Pemetrexed-conjugated hyaluronan for the treatment of malignant pleural mesothelioma

Article abstract of DOI:10.1016/j.ejps.2019.105008

Pemetrexed (PMX) is a multi-targeted antifolate drug used for the treatment of malignant pleural mesothelioma (MPM) and non-small cell lung cancer. Hyaluronan (HA) in blood is well known as a disease marker of MPM. We synthesized PMX-conjugated hyaluronan

Full text of DOI:10.1016/j.ejps.2019.105008

Accepted Manuscript
Pemetrexed-conjugated hyaluronan for the treatment of malignant
pleural mesothelioma
Yuki Amano, Seiichi Ohta, Kazuma L. Sakura, Taichi Ito
PII:
DOI:
Article Number:
Reference:
S0928-0987(19)30271-4
https://doi.org/10.1016/j.ejps.2019.105008
105008
PHASCI 105008
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
31 May 2019
4 July 2019
10 July 2019
Please cite this article as: Y. Amano, S. Ohta, K.L. Sakura, et al., Pemetrexed-conjugated
hyaluronan for the treatment of malignant pleural mesothelioma, European Journal of
Pharmaceutical Sciences, https://doi.org/10.1016/j.ejps.2019.105008
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Page 1
Pemetrexed-conjugated hyaluronan for the
treatment of malignant pleural mesothelioma
565-0871,
E-mail address: taichi@m.u-tokyo.ac.jp

ACCEPTED MANUSCRIPT
Page 2
Abstract:
Keywords:
Anti-tumor, Drug conjugates, Pemetrexed, Malignant pleural mesothelioma

ACCEPTED MANUSCRIPT
Page 3
thymidylate synthase (TS), dihydrofolate reductase (DHFR)
and glycinamide ribonucleotide formyltransferase (GARFT), leading to the
interference of DNA synthesis (
is a relatively new
. PMX
compared with other drugs such as methotrexate
(MTX), whose polymer conjugation has actively been investigated for long time
(
ń
. PMX was approved by FDA in 2004 for treatment of malignant
pleural mesothelioma (MPM), and now also be used for non-small cell lung cancer
(
.

ACCEPTED MANUSCRIPT
Page 4
Conjugation of PMX to polymers, as well as its loading to drug carriers, can be a
promising approach to improve its therapeutic efficacy. This strategy is expected to
improve the poor water solubility and short retention time, which are main drawbacks
of PMX. The synthesis of two polymer conjugates have been reported so far;
hydroxyethyl starch (
a glycosaminoglycan that is one of the major components
of extra cellular matrix. It is well known that HA in pleural effusion is utilized
clinically as a prognostic marker in
. The viscosity of the plural effusion is extremely high in some patients
of MPM due to its high concentration of HA. Thus, it is speculated that the turnover
of HA would be relatively active compared to normal tissues (
.
Generally speaking, HA is internalized by cells via binding to its receptors such as
CD44 and subsequently inducing receptor-mediated endocytosis in its turnovers
process both in normal tissues and cancer tissues (
. Because CD44 is overexpressed in tumor cells with high affinity state,
it is expected that HA is aggressively taken up and then metabolized via this transport
pathway in cancer cells. By taking advantage of these characteristic interactions with

ACCEPTED MANUSCRIPT
Page 5
tumors, HA has been studied as a targeted drug carrier for wide range of cancers
(
.
HA with different molecular weights
were first derivatized with
(ADH), and then conjugated to
PMX. Their effect on enzyme inhibition activity, cellular internalization, and growth
inhibition against MPM cell lines were examined in vitro. Their anti-tumor activity
was also evaluated using a mouse model of MPM.
×
Hydrate

ACCEPTED MANUSCRIPT
Page 6
Dihydrofolate Reductase Assay Kit was
purchased from
Fetal
bovine serum was purchased from Biosera, Inc., (Villebon sur Yvette, France), and
epidermal growth factor was purchased from PeproTech Ltd. (London, UK). Dialysis
membrane (Spectra/Por, MWCO = 6000–8000 Da) was purchased from Spectrum
Laboratories Inc. (Rancho Dominguez, CA, USA). The human pleural mesothelial
cell line, MeT-5A, was purchased from American Type Culture Collection (Rockville,
MD, USA). Recombinant Human Thymidylate Synthase protein (ab95378) was
purchased from Abcam (Cambridge, UK).
HA-adipic dihydrazide (HA-ADH) was synthesized as described previously
(
.
·

ACCEPTED MANUSCRIPT
Page 7
°C until use in further synthesis.
HA-ADH-PMX was synthesized by reacting HA-ADH with 2 molar equivalents of
NHS-PMX to ADH. The activated PMX was reacted with 0.2 g of HA-ADH
dissolved in 26.66 mL of 0.1 M NaHCO3 solution at room temperature for 18 h. The
resultant solution was dialyzed

ACCEPTED MANUSCRIPT
Page 8
JEOL
JNM-A500, JEOL), FT-IR (FT/IR-4200ST, JASCO)
were dissolved in D2O and PMX was dissolved in
1
DMSO-d6 for H NMR analysis. FT-IR spectra were measured using a potassium
bromide (KBr) tablet of each polymer. UV-vis absorption spectra of polymers were
measured in phosphate buffer saline (pH 7.4). The absolute molecular weight and
root-mean-square radius were determined by SEC-MALS (DAWN8+, Wyatt
Technology Corp.). Separation was performed at room temperature using Shodex
OHpak LB-806M column (Showa Denko Co., Ltd.) at a flow rate of 0.5 mL/min.
Sodium phosphate buffer (pH 6.7), composed of 0.05 M phosphate buffer containing
0.2 M NaCl was used as a mobile phase. Mw and radius values were calculated by
Astra v7.1 software, using a dn/dc value of 0.153 (
.
at pH 7.4
TrisHCl, and 0.75 mM of NaEDTA (Solution A). THF was dissolved in solution
A at 1.1 mM (Solution B).

ACCEPTED MANUSCRIPT
Page 9
dUMP was
dissolved in solution A at 100 mM (Solution D).

ACCEPTED MANUSCRIPT
Page 10



ACCEPTED MANUSCRIPT
Page 11
epidermal
fetal bovine serum
growth factor

ACCEPTED MANUSCRIPT
Page 12
μ
μ
μ
μ

ACCEPTED MANUSCRIPT
Page 13
μ
μ
μ
μ

ACCEPTED MANUSCRIPT
Page 14
HA-ADH-PMX was then synthesized by reacting HA-ADH with
NHS-PMX).

ACCEPTED MANUSCRIPT
Page 15

ACCEPTED MANUSCRIPT
Page 16

ACCEPTED MANUSCRIPT
Page 17

ACCEPTED MANUSCRIPT
Page 18

ACCEPTED MANUSCRIPT
Page 19

ACCEPTED MANUSCRIPT
Page 20
′

ACCEPTED MANUSCRIPT
Page 21

ACCEPTED MANUSCRIPT
Page 22
°
Table 2. Determined Vmax, Km,
37 °C)
′ and Ki from DHFR inhibition assay (pH =7.5, at
Vmax [min-1]
Km [μM]
′ [μM]
―
Ki [μM]
―
1.2×10-2
0.58
PMX (-)

ACCEPTED MANUSCRIPT
Page 23
2.4×10-2
9.4×10-3
1.3×10-2
25
―
PMX
HA-ADH-PMX
(76 kDa)
―
―
1.4
HA-ADH-PMX
(130 kDa)
1.1×10-2
3.5
FRα-mediated endocytosis
via interaction with PMX (
pathways.
would be possible internalization

ACCEPTED MANUSCRIPT
Page 24
less
polymer entanglement or low molecular size (Table 1).
μ

ACCEPTED MANUSCRIPT
Page 25
μ

ACCEPTED MANUSCRIPT
Page 26

ACCEPTED MANUSCRIPT
Page 27
The antitumor effect of HA-ADH-PMX was evaluated against MPM mouse model
using AB22 cells. Seven days after AB22 cells inoculation in pleural cavity, PMX and
HA-ADH-PMXs were injected intrapleurally. The survival time of mice were

ACCEPTED MANUSCRIPT
Page 28
recorded until all mice died. Median survival times were 39, 40, 50 and 52 days in
HA-ADH-PMX (130 kDa), control without material injections, HA-ADH-PMX (76
kDa), and native PMX group, respectively. The weight changes of mice were shown
in Table S1 in supplementary information. Survival time of HA-ADH-PMX (130
kDa) group was comparable with that of control. On the other hand, despite of the
lower in vitro cytotoxicity than HA-ADH-PMX (130 kDa), the average survival time
of HA-ADH-PMX (76 kDa) group was longer than control, and almost the same with
that of native PMX group, although there was no significant difference.
In the clinical standard regimen, PMX is first administered intravenously at 500
mg/m2, followed by the intravenous administration of cisplatin at 75 mg/m2 on day 1
of each 21-day cycle. Following this clinical standard protocol, many researches
administered 10 mg/kg of PMX, either intravenously or intrapleurally, once every 3
days for 2-6 times in animal experiments (
. On the other hand, in this research, we administered 60
mg/kg of PMX in a single shot, assuming endoscopic administration of materials into
pleural cavity due to the several advantages of intrapleural chemotherapy (
. Furthermore, it was expected that HA-ADH-PMX could retain in
pleural cavity because of its high viscoelasticity. Although our results showed no
significant difference among all groups, there is a possibility that therapeutic effect of
HA-ADH-PMX will be increased by multiple administrations in the same way as
previous researches.

ACCEPTED MANUSCRIPT
Page 29
HA-ADH-PMX (130 kDa) had higher cytotoxicity than
HA-ADH-PMX (76 kDa) in vitro (Figures 6-7), the in vivo antitumor effect of
HA-ADH-PMX (76 kDa) was higher than that of HA-ADH-PMX (130 kDa) (Figure
8). This different tendency between in vitro and in vivo would be explained by the
combined effect of the transport kinetics and enzyme inhibitory activity. Whereas
HA-ADH-PMX (76 kDa) showed higher cellular uptake (Figure 5), inhibitory activity
against
enzymes was higher in HA-ADH-PMX (130 kDa)