Proteoglycans as target for an innovative therapeutic approach in chondrosarcoma: Preclinical proof of concept

Article abstract of DOI:10.1158/1535-7163.MCT-16-0003

To date, surgery remains the only option for the treatment of chondrosarcoma, which is radio- and chemoresistant due in part to its large extracellular matrix (ECM) and poor vascularity. In case of unresectable locally advanced or metastatic diseases with a poor prognosis, improving the management of chondrosarcoma still remains a challenge. Our team developed an attractive approach of improvement of the therapeutic index of chemotherapy by targeting proteoglycan (PG)-rich tissues using a quaternary ammonium (QA) function conjugated to melphalan (Mel). First of all, we demonstrated the crucial role of the QA carrier for binding to aggrecan by surface plasmon resonance. In the orthotopic model of Swarm rat chondrosarcoma, an in vivo biodistribution study of Mel and its QA derivative (Mel-QA), radiolabeled with tritium, showed rapid radioactivity accumulation in healthy cartilaginous tissues and tumor after [3H]-Mel-QA injection. The higher T/M ratio of the QA derivative suggests some advantage of QA-active targeting of chondrosarcoma. The antitumoral effects were characterized by tumor volume assessment, in vivo 99mTc-NTP 15-5 scintigraphic imaging of PGs, 1H-HRMAS NMR spectroscopy, and histology. The conjugation of a QA function to Mel did not hamper its in vivo efficiency and strongly improved the tolerability of Mel leading to a significant decrease of side effects (hematologic analyses and body weight monitoring). Thus, QA conjugation leads to a significant improvement of the therapeutic index, which is essential in oncology and enable repeated cycles of chemotherapy in patients with chondrosarcoma.

Full text of DOI:10.1158/1535-7163.MCT-16-0003

Proteoglycans as target for an innovative therapeutic approach in chondrosarcoma: preclinical
proof of concept
Caroline Peyrode1,2,≠ and Valérie Weber1,2,≠, Aurélien Voissière1,2, Aurélie Maisonial-Besset1,2,
Aurélien Vidal5, Philippe Auzeloux1,2, Vincent Gaumet1,2, Michèle Borel1,2, Marie-Mélanie Dauplat4,
Mercedes Quintana1,2, Françoise Degoul1,2, Françoise Rédini3, Jean-Michel Chezal1,2, Elisabeth Miot-
Noirault1,2
1.
Clermont Université - Université d’Auvergne - Imagerie Moléculaire et Thérapie Vectorisée -
F-63000 Clermont-Ferrand - FRANCE
2.
3.
4.
5.
≠
INSERM - UMR990 - F-63005 Clermont-Ferrand - FRANCE
INSERM - UMR S957 - Nantes Atlantique Université, 44035 Nantes - FRANCE
CLCC Jean-Perrin - 63001 Clermont-Ferrand Cedex - FRANCE
Arronax - CS 10112 - 44817 Saint Herblain Cedex - FRANCE
Both authors contributed equally to this work.
Short title : Proteoglycans as target for therapy of chondrosarcoma
Keywords : Chondrosarcoma, targeting, proteoglycans, melphalan, therapy
Financial support : Researchers assigned to our laboratory UMR 990 Inserm/UdA received a financial
support from Ligue contre le cancer Auvergne, CPER and PRTK INCa/DGOS 2015-051 for
coordination of all the work presented here.
Corresponding author :
Caroline Peyrode - UMR 990 Inserm/UdA - 58 rue Montalembert, BP 184, 63005 Clermont-Ferrand
Cedex – FRANCE
Telephone : 0033 4 73 17 80 30, Fax : 003 4 73 15 08 01
Email : caroline.peyrode@udamail.fr
Conflicts of interest : No potential conflicts of interest were disclosed
Word count: Astract : 236 words
Text (excluding references): 4899 words
Number of figures : 5
Number of tables : 1
1

Abstract
To date, surgery remains the only option for the treatment of chondrosarcoma (CHS), which is
radio- and chemoresistant due in part to its large extracellular matrix (ECM) and poor vascularity. In
case of unresectable locally advanced or metastatic diseases with a poor prognostic, improving the
management of CHS still remains a challenge. Our team develops an attractive approach of
improvement of the therapeutic index of chemotherapy by targeting proteoglycan(PG)-rich tissues
using a quaternary ammonium (QA) function conjugated to melphalan (Mel). First of all, we
demonstrated the crucial role of the QA carrier for binding to aggrecan by Surface Plasmon
Resonance. In the orthotopic model of swarm rat CHS, an in vivo biodistribution study of Mel and its
QA derivative (Mel-QA), radiolabelled with tritium, showed rapid radioactivity accumulation in
healthy cartilaginous tissues and tumor after [3H]-Mel-QA injection. The higher T/M ratio of the QA
derivative suggests some advantage of QA active targeting of CHS. The antitumoral effects were
1
characterized by tumor volume assessment, in vivo 99mTc-NTP 15-5 scintigraphic imaging of PG, H-
HRMAS NMR spectroscopy and histology. The conjugation of a QA function to Mel doesn’t
hampered its in vivo efficiency and strongly improves tolerability of Mel leading to a significant
decrease of side effects (hematological analyses and body weight monitoring). Thus QA conjugation
leads to a significant improvement of the therapeutic index, which is essential in oncology and enable
repeated cycles of chemotherapy in patients with CHS.
2

Introduction
One of the key problems in chondrosarcoma (CHS), the second most common type of skeletal
malignancy after osteosarcoma, is the lack of response to both chemotherapy and radiotherapy, giving
rise to poor patient outcomes and leaving surgery as the only effective curative treatment. The type of
required surgical procedure may vary according to the grade, the initial location and the extent of the
disease. Large en-bloc dramatic resection or amputation, often necessary to allow the local control of
the tumor and to avoid metastases, are at the origin of strong disability and morbidity (1).
Nevertheless, wide resection is not always possible in large tumors and critical anatomic locations
such as pelvis, axial skeleton and also in many cases of local recurrence. The clinical behavior and the
prognosis for patients with CHS vary widely with a 10-year survival rate ranging from 29 to 83%
depending on the tumor grades (2,3).
For CHS, the current clinical challenge is to prevent recurrences and to find better treatment options
for patients with inoperable primary or recurrent disease, and metastases. Published data regarding the
sensitivity of CHS to cytotoxic agents are scarce in both pre-clinical and clinical reports. A recent
study has shown that some CHS cell lines may display moderate sensitivity to doxorubicin and
cisplatin, the most commonly used chemotherapeutic agents in sarcomas (4). In 2012, in a large
retrospective and multicenter series, chemotherapy using anthracycline was associated with a median
progression free survival of 4.7 months in the first-line (2,3). The study of Van Maldegen et al. also
demonstrated that the survival rate in patients with unresectable CHS was also significantly higher in
the case of systemic treatments (doxorubicine-based chemotherapy, and nonchemotherapy-based
agents such as imatinib and sirolimus) compared to patients managed with best supportive care only
(overall survival at three years: 26% versus 8% respectively, p < 0.05) (5). According to such poor
outcome and no global improvement in survival of patients with CHS over the past thirty years,
personalized medicine including a combination of targeted therapies will most likely be the best
approach to tackle the resistance of human CHS.
Treatments targeting tumor microenvironment have therefore been suggested as complements to
surgical excision (6,7). Nevertheless, due to the large amount of extracellular matrix (ECM) and the
3

poor vascularity of CHS, the anticancer agents have to diffuse over a relatively long distance in order
to reach tumor cells and be effective (5).
The best approach for a personalized medicine of CHS might be to take advantages of such phenotypic
microenvironmental features, by developping an attractive approach of targeting the
proteoglycan(PG)-rich cartilaginous tissues using a quaternary ammonium (QA) function as a carrier
for drugs or radionuclides. The positively charged QA interacts with the sulfate and carboxylate
groups present in PG highly expressed in the chondrogenic ECM (8). With this strategy, we
successfully delivered imaging agents as well as therapeutic drugs (such as anti-inflammatory drugs,
metalloproteinase inhibitors, cytotoxics and more recently radiosensitizing nanoparticles (8–13))
selectively to PG-rich tissues such as cartilage and CHS. In particular, the suitability and high
sensitivity of the radiotracer N-[triethylammonium]-3-propyl-1,4,7,10,13-pentaazacyclopentadecane
radiolabeled with 99mTc (99mTc-NTP 15-5) was validated for in vivo diagnosis and follow up of CHS
via scintigraphic imaging (14–16). This radiotracer is currently transfered into clinic for a first
evaluation in humans. In parallel we developed a therapeutic approach with a QA derivative of
melphalan (Mel-QA), an alkylating agent, that showed promising results from in vitro and in vivo
preclinical experiments (13). The aim of the present study was to characterize this therapeutic strategy.
First of all, the binding ability of Mel-QA to aggrecan (major PG in CHS) was demonstrated
by Surface Plasmon Resonance (SPR) respectively to its non-targeted equivalent (Mel). Quantitative
whole body autoradiography showed rapid accumulation of radioactivity in cartilaginous tissue after
injection of the tritium radiolabeled Mel-QA, as compared to Mel. The main interest of the QA vector
was to strongly attenuate side effects, monitored by hematological analyses and body weight, while
1
maintaining a tumor growth inhibition assessed by in vivo 99mTc-NTP 15-5 scintigraphic imaging, H-
HRMAS NMR spectroscopy and histological analyses of biopsies.
The work reported here clearly validates the relevance of PG as target for an innovative therapeutic
approach in CHS.
4

Materials and methods
Chemical synthesis
Mel was used as its hydrochloride form after treatment of Mel (Interchim) with a solution of 2
N hydrochloric acid in diethyl ether. The QA conjugate of Mel (Mel-QA) was prepared as already
published (13,17) and fully characterized (Supplementary Fig. S1).
The NTP 15-5 and its non-targeted equivalent (i.e. 15-5) were synthesized and radiolabeled with
99mTc as previously described (18).
Surface Plasmon Resonance (SPR) binding assays
To demonstrate the binding affinity between the QA entity and aggrecan, we used the SPR
method with aggrecan immobilized onto the surface of the sensor chip. When studied compounds
interact with aggrecan, they form a complex which modifies the refractive index. This change is
measured in real-time and the signal obtained is plotted in resonance unit (RU) versus time (19,20).
SPR assays were carried out on a Biacore T200 instrument with CM4 sensor chips (GE Healthcare).
Sensor chip CM4 has a dextran matrix with a low degree of carboxylation which may be of value for
reducing non-specific binding with highly positive charged analytes and may be useful for kinetic
analysis. To immobilize the ligand, sensor chip was firstly activated using a 1:1, v:v, mixture of a 0.2
M aqueous 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt solution and a 0.5 M
aqueous N-hydroxysuccinimide (amine coupling kit, GE healthcare) solution at a flow rate of 5
μL/min for 10 min. After that, aggrecan from bovine articular cartilage (Sigma Aldrich) was coated to
the sensor chips at 400 μg/mL in running buffer HBS-P+ 1X (0.01 M HEPES, 0.15 M NaCl, 0.05%,
v/v, surfactant p20, pH 7.4) containing 6 mM of hexadecyltrimethylammoniumbromide (CTAB) with
a flow rate of 5 μL/min to a level of ∼500 RU. Unoccupied binding sites were blocked using an
aqueous 1 M ethanolamine solution (pH 8.5) (Amine coupling kit, GE Healthcare) with a flow rate of
5 μL/min for 10 min. Mel and Mel-QA were injected with a flow rate of 30 μL/min in increasing
concentrations (0 to 2 mM) using five 600-s injections and a 200-s dissociation time. Following each
experiment, flow cells were regenerated twice with NaCl 2 M for 150 s. The interactions were
recorded as the difference in response between the immobilised aggrecan flow cell and a
5

corresponding control flow cell (activated and blocked, but without immobilised aggrecan). Kinetic
and statistic parameters (KD and Chi2) were determined by Biacore T200 Evaluation software by
fitting the steady state values at equilibrium (Req). Experiments were performed in triplicate.
Orthotopic model of Swarm Rat Chondrosarcoma
The Swarm Rat Chondrosarcoma (SRC) line (Dr. Patrick A. Guerne, Geneva, Switzerland)
was delivered as tissue fragments which were frozen until use. Protocols were led under the
authorization of the French Directorate of Veterinary Services and in accordance with the 2010/63/UE
Directive and were conducted as previously described (13) using male Sprague-Dawley rats (Charles
River). All experiments were conducted after ethics committee approval (C2E2A, n°CE 82-12).
Allograft transplantation of a 10 mm3 SRC fragment was performed on the right paw, the other paw
being used as the contralateral control. These SRC fragments were collected from well-developed
tumors on the paratibial area of donor Sprague-Dawley rats.
Radiosyntheses of [3H]-Mel and [3H]-Mel-QA
An excess of N-t-Boc-4-amino-L-phenylalanine ethyl ester (1) was alkylated by reaction with
[3H]-2-bromoethanol ([3H]-3), previously obtained in situ from ethyl bromoacetate (2) (2 mmol) and
[3H]-NaBH4 (3.7 GBq, diluted with 2.2 mmol of unlabeled NaBH4). Then the crude reaction mixture
containing amine 1 and N-t-Boc-4-(2-hydroxy-[2-3H]ethyl)amino-L-phenylalanine ethyl ester ([3H]-4)
reacted with ethylene oxide (approx. 3 mL) in acetic acid to provide the corresponding 4-[bis(2-
hydroxy-[2-3H]ethyl)]amino-phenyl derivative ([3H]-5) in 50% radiochemical yield (2.15 GBq, 2
mmol). Appel chlorination with triphenylphosphine (5.9 mmol) and carbon tetrachloride (2 mL) in
dichloromethane (20 mL) gave, after silica gel chromatographic purification, the corresponding
tritiated dichloro derivative [3H]-6 (0.96 mmol, 962 MBq) in 48% chemical yield. Then, the ester and
the t-Boc protecting group of compound [3H]-6 were hydrolyzed with an aqueous 6 N hydrochloric
acid solution (5 mL) to provide the hydrochloride salt of tritiated Mel ([3H]-Mel, 0.96 mmol, 740
MBq) in quantitative chemical yield. On the basis of this radiolabeling sequence, [3H]-Mel was
isolated with a specific activity of 777 MBq/mmol and a radiochemical purity > 98%, as determined
by HPLC analyses (Supplementary Fig. S2). The overall yield of this radiochemical procedure was
20%.
6

For the preparation of tritiated Mel-QA, a five-step radiosynthesis starting from hydrochloride
salt of [3H]-Mel was developed. Thus, after Boc protection of the amino group of [3H]-Mel (0.69
mmol, 573 MBq), N-protected Mel was immediately coupled with 3-(dimethylamino)propylamine
(0.83 mmol) in the presence of dicyclohexylcarbodiimide (DCC, 1.03 mmol) and 1-
hydroxybenzotriazole (HOBt, 0.74 mmol) to give the corresponding amide intermediate [3H]-7 in 75%
chemical yield after purification by column chromatography. Treatment with methyl iodide (2.3
mmol) in ethanol gave the QA compound [3H]-8 in quantitative yield. Finally, the t-Boc-protecting
group was removed using a 2 N ethanolic hydrochloric acid solution (12 mL). A chromatography over
a chloride ion-exchange resin (Dowex® 1 × 8−200 mesh, Aldrich) provided the desired QA derivative
of Mel ([3H]-Mel-QA, 0.46 mmol, 407 MBq) in 75% overall chemical yield from [3H]-Mel.
Finally, [3H]-Mel-QA was isolated with a specific activity of 814 MBq/mmol and a radiochemical
purity > 97%, as determined by HPLC analyses (Supplementary Fig. S2). The overall yield of this
radiosynthesis was 15% based on [3H]-NaBH4.
Tissue distribution study of [3H]-Mel and [3H]-Mel-QA conjugate by whole body
autoradiography and urinary excretion
This study was performed on thirty SRC bearing rats at stage day 20 post implantation.
Animals were randomized into two groups (n=15 per group) and received i.v. injection of 2.5 MBq (in
200 µL of physiological saline) of [3H]-Mel or [3H]-Mel-QA.
At several time-points after injection (5 and 15 min, then 1, 6 and 24 h), intracardiac blood
collection were performed (3 rats from each group per time point) under anesthesia by inhalation of
isoflurane (CSP) in air (1.5%, 1 L/min). Animals were then immediately sacrificed by carbon dioxide
inhalation, frozen in liquid nitrogen and embedded in blocks of carboxymethyl cellulose 2%, that were
sagittally sectioned into 40 µm slices at -22 °C with a cryomicro-tome (Reichert-Jung Cryopolycut).
Each slice was dehydrated for 48 h in a cryochamber. Whole body slices (25 slices per animal and per
time point) were then exposed for 1000 min in a digital autoradiographic analyser (βImager®,
Biospace Mesures). Surfacic activity (counts in cpm/mm2) was quantified in regions of interest (ROIs)
delineated over organs of interest including tumors, corrected for radioactive decay and expressed as
% of Injected Dose (ID)/g of tissue, after calibration with tritium microscale standards
7

(Autoradiographic [3H] Microscale®, Amersham Biosciences). Tumor-to-muscle ratio was calculated
as follows: T/M = (counts per min/mm2 in tumor) / (counts per min/mm2 in muscle).
In a parallel experiment, groups of six rats received [3H]-Mel or [3H]-Mel-QA, and were housed in
metabolic cages for urine collection from 6 h to 24 h after injection.
Radioactivity of urine or blood samples was directly measured after addition of Packard Ultima Gold
cocktail (PerkinElmer) in a Wallac Winspectral 1414 liquid scintillation spectrometer (PerkinElmer).
Tumor growth inhibition assessment
Experiments were conducted on SRC bearing rats being randomized into three groups
(control, Mel-QA and Mel), with a number of 6 to 8 animals/group. Treated rats received three doses
of Mel or Mel-QA (16 µmol/kg per injection) by i.v. route at four-day intervals (q4d x 3 schedule),
beginning on day 8 post-implantation. Controls received the excipient by i.v. route according to the
same schedule. Animal weight and tumor volume were recorded twice a week. The Tumor Volume
(TV) of each tumor was estimated using the formula: TV (mm3) = (L x W2)/2 where L is the length in
mm, and W the width in mm.
At the end of the study, tumors were removed, fixed in 10% buffered formalin and then embedded in
paraffin. Slices (5 µm) were then stained with hematoxylin-eosin.
Antitumor efficacy assessed by in vivo 99mTc-NTP 15-5 scintigraphy imaging targeting PG
Antitumor efficacy was monitored by scintigraphy imaging, using the radiotracer 99mTc-NTP
15-5 developed by our group at day 7 (one day before treatment) and day 30 (two weeks after
treatment) (15). Acquisitions were performed 30 min after i.v. tracer administration (25 MBq/animal),
with a 10-min planar acquisition for each posterior paw positioned over the parallele-hole collimator
of a small-animal γ camera (Gammaimager®, Biospace) and with a 15% window centered on the 140-
keV photopeak of 99mTc. All the scans were evaluated by the same experienced investigator, using
fixed-size ROIs delineated over tumor and muscle patterns. For each ROI, total activity, count in cpm
(counts per min) per pixel were obtained. Tumor-to-muscle ratio was calculated: T/M = (counts per
min/mm2 in tumor) / (counts per min/mm2 in muscle).
8

To assess the in vivo specificity of 99mTc-NTP 15-5 imaging, additional SRC animals (n = 10)
were injected with 99mTc-NTP 15-5 or its non-targeted equivalent (99mTc-15-5) at day 20 post
implantation when tumors exhibited a mean volume of 949 ± 223 mm3. Dynamic Planar Imaging
(DPI) was performed to determine the kinetics of the tracer distribution in tumor. SRC animals were
i.v. injected via the tail vein with 25 MBq of 99mTc-NTP 15-5 or 99mTc-15-5 simultaneously to the
starting of a 120 min duration list mode acquisition using a 5 min sampling time. Time activity curves
were obtained from ROIs drawn around tumor and muscle with all measured activities corrected for
radioactive decay and T/M ratio calculated at each time point.
Antitumor efficacy analysis by 1H HRMAS NMR spectroscopy profile
1H HRMAS spectroscopy profile analyses were performed in a parallel study following the
same protocole with additional animals (4 animals per group). Tumors were excised one week after
the final injection, rapidly frozen in liquid nitrogen and stored at -80 °C until analysis.
All experiments were performed on frozen tumor samples flushed for a few seconds in cooled D2O. 1H
NMR spectroscopy was performed on a small-bore Bruker DRX 500 magnet (Bruker) equipped with
an HRMAS probe. Samples were then set into 4-mm-diameter, 50 µL free volume ZrO2 rotor tubes
without upper spacer. A total of 3 µL of D2O containing 0.75% 3-(trimethylsilyl)propionic-2,2,3,3-d4
acid (TSP; Sigma), used for peak referencing was added to the rotor tubes to lock the spectrometer.
Rotors were spun at 4 kHz in order to keep the rotation sidebands out of the acquisition window. A
Bruker cooling unit was used to maintain the sample temperature at 277 K to minimize tissue
degeneration. One-dimensional 1H NMR sequence was a saturation recovery sequence. The
resonances were assigned based on the known chemical shifts of the major structural groups using
tissues data (21). The relative content of metabolites was estimated by peak area integration. To
compare the metabolic profiles between experimental groups, each peak was normalized with the
respect of the total integral of the spectrum in the 0.5 to 4.7 ppm region (21–23). Sample stability
inside the rotor was assessed at a temperature of 277 K over several hours by a series of analyses.
During the 48 min overall acquisition period, no significant changes in NMR signals of biological
material were observed. Each experiment was performed in triplicate.
9

Side effect assessment
Side effects of treatments with Mel or Mel-QA were assessed by both animal’s weight and
hematological parameter measurements.
-
Weight variation was calculated according to the formula:
% = ((Weight at Day x -Weight at Day 4)/Weight at Day 4) x100.
- Hematological parameters were assessed at day 18, i.e two days after the end of treatments.
The blood (50 µL by retro-orbital puncture) of each animal was removed and processed on a Melet
Schloesing MS9-5 Hematology Analyzer (Diamond diagnostics).
Statistical analyses
Statistical analyses were conducted using the GraphPad Prism 5 software. Data are reported as
mean SD. Results were analyzed by ANOVA followed Tukey’s post-test. We considered p values <
0.05 to indicate statistical significance (* p < 0.05, ** p < 0.01 and *** p < 0.001).
10

Results
Evaluation of the QA binding to PG by Surface Plasmon Resonance (SPR)
Studies were performed for Mel-QA respectively to Mel injected in increasing concentrations
(0 to 2 mM). The sensorgrams obtained after signal subtraction of control was displayed versus time.
As illustrated in Figure 1 and regards to KD values, Mel-QA exhibited affinity to aggrecan (KD = 2.25
± 0.92 mM) in contrast to Mel demonstrating that QA vector allows aggrecan binding.
In vivo biodistribution studies of [3H]-Mel and [3H]-Mel-QA
Radiosyntheses of [3H]-Mel and [3H]-Mel-QA
The radiosyntheses of [3H]-Mel and [3H]-Mel-QA are shown in Figure 2A. Both Mel-QA and
3
Mel were radioisotopically labeled on the alkylating moiety, also H radionuclide was introduced in
the chloroethyl groups. [3H]-Mel was synthesized from N-t-Boc-4-amino-L-phenylalanine ethyl ester
according to a slightly modified sequence previously reported in the literature (24). For the preparation
of [3H]-labeled Mel-QA, the radiosynthetic sequence starting from [3H]-Mel was adapted from the
five-step synthesis developed for the corresponding non-radioactive derivative and 14C-labeled Mel-
QA (17,25). Both [3H]-Mel and [3H]-Mel-QA were successfully obtained with a specific activity of
814 MBq/mmol and a radiochemical purity > 97 %, as determined by radio-RP-HPLC analyses.
QA functionalization allows tumor uptake of Mel
The distribution of radioactivity in several tissues after i.v. injection of 2.5 MBq of [3H]-Mel
or [3H]-Mel-QA in SRC animals is expressed as the percentage of injected dose per gram of tissue (%
ID/g) (Table 1). After injection of [3H]-Mel-QA, radioactivity rapidly accumulates in PG-rich tissues
such as cartilages and tumor (Figure 2C). Five min post injection (p.i.) of [3H]-Mel-QA, the T/M value
was significantly higher (p < 0.05) compared to [3H]-Mel (T/M values 5 min p.i.: 5.77 1.11 for [3H]-
Mel-QA versus 2.22 1.20 for [3H]-Mel) (Figure 2B).
Furthermore, higher levels of each drug were found in the metabolizing and elimination organs, such
as liver and kidneys. As expected for small hydrophilic compounds, Mel and Mel-QA are cleared in
the kidney (i.e. about 16% of cumulative renal excretion) and no significant difference was observed
between the two compounds at 24 h p.i. (Table 1). However radioactivity was observed in liver after
[3H]-Mel-QA injection as compared to [3H]-Mel.
11

Finally, radioactivity in the blood was higher after injection of [3H]-Mel than [3H]-Mel-QA (Table 1).
In vivo antitumor efficacy and side effects of Mel and Mel-QA in SRC model
Mel-QA delays CHS growth in vivo
Mel-QA and Mel were given at 16 µmol/kg according to a q4d x 3 schedule beginning on day
8 post tumor-implant. The control group received i.v. saline injection.
Based on the tumor volume monitoring, a significant inhibition of tumor growth (p < 0.01)
was observed for both Mel-QA and Mel treated rats compared to the control group, but no significant
difference was observed between the two treated groups (Figure 3A).
In vivo antitumor effects were also assessed by 99mTc-NTP 15-5 scintigraphic imaging, as this
radiotracer was previously demonstrated to provide suitable criteria for monitoring CHS (14–16). As
illustrated in Figure 3B, quantitative analysis of in vivo 99mTc-NTP 15–5 imaging of tumors evidenced
significant changes in radiotracer uptake in treated animals versus controls. A significant increase of
84% of T/M ratio (p < 0.05) was observed in non-treated rats between day 7 (1 day before treatment)
and day 30 (14 days after the last drug injection) whereas this ratio remained overall constant in Mel
or Mel-QA treated rats. Furthermore, to assess the in vivo specificity of 99mTc-NTP 15-5 imaging and
to exclude the possibility of unspecific uptake in tumoral tissue, 99mTc-NTP 15-5 and its non-targeted
equivalent (99mTc-15-5) were injected by i.v. route in additional SRC bearing rats (n = 10). Time
activity curves are shown in Figure 3C in term of T/M ratio. From 5 min p.i. of 99mTc-NTP 15-5, T/M
ratio gradually increased and reached a plateau as soon as 30 min which was maintained at least for 1
h (T/M values 1 h p.i.: 2.25 0.11), allowing tumor visualization with an excellent contrast (Figure
3D). On the contrary, T/M ratio for 99mTc-15-5 was around 1 throughout the course of the study.
According to these results, we demonstrated the potential of 99mTc-NTP 15-5 as a “methodology
companion” of targeted therapies of CHS.
To characterize the antiproliferative effects, tumors were removed one day after the end of the
treatments and anatomopathological analyses were performed. For both treated groups,
histopathological analyses showed signs of tumor regression, such as necrotic cells, fibro-
inflammatory component and increased vascularization. These signs were observed for 57% and 40%
of tumors in the Mel-QA and Mel groups, respectively (Figure 4A). Furthemore mitotic activity index
12

values showed a significant decrease (p << 0.01) in the proliferating activity in the two treated groups
(Figure 4B). In contrast, the tumors of non-treated rats were characterized by hypercellularity with a
high mitotic activity index.
Finally, in a parallel study, SRC bearing rats were treated following the same protocol and
1
tumors were excised one week after the final injection for H-HRMAS NMR spectroscopy analyses
(Figure 4C) which confirmed necrosis and apoptosis. Results revealed a significant increase (p < 0.01)
in the spectral intensity ratio of methylene (CH2) resonance (at 1.28 ppm) to methyl (CH3) resonance
(at 0.9 ppm) in the Mel-QA treated group as compared to controls (Figure 4D). This ratio, which is
considered as an apoptosis marker (26,27), was also significantly higher (p < 0.01) than in the Mel-
treated group (Mel-QA: 2.34 ± 0.22 versus Mel: 1.47 ± 0.08). In the same way, a significant increase
in CH2(lip)/total creatine(tCr) ratio, which is considered as a necrosis marker (28), was observed in the
Mel-QA group (Mel-QA: 11.5 ± 2.07 versus Mel: 5.0 ± 1.26).
Significant reduction of side effects with QA derivative
The body weight of each rat was regularly recorded and hematological parameters were
assessed 48 h after the last dose of both compounds (Figure 5). Mel-treated rats exhibited significant
weight loss starting from the second injection which reached 15% at day 23 post implantation (Figure
5A). Furthermore, the clinical score including diarrhea, rough coat, closed eyes, lethargy and bleeding
were determined for each animal throughout time course of study: we assigned a score of 1 (presence)
or 0 (absence) for each of these criteria. One day after the last dose of treatment, none clinical side
effects were observed for Mel-QA treated rats and controls, in contrast to Mel-treated rats with an
average clinical score of 2.7 2.1. For Mel treated rats, these symptoms were associated with a severe
leucopenia, a common side effect of alkylating agents, which was marked by a significant decrease in
white blood cells and lymphocytes numbers (Figure 5B). Furthermore, in comparison with Mel-treated
animals, an improvement in hematological parameters was observed for Mel-QA treatment, i.e.
attenuation of leucopenia with, more specifically, a significant attenuation of lymphopenia (p < 0.01).
13

Discussion
CHS, with an estimated incidence of 0.2 in 100 000 patients per year, is a rare disease with
most patients being cured by a surgical excision. However, in case of unresectable locally advanced,
inoperable or metastatic disease, chemotherapy and radiotherapy appear largely ineffective. The poor
prognostic and lack of effective treatments markedly highlight a pressing need to develop new
therapeutic approaches (1,5).
Since efficacy of antineoplastic treatments is mainly hampered by toxic side effects, improving their
therapeutic index, by reducing adverse effects and increasing drug accumulation in tumor tissue,
remains a challenge in drug development. Among the numerous strategies aimed at such objectives,
“carrier” with preferential binding to one or more tumor targets may offer promising therapeutic
opportunities. This was the strategy employed here for experimental CHS: a QA function (i.e. a carrier
exhibiting affinity for the high negative fixed charged density of PG) was conjugated to Mel. In this
way, we expected to improve its therapeutic index by reducing systemic toxicity while keeping an
anti-tumor activity.
First of all, the role of QA function for PG binding was demonstrated in vitro by SPR with
aggrecan, the major PG component of CHS. As expected, binding of the QA-functionalized derivative
of Mel to aggrecan was characterized with a KD value in the millimolar range (2.25 0.92 mM) which
is not observed for Mel. These results confirmed our hypothesis that the Mel-QA conjugate establishes
ionic interactions with the high negative fixed-charge density of aggrecan. The next step was then to
confirm in vivo the interaction of both Mel-QA and Mel with the ECM of CHS by a biodistribution
study using the SRC model. This preclinical model reproduces the histological and clinical behaviors
of the human grade II disease (29–32). For biodistribution study, 3H radionuclide was introduced with
excellent radiochemical purities (> 97%) on the common alkylating group to allow comparative
biodistribution of both compounds. Quantitative whole body autoradiography showed rapid
distribution of radioactivity after [3H]-Mel-QA i.v. administration: Radioactivity rapidly accumulated
in healthy cartilaginous tissues and tumor. A high accumulation of radioactivity was also observed in
the kidney and liver, as previously reported for Mel-QA and Mel in healthy rats (25). This
accumulation of radioactivity in liver could be ascribed to metabolism but specific PG binding could
14

not be excluded. As a parenchymal organ, liver contains mainly Heparan Sulfate-GAGs with
additional minor amounts of Dermatan Sulfate-GAGs, Hyaluronic Acid (HA) and Chondroitin
Sulfate-GAGs which are barely detectable (33,34). According to us, since autoradiography showed
radioactivity of all radiolabeled derivatives (unchanged compound as well as its metabolites), liver
signal could also be mainly attributable to metabolism and bile excretion of Mel-QA since previous
distribution, in healthy rats, of [14C]-Mel-QA evidenced fecal biliary excretion of around 30% (25).
These results are also consistent with published data of Mel (35,36).
In the SRC model, there is a tendency for radioactivity to accumulate at higher level in tumor after [3H]-
Mel-QA injection as compared to [3H]-Mel but statistically non significant due to the variability
between animals. To correct the inter-rat variability, T/M ratio was calculated for each animal. If
radioactivity is expected to accumulate specifically in tumor tissue, and not only due to blood flow,
tumor to muscle ratio should be expected to be high than 1. The mean T/M ratio for Mel-QA at 5 min
post-injection (5.77 1.11) was significantly higher than for [3H]-Mel (2.22 1.20, p < 0.05). It could
be due to an enhanced accumulation in tumor as well as a lower circulating activity of the radiolabeled
Mel-QA.
Finally, we confirmed the interest of PG targeting strategy with the QA function for CHS therapy
notably in term of therapeutic index, essential in oncology. Combining tumor volume assessment, in
1
vivo 99mTc-NTP 15-5 scintigraphic imaging of PG, H-HRMAS NMR spectroscopy and histological
analyses of biopsies, this work demonstrates that the conjugation of a QA function to Mel does not
hamper its in vivo efficiency. More importantly, a significant decrease of side effects in terms of body
weight and haematological profile was observed for animals treated with Mel-QA as compared to
those receiving Mel. Thus QA function leads to a significant improvement of the therapeutic index.
The excellent tolerability of Mel-QA could enable repeated cycles for therapeutic effects in patients
with CHS.
The tumor growth inhibition observed in the SRC model treated by Mel-QA raises the
question of the mechanism at the origin of this effect. Assessment of alkylating capacity using the 4-
(4-nitrobenzyl)pyridine assay, as previously described (17), proved that an alkylating activity was
maintained for Mel-QA with an alkylation rate of 52.5 4.9 x 10-5 min-1 compared to 20.9 2.6 x 10-5
15

min-1 for Mel. (Supplementary Table S1). Nevertheless, does the biological activity observed only
attributable to DNA alkylation, like for Mel (37) or to other events, as for example the degradation of
PG. Indeed, we think that the mechanism of action of Mel-QA is more complex than a simple
alkylation and may be due to a degradation of the PG of ECM. We previously demonstrated that
treatments with Mel-QA induce changes in the expression and degradation of the PG (13). PG are an
important component of the tumor microenvironment, which represents a complex and highly
dynamic media basically composed of non-malignant cells as well as a ECM consisting of fibrous
structural proteins, fibrous adhesive proteins and PG. Due to their complex structure, PG play an
important role in cell–cell and cell–ECM interactions and signaling in a variety of cellular functions
(motility, adhesion, growth) (38,39). Modifications of PG contribute to altered composition of ECM
and also could explain the reduced tumor growth observed with Mel-QA in SRC bearing rats.
Concerning CHS, the importance of PG in SRC growth was firstly suggested by Oegema et al who
hypothesized that reducing the PG content was a way to decrease accessibility of growth factors and to
increase the immunological cell infiltration (40).
Recently, tumor microenvironment has been gradually recognized as a key contributor for cancer
progression and drug resistance (41,42). PG appear as major partners for many organ integrity and
function, and might represent interesting targets in other malignant pathological processes, such as
head and neck carcinomas (43,44). Since QA strategy has demonstrated its potential for CHS
management through ECM interaction, it will be of great interest to take advantage of another
phenotypic feature of CHS microenvironment that is hypoxia (7,45,46). Exploiting hypoxia is well-
documented in cancer therapy through hypoxia-activated prodrugs and inhibitors of molecular targets
upon which hypoxic cell survival depends. Therefore, combining PG targeting and hypoxia-activated
prodrugs could serve as basis for innovative and selective, microenvironmentally targeted CHS
therapy.
Acknowledgments:
Authors thank Delphine Skrzydelski and Romain Vives and especially Sophie Besse for its technical
expertises and help for in vivo treatments and heamatological sampling.
16

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20

Comparative distribution of radioactivity after i.v. administration of [3H]-Mel-QA or [3H]-Mel. (a % of ID/g tissue, b % of ID)
Tissue :
[3H]-Mel
Tumora
Cartilagea
0.93 ± 0.01
1.97 ± 0.12**
ND
Livera
Kidneya
Lunga
Musclea
Bloodb
1.79 ± 1.62
2.73 ± 0.48
1.84 ± 1.44
3.35 ± 0.01
2.05 ± 0.77
1.62 ± 0.62
1.26 ± 0.01
0.72 ± 0.20
1.04 ± 0.01
0.87 ± 0.01
7.87 ± 6.64
17.23 ± 4.82
6.43 ± 3.34
10.28 ± 10.54
3.66 ± 0.42
13.28 ± 4.68
1.26 ± 0.13
2.70 ± 0.02*
1.25 ± 0.38
1.37 ± 0.05
29.48 ± 25.39
41.80 ± 3.43
35.13 ± 19.07
37.50 ± 0.05
29.13 ± 7.09
21.71 ± 4.17
12.01 ± 0.91
9.84 ± 0.96
9.35 ± 2.55
5.60 ± 0.01
6.15 ± 3.82
3.43 ± 0.81
4.90 ± 2.06
19.58 ± 0.00
3.31 ± 0.30
2.05 ± 2.05
1.09 ± 0.01
1.56 ± 0.17
0.88 ± 0.01
0.87 ± 0.01
1.10 ± 0.55
0.48 ± 0.04
1.57 ± 0.97
0.85 ± 0.58
1.26 ± 0.17
0.53 ± 0.18
0.68 ± 0.17
0.46 ± 0.20
0.60 ± 0.05
0.43 ± 0.11
0.24 ± 0.05
0.09 0.00*
0.20 ± 0.07
0.00 0.00
0.07 0.05
0.05 0.03
0.04 0.00
0.03 0.01
0.08 0.03
0.03 0.00
5 min
15 min
1 h
[3H]-Mel-QA
[3H]-Mel
[3H]-Mel-QA
[3H]-Mel
3.38 ± 0.12**
ND
[3H]-Mel-QA
[3H]-Mel
ND
ND
6 h
[3H]-Mel-QA
[3H]-Mel
ND
ND
24 h
[3H]-Mel-QA
ND
Cumulative radioactivity excreted in urine after i.v. administration of [3H]-Mel-QA or [3H]-Mel (% of ID)
Time (h)
[3H]-Mel
[3H]-Mel-QA
0-6
6-24
4.31
5.90
0-24
16.65
15.91
12.34
10.01
Table 1 : Comparative distribution of radioactivity in tumor, cartilage, liver, kidney, lung, muscle and blood, and cumulative radioactivity excreted in urine
after i.v. administration of [3H]-Mel or [3H]-Mel-QA. ND: Non detectable. *p < 0.05, **p < 0.01

Figure Legends
Figure 1:
Affinity kinetics between aggrecan and Mel-QA or Mel by SPR. Representative examples of
sensorgrams for the affinity kinetic analysis of Mel-QA and Mel. The dissociation constant (KD) of
each example are given.
Figure 2:
Conjugation with QA allows a quick accumulation of Mel in tumor. A. Radiolabeling of [3H]-Mel
and its QA conjugate ([3H]-Mel-QA). B. Tumor/Muscle ratios 5 min, 1 h and 6 h after i.v.
3
3
*
administration of [ H]-Mel and [ H]-Mel-QA. p < 0.05. C. Representative two-dimensional images
obtained of rat whole-body slices showing the tissue distribution of radioactivity at 5 min post
injection (p.i.) after i.v. administration of [3H]-Mel-QA (C1) or [3H]-Mel (C2). T: Tumor; AC :
Articular cartilage.
Figure 3:
- Evaluation of the in vivo antitumor activity of Mel-QA and its non-targeted equivalent (Mel).
A. In vivo assessment of tumor growth. Mel and Mel-QA were given by i.v. route at 16 µmol/kg
according to a q4d x3 schedule. Tumor Volume (TV) was monitored regularly. * Significativity was
calculated versus control group. B. Quantitative analysis of 99mTc-NTP 15-5 accumulation in tumors
versus muscle at day 7 and day 30 post implant.
- Evaluation of the specificity of 99mTc-NTP 15-5 imaging. C. Tumor/Muscle ratio of 99mTc-NTP
15-5 and 99mTc-15-5 at different time points after i.v. injection in SRC bearing rats. D. Structure of
99mTc-NTP 15-5 and its non-targeted equivalent 99mTc-15-5 with representative in vivo scintigraphic
images of the tumor-bearing hind limb obtained 1 h post i.v. injection of 25 MBq of 99mTc-NTP 15-5
(D1) or 99mTc-15-5 (D2). FC : Femoral Condyle, PT: Plateau Tibial, T: Tumor
1

Figure 4:
- Anatomopathological analysis one day after the last injection
A. Histopathologic analysis, haematoxylin and eosine, original magnification: x 10; A1. Control: The
tissue shows an important mitotic activity (M) without fibro-inflammatory component; A2. Melphalan-
treated rat and A3 Melphalan-QA treated rat: Both tissues show a lot of necrotic cells (NC) associated
with a fibro-inflammatory component. B. Mitotic activity index was evaluated as the number of
mitotic figures per fields.
1H HRMAS profiling analyses one day after the last injection
-
C. Representative 1H NMR spectra of SRC tumors of treated and non treated rats one day after the end
of treatment. Major assignments : a: CH3 signal of fatty acids residues of mobile lipids (0.90 ppm), b:
CH2 signal of fatty acids residues of mobile lipids (1.28 ppm) and c : CH3 signal of creatine (3.03
ppm). D. CH2(lip)/CH3(lip) ratio and CH2(lip)/Total creatine (tCr) for each group.
Figure 5: QA conjugation strongly attenuates side effects.
A: Percent change in body weight from day 4 post implantation. While there is no weigth loss in the
Mel-QA treated group, a significant weight loss is observed in the Mel-treated group starting from the
second injection. B: Haematological parameters 48 h after the end of the treatment (Day 18). QA
conjugation allows a significant attenuation of lymphopenia.
2

Figure 1
2 m M
2 m M
1 .5 m M
1 m M
1 .5 m M
1 m M
0 .8 m M
0 .6 m M
0 .5 m M
0 .4 m M
0 .2 m M
0 m M
0 .8 m M
0 .6 m M
0 .5 m M
0 .4 m M
0 .2 m M
0 m M
M e l-Q A
M e l
8 0
6 0
4 0
2 0
8 0
6 0
4 0
2 0
2 0 0
4 0 0
6 0 0
8 0 0
2 0 0
4 0 0
6 0 0
8 0 0
T im e (s)
T im e (s)
KD = 3.3 x 10-3 M
KD = 302 M
Chi2 = 1,25
Chi2 = 0,894

Figure 2
A
B
8
7
6
5
4
3
2
1
0
Mel-QA
Mel-AQ
Mel
Mel
*
5
60
360
p < 0.05
Time after i.v. administration (min)
*
a Reagents and conditions: (i) NaBH4, [3H]-NaBH4 (3.7 x 103 MBq), EtOH, rt, 5 h; (ii) EtOH,
90 °C, 17h; (iii) ethylene oxide, acetic acid, rt, 24h; (iv) PPh3, CCl4, DCM, rt, 18h; (v) 6 N
HCl, 90 °C, 4h30; (vi) 1) (Boc)2O, TEA, MeOH, rt, 1h30 ; 2) NH2(CH2)3N(CH3)2, DCC,
HOBt, DCM, rt, 18h; (vii) CH3I, EtOH, rt, 18h; (viii) 1) HCl/EtOH, rt, 3h
; 2)
Dowex(chloride) 1 × 8−200.
C
C1
C2
T
AC
AC
AC
T
Radioactivity distribution of [3H]-Mel at 5 min p.i.
40 µm-thick sagittal slice
Radioactivity distribution of [3H]-Mel-QA at 5 min p.i.
40 µm-thick sagittal slice
T
AC
AC
AC
T
Corresponding anatomopathological slice
Corresponding anatomopathological slice

Figure 3
A
B
*
3
Day 7 Day 30
2.5
2
Injections
1.5
1
0.5
0
**
**
*
Vehicle
Mel-AQ
Mel
**
**
p < 0.05
*
**
p < 0.05
p < 0.01
*
**
C
FC
TP
D
99mTc-15-5
T
99mTc-NTP 15-5
D1
D2
99mTc-NTP 15-5
T
**
** ** **
** **
** **
**
p < 0.05
p < 0.01
*
**
99mTc-15-5

Figure 5
A
B
**
M
**
NC
NC
2.34
3.5
3
F
M
NC
NC
NC
2.5
2
M
1.5
1
F
0.18
p < 0.05
p < 0.01
0.5
0
0.02
*
**
A1: Control
A2: Mel treated rat
A3: Mel-QA treated rat
Mel-QA Mel Vehicle
b: CH2 (lip)
a: CH3 (lip)
Vehicle
Mel
Mel-QA
C
D
c: tCreatine
CH2(lip)/CH3(lip)
CH2(lip)/tCr
**
3. Mel-QA
**
**
3
2.5
2
18
16
14
12
10
8
6
4
2
0
**
b
c
c
a
2. Mel
*
b
1.5
1
a
1. Control
0.5
0
p < 0.05
p < 0.05
*
*
** p < 0.01
** p < 0.01

Figure 5
A
B
Mel treated rats
Mel-QA treated rats
Red Blood Cells
(x 106/µL)
Controls
Mel-QA treated rats
Mel treated rats
Controls
Injection
White Blood Cells
% of Lymphocytes
Hemoglobin (g/dL)
(x 103/µL)
***
*
**
**
70
60
50
40
30
20
10
0
12
10
8
8
7
6
5
4
3
2
1
0
16
14
12
10
8
90
80
70
60
50
40
30
20
10
0
**
6
6
p < 0.05
4
*
4
p < 0.01
**
**
**
2
2
**
**
4
9
14
19
24
-10
0
0
**
p < 0.05
p < 0.01
p < 0.001
Days post implantation
-20
*
**
***
**