12b80 - Hydroxybisphosphonate Linked Doxorubicin: Bone Targeted Strategy for Treatment of Osteosarcoma

Article abstract of DOI:10.1021/acs.bioconjchem.9b00210

To reply to as yet unmet medical needs to treat osteosarcoma, a form of primary bone cancer, we conceived the 12b80 compound by covalently conjugating antineoplastic compound doxorubicin to a bone targeting hydroxybisphosphonate vector and turned it into a prodrug through a custom linker designed to specifically trigger doxorubicin release in acidic bone tumor microenvironment. Synthesis of 12b80 was thoroughly optimized to be produced at gram scale. 12b80 was evaluated in vitro for high bone support affinity, specific release of doxorubicin in acidic condition, lower cytotoxicity, and cellular uptake of the prodrug. In vivo in rodents, 12b80 displayed rapid and sustained targeting of bone tissue and tumor-associated heterotopic bone and permitted a higher doxorubicin payload in tumor bone environment compared to nonvectorized doxorubicin. Consequently, 12b80 showed much lower toxicity compared to doxorubicin, promoted strong antitumor effects on rodent orthotopic osteosarcoma, displayed a dose-response therapeutic effect, and was more potent than doxorubicin/zoledronate combination.

Full text of DOI:10.1021/acs.bioconjchem.9b00210

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Article
12b80 - Hydroxybisphosphonate linked doxorubicin:
bone targeted strategy for treatment of osteosarcoma
Emmanuelle David, Sébastien Cagnol, Jean-Yves Goujon, Maxim Egorov, Julien
Taurelle, Charlène Benesteau, Laëtitia Morandeau, Christelle Moal, Marie Sicard, Samuel
Pairel, Dominique Heymann, Francoise Redini, François Gouin, and Ronan Le Bot
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00210 • Publication Date (Web): 02 May 2019
Downloaded from http://pubs.acs.org on May 4, 2019
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Bioconjugate Chemistry
TITLE
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12b80 - Hydroxybisphosphonate linked doxorubicin: bone targeted
strategy for treatment of osteosarcoma
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AUTHOR NAMES
Emmanuelle David*○, Sébastien Cagnol○, Jean-Yves Goujon○, Maxim Egorov○, Julien Taurelle○,
Charlène Benesteau○, Laëtitia Morandeau○, Christelle Moal○, Marie Sicard○, Samuel Pairel○,
Dominique Heymann•, Françoise RediniΨ, François Gouinǂ and Ronan Le Bot○.
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AUTHOR ADDRESS
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○Atlanthera, 3 rue Aronnax, 44821 Saint Herblain, France. Institut de Cancérologie de l’Ouest, Inserm, CRCINA, Uni-
versité de Nantes, Université d’Angers. Blvd Jacques Monod, 44805 Saint-Herblain, France. Ψ INSERM UMR1238, Uni-
versité de Nantes, Sarcomes osseux et remodelage des tissus calcifiés, Faculté de médecine, 44035 Nantes, France.
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Departement de chirurgie, centre Léon Bérard, 28 rue Laënnec, 69008 Lyon, France.
KEYWORDS
bisphosphonates, bone targeting, doxorubicin, drug conjugation, drug delivery, osteosarcoma.
ABSTRACT: To reply to as yet unmet medical needs to treat osteosarcoma, a form of primary bone cancer, we conceived
the 12b80 compound by covalently conjugating antineoplastic compound doxorubicin to a bone targeting hydroxybisphos-
phonate vector and turn it into a prodrug through a custom linker designed to specifically trigger doxorubicin release in
acidic bone tumor microenvironment. Synthesis of 12b80 was thoroughly optimized to be produced at gram scale. 12b80
was evaluated in-vitro for high bone support affinity, specific release of doxorubicin in acidic condition, lower cytotoxicity
and cellular uptake of the prodrug. In-vivo in rodents, 12b80 displayed rapid and sustained targeting of bone tissue and
tumor-associated heterotopic bone and permitted a higher doxorubicin payload in tumor bone environment compared to
non-vectorized doxorubicin. Consequently, 12b80 showed much less toxicity compared to doxorubicin, promoted strong
antitumor effects on rodent orthotopic osteosarcoma, displayed a dose-response therapeutic effect and was more potent
than doxorubicin/zoledronate combination.
INTRODUCTION
methotrexate (called MAP); etoposide or ifosfamide can
also be used in addition4,5. Despite efforts to improve
treatments, 5-year event-free survival (EFS) rates have
remained below 70% for patients with localized tumors6.
Prognosis is worse for patients with unresectable primary
disease or metastatic disease with 5-year EFS rates around
30%7,8. Because the survival rates have not significantly
changed in the past 30 years (10-year survival rate in-
creased from only 57.7 to 61%9), new therapeutic ap-
proaches are needed. To reply to as yet unmet medical
need, our rational was to covalently bind a bone targeting
vector to a potent gold standard antineoplastic compound
to bring and deliver it in higher quantity in tumor bone
environment to improve efficacy and decrease systemic
distribution and associated toxicity. Our proof of principle
Osteosarcoma is the most common primary bone tumor, a
rare form of cancer, which accounts for less than 0.2% of
all cancers and represents less than 1500 and 1000 pa-
tients diagnosed each year respectively in Europe and
USA1,2. Osteosarcoma can occur at any age but has a higher
incidence in patients under 24, with a peak during puber-
tal growth around 153. The disease has an early and highly
invasive and distant metastatic risk (particularly to lungs)
associated with a poor outcome. Although it can develop in
any bone, 80% of osteosarcomas affect the bone tissue
with rapid growth such as the extremities of long bones
(femur, tibia, humerus). Osteosarcoma derives from osteo-
blasts and is characterized both by bone formation and
osteolytic lesions. Indeed, tumor cells promote formation
of tumor osteoid (immature bone tissue) and tumoral
heterotopic bone tissue. In addition, osteosarcoma cancer
cells activate osteoclasts leading to osteolysis (bone re-
sorption) and acidic tumor microenvironment. Current
strategy of treatment for osteosarcoma includes neoadju-
vant chemotherapy, limb salvage surgery and adjuvant
chemotherapy. The most effective chemotherapy regimen
is the combination of doxorubicin, cisplatin and high-dose
was demonstrated with 12b80:
a
cleavable hy-
droxybisphosphonate (HBP) bound doxorubicin. A HBP
was chosen as the bone targeting vector thanks to its
strong affinity to bone tissue10,11. Furthermore, HBPs have
been used to treat bone metastasis malignancies and
postmenopausal osteoporosis as a preventive therapy
against skeletal-related events12,13. Some research was
carried out to improve the systemic anticancer properties
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of hydroxybisphosphonates themselves by masking the doxorubicin demonstrated efficacy in a model of bone
metastases30. Those publications strongly support the
interest of targeting bone tumor with HBP conjugated
drugs and the need for new efficient synthetic approaches
to the HBP bifunctional molecules. Most of the studies
describing HBP- and BP-linked doxorubicin are based on
synthetically more accessible BP vectors31,32. But even in
these cases, only small amounts of final products were
obtained, sometimes without pure final product isolation,
and with rather low solubility (1-5 mM)31 which was prob-
ably not sufficient for detailed physicochemical and biolog-
ical property studies. Moreover, the use of the acid sensi-
tive acylhydrazone linker is associated to a fast cleavage
rate that could disadvantage prodrug safety and large scale
chemical production. In contrast, we designed a thiocarbo-
hydrazone type imine linker for 12b80 molecule to reduce
systemic toxicity, to increase the aqueous solubility (up to
255 mM)33 and to improve the stability during large scale
production. The synthesis of 12b80 compound was thor-
oughly optimized to be produced at gram scale. Thus, the
aim of this paper was to describe the improved synthesis
of 12b80, its properties and its efficacy for the treatment of
osteosarcoma.
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phosphonic acid through organic protecting groups and
introducing hydrophobic functions in the side chain to
reduce their polarity, osseous affinity and to improve the
soft tissue uptake14. Structure-antitumor activity relation-
ship investigation of non-protected hydroxybisphospho-
nates themselves resulted in compound showing in-vitro
anti-proliferative activity against tumor cell lines with IC50
value of 9.5 x 10-5 M 15. In the actual work doxorubicin was
selected as an optimal antineoplastic agent to vectorize
since it is considered as the gold standard in sarcoma ther-
apy, including osteosarcoma, but shows a limited thera-
peutic index due to severe cardiac toxicity. Furthermore,
we benefited from the natural fluorescence of doxorubicin
due to its anthracycline chromophore group, to facilitate
distribution study. The innovation of 12b80 lies in its cus-
tomized linker designed to take advantage of the acidity of
bone tumor microenvironment to specifically trigger the
doxorubicin release. This linker also contributes consider-
ably to the high hydrosolubility of 12b80. Bisphosphonate
(BP) molecules have been studied extensively since the
1960's and a multitude of BP-based products are commer-
cially available16. Mechanistically, BPs exhibit affinity to-
ward bone by chelating calcium ions present in bone hy-
droxyapatite (HA). The involvement of phosphonate
groups in the HA binding mechanism was further evaluat-
ed to investigate the effect of phospho-esters on the ability
of the BP to bind HA10. One of the varieties of BP are HBPs
having a hydroxyl group on the carbon atom of the BP
moiety participating in the binding of these compounds to
the mineralized bone matrix17 and rising the affinity for HA
crystals by tridentate binding18. For this reason, HBP vec-
tors are more efficient for the bone targeting than BP.
Classic methods of simple HBP compound synthesis were
reviewed and are rather drastic to be convenient for multi-
functional HBP molecule synthesis19. That is why only few
attempts to synthetize complex individual bifunctional
HBP molecules vectorizing a potent API are published.
Such molecules have advantages of strictly defined chemi-
cal structure, API content, physicochemical characteristics
and purity. Meanwhile the study of non-HBP polymer self-
assembling nanoparticle delivery systems, vectorizing
covalently bounded doxorubicin20,21 and daunorubicin22
led to improve their in-vitro efficacy. Redox tumor-
sensitive non-HBP nanoparticle carrier for free methotrex-
ate encapsulation has been reported23. For nanoparticles
coated with HBP groups, the osseous affinity24 and anti-
cancer activity of supported doxorubicin25 in the bone
have been demonstrated. Development of bone targeting
anticancer therapy is a promising strategy26,27. Some pro-
jects were conducted on bisphosphonate-conjugated
antineoplastic agents but only few of them have shown
evidence of antitumor activity in osteosarcoma models.
The first one was based on HBP vectorized liposomes
charged with non-modified doxorubicin28. The second one,
the MBC-11 molecule which consists of the HBP etidronate
conjugated to the antineoplastic agent cytarabine, has been
tested clinically and was the first bisphosphonate conju-
gate to demonstrate efficacy in first-in-human phase I on
cancer induced bone disease29. Furthermore, HBP-linked
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RESULTS AND DISCUSSION
Synthesis and structure. Our previous approaches to the
synthesis of various HBP-conjugated anticancer drugs
showed that in the case of the doxorubicin, the aqueous
solubility of the final HBP compound was limited34. There-
fore, we designed new HBP conjugated doxorubicin mole-
cules, for improvement of aqueous solubility33 and suc-
ceeded in the 12b80 molecule. 12b80 molecule consists of
three building blocks: the Doxorubicin, the Linker and
the HBP vector conjugated together through imine bonds
(Figure 1). The custom designed Linker played a key role
in both acid sensitive doxorubicin release and high aque-
ous solubility of 12b80. The synthesis of the Vector has
been
described
previously34.
was alkylated with 1,3-
Briefly,
the
3-
hydroxybenzaldehyde
1
dibromopropane and the aldehyde group of 2 was protect-
ed with an acetal group to give the intermediate 3. Substi-
tution of bromine atom with methylamine followed by the
Mikael addition of acrylic acid on 4 gave the acid 5, the
total yield was 65% over 4 steps. The conversion of the
carboxyl of 5 to the HBP group was initially carried out
using the mild catecholborane activation method19,34–36
with highly nucleophilic tris(trimethylsilyl)phosphite37.
Though this method is efficient, in the present work espe-
cially for the Vector we improved the yield of this step
from 40% to 60% via the pivalic acid mixed anhydride
method of HBP derivatives synthesis that we developed
earlier33. This approach allowed also to bypass the chro-
matographic purification of the Vector and scale up the
synthesis to 32 g of the final product. We reported the
Linker synthesis starting from the anhydride 6 earlier33. In
the present work we optimized its isolation procedure for
the large-scale synthesis (up to 45 g) using original eth-
ylene glycol/MeCN mixture that allowed to reach higher
concentration and purity during Linker isolation step.
Initially, 12b80 molecule was synthesized by a simultane-
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Bioconjugate Chemistry
ous coupling of the Linker to the Doxorubicin and the
Vector33. To obtain a higher conversion of Doxorubicin,
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the synthesis was optimized: the Linker-Vector was first
synthesized, which was thereafter coupled with the Doxo-
rubicin resulting in at least 1.6 times higher yield and
better reproducibility. Final 12b80 compound was ob-
tained as a disodium salt readily soluble in the water. The
structure of 12b80 molecule was proved by 1D and 2D
NMR spectroscopy methods. The configurations of the
imine bonds were proved by the NOEs analysis (Figure 2).
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Figure 1. Optimized synthesis of the 12b80 molecule.
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Figure 2. Key NOEs in the 12b80 molecule (NOESY, 1%TFA-
D/DMSO-D6, 400 MHz).
Figure 3: In-vitro binding and release of 12b80. A: Binding
assay to hydroxyapatite (HA) and bovine bone. Rate of bind-
ing of 12b80 (red) and doxorubicin (black) on supports was
determined by HPCL analysis of supernatant after 30 minutes
of contact. Experiments were performed in triplicate and
results are expressed in Mean ± SD. B: Tumor-associated
acidic pH triggered doxorubicin release from 12b80. Doxoru-
bicin release from hydroxyapatite-fixed 12b80 was studied in-
vitro and measured by HPLC at 4 different pH: DPBS (pH 7.4)
or citrate buffer (pH 6, 5, 4). Tumor-associated pH is ± 6 and
tumor-associated osteolysis pH is ± 4
Binding of 12b80 to HA and bone. The binding of 12b80
was evaluated on 2 supports, HA and natural bovine bone,
in comparison with doxorubicin. The rate of binding of
12b80 was respectively 47% and 10% on HA and bone
within 30 minutes (Figure 3A). In comparison, the rate of
binding of doxorubicin was respectively 8% and 0.3% on
HA and bone. 12b80 exhibited rapid fixation and high
affinity for synthetic and natural calcium phosphate com-
pared to doxorubicin. These results were in accordance
with published data on HBP affinity for HA30,31
.
In-vitro cytotoxicity of 12b80. The effects of doxorubicin
and 12b80 treatment on cell viability were compared in-
vitro on normal mouse fibroblast L929 cells and on osteo-
sarcoma-derived rat OSRGa cells after 72 h of treatment
with doses ranging from 5 nM to 50 µM. As shown in Fig-
ure 4, IC50 of doxorubicin was around 134 nM in OSRGa
cells and 239 nM in L929 cells, while IC50 of 12b80 was
around 1.1 µM in OSRGa cells and 3.1 µM in L929 cells.
Those results clearly indicated that 12b80 treatment was
less toxic than non-vectorised doxorubicin, with a 8.3 fold
higher and 12.9 fold higher IC50 for 12b80 in comparison
to doxorubicin in cancerous OSRGa cells and fibroblast
cells respectively. Those results supported the postulate
that conjugation of doxorubicin to HBP vector through
12b80 molecule could turn doxorubicin into a less toxic
prodrug.
Doxorubicin release from 12b80-HA at different pH medi-
um. The release of doxorubicin from HA coated with 12b80
was investigated by HPLC in medium with different pH. As
shown in Figure 3B, there was a direct relationship be-
tween acidity and release rate of doxorubicin. Less than
2% of doxorubicin was released from support within 3
days at a neutral pH (7.4: DPBS), in contrast, over 11% of
doxorubicin was cleaved from 12b80 at the same time at a
pH of 4. Intermediary release levels were observed at pH 5
and 6. Linker structure with imine bonds was indeed de-
signed to be sensitive to low pH, to take advantage of the
acidic pH associated with tumor cell microenvironment
(pH between 6 and 7)38,39 and with the osteoclast-
associated bone resorptive activity pH in the range of 440.
In-vitro release studies with 12b80 highlighted the ability
of acidic conditions associated with bone tumor to trigger
the continuous release of doxorubicin from 12b80.
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Figure 4: Comparison of doxorubicin and 12b80 treatments
on cell viability. Exponentially growing immortalized fibro-
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blastic mouse L929 cells or osteosarcoma-derived rat OSGRa
cells were treated for 72 h with doxorubicin (Doxo), 12b80 or
their respective solvent (control) at doses from 5 nM to 50
µM. Results represented as Mean ± SD of triplicats.
Cellular intake of 12b80 in-vitro. Analysis of intra cellular
drug uptake in OSRGa cells by detection of doxorubicin-
associated fluorescence showed that fluorescence associ-
ated with free doxorubicin started to be detectable 1 h
after treatment to gradually increase in fluorescence inten-
sity throughout the 48 h of treatment. Localization of the
fluorescence signal was mostly in the nuclear compart-
ment after 1 h and 4 h of treatment and delocalized from
the nuclear to the peri-nuclear compartment after 8 h to be
mostly localized at the periphery of nucleus after 24 h of
treatment (Figure 5). Such cytosolic distribution of doxo-
rubicin has previously been shown to be associated with P-
Glycoprotein mediated drug resistance41. In contrast, up-
take of 12b80-associated fluorescence started to be de-
tectable after 8h of treatment. Fluorescence signal re-
mained weak and only increased between 24 h and 48 h of
treatment suggesting a lower cellular uptake of 12b80 in
comparison with doxorubicin. Fluorescence signal associ-
ated with 12b80 was only detectable in the perinuclear
compartment of cells. We postulated that perinuclear lo-
calization of 12b80 could be a hallmark of fluid phase en-
docytosis mechanism related to cellular entry of HBP mol-
ecules42–44. Since most of the proapoptotic effects of doxo-
rubicin are associated with DNA damage, lower cell uptake
and absence of detectable nuclear localization of 12b80
molecule could contribute to the lower in-vitro toxicity of
HBP-conjugated doxorubicin prodrug.
Figure 5: Comparison of doxorubicin and 12b80 cellular up-
take and distribution. A: OSRGa cells have been treated with 1
µM doxorubicin for 1 h (a), 4 h (b), 8 h (c), 16 h (d), 24 h (e) or
48 h (f). B: OSRGa cells have been treated with 1 µM 12b80 for
1 h (a), 4 h (b), 8 h (c), 16 h (d), 24 h (e) or 48 h (f). Fluores-
cence of doxorubicin and 12b80 has been evaluated by fluo-
rescence microscopy on living cells. Scale bar = 20 µm.
Range finding of doses of 12b80 in mice. Usable doses of
12b80 in mice were evaluated and compared to doxorubi-
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cin (Figure 6). The relationship between dose and toxicity noticeable between therapeutic doses of 12b80 (32.5
1
could be verified after single intravenous injection of doxo-
rubicin from 10 to 32.5 µmol/kg, indeed, single doses of 10
or 15 µmol/kg were well tolerated whereas doses of 25 or
32.5 µmol/kg induced severe body weight loss. A correla-
tion between dose and toxicity was also established for
12b80 after single intravenous injection from 32.5 to
100µmol/kg: single doses of 32.5 or 50 µmol/kg were well
tolerated whereas doses of 75 µmol/kg or 100 µmol/kg led
to severe body weight loss. At the equivalent dose of 32.5
µmol/kg, 12b80 (without any effect on body weight) was
not toxic in contrast to doxorubicin treatment, which re-
quired euthanasia of animals on D6. As a result, 15
µmol/kg (corresponding to 8.7 mg/kg) was the MTD (max-
imum tolerated dose) of doxorubicin, in accordance with
published data45,46, while the MTD of 12b80 was set to be
50 µmol/kg. Thus, MTD of 12b80 was more than 3 fold
higher than doxorubicin on mice after single injection.
Reduction of systemic toxicity of 12b80 compared to doxo-
rubicin could be explained by the specific bone targeting
with HBP, and by the prodrug properties with reduction of
intracellular intake of complete 12b80. As a result, 12b80
could be used at higher doses than doxorubicin in order to
improve its efficacy. Toxicity studies after repeated doses
(9 injections) indicated that the maximum usable dose of
12b80 was set at 32.5 µmol/kg (data not shown) whereas
is set at 3.4 µmol/kg (corresponding to 2 mg/kg/injection,
and 18mg/kg cumulative dose) for doxorubicin47, which
represents around a 10 fold ratio.
µmol/kg) and doxorubicin (3.4 µmol/kg). Not only the
maximal concentration in bone at first timepoint was high-
er with 12b80 than with doxorubicin at an equivalent dose,
but 12b80 was also more slowly eliminated from the bone
tissue. Elimination of fluorescence after 12b80 treatment
from the bone was studied for 9 months post-single injec-
tion (Figure 7B). Fluorescence intensity in the bone after 3
weeks and 2 months post-treatment represented respec-
tively around 50% and 10% of initial intensity on D1, then
remained stable from 2 to 9 months after a single injection.
Thus, fluorescent compounds were still detectable in bone
9 months after single 12b80 injection. In the tumor, con-
centration of fluorescent compounds was much higher
after 12b80 treatment at 32.5µmol/kg than after doxoru-
bicin treatment at 3.4 µmol/kg (Figure 7C). At timepoints
24 h and 72 h, fluorescence intensity in tumor was around
10 times higher after 12b80 treatment at 32.5 µmol/kg
than after doxorubicin treatment at 3.4 µmol/kg (both at
therapeutic dose). In a second step, a histological analysis
was carried out on tumor at 24 h post-treatment to
demonstrate the distribution of 12b80 on the tumor in
comparison with a doxorubicin-treated mouse tumor (Fig-
ure 7D). In the tumor, the level of fluorescent compound
seemed much higher after 12b80 treatment at 32.5
µmol/kg than after doxorubicin treatment at 3.4 µmol/kg
at timepoint 24 h. Furthermore, 12b80 was specifically
distributed in mineral nodes inside the tumor (fluores-
cence area was colocalized with mineralized areas),
whereas doxorubicin distribution was more diffuse. Con-
centration of 12b80 in bone and tumor could not be
achieved with doxorubicin. Study with radiolabeled 12b80
over 2 months showed an increasing uptake in bone be-
tween 6 and 24 h; thereafter, the activity level remained
relatively constant to 72 h and then slowly decreased (Fig-
ure 7E). Half-life in bone was around 20 days, and skeleton
represented the main target organ on D59. At timepoint 6
h, nearly 10% of injected dose (ID) was distributed in skel-
eton and was consistent with published data on HBP prod-
ucts48–50. All these results demonstrated a rapid distribu-
tion of 12b80 in bone tissue in a few hours and a slow
elimination within several months. These experiments
confirmed the improvement in targeting both bone and
bone tumor with doxorubicin when linked to HBP, in terms
of concentration and of duration of exposure.
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Figure 6: 12b80 prevented systemic toxicity of doxorubicin on
body weight. Body weight was monitored following single IV
injection of doxorubicin or 12b80 on D0 on Swiss mice (n=3
per group, Mean ± SD).
12b80 distribution in mice. Compound concentration es-
timated by fluorescence of doxorubicin and derivatives in
the tibia was higher after single 12b80 treatment at 32.5
µmol/kg (therapeutic dose) than after doxorubicin treat-
ment at 32.5 µmol/kg (highly toxic dose) at all timepoints
from 4 h to 72 h (Figure 7A). The difference was even more
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Figure 7: 12b80 distribution in mice. A: 12b80 increased targeting of doxorubicin to bone tissue. Fluorescence* signal was meas-
ured by HPLC in the tibia of NMRI Nude mice 4 h to 72 h after single IV injection (Mean ± SD, n=3 per timepoint). B: 12b80 was
slowly eliminated from bone. Fluorescence* signal was measured by HPLC in femur of C57BL/6 mice 1 day to 9 months after single
IV injection of 12b80 at 32.5 µmol/kg (Mean ± SD, n=3 per timepoint). C: 12b80 increased targeting of doxorubicin to osteosar-
coma tumor. Fluorescence* signal was measured by HPLC in orthotopic osteosarcoma tumor (OSRGa) of NMRI Nude mice, 24 or 72
h after single IV injection (Mean ± SD, n=3 per timepoint). D: 12b80 was specifically distributed in mineral nodes inside the tumor.
Histological analysis of tumor was carried out on osteosarcoma-bearing NMRI Nude mice (OSRGa) 24 h after single IV treatment
with 12b80 at 32.5 µmol/kg or doxorubicin at 3.4 µmol/kg. Slides were stained with Van Kossa and fluorescence was observed at
3
specific channel (fluorescence was excited at 532-554nm and emission was collected at 573-613nm). E: H-12b80 distribution in
3
skeleton of mice. Distribution of H-12b80 was assessed over 59 days after single IV injection at 32.5 µmol/kg to male Swiss mice
(Mean ± SD, n=3 per timepoint). Timepoints: 30 min, 2 h, 6 h, 24 h, 72 h, D21 and D59. Radioactivity was counted in femur and
estimated in total skeleton with formula: %ID in skeleton = % ID in femur / 0.02845. (* 3 Fluorescent compounds were detectable
by HPLC after bone tissue processing: doxorubicin, doxorubicinone and anthraquinone).
12b80 efficacy in osteosarcoma model in mice. Efficacy of
treatment with cycles of repeated injections of 12b80 was
evaluated in OSRGa osteosarcoma model. Indeed, this
therapeutic scheme every 3 weeks was based on conven-
tional cycle with doxorubicin in human51. Anti-tumor effi-
cacy of 12b80 was clearly correlated to injection dose and
cumulative dose, with an increase of efficacy from 120
µmol/kg (2x20 µmol/kg x 3 cures) to 293 µmol/kg (3x32.5
µmol/kg x 3 cures) (Figure 8A). Thus, cures of 3 consecu-
tive daily injections every 3 weeks at 32.5 µmol/kg were
defined as the best regimen. Efficacy of 12b80 cycles
(3x32.5 µmol/kg) was compared to combination of doxo-
rubicin + zoledronate with the same therapeutic scheme
(therapeutic doses respectively 3x3.4 and 3x0.34 µmol/kg;
i.e. 3x2 mg/kg and 3x100 µg/kg) in the same osteosarcoma
model (Figure 8B). 12b80 and doxorubicin were both in-
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Page 8 of 25
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combination treatment of doxorubicin and zoledronate,
12b80 had a significantly more pronounced antitumor
response. On D47, 12b80 treatment reduced tumor volume
by 57% compared to doxorubicin/zoledronate combina-
tion. 12b80 treatment promoted strong antitumor re-
sponse highlighted by a reduction of tumor growth and by
an inhibition of tumor-associated bone remodeling (reduc-
tion of both osteolytic lesions and heterotopic bone for-
mation) (Figure 8C). All together these results demon-
strated that maximum efficacy of 12b80 was higher than
maximum efficacy of doxorubicin resulting in an increased
efficacy/toxicity ratio.
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Figure 8: 12b80 efficacy in mice. A: 12b80 treatment reduced tumor growth in osteosarcoma model and displayed a dose-response
therapeutic effect. Orthotopic paratibial OSRGa tumors were implanted on D0 in NMRI Nude mice and IV treatment with 12b80 or
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DPBS vehicle were administered in cycle every 3 weeks from D1. n=10 mice per group. Mean ± SEM. Statistical analysis at endpoint
on D56: Mann Whitney test. * p<0.05, ** p<0.01, *** p<0.001. B: 12b80 was more potent than combination of doxorubicin and
zoledronate on osteosarcoma tumor growth. Orthotopic paratibial OSRGa tumors were implanted on D0 in NMRI Nude mice. Mice
received IV treatment with DPBS vehicle, 12b80 or doxorubicin+zoledronate every 3 weeks from D6. n=6 mice per group. Mean ±
SEM. Statistical analysis on D47: Mann Whitney test. * p<0.05, ** p<0.01, *** p<0.001. C: 12b80 inhibited osteosarcoma-associated
bone remodeling. Orthotopic paratibial OSRGa tumors were implanted on D0 in NMRI Nude mice. Mice received IV treatment with
DPBS vehicle, 12b80 or doxorubicin+zoledronate every 3 weeks from D6. Radiography of tumor limbs (tibias) was performed on
D48 on all mice (n=6 per group).
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dried under vacuum at room temperature. The Vector was
1
CONCLUSIONS
obtained as a white powder (32.2 g, purity 82% by H
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The 12b80 molecule consists of three building blocks: the
doxorubicin bound by the Linker to the bone targeted HBP
Vector through imine bonds. The innovation was based on
designed Linker-Vector that played a key role in the acid
sensitive doxorubicin release and the high aqueous solubil-
ity of the compound. 12b80 displayed rapid and sustained
targeting of bone tissue and tumor-associated heterotopic
bone and permitted a higher doxorubicin payload in tumor
bone environment. Doxorubicin release from 12b80 was
dependent on acidic pH associated with active bone tumor
environment. 12b80 showed a much lower toxicity com-
pared to doxorubicin. 12b80 promoted strong antitumor
effects on rodent orthotopic osteosarcoma, displayed a
dose-response therapeutic effect and was more potent
than doxorubicin/zoledronate combination. Thus, 12b80 is
the first example of cleavable form of HBP-bound doxoru-
bicin with optimized chemical synthesis, detailed physico-
chemical study, and antitumoral efficacy in preclinical
models, that represents a promising new therapy for pa-
tients with osteosarcoma, a rare disease with unmet medi-
cal needs. Therapeutic indications of 12b80 could presum-
ably be extended to other kinds of tumors that develop in
bone environment, such as Ewing sarcoma, myeloma, but
also in bone metastases secondary to breast or prostate
cancer and could benefit to substantially more patients.
NMR, 60% yield). HRMS: [M - H]- m/z = 410.0779; calc. for
1
C14H22NO9P2 = 410.0775. H NMR (D2O, 400 MHz): δ 9.74
(s, 1H), 7.41 (m, 3H), 7.21 (d, 1H), 4.09 (t, 2H), 3.29 (br m,
4H), 2.76 (s, 3H), 2.21 (br m, 2H), 2.13 (br m, 2H). 31P NMR
(D2O, 162 MHz): δ 17.25.
Linker. Thiocarbohydrazide (66 g, 630 mmol, 4 eq) was
solubilized in DMSO (400 mL) at 85 °C under argon using
magnetic stirring. After solubilization the solution was
cooled to room temperature without stirring. Compound 6
(40 g, 120 mmol, 1 eq) was solubilized in DMSO (216 mL)
at 85 °C under argon using magnetic stirring. The hot solu-
tion of compound 6 was added by syringe to the solution of
thiocarbohydrazide at room temperature in 10 min using
vigorous stirring (1000 rpm). Reaction mixture was stirred
for 45 min and then diluted with ethylene glycol (200 mL).
Then acetonitrile was added (2.4 L) using vigorous stirring
(1000 rpm), which resulted in the precipitation of the
Linker. The mixture was stirred for 15 min. The precipi-
tate was filtered off and washed twice with a mixture of
ethylene glycol/MeCN=1/3 (100 mL) and then 4 times
with MeCN (100 mL). The solid was dried under vacuum
on rotavapor at 30 °C. The residual solid was solubilized in
ethylene glycol (240 mL) at 40 °C. MeCN (720 mL) was
gradually added to the brown solution obtained using
vigorous stirring (1000 rpm) to precipitate the Linker.
The mixture was stirred for 15 min at room temperature.
The precipitate was filtered off and washed 3 times with a
mixture of ethylene glycol/MeCN=1/3 (100 mL) and then
3 times with MeCN (100 mL). The solid was transferred to
a round bottom flask, MeCN (200 mL) was added and the
mixture was stirred for 15 min at room temperature. The
off-white Linker (45 g, 80 % yield) was filtered off,
washed twice with MeCN (100 mL) and dried under vacu-
um on rotavapor at 30 °C. HRMS: [M - H]- m/z = 467.1247;
EXPERIMENTAL PROCEDURES
Synthesis. The methods of synthesis of compounds 2 - 5
are available in the Supporting Information.
Vector. The dry acid 5 (30 g, 97 mmol, 1 eq) was solubil-
ized in dry THF (150 mL, water bath 55°C) under argon
using magnetic stirring. Triethylamine (13.5 mL, 97 mmol,
1 eq) was added and the reaction medium was cooled
down to 10 °C by using an ice bath. Pivaloyl chloride (11.8
mL, 97 mmol, 1 eq) was added dropwise and the reaction
medium was stirred for 20 min under ice bath cooling. The
reaction medium was filtered under argon to remove tri-
ethylamine salt, the P(OSiMe3)3 (97.2 mL, 291 mmol, 3 eq)
was quickly added and the reaction medium was stirred
for 1 h at room temperature under argon. Methanol (100
mL) was added and the reaction medium was stirred for
10-15 min. Ether (500 mL) was added, the precipitate was
separated and washed 3 times with ether (50 mL). The
product was dried under vacuum and solubilized in water
(25 mL) and after 15 min 12N NaOH (around 16 mL, 1 eq)
was added until reaching pH=7. Methanol was added (250
mL) and precipitate was obtained by addition of ether
(400 mL). The solid was filtered off, washed with ether and
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calc. for C12H23N10O6S2 = 467.1249. H NMR (D2O, 400
MHz): δ 3.87 (2H, s), 3.70 (2H, s), 3.24 (2H, s).
Linker-Vector. The solution of the Vector (1.7 g, 3.7
mmol, 1 eq) and TFA (1 mL) in DMSO (23 mL) was added
dropwise (3 min) to the solution of the Linker (4.5 g, 9.6
mmol, 2.5 eq) in DMSO (12 mL) at room temperature un-
der vigorous stirring. The reaction mixture was stirred 5
min at room temperature then diluted with THF (175 mL),
stirred 15 min. The precipitate was filtered off, washed
with THF, dried under vacuum at 30 °C and solubilized in
DMSO (23 mL) at 40 °C. Under the vigorous stirring the
THF (60 mL) was added. After 15 min the precipitate was
filtered off, washed with THF, and dried under vacuum at
30 °C. The Linker-Vector was obtained (2.6 g, 80% yield)
as a white powder. HRMS: [M - H]- m/z = 860.2009; calc.
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for C26H44N11O14P2S2 = 860.1991. H NMR (D2O, 400 MHz):
δ 8.15-6.85 (5H, m), 4.25-2.95 (18H, m), 2.87 (3H, s), 2.34
(2H, m), 2.23 (2H, m). 31P NMR (D2O, 162 MHz): δ 17.73.
12b80. Doxorubicin hydrochloride (500 mg, 0.86 mmol, 1
eq) and the Linker-Vector (1600 mg, 1.85 mmol, 2.2 eq)
were mixed under argon in the solution of 1% TFA/DMSO
(20 mL). The solution was stirred 4 h at room temperature
under argon. The THF (80 mL) was added, the reaction
mixture was stirred 15 min and then the precipitate was
filtered, washed with THF and dried under vacuum at
30°C. Crude 12b80 was obtained as a red solid (1.8 g) and
solubilized in 0.5M NaHCO3 (15 mL). The solution was
injected with a syringe in the C18 column (220 g) and
eluted in the gradient from 3% EtOH to 15% EtOH. Frac-
tions with a purity >90% (HPLC control) were mixed, con-
centrated under vacuum at 30 °C and lyophilized. 12b80
was obtained as a sodium salt (450 mg, 36% yield, HPLC:
95%). HRMS: [M + H]+ m/z = 1387.3812; calc. for
Binding assay. Hydroxyapatite was purchased from Biorad.
Bovine bone (femur) was crushed after freezing in liquid
nitrogen. 50 mg of tissue or HA were suspended in a 12b80
solution (1.4 mM; 5 mL). The mixture was stirred 30 min
at room temperature (360° rotation stirrer). After centrif-
ugation, the supernatant was removed and analyzed by
HPLC-UV to determine the concentration and then to cal-
culate the rate of binding.
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Drug release studies. 90 mg of HA were suspended in a
12b80 solution (1.1 mM; 6 mL) and stirred 30 min at room
temperature (360° rotation stirrer). HA-bound 12b80 was
washed with 0.9% NaCl (3x3 mL), filtered and washed
with methanol, and dried under vacuum. 5 mg of 12b80-
HA was suspended in 500 µL of solution in Eppendorf tube
at 37 °C in an oven. 4 solutions were used: DPBS (pH 7.4)
or citrate buffer (pH 6, 5, 4). All the solvent was removed
at 24 h, 48 h and 72 h, diluted (1/2) with 7 mM EDTA and
analyzed by HPLC. 500 µL of solution was added to the
residual 12b80-HA between each timepoint.
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C53H73N12O24P2S2 = 1387.3772. H NMR (1%TFA-D/DMSO-
D6, 400 MHz): δ 14.07 (1H, s), 13.36 (1H, s), 11.97 (1H, s),
11.24 (1H, s), 10.53 (1H, s), 10.48 (1H, s), 10.30 (1H, s),
9.80 (1H, s), 8.05 (1H, s), 7.95 (2H, s), 7.84 (2H, m), 7.68
(1H, dd), 7.46 (1H, s), 7.40 (1H, d), 7.33 (1H, t), 7.02 (1H,
d), 5.30 (1H, s), 4.99 (1H, s), 4.61 (1H, t), 4.5-3.7 (12H, m),
3.6-2.85 (14H, m), 3.01 (1H, d), 2.81 (3H, s), 2.27 (4H, m),
2.13 (2H, m), 1.85 (1H, t), 1.65 (1H, d), 1.18 (3H, d). 31P
NMR (1%TFA-D/DMSO-D6, 162 MHz): δ 18.23.
Cell line. Rat OSRGa cell line was established from an ini-
tially radio-induced osteosarcoma52. OSRGa cells were
grown in DMEM cell culture medium supplemented with
10% fetal bovine serum in a humidified 5% CO2 chamber
at 37 °C. Mouse fibroblast L929 cells were grown in RPMI
cell culture medium supplemented with 10% fetal bovine
serum in a humidified 5% CO2 chamber at 37 °C.
Safety. As for cytotoxic agents, doxorubicin and 12b80
were handled with care using gloves, masks, lab coats and
glasses for solutions, and under chemical fume hood in
addition for powder.
Cell viability assay. Cell viability was quantified by measur-
ing metabolic activity with WST-1 assay (Roche). Briefly,
OSRGa cells were plated into 96-Well-Plates at an initial
density of 2000 cells/well and L929 cells were plated at an
initial density of 1000 cells/well. Cells were grown in a
humidified 5% CO2 chamber at 37 °C in their respective
cell culture media. After 24 h, cells were treated for 72 h
with doxorubicin (dissolved in water) or with 12b80 (dis-
solved in DPBS) at concentrations ranging from 5 nM to 50
µM, or with their respective control solvent. Cell viability
was evaluated after 72h by measuring the reduction of
tetrazolium salt WST-1 to formazan at 37 °C. Absorbance
of the reduction reaction was read at 420 and 480 nm.
Measure was stopped when the absorbance of solvent
treated cells reached a value between 1.5 and 2. Each con-
dition was performed in triplicate wells.
Physicochemical characterization. Flash chromatography
purification was conducted using a Gilson PLC2020 system
using C18 cartridges from Macherey Nagel. Thin layer
chromatography (TLC) was performed on Merck Silica Gel
60 F254 plates, and the developed plates were visualized
under a UV lamp at 254 nm. NMR spectra were recorded at
1
400 MHz for H and 163 MHz for 31P at 303 K on samples
dissolved in the appropriate deuterated solvent. Used
references were the deuterated solvent signal for residual
1H. Chemical displacement values (δ) are expressed in
parts per million (ppm) and coupling constants (J) in hertz
(Hz). HRMS analyses were carried out on Orbitrap mass
spectrometer (ESI). HPLC studies were conducted using
Waters system with a Phenomenex Luna Column 5u
C18(2) 100Å 150x4.6 mm. Chromatographic conditions
were as follows: flow 1.0 ml/min; mobile phase A - ammo-
nium acetate 20 mM and 0,02% EDTANa2 and NH4OH
(pH=8.7), B - acetonitrile, injection volume 20 µL; gradient,
initial mobile phase 5% B, 0-10 min increased to 70%, 10-
12 min 70%B, 12-14 min decreased to initial mobile phase.
Fluorescence detection was performed at 480 nm for exci-
tation and 558 nm for emission (2475 FLR detector Wa-
ters), UV detection was performed at 275 nm. All reagents
were purchased from Sigma-Aldrich or TCI and were used
as supplied by the manufacturer.
In-vitro comparison of doxorubicin and 12b80 cellular
drug uptake and distribution. OSRGa cells were plated on
glass coverslip into 12 wells plate at a concentration of
25000 cells/well. Cells were incubated in a humidified 5%
CO2 chamber at 37 °C. After 96 h, cells were treated with 1
µM of 12b80 or doxorubicin for 1, 4, 8, 16, 24 or 48 h at 37
°C. Fluorescence images were obtained with a Nikon
Eclipse 80i microscope on living cells at a 400x magnifica-
tion. Doxorubicin-associated fluorescence was excited at
532-554 nm and emission was collected at 573-613 nm.
Animals. All procedures and experimental protocols used
in this study were in compliance with the 2010/63/EU
directive of the European Parliament, for the protection of
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animals used for scientific purposes. All experiments were tion of 12b80 (32.5 µmol/kg) or doxorubicin (3.4
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approved by French Research Ministry (project authoriza-
tion APAFIS#2193 and MESR#01489-01) and were in
accordance with the institutional guidelines of the Region-
al Ethical Committee for animal experiment (CEEA PdL06).
Mice were purchased from Janvier Labs and were acclima-
tized for a minimum of 7 days prior to the beginning of the
study. 5 to 6 week old male Swiss mice, male and female
C57BL/6 mice, and female NMRI Nude mice were included
in protocols. All animals were housed in pathogen-free and
controlled conditions (temperature 22±2 °C, humidity
55±10%) on a 12 h light-dark cycle, in cages of 3 to 5 ani-
mals. All animals had free access to 0.2 µm filtered water
and were fed ad libitum with a commercial chow. Animals
were identified by individual ear tags, randomly distribut-
ed in 3 to 10 animals per group and monitored every day
for clinical status. As a general measure of gross toxicity,
body weight monitoring was performed twice a week.
Euthanasia was performed by cervical dislocation under
isoflurane inhalation anesthesia.
µmol/kg) associated with zoledronate (0.34 µmol/kg)
from D6 on 3 consecutive days every 3 weeks and were
euthanized on D48.
Histological analysis. For analysis of distribution in tumor,
tissues were sampled 24 h post-injection, fixed in 4% for-
malin, dehydrated in 70% ethanol and with automatized
system (Leica), embedded in paraffin and stored at 4 °C.
Each sample was sectioned using a microtome (Microm
Microtech). 4-µm-thick sections were cut and affixed on
treated histological slides (Super frost plus – Thermo sci-
entific). Fluorescence was observed on sections in a specif-
ic channel (fluorescence was excited at 532-554 nm and
emission was collected at 573-613 nm) to localize 12b80-
and/or metabolites-associated fluorescence. Sections were
then stained with Van Kossa to localize tumor mineral
nodes. Nanozoomer (Hamamatsu) was used for images
acquisition.
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HPLC analysis of biodistribution. For analysis of distribu-
tion in tibia, femur and tumor, samples were collected,
stored at -20 °C and crushed in liquid nitrogen with a mor-
tar. A weighed quantity of tissue (50 to 200 mg) was dis-
persed in 6N HCl (0.75 mL) stirred at ambiant temperature
for 18 h. The mixture was extracted with a solution of
CHCl3 and MeOH (9/1; 0.75 mL). The organic layer was
collected and the volatiles were removed under air flow.
The residue was solubilized in CH3CN (200 µL), centri-
fuged and analysed by HPLC (waters system). Total fluo-
rescence was presented in nmol/g of tissue as sum of con-
centration of main products in samples: doxorubicin, dox-
orubicinone and anthraquinone.
Experimental osteosarcoma model. Osteosarcoma model
was induced by injection of OSRGa rat osteosarcoma cell
line to NMRI nude mice. 2x106 cells in 15 µL of DPBS were
injected in close contact with the right tibia, after light
periost scratching, on mice anesthetized by isoflurane
inhalation (air 0.5 L/min and isoflurane 2%). Tumor vol-
ume was quantified twice a week by measuring two per-
pendicular diameters with a Vernier caliper and calculated
with the formula: (l² x L)/2 (l, the smallest and L, the larg-
est diameter). Mice with tumor volumes exceeding 2500
mm3 were sacrificed. Radiography of the right tibia was
performed on each animal using a radiography unit (Fax-
itron) after euthanasia.
3
Radiolabelling. H-12b80 was synthesized starting from
Treatment of rodents. 12b80 powder was solubilized in
DPBS. Doxorubicin powder was purchased from AOKchem
and solubilized in 0.9% NaCl. Zoledronate was purchased
from TCI and solubilized in DPBS. Mice were treated with
intravenous bolus injection of 12b80 or doxorubicin at 10
mL/kg in tail vein using 29G insulin syringe. Zoledronate
was administered by subcutaneous injection at 1 mL/kg.
For MTD evaluation, Swiss mice were treated on D0 by
single injection of doxorubicin (10 to 32.5 µmol/kg) or
12b80 (32.5 to 100 µmol/kg) and euthanized on D27. For
analysis of distribution in healthy femurs, C57BL/6 mice
were treated on D0 by single injection of 12b80 (32.5
µmol/kg) and sacrificed between 1 day and 9 months post-
injection. For analysis of distribution in tumors and
healthy tibias, OSRGa tumors were inoculated on NMRI
nude mice on D0, mice received single injection of doxoru-
bicin (3.4 or 32.5 µmol/kg) or 12b80 (32.5 µmol/kg) on
D33 and were euthanized 4 h to 72 h post-injection. For
dose-response efficacy study, OSRGa tumors were inocu-
lated on NMRI nude mice on D0, mice were treated with
12b80 (20 or 32.5 µmol/kg) from D1 on 2 or 3 consecutive
days every 3 weeks and were sacrificed between D53 and
D66 according to tumor volume. For antitumor efficacy
comparison to reference treatment, OSRGa tumors were
inoculated on NMRI Nude mice on D0, mice received injec-
3H-doxorubicin (Moravek) using the same strategy de-
3
scribed in Figure 1. H-12b80 was administered intrave-
nously at 32.5 µmol/kg to male Swiss mice. The distribu-
3
tion of H-12b80 in bones was assessed over 59 days. 3
mice were sacrificed at each timepoint and femurs were
collected, rinsed with 0.9% NaCl, blot-dried and weighed
using a precision scale and put into scintillation vial. 1 mL
of 6N HCl was added in each sample and vial was stirred at
room temperature for 72 to 96 h. Then, 1.5 mL of solubiliz-
ing solution (Solvable™ Tissue Solubilizer - Perkin Elmer)
was added and vials were placed overnight at 56 °C. The
discoloration was performed by addition of 0.3 mL 30%
hydrogen peroxide. 10 mL of liquid scintillation cocktail
(LLT Ultima Gold - Perkin Elmer) was then added and vials
were placed in darkness before counting to allow decay of
luminescence. Then, tubes were placed under an automatic
TDCR liquid scintillation counter (Hidex 300 SL) calibrated
for tritium radionuclide. 40 seconds per sample was need-
ed to count the radioactivity. Radioactivity was given in
disintegrations per minute (dpm). The radioactivity in
bone samples was expressed as percentage of the injected
dose (ID) per gram of bone. % of ID in total skeleton was
estimated with following formula: %ID skeleton = % ID
femur / 0.02845. This formula was based on the analysis of
weight of femur vs weight of total skeleton in mice from
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concentration; ID: injected dose; MTD: maximum tolerated
dose.
the same strain and same age. In this study of 4 week old
mice, femurs represented 2.845% of total skeleton weight.
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Statistical methods. In-vitro studies of binding, drug re-
lease and cell viability were performed in triplicate and
presented as Mean ± SD. In-vivo studies of MTD or distribu-
tion were conducted on 3 animals per group and
timepoint; body weight or total fluorescence were pre-
sented in Mean ± SD, but no statistical analysis was per-
formed on these small samples. In-vivo studies of efficacy
were conducted on 10 mice per group (dose-response
efficacy study) or 6 mice per group (efficacy comparison to
reference treatment) and tumor volumes were presented
as Mean ± SEM. Statistical analysis was presented for the
last measurement of tumor volume, by Mann Whitney test.
Statistical analysis was performed using Prism3 and excel
softwares. Probability values of less than 0.05 (two-tailed)
were used as the critical level of significance for all tests.
Mean values were annotated with an asterisk symbol (* p-
value < 0.05, ** p-value < 0.01 and *** p-value < 0.001).
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* Emmanuelle David. Atlanthera, 3 rue Aronnax, 44821 Saint
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R. Le Bot conceived the project. M. Egorov designed the syn-
thetic and purification work which was carried out by J.Y.
Goujon, L. Morandeau and M. Egorov. J.Y. Goujon developed
the analytical and bioanalytical methods which were carried
out by M. Egorov, L. Morandeau and J.Y. Goujon. S. Cagnol
designed the in-vitro biological studies, which were conducted
by J. Taurelle, M. Sicard and S. Cagnol. E. David monitored the
project planning and designed the in-vivo biological studies,
which were carried out by J. Taurelle, C. Moal, M. Sicard, C.
Benesteau, S. Pairel and E. David. E. David wrote this paper,
with the contribution of S. Cagnol, J.Y. Goujon and M. Egorov.
D. Heymann, F. Redini and F. Gouin were part of the scientific
committee for the project. All authors have given approval to
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Bioconjugate Chemistry
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Bioconjugate Chemistry
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Bioconjugate Chemistry
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ACS Paragon Plus Environment