiI Intensity (P value)

48h ROI mean DiI Intensity (P value)

24h DiI Area Fraction (P value)

48h DiI Area Fraction (P value)

CLX-661

0.0229

0.0006

0.0003

< 0.0001

CLX-703

0.1319

0.0025

0.0001

0.0027

CLX-706

0.0012

1 uM (unpublished results). These results indicate that a mock nanoparticle comprised of carboxymethyl cellulose and PEG fragments of the conjugate is not responsible for the activity of these compounds in the cancer cell lines studied.

For studies run in GBM cell lines, Cisplatin served as a positive control and is known to be of low activity in these cell lines. IC50’s in the low µM range were expected. Select CBZed-Nano formulations (CLX-661 and CLX-861) with and without DiI were also characterized and found to be similar in potency as controls with an IC50 in a narrow range of 1.6-2.2 nM.

It is important to note that free CBZ is instantly bioavailable, whereas the CBZed-Nano formulations would be expected to provide a slow and sustained release of drug payload over time. The CBZed-Nano formulations would be predicted to be marginally less active than free CBZ drug. The in-vitro results suggest that CBZed-Nano formulations are potent and thus promising candidates for in-vivo efficacy studies in several cancer cell lines including prostate and GBM.

Pharmacokinetics (Orthotopic LN-229 model). In this screening PK study, the CBZed-Nano formulations CLX-861, CLX-706, free CBZ and a saline vehicle control were administered intravenously (i.v.) in an orthotopic LN-229 GBM xenograft model in female BALB/c nude mice. The single dose was administrated at day 7 after tumor inoculation. Perfused brain and plasma samples were collected at pre-determined time points (30min, 5h and 24h post dose) and

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were evaluated by LC-MS for free CBZ, indicative of CBZ released from CBZed-Nano formulations and bound CBZ, indicative of circulating intact polymer conjugate. The LC-MS results presented in Figure 8 depict the mouse PK and brain distribution profiles obtained from Balb/c nude mice dosed with free CBZ (25 mg/Kg) and CBZed-Nano formulations derived from CLX 861 (100 mg/Kg) and CLX 706 (100 mg/Kg) polymers.

Figure 8. Pharmacokinetic plots of CBZ and CBZed-Nano formulations in plasma and brain of mice with intracranial LN-229 tumors are shown over 24h. Unconjugated free CBZ(A) was measured in plasma and brain. For CBZed-Nano formulations derived from CLX-861(B) and CLX-706 (C), coupled and released CBZ are shown as measured in plasma or brain. The lower limit of quantitation (LLOQ) of free unconjugated or released CBZ was 25 and 30 ng/mL for plasma and brain and the LLOQ of total CBZ was 25 and 300 ng/mL for plasma and brain, respectively.

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From this in-vivo orthotopic PK screening study it was established that CBZ was detectable in both brain and plasma. Free CBZ and CBZ released from CBZed-Nano formulations in plasma was rapidly depleted over 24h, whereas bound CBZ derived from CBZed-Nano formulations CLX-861 and CLX-706 exhibited better retention in brain relative to plasma over the same 24h interval. The amount of released CBZ in brain from CBZed-Nano formulations could not be assessed as levels were below the limit of quantitation.

The current findings provide further evidence that CBZed-Nano can accumulate in the brain as was also suggested from imaging studies with encapsulated DiI and indicates the potential use of these formulations for the treatment of GBM.

LN-229 GBM Efficacy Study. In this study, the therapeutic efficacy of the CBZed-Nano formulations in the treatment of an orthotopic LN-229 GBM xenograft model in female BALB/c nude mice was evaluated. The study was executed delivering 3 doses at 3 dose levels (25, 100 and 150 mg/Kg) on a weekly schedule of CBZed-Nano formulations derived from the CLX 861, which provides nanoparticles nominally in the 60-70 nanometer size range and CLX 706, a polymer providing smaller nanoparticles in the range of 35-40 nanometers. A control group dosed free CBZ following the same administration schedule at 25 mg/Kg. Both formulations appeared promising from IC50 screening in the LN-229 cell line relative to free CBZ and the orthotopic PK study in the Balb/c mouse model provided further evidence that these formulations could cross the BBB and exhibit retention in brain tissue as compared to free CBZ.

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Survival Analysis. For the assessment of antitumor activity any mice deaths related to toxicity of drug were excluded. The analysis of survival times was defined as the time from the day post tumor inoculation to the day of animal death or to date where animal was euthanized. For each group the median survival time (MST) and statistical significance of the increased life span (P values) were calculated relative to the vehicle control group. The summary analysis of survival times is provided in Table 5. The highlighted result shown for Group-3, CLX 861 (100 mg/Kg) was determined to be significant (P < 0.05) using the Independent-Samples T Test.

Table 5. Antitumor Activity of CBZed-Nano by Survival Time Analysis

P Value Treatment

MST (days) vs group-1

Group-1 Control (Saline)

59(27-69)

-

Group-2 CLX 861 (25mg/Kg)

59(32-98)

0.251

Group-3 CLX 861 (100mg/Kg)

86(17-98)

0.022

Group-4 CLX 861 (150mg/Kg)

40(23-86)

0.310

Group-5 CLX 706 (25mg/Kg)

33(25-98)

0.232

Group-6 CLX 706 (100mg/Kg)

35(25-85)

0.895

Group-7 CLX 706 (150mg/Kg)

NA(28-98)

0.086

Group-8 CBZ (25mg/Kg)

23(11-98)

0.499

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For the CBZed-Nano formulations screened in this orthotopic GBM efficacy study only the composition derived from the CLX-861 polymer dosed at 100mg/Kg showed significant prolongation of the life span (P = 0.022) compared with the vehicle control group.. Of the CBZed-Nano formulations derived from CLX-706 polymer the group dosed at 150 mg/Kg provided the best result although this group did not reach significance (P = 0.086) relative to the vehicle control group. The loss of 3/10 mice from the free CBZ group (at 25 mg/Kg) during the initial dosing interval (after 1’st dose) was ascribed to toxicity of the drug at this dose level. Survival curves were constructed for each group however as only specific CBZed-Nano formulations derived from CLX-861 polymers were shown to be significant the corresponding Kaplan-Meier plot as shown in Figure 9 depict results from those formulations.

Figure 9. Kaplan-Meier survival curves for CBZed-Nano formulations derived from CLX-861 polymers dosed at 150, 100 and 25 mg/Kg, CBZ dosed at 25 mg/Kg and vehicle control. Groups were dosed weekly starting 7 days post inoculation (Q7D x 3).

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Body Weight Analysis. An attractive feature of the CBZed-Nano formulation technology is the potential for improved safety and tolerability for drugs with known toxicities. In this study the impact of weekly drug administration on body weight as determined by percent change from baseline provides a useful means to make meaningful comparisons. A summary of body weight loss for CBZed-Nano formulations and free CBZ as a percentage relative to the vehicle control (saline) group are shown in Table 6.

Table 6. Body Weight Loss of Different Groups on Day 22 (1 day after third dose) P Value Treatment

BWL(%)

Mice Number vs group-1

Group-1 Vehicle Control

-1.63

10

-

Group-2 CLX 861 (25mg/Kg)

-1.94

10

0.849

Group-3 CLX 861 (100mg/Kg)

-8.13

10

0.022

Group-4 CLX 861 (150mg/Kg)

-14.91

10

10%) for the highest dosed groups (150 mg/Kg), moderate ( 10%), the intermediate group experiencing moderate change (5-10%) and the low dose group dropping marginally (< 5%). Recovery in weight post dosing generally spanned several days for the low and intermediate dosed groups whereas a week was required for the highest dosed groups. Issues related to tolerability of the free CBZ drug at the 25 mg/Kg dose are apparent from these plots with % body weight losses

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equaling and trending similarly to those found for the highest dosed CBZed-Nano formulations at 150 mg/Kg. These findings clearly indicate that CBZed-Nano formulations are better tolerated and require shorter recovery times at doses up to 4X higher than free CBZ in this model. These results suggest that CBZed-Nano nanoparticles would likely have a much wider therapeutic window compared to free CBZ drug.

DISCUSSION We report here on three novel CBZed-Nano compositions, CLX-861, CLX-706, and CLX-703 which are synthetic analogs of the prototype nanoparticle composition CLX-661. The CLX-861 polymer was synthesized with optimized chemistry using a scalable continuous flow process as compared to CLX-661 resulting in nanoparticles of comparable size (60-70 nm) to CLX-661 (60-70 nM). By varying conditions of synthesis and assembly, we also produced smaller species of particles; CLX-703 (28-40 nm) and CLX-706 (20-25 nm), which we reasoned might be more effective in penetrating tissue and delivering a taxane payload to the brain. In vitro characterization confirmed that all CBZed-Nano formulations were active and potent with similar (low nM) IC50’s in both PC3 and GBM cell lines. In vitro plasma stability was notably higher for nanoparticles derived from CLX-703 than for the other three compositions for reasons that are not apparent from analysis of physical characteristics and chemical composition. However, based on this and the absence of efficacy results for CLX-703 in our in-vivo orthotopic PC3 model relative to CLX-661 and CLX-861 we de-prioritized this formulation from further in vivo study. Although we do not have complete mechanistic data available at this time we believe

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it would be important for drug loaded nanoparticles that are able to cross the BBB to be labile to hydrolysis in order to liberate the drug at the tumor site in sufficient concentration to be efficacious. Considering the tunable features of the Cellax technology we believe this to be an interesting area for further exploration. In vivo, in an intracranial U87MG GBM mouse model, fluorescently labeled CBZed-Nano formulations showed a propensity to accumulate in the brain, especially in the region of intracranial brain tumors. The DiI dye fluorescence acts as a surrogate for drug penetration and demonstrates consistent uptake in tumor tissues. While some of the 24h time points did not reach statistical significance relative to normal brain tissue, which may be due in part to the long circulating properties of this formulation, we did see significant uptake of multiple CBZed-Nano formulations within tumor regions. Differing DiI intensity could be accounted for by differing amounts of DiI labeling between formulations (which was normalized in this study), but also differences in clustering of extravasated nanoparticles. Changes in area fraction of dye could be interpreted as variations in extravasation of DiI particles between different formulations, which could occur due to slightly different formulation dependent biodistribution. This may be due to nanoparticle-blood vessel – tumor interactions. This selective accumulation, which may result from a compromised BBB in these regions, prompted us to investigate the distribution in a screening pharmacokinetics study of representative nanoparticle types CLX-861 and CLX-706 in plasma and in the brain of mice bearing orthotopic brain tumors. Intravenous administration of both particle types produced measurable levels of conjugated CBZ in the brain, with relatively long residence times.

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Finally, in the orthotopic GBM LN229 model, we found that the CLX-861 CBZed-Nano formulation was tolerable and efficacious, providing statistically superior improvement in survival at a 100 mg/Kg (total CBZ) dose relative to vehicle control or unconjugated free CBZ drug. The free CBZ control showed poor tolerability at the 25 mg/Kg dose and resulted in the loss of 3 of 10 mice due to toxicity at this dose and clearly would not be tolerated at a 100 mg/Kg dose. Surprisingly, the smaller CLX-706 nanoparticles did not demonstrate statistical benefit in the efficacy study relative to controls and free CBZ drug. This lack of improved therapeutic index in the smaller particles did not result from reduced plasma stability relative to CLX-861, but may have been a consequence of poorer selectivity in targeting tumor versus normal tissue. Interestingly, at the highest CLX-861 dose tested, 150 mg/Kg, survival of the GBM mice was also poorer than controls, perhaps reflecting an upper limit of tolerability for these particles, based on an as yet unidentified toxicity. Potential toxicity concerns related to the carboxymethyl cellulose or PEG components of the Cellax-CBZ conjugates have not been the subject of the present study, however it is known that small lipophilic molecules of suitable molecular weight and charge may cross from the blood into the CNS with most all molecules of mw > 500 unable to cross the BBB.16 Future work focusing on the stability, permeation and invivo toxicity of smaller fragments of the efficacious CBZed-Nano formulation, which was derived from the CLX-861 conjugate would be expected to address this.

CONCLUSIONS The initial results in glioma models with CBZed-Nano formulation CLX-861 as a representative Cellax nanoparticle are promising and indicate further work on dose and schedule are warranted.

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This formulation would be the preferred candidate to advance into formal toxicity studies and be the focus of future work. More generally, if clinically validated, the Cellax technology has the potential to improve the therapeutic potential of a wide variety of cancer chemotherapeutic agents in GBM, metastatic brain disease, and other indications.

EXPERIMENTAL PROCEDURES Polymer Synthesis. Cellax-CBZ polymer (prototype CLX-661) is synthesized from an acetylated carboxymethylcellulose polymer (CMC-Ac), Cabazitaxel (CBZ), and polyethylene glycol (PEG) as previously described,13 CLX-861, CLX-703 and CLX-706 are analogues synthesized using a variation of CLX-661 chemistry with additional modifications to reaction conditions.

NMR Quantification of Nanoparticle Dose and Polymer Composition. The chemical composition of the polymer conjugates (CLX-661, CLX-703, CLX-706 and CLX-861) and dose of CBZed-Nano particle formulations were determined by quantitative 1H-NMR (qNMR) using 2-methyl 5-nitro benzoic acid as an internal standard. CBZ content was confirmed by determining the taxane ring response (as determined by integration of the signal) within the sample relative to the internal standard employing a CBZ calibration. Likewise, PEG content was confirmed by comparing the ethylene glycol response within the sample relative to the internal standard employing a PEG calibration. Dose concentrations from CBZed-Nano

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formulations were measured similarly, after being lyophilized, dissolved with a DMSO-d6 solution containing internal standard and sonicated.

CBZed-Nano Formulation, DiI Encapsulation and Concentration. Nanoparticles derived from Cellax-CBZ polymers, with or without DiI were prepared by a controlled nanoprecipitation process through a two-channel microfluidic system (NanoAssemblr, Precision Nanosystems, Canada) adapting a procedure originally developed for Cellax-Docetaxel nanoparticles.14 For imaging studies, a fluorescent dye (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate) DiI (Sigma 42364) compound can be stably encapsulated in CBZed-Nano formulations. Particles were formed with the following parameters; flow rate ratio of 3:1 saline:polymer (or polymer-DiI mixture), total flow rate of 18 mL/min., quenching in equal parts saline and concentrations of 30 mg polymer/g MeCN for CLX-706, 10 mg polymer/g MeCN for CLX-703 and CLX-661. For formulations containing DiI each polymer solution contained 1% DiI by mass which was taken from a well-mixed stock DiI solution of 10 mg/mL in MeCN. Product was dialyzed to remove MeCN, sterile filtered and concentrated.

Concentration Determination of DiI in CBZed-Nano Formulations. To calculate DiI concentration a DiI calibration curve (calibration points: 80, 16, 3.2, 0.64, 0.128, 0.0256, 0.00512, and 0.0 ug/mL) was generated through serial dilution using MeCN as diluent and placed into a 96 polystyrene black well plate. Fluorescence on the microplate was measured by means of a microplate reader (Excitation λ=535 nm, Emission λ=590 nm).

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Dynamic Light Scattering Measurements. Particle sizes were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (model # ZEN3600) instrument at room temperature. Samples were diluted with normal saline until clarified (clear) and measured at a scattering angle of 173 degrees with a 633 nm He-Ne laser. The sizes reported are averages of three measurements. The measurements provided are the mean of three measurements of 10 runs each. Reported Z-average diameters are the intensity-weighted diameters obtained from dynamic light scattering measurements and PDI is the polydispersity index obtained from the cumulants fitting program, and reported by the DLS instrument.

TEM Microscopy. Nanoparticle samples were negatively stained with a 2% aqueous solution of uranyl acetate (UA) on a carbon copper grid and evaluated via a Hitachi 7000 TEM (Schaumburg, IL) using an acceleration voltage of 75 kV and a direct magnification of 70,000x.

In-vitro Drug Release and LC-MS Analysis. LC-MS (AB SCIEX QTRAP 5500) was used to analyze drug release from CBZed-Nano formulations suspended in mouse plasma (Balb/c) containing sodium heparin (Innovative Research). Complete details for the methods used to prepare samples, internal and calibration standards and isolate samples for analysis including a full description of the LC-MS methods and spectroscopic parameters have been previously reported.5

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In-vitro Activity Screening in PC3 and GBM Cell Lines. Determination of the IC50 of CBZed-Nano formulations 661, 703, 706 and free CBZ on the PC3 prostate cancer cell line were obtained using the ATPLite proliferation assay. This assay has been used to determine the potency of CBZed-Nano formulations derived from CLX-661 as previously reported13 and in this study has also been employed to evaluate select CBZed-Nano formulations prepared with DiI. Determination of the IC50 of CBZed-Nano formulations 861 and 706 and free CBZ on select human cancer GBM cell lines LN-229 and U118MG was accomplished using the CellTiterGlo® luminescent cell viability assay (Promega, Cat. No.: G7572 at CrownBio international R&D center, ChangPing District, Beijing, P.R. China 102200). These two cell lines were selected for evaluating the potency of CBZed-Nano formulations since a corresponding orthotopic human GBM cancer xenograft model suitable for efficacy evaluation had been optimized in both cell lines. Each cancer cell line was tested with free unconjugated CBZ, CBZed-Nano formulations (CLX-861 and CLX-706), a standard chemotherapy drug (Cisplatin) as reference control, vehicle used for test article and culture medium as a negative control. All cells were cultured in DMEM media (GIBCO, USA) supplemented with 10% FBS at 37°C, 5% CO2 and 95% humidity. CBZed-Nano formulations derived from CLX-861 and CLX-706 were supplied by OICR (Canada), CBZ (Bolon Pharmachem, China) and Cisplatin (Qilu Pharma, China). On day 1, the cells were seeded. On day 2, drug formulations were dosed. For readout the CellTiter-Glo® reagent was added and the suspension was agitated for 2 minutes. The suspension was allowed to incubate at room temperature for 10 mins and luminescence was recorded using an Envision reader. The data were plotted using GraphPad Prism 5.0. In order to

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calculate IC50, a dose-responsive curve was fitted using nonlinear regression with a sigmoidal dose response model.

Pharmacokinetic Study in Orthotopic LN-229 GBM Xenograft Model. The LN-229 cell line was selected for modelling since IC50 results obtained from several GBM cell lines tested indicated that CBZed-Nano formulations had the highest potency in this cell line. CBZed-Nano formulations (CLX-861 and CLX-706) were singly dosed at 100 mg/Kg and free drug CBZ at 25 mg/Kg respectively to female Balb/c nude mice with 3 animals per group. Pharmacokinetic parameters were calculated from plasma and brain tissue samples after intravenous (i.v.) bolus administration of CBZed-Nano formulations (CLX-861 and CLX-706), free drug (CBZ) and a saline vehicle control, based on blood and brain tissue collection at 30 minutes, 5 hs and 24 h intervals post dosing. Female BALB/c nude mice (Shanghai Lingchang Bio-Technology Co. Ltd (LC, Shanghai, China)), 6-8 weeks old, weighing approximately 18-22g were employed in this study. The LN-229 cell line was maintained as a monolayer culture in DMEM media supplemented with 10% fetal bovine serum (FBS) at 37°C in an atmosphere with 5% CO2. Tumor cells were sub-cultured 2 times per week and cells in the exponential growth phase were harvested and counted for tumor inoculation. Mice were first anesthetized by intramuscular injection of ketamine / xylazine. The head skin of the mouse was sterilized with 75% alcohol. A 2 to 3 mm length incision was made at the right of the midline and anterior to the interaural line. Mice were then inoculated intracranially with 1x 105 tumor cells in 2 µL of PBS at the right frontal lobe, 2 mm lateral from the bregma and 0.5 mm from the anterior at a depth of 3.5 mm. The incision was stitched using No.6 suture and then sterilized with povidone iodine solution.

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The mice were kept warm until they recovered from the anaesthesia. The treatments were started at day 7 post inoculation. Prior to inoculation, before grouping and treatment, all animals were weighed and body weight was used to randomize animals into specified groups in order to minimize systematic error.

Blood and Brain Collection Procedure. Approximately 200 uL of whole blood was collected via retro-orbital puncture and placed into labelled K2-EDTA tubes. Tubes were maintained on ice and centrifuged at 8,000 rpm for 5 minutes. Plasma samples (50 µL) were transferred to labelled EP tubes and stored at -80oC prior to shipping. After blood collection was completed animals were perfused with a saline solution administered via infusion needle into the left ventricle with needle fixed in place with a hemostat. Perfusion was initiated with saline to flush the residual cycling blood. When saline was clear, perfusion was deemed complete. Once complete the skin above the skull was removed by fixing the skull with tweezers and using an eye scissor to cut open the skull from the foramen magnum to the eye socket on both sides. The skull was raised to expose the brain tissue, the meningeal was removed and all vessels and nerves cut. Elbow tweezers were used to insert the bottom of rhinencephalon and the whole brain was then removed. To labelled pre-weighed tubes the recovered brain was placed, the weight recorded and sample snap frozen. Samples were stored at -80oC prior to shipping.

Bioanalytical Analysis of Plasma and Brain Tissue Samples. For analysis of plasma 20 µL was aliquoted to two micro-centrifuge tubes. One set was used for total CBZ analysis. Another set was used for released CBZ analysis. For total CBZ analysis, 10

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µL of morpholine was added to each aliquot to hydrolyze and liberate the covalently linked CBZ. The morpholine treated samples were incubated for 2 h in a 40°C water bath. After the incubation step, a 90 µL volume of ice-cold quenching solvent (acetonitrile containing 0.5% formic acid and 100 ng/mL of internal standard CBZ-D6) was added to samples. For released CBZ analysis, 10 µL of saline was added to each aliquot followed by a 90 µL volume of ice-cold quenching solvent. Calibration standards were prepared by spiking appropriate concentrations of standard CBZ solution to aliquots of blank plasma or tissue homogenate. 10 µL of saline was added to each aliquot follow by a 90 µL volume of ice-cold quenching solvent. All samples obtained for CBZ analysis and calibration standards were vortexed, centrifuged at 14000 rpm for 6 min, and the supernatant collected and analyzed by LC-MS-MS. Pharmacokinetic parameters were obtained by non-compartmental modelling analysis with Phoenix WinNonlin Software® (Pharsight Corp, Mountain View, CA). For analysis of brain tissue samples the homogenized brain was centrifuged at 2,000 rpm for 3 min and placed on ice for 30 minutes to remove major bubbles and large aggregates. 40 µL of brain homogenates were aliquoted to two micro-centrifuge tubes. One set was used for total CBZ analysis. Another set was used for released CBZ analysis. For total CBZ analysis, 20 µL of morpholine was added to each aliquot to hydrolyze and liberate the covalently linked CBZ. The morpholine treated samples were incubated for 2 h in a 40oC water bath. After the incubation step, a 120 µL volume of ice-cold quenching solvent (acetonitrile containing 0.5% formic acid and 100 ng/mL of internal standard CBZ-D6) was added to all samples. For released CBZ analysis, 20 µL of saline was added to each aliquots follow by a 120 µL volume of ice-cold quenching solvent.

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Calibration standards were prepared by spiking appropriate concentrations of free CBZ stock solution to aliquots of blank brain homogenate. To each aliquot 20 µL of saline was added followed by a 120 µL volume of ice-cold quenching solvent. All samples obtained for CBZ analysis and calibration standards were vortexed, centrifuged at 14000 rpm for 6 min, and the supernatant collected and analyzed by LC-MS-MS. Quantitative analysis of CBZ was done by LC-MS-MS (AB Sciex Q-Trap 5500 mass spectrometer equipped with Agilent 1200 series HPLC system). Target ions were set at 836 > 555 (m/z) and 842 > 561 (m/z) for CBZ and the internal standard, CBZ-D6, respectively. Calibration curves were generated using peak area ratios of CBZ to internal standard versus the known CBZ concentrations employing a linear regression analysis with a weighting scheme of 1/concentration. A calibration curve of free CBZ (no morpholine treatment) was plotted and used to calculate all quality control and sample concentrations. Pharmacokinetic parameters were obtained by non-compartmental modelling analysis with Phoenix WinNonlin Software® (Pharsight Corp, Mountain View, CA).

In Situ Quantification of CBZed-Nano in Orthotopic U87MG GBM Tumor Tissue. Female NCr nu/nu mice, age 8-12 weeks, were implanted intracranially on day 1with 1x106 U87MG cells at Charles River Labs (Morrisville, NC). On day 12, 100 mg/Kg of nanoparticle preparation (CLX 661, CLX 703, or CLX 706) was administered i.v.. Animals were sacrificed at 24h post-dose, and brains were removed and divided into 2 sagittal sections cryopreserved with OCT. Quadracep muscle tissue was also cryopreserved in Oct to act as a control. Brain sections were scanned for DiI fluorescence via TissueScope 4000 (Huron Technologies, Waterloo ON), followed by H&E staining of same tissue section, digitized to a pyramid bigTiff

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file. The DiI fluorescence image was aligned to its corresponding H&E image via rigid registration using a custom built MATLAB script, and aligned tiff images were loaded into Definiens Tissue Studio 4.0 software for quantification. Tumor region(s) of interest were defined by training the software to recognize tumor tissue using a machine-learning classifier on the H&E image, with manual quality correction to ensure appropriate region identification. Image quantification intensity thresholds were chosen to quantify the area of CBZed-Nano / DiI marker present within each region (Tumor, normal brain), 3 thresholds corresponding to more permissive (low, intensity of 60 (arbitrary units)) to more restrictive (high, intensity of 150), were chosen, and areas were calculated for each sample and region of interest (Tumor, normal brain). Total tumor and normal brain area for each region of interest (ROI) were used to calculate the area fraction covered by DiI positive signal. In addition, mean DiI signal within the entire region of interest, and within the DiI positive volume, is reported. As DiI fluorescence calibration by dilution series produced inconclusive results (data not shown), intensities were corrected by the ratio of the dye concentrations present within the particles. Thus, the "Normalized nanoparticle intensity" corresponds to the DiI intensity multiplied by the ratio of CLX-703 / CLX (661 or 706), to normalize the intensity to "effective nanoparticle concentration".

Orthotopic LN-229 GBM Efficacy Model. An in vivo efficacy study of CBZed-Nano formulations CLX 861 and CLX 706 versus free unconjugated drug (CBZ) and a vehicle control (saline) in the treatment of orthotopic LN-229 GBM xenograft model in female BALB/c nude mice (Shanghai Lingchang Bio-Technology Co. Ltd (LC, Shanghai, China) was evaluated at

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CrownBio international R&D center, ChangPing District, Beijing, P.R. China 102200). Eight study groups with N=10 animals/each were treated once every seven days for 3 weeks, i.v., as follows by group number: (1) vehicle, (2) 25 mg/Kg CLX 861, (3) 100 mg/Kg CLX 861, (4) 150 mg/Kg CLX 861, (5) 25 mg/Kg CLX 706, (6) 100 mg/Kg CLX 706, (7) 150 mg/Kg CLX 706, (8) 25 mg/Kg CBZ.

Observations, Handling and Termination of Mice and Data Collection. Tumor cell inoculation was executed following the same procedure as described in the section describing the Pharmacokinetics study. In this study the animals were checked daily for morbidity and mortality. At the time of routine monitoring, the animals were checked for any effects of tumor growth and treatments on normal behavior such as mobility, visual estimation of food and water consumption, body weight gain/loss (body weights were generally measured twice weekly and more frequently (daily) once body weight losses approached 15-20%, eye/hair matting and any other abnormal effects. Deaths and clinical signs were observed and recorded for each mouse in order to separate any toxicity related deaths, which may be attributed to drug as opposed to have resulted from disease progression. Toxicity related deaths in mice were considered to occur within 2 weeks of last dose of drug administered. Animals with body weight loss (BWL) induced by the therapy of > 20% post dosing were given a dose holiday until BWL recovery improved to 20%, if animals were found to be having other severe clinical signs (e.g. prolonged diarrhea, persistent anorexia, labored respiration, lethargy or failure to respond to gentle stimuli) or in the case where

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animals could not obtain adequate food or water. The major endpoints in this study were animal survival and body weight. Animals were checked daily for disease progression and for drug-related toxicities and animals that showed deteriorating and moribund condition were euthanized with CO2. The survival of all animals was followed and Median Survival Time (MST) was calculated for each group. The increase in life-span (ILS) was calculated as follows: ILS (%) = 100 x [(Median Survival Time of drug treated group/Median Survival Time of vehicle group) – 1] (%). The log-rank test was used to compare survival curves between groups and Independent-Samples T Test was used to compare the body weight change between groups. The method detailed under pharmacokinetic study in orthotopic GBM xenograft model was followed for this procedure.

Review of Animal Study Designs. Protocols for animal studies described herein were reviewed and approved by the institutional animal care and use committee (IACUC) at the relevant institutions (Charles Rivers and Crown Biosciences). All animal research conducted adhered to the ’’Principles of Laboratory Animal Care’’ (NIH publication #85-23, revised in 1985).

ASSOCIATED CONTENT Supporting Information Chemistry scheme and reaction conditions employed to prepare Cellax-CBZ polymers (CLX661, CLX-703, CLX-706 and CLX-861), specifications of polymers and CBZed-Nano formulations derived from Cellax-CBZ polymers; summary of the qNMR method for

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determining wt% of CBZ and PEG in Cellax-CBZ conjugate polymers and in CBZed-Nano particles; LC-MS method for quantifying residual unconjugated free CBZ and PEG; LC-MS parameters used to quantify CBZ in plasma release studies; A derivation of the equations used to calculate CBZTotal, CBZRecovered and CBZCoupled for the bioanalytical analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors † E-mail: Fight Against Cancer Innovation Trust, Toronto, Ontario, Canada; [email protected]; Phone: 613-979-3235, Triphase Accelerator, La Jolla, California, USA; [email protected]; Phone: 650-303-4544.

Notes: The authors declare no competing financial interest.

ACKNOWLEGDEMENTS

We thank Dr. Ahmed Aman in the Medicinal Chemistry Platform at the Ontario Institute for Cancer Research for his assistance with the bioanalytical chemistry method development. This study was conducted with support from the Ontario Institute for Cancer Research (OICR) and the Fight Against Cancer Innovation Trust (FACIT) through funding provided by the Government of Ontario. The authors would like to dedicate this manuscript to Dr. Mark Ernsting, who was one of the key inventors of the Cellax technology. Dr. Ernsting passed away in December 2015.

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ABBREVIATIONS

DAB, 10-deacetylbaccitin 3; DTX, Docetaxel; CMC-Ac, acetylated carboxymethyl cellulose polymer; CBZ, Cabazitaxel; CBZ-D6, deuterated Cabazitaxel; PEG, polyethylene glycol; Pgp, Pglycoprotein; ATP, adenosine triphosphate; GMP, good manufacturing practices; IC50, half maximal inhibitory concentration; PC3 cells, a human prostatic carcinoma cell line; DiI, 1,1’dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate; qNMR, quantitative nuclear magnetic resonance; LC-MS, liquid chromatography/mass spectroscopy; MeCN, acetonitrile; DMF, dimethyl formamide; EDC.HCl, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloric acid; NHS, N-Hydroxy succinimide; DMAP, 4-Dimethylaminopyridine; DLS, dynamic light scattering; PDI, polydispersity index; TEM, transmission electron microscopy; DMEM, Dulbecco's Modified Eagle Medium; FBS, fetal bovine serum; K2-EDTA, dipotassium ethylenediaminetetraacetic acid; MALS, multi-angle light scattering; GPC, gel permeation chromatography; BWL, body weight loss; MST, Median Survival Time.

REFERENCES

(1) Ernsting, M. J., Tang, W. L., MacCallum, N., and Li, S. D. (2011). Synthetic modification of carboxymethylcellulose and use thereof to prepare a nanoparticle forming conjugate of docetaxel for enhanced cytotoxicity against cancer cells. Bioconj. Chem. 22, 2474-2486.

(2) Ernsting, M. J., Murakami, M., Roy, A., and Li, S. D. (2013). Factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles. J. Controlled Release 172, 782-794.

(3) Li, S. D., Ernsting, M. J., and Tang, W. L. (2013). U.S. Patent No. 8,591,877. Washington, DC: U.S. Patent and Trademark Office.

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