Synthesis of Poly[ N -(2-hydroxypropyl)methacrylamide] conjugates of inhibitors of the ABC transporter that overcome multidrug resistance in doxorubicin-resistant P388 cells in vitro

Article abstract of DOI:10.1021/bm500649q

The effects of novel polymeric therapeutics based on water-soluble N-(2-hydroxypropyl)methacrylamide copolymers (P(HPMA)) bearing the anticancer drug doxorubicin (Dox), an inhibitor of ABC transporters, or both, on the viability and the proliferation of the murine monocytic leukemia cell line P388 (parental cell line) and its doxorubicin-resistant subline P388/MDR were studied in vitro. The inhibitor derivatives 5-methyl-4-oxohexanoyl reversin 121 (MeOHe-R121) and 5-methyl-4-oxohexanoyl ritonavir ester (MeOHe-RIT), showing the highest inhibitory activities, were conjugated to the P(HPMA) via the biodegradable pH-sensitive hydrazone bond, and the ability of these conjugates to block the ATP driven P-glycoprotein (P-gp) efflux pump was tested. The P(HPMA) conjugate P-Ahx-NH-N=MeOHe-R121 showed a dose-dependent increase in the ability to sensitize the P388/MDR cells to Dox from 1.5 to 24μM, and achieved an approximately 50-fold increase in sensitization at 24μM. The P(HPMA) conjugate P-Ahx-NH-N=MeOHe-RIT showed moderate activity at 6μM (～10 times higher sensitization) and increased sensitization by 50-fold at 12μM. The cytostatic activity of the P(HPMA) conjugate P-Ahx-NH-N=MeOHe-R121(Dox) containing Dox and the P-gp inhibitor MeOHe-R121, both bound via hydrazone bonds to the P(HPMA) carrier, was almost 30 times higher than that of the conjugate P-Ahx-NH-N=Dox toward the P388/MDR cells in vitro. A similar result was observed for P-Ahx-NH-N=MeOHe-RIT(Dox), which exhibited almost 10 times higher cytostatic activity than P-Ahx-NH-N=Dox.

Full text of DOI:10.1021/bm500649q

Article
pubs.acs.org/Biomac
Synthesis of Poly[N‑(2-hydroxypropyl)methacrylamide] Conjugates
of Inhibitors of the ABC Transporter That Overcome Multidrug
Resistance in Doxorubicin-Resistant P388 Cells in Vitro
,†
‡
†
†
†
†
‡
‡
̌
̌
V. Subr,* L. Sivak, E. Koziolova, A. Braunova, M. Pechar, J. Strohalm, M. Kabesova, B. Ríhova,
́
́
́
̌
́
́
K. Ulbrich,† and M. Kovar‡
́
̌
†Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic v.v.i., Heyrovsky Sq. 2, 162 06 Prague 6, Czech
Republic
‡Institute of Microbiology, Academy of Sciences of the Czech Republic v.v.i., Víden
ska 1083, 142 20 Prague 4, Czech Republic
̌ ́
S
* Supporting Information
ABSTRACT: The effects of novel polymeric therapeutics based on water-
soluble N-(2-hydroxypropyl)methacrylamide copolymers (P(HPMA)) bear-
ing the anticancer drug doxorubicin (Dox), an inhibitor of ABC transporters,
or both, on the viability and the proliferation of the murine monocytic
leukemia cell line P388 (parental cell line) and its doxorubicin-resistant
subline P388/MDR were studied in vitro. The inhibitor derivatives 5-methyl-
4-oxohexanoyl reversin 121 (MeOHe-R121) and 5-methyl-4-oxohexanoyl
ritonavir ester (MeOHe-RIT), showing the highest inhibitory activities, were
conjugated to the P(HPMA) via the biodegradable pH-sensitive hydrazone
bond, and the ability of these conjugates to block the ATP driven P-
glycoprotein (P-gp) efflux pump was tested. The P(HPMA) conjugate P-Ahx-NH-NMeOHe-R121 showed a dose-dependent
increase in the ability to sensitize the P388/MDR cells to Dox from 1.5 to 24 μM, and achieved an approximately 50-fold
increase in sensitization at 24 μM. The P(HPMA) conjugate P-Ahx-NH-NMeOHe-RIT showed moderate activity at 6 μM
(∼10 times higher sensitization) and increased sensitization by 50-fold at 12 μM. The cytostatic activity of the P(HPMA)
conjugate P-Ahx-NH-NMeOHe-R121(Dox) containing Dox and the P-gp inhibitor MeOHe-R121, both bound via hydrazone
bonds to the P(HPMA) carrier, was almost 30 times higher than that of the conjugate P-Ahx-NH-NDox toward the P388/
MDR cells in vitro. A similar result was observed for P-Ahx-NH-NMeOHe-RIT(Dox), which exhibited almost 10 times higher
cytostatic activity than P-Ahx-NH-NDox.
inhibitors of ABC transporters have been described. Third
generation inhibitors, including elacridar, zosuquidar, and
tariquidar, effectively block the efflux function of the ABC
transporters at nanomolar concentrations.2,4,7,8
INTRODUCTION
■
One of the most serious problems for chemotherapy is the
gradual development of resistance of the cancer cells, which are
originally highly sensitive, to treatment with the wide spectrum
of anticancer drugs commonly used in cancer chemotherapy.
This phenomenon is called acquired multidrug resistance
(MDR) and is the result of the overexpression of adenosine
triphosphate-binding cassette proteins (ABC transporters) in
tumor cells. MDR is responsible for a significant proportion of
chemotherapy failure in cancer treatment.1−5 The energy-
dependent efflux of various hydrophobic anticancer drugs, such
as anthracyclines, vinca alkaloids, taxanes, and others, by the
ABC transporters reduces the intracellular concentration of the
drugs below the effective cytotoxic threshold and is the major
mechanism of MDR. In humans, 48 genes encoding ABC
transporters have been classified. P-glycoprotein (P-gp), breast
cancer resistance protein (BCRP), and multidrug resistance
proteins (MRPs) are considered to be the most important of
these genes.4,6
In addition to these molecules, a series of short peptide P-gp
inhibitors called reversins have been synthesized and described.
They consist of di- and tripeptide derivatives that share
common physicochemical and structural features, such as bulky
aromatic and/or alkyl groups. Reversin 121 (Boc-Asp(OBzl)-
Lys(Z)-OtBu) is an aspartyl lysine (Asp-Lys) dipeptide
derivative, and reversin 205 (Nα,Nε-bis[Boc-Glu(OBzl)-Lys-
OMe) is a tripeptide derivative with a high inhibitory activity
for P-gp.9 The potentials of reversin 121 and reversin 205 have
been studied in MDR1-expressing tumor cells in vitro,10,11 and
reversin 121 has been studied in a murine pancreatic tumor cell
line Panc02 in vivo.12
Several analogues of reversin 121 and reversin 213 were
synthesized to evaluate their ability to inhibit P-gp-mediated
Inhibition of the ABC transporters by low-molecular-weight
inhibitors seems to be one of the most promising approaches
for overcoming MDR in cancer cells. Three generations of
Received: May 5, 2014
Revised: June 25, 2014
Published: June 30, 2014
© 2014 American Chemical Society
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Table 1. Physicochemical Characteristics of the Inhibitor Derivatives
elemental analysis, calcd/found
inhibitor derivative
mp [°C]
ESI-MS m/z (M − Na)+
C
H
N
S
reversin 121
OPe-R121
87−88
57−58
70−71
62−64
73−74
54−56
49−51
52−54
664.1
63.83/64.16
63.83/63.53
64.75/64.68
64.75/65.03
66.74/66.51
64.8/64.68
62.39/61.81
62.39/62.00
62.69/62.12
64.07/63.53
62.57/61.63
n.d.
7.09/7.80
7.09/6.9
7.40/7.36
7.40/7.27
6.75/6.67
7.53/7.88
6.90/7.48
6.90/6.87
6.80/7.34
6.41/6.59
7.04/7.63
n.d.
6.57/6.79
6.57/6.56
6.29/6.31
6.29/6.18
5.99/5.92
7.75/7.58
9.92/9.66
9.92/9.60
10.07/10.31
9.54/9.33
10.87/10.04
n.d.
662.33
690.33
690.33
724.17
745.17
869.42
869.17
841.58
903.50
902.41
847.50
OHe-R121
MeOHe-R121
OPB-R121
Ma-Ahx-R121
OHe-RIT
7.57/7.58
7.57/7.12
7.44/7.22
7.28/7.44
7.11/6.81
n.d.
MeOHe-RIT
OPe-RIT
a
n.d.
OPB-RIT
n.d.
Ma-Ahx-RIT
MeOHe-R205
51−53
109−112
a
n.d. = not determined.
drug efflux in NIH3T3 mouse cells or K562/R7 human
erythroleukemic cells, which overexpress P-gp. These analogues
retained good activity compared to cyclosporin A and the
original reversins.13,14
The aim of this study was to investigate the potential of novel
polymeric therapeutics based on water-soluble P(HPMA)
copolymers bearing either an anticancer drug, an inhibitor of
ABC transporters or both. Binding of the anticancer drug or an
inhibitor of the ABC transporters to the polymeric carrier
should significantly prolong their blood circulation, improve
their solubility and overcome nonspecific side effects on healthy
tissue due to passive accumulation in solid tumors via the
enhanced permeability and retention (EPR) effect.32 The P-gp
inhibitor in the conjugate will further increase the intracellular
load of the cancerostatic drug in MDR cancer cells. Here, we
tested the inhibitory activity of low-molecular-weight deriva-
tives of reversin 121, reversin 205 (Nα, Nε-bis[Boc-Glu(OBzl)-
Lys-OMe), and ritonavir and the P(HPMA) conjugates
containing these derivatives, bound via a biodegradable pH-
sensitive hydrazone bond, in the Dox-resistant P388/MDR
mouse leukemia and the sensitive parental P388 cell lines.
Omelyanenko et al.15 have hypothesized that the admin-
istration of anticancer drugs as water-soluble polymeric
conjugates will be inaccessible to the energy-driven P-gp-
mediated efflux pump, thus increasing the intracellular drug
concentration in P-gp-expressing MDR cells when compared to
the free drug.
Polymer-bound drugs,16,17 micelles,18−20 liposomes,21,22 and
nanoparticles23−25 are internalized by endocytosis through
endosomes/lysosomes and ultimately accumulate inside the
cell. Thus, these forms of drugs should be inaccessible to the
energy-dependent efflux pump localized at the cytoplasmic
membrane.
The results obtained by Minko et al. indicate that water-
soluble anticancer drug conjugates based on a N-(2-
hydroxypropyl)methacrylamide copolymer P(HPMA) contain-
ing doxorubicin (Dox) bound via the lysosomally degradable
GlyPheLeuGly spacer are partially able to avoid the P-gp-
mediated efflux of Dox in the Dox-resistant A2780/AD human
ovarian carcinoma cell line.26−29
Zhao et al. described the synthesis of liposomes loaded with
epirubicin and their biological activity in MDA-MB 435 cells
and their MDR counterparts (MDA-MB 435/ADR) in vitro.
They also described a tumor growth inhibition model in vivo.
Epirubicin-loaded liposomes effectively inhibited growth in
both cell lines.22
Hydrolytically degradable hydrogels based on N-(2-
hydroxypropyl)methacrylamide (HPMA) loaded with Dox
and cyclosporin A were also used in combined therapy against
the mouse P388/MDR leukemia, and a synergistic effect of the
cytostatic drug and the chemosensitizing agent was observed.
The tumor volumes were reduced to approximately 50% after
the implantation of a HPMA hydrogel containing four times
the maximum tolerated dose (MTD) of Dox and cyclosporine
A (CsA).30
MATERIALS AND METHODS
■
Materials. Methacryloyl chloride, 1-amino-propan-2-ol, 2,2′-azobis-
(2-methylbutyronitrile) (AIBN), 4,5-dihydro-thiazole-2-thiol (TT),
Boc-hydrazide, 2-cyanopropan-2-yl benzodithioate (CTA), 4-oxo-
pentanoic acid, 6-oxo-heptanoic acid, succinic anhydride, iron(III)
acetylacetonate, isopropylmagnesium chloride (2 M solution in THF),
4-(dimethylamino)pyridine (DMAP, N-ethyl-N′-(3-(dimethylamino)-
propyl)carbodiimide hydrochloride (EDC), N,N′-dicyclohexyl carbo-
diimide (DCC), (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate (PYBOP), 1-hydroxybenzotriazole hydrate
(HOBT), 2,4,6-trinitrobenzenesulfonic acid (TNBSA), N,N-diisopro-
pylethylamine (DIPEA), triethylamine (Et3N), N,N-dimethylforma-
mide (DMF), dimethyl sulfoxide (DMSO), tert-butyl alcohol (t-
BuOH), dichloromethane (DCM), and silica gel 60 were purchased
from Sigma-Aldrich, Czech Republic. N-α-t-Boc-L-aspartic acid β-
benzyl ester (Boc-Asp(OBzl)-OH), L-aspartic acid β-benzyl ester (H-
Asp(OBzl)-OH), N-ε-CBZ-L-lysine t-butyl ester hydrochloride (H-
Lys(Z)-OtBu·HCl), 4-Fmoc-hydrazinobenzoyl AM Nova Gel, Fmoc-
Lys(ivDde)-OH, and Fmoc-Glu(OBz)-OH were purchased from
Novabiochem. 4-(2-Oxopropyl)-benzoic acid was purchased from
Rieke Metals Inc.. Ritonavir was purchased from ChemPacific Corp.
Doxorubicin hydrochloride (Dox·HCl) was purchased from Meiji
Seiko, Japan.
Pluronic P85 block copolymers loaded with daunorubicin
were studied in the P-gp-overexpressing human ovarian
carcinoma cells, SKVLB, and non-MDR SKOV3 cells. A
dramatic increase in the daunorubicin cytotoxic activity (up to
700 times) was observed in the presence of 0.01%−1%
copolymer in the case of the SKVLB cells.18,19,31
All other chemicals and solvents were of analytical grade. The
solvents were dried and purified using conventional procedures and
distilled before use.
Synthesis of Monomers. N-(2-Hydroxypropyl)methacrylamide
(HPMA) was synthesized via the reaction of methacryloyl chloride
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with 1-aminopropan-2-ol in dichloromethane in the presence of
sodium carbonate.33
of EDC (0.82 g; 4.30 mmol). The yield was 1.4 g (37%) of an oily
product, which was used in the next step without purification.
Synthesis of 1-[4-(2-Thioxo-thiazolidine-3-carbonyl)-phenyl]-
propan-2-one (OPB-TT). The OPB-TT was prepared by the reaction
of 4-(2-oxopropyl)-benzoic acid (1.02 g 5.74 mmol) with 4,5 dihydro-
thiazole-2-thiol (0.69 g, 5.74 mmol) in the presence of EDC (1.43 g,
7.47 mmol). HPLC analysis showed a single peak at 305 nm with a
retention time of 9.12 min., mp = 143−146 °C. Elemental analysis:
calcd/found C = 55.89/54.43%; H = 4.69/4.24%; N = 5.01/5.29%; S
= 22.96/22.80%. ESI-MS: m/z = 302.0 (M − Na)+.
6-(2-Methyl-acryloylamino)-hexanoic acid (Ma-Ahx-COOH) was
prepared as previously described.34
2-Methyl-N-[6-oxo-6-(2-thioxo-thiazolidin-3-yl)-hexyl]-acrylamide
(Ma-Ahx-TT) was prepared as previously described.35
N′-[6-(2-Methyl-acryloylamino)-hexanoyl]-hydrazinecarboxylic acid
tert-butyl ester (Ma-Ahx-NH-NH-Boc) was prepared by reaction of
Ma-Ahx-COOH (3.0 g, 15.1 mmol) with Boc-hydrazide (2.09 g, 15.80
mmol) in the presence of EDC (3.75 g, 19.58 mmol) in DCM (20
mL). The reaction mixture was stirred for 3 h at room temperature
and then extracted with distilled water (3 × 20 mL) and with an
aqueous solution of NaHCO3 (2 wt %). The organic layer was
separated and dried over Na2SO4. The DCM was evaporated, and the
product was crystallized from ethyl acetate. Yield: 2.13 g (45.2%).
HPLC analysis showed a single peak at 220 nm with a retention time
of 8.05 min., mp =139 °C. Elemental analysis: calcd/found C = 57.49/
57.34%; H = 8.68/8.86%; N = 13.41/13.06%. ESI-MS: m/z = 335.96
(M − Na)+.
Synthesis of the Inhibitor Reversin 121 and Derivatives of
Reversin 121. Synthesis of Boc-Asp(OBzl)-Lys(Z)-OtBu (Reversin
121). Reversin 121 was prepared by the reaction of Boc-Asp(OBzl)-
OH and H-Lys(Z)-OtBu·HCl as described earlier.9 Boc-Asp(OBzl)-
OH (0.1 g, 0.31 mmol), H-Lys(Z)-OtBu·HCl (0.115 g, 0.31 mmol),
HOBT (0.044 g, 0.32 mmol), and Et3N (45 μL, 0.32 mmol) were
dissolved in DMF (5 mL), and the solution was cooled to −10 °C.
DCC (0.07 g, 0.34 mmol) was dissolved in DMF (5 mL), and after
cooling to −10 °C the two solutions were mixed. The reaction mixture
was left overnight at 5 °C. The DMF was evaporated, and the solid
residue was dissolved in ethyl acetate. The precipitated dicyclohexyl
urea was removed by filtration, and the solution was extracted with an
aqueous solution of citric acid (10% w/v) followed by extraction with
a saturated solution of NaHCO3. After drying the ethyl acetate phase
over Na2SO4, the reversin 121 was crystallized from the ethyl
acetate:hexane mixture. HPLC analysis gave a single peak at 220 nm
with a retention time of 10.93 min. The other characteristics, including
melting point, elemental analysis, and ESI-MS, are summarized in
Table 1. The 1H NMR spectra (300 MHz, DMSO, 296 K) are
provided in the Supporting Information.
1
The H NMR spectra (300 MHz, DMSO, 296 K) of OPe-TT,
OHe-TT, and OPB-TT are provided in the Supporting Information.
Synthesis of OPe-Asp(OBzl)-Lys(Z)-OtBu (OPe-R121). H-Asp-
(OBzl)-OH (0.5 g, 2.24 mmol) and NaHCO3 (0.19 g, 2.24 mmol)
were dissolved in a mixture of THF (10 mL) and distilled water (5
mL), and OPe-TT (0.49 g, 2.24 mmol) was then added. The reaction
was carried out at room temperature for 24 h. The THF was
evaporated, and the water phase was extracted with ethyl acetate (3 ×
20 mL). The reaction mixture was acidified with HCl to pH = 2 and
extracted with ethyl acetate (3 × 20 mL). The organic phase was dried
over Na2SO4, and the ethyl acetate was evaporated. OPe-Asp(OBzl)-
COOH was obtained as oily product (0.725 g) and was used in the
next step without purification.
OPe-Asp(OBzl)-COOH (0.725 g, 2.24 mmol), H-Lys(Z)-Ot-
Bu.HCl (0.93 g, 2.48 mmol), HOBT (0.34 g, 2.48 mmol), and
DIPEA (0.42 mL, 2.24 mmol) were dissolved in DMF (5 mL), and
EDC (0.57 g, 0.30 mmol) was then added. The reaction mixture was
stirred for 24 h at room temperature. The DMF was evaporated, and
the residue was dissolved in DCM and extracted with a 2 wt %
aqueous solution of KHSO4, NaHCO3, and distilled water. The
organic phase was dried over Na2SO4, and the solvent was evaporated.
The oily crude product was dissolved in a 60:40 mixture of
acetonitrile/distilled water, and the pure product was obtained by
preparative HPLC on a Biospher PSI 120 C18 250 × 25 mm (Watrex)
column with UV detection at 220 nm using the mobile phase water/
acetonitrile with an acetonitrile gradient from 50% to 100%. The flow
rate was 5 mL/min. The fractions with the pure product were
collected and evaporated under vacuum. OPe-R121 was recrystallized
from an ethyl acetate/hexane mixture. HPLC analysis showed a single
peak at 220 nm with a retention time of 10.18 min. The other
characteristics, including melting point, elemental analysis, and ESI-
Synthesis of 1-(2-Thioxo-thiazolidin-3-yl)-pentane-1,4-dione
(OPe-TT). 4-Oxo-pentanoic acid (2.0 g, 17.2 mmol) and 4,5 dihydro-
thiazole-2-thiol (2.05 g, 17.2 mmol) were dissolved in DCM, and N-
ethyl-N′-(3-(dimethylamino)propyl)carbodiimide hydrochloride
(EDC) (3.95 g, 20.6 mmol) was added. The reaction mixture was
stirred for 3 h at room temperature and then extracted with distilled
water (3 × 20 mL) and with an aqueous solution of NaHCO3 (2 wt
%). The organic layer was separated and dried over Na2SO4. The
DCM was evaporated, and the product was crystallized from ethyl
acetate. Yield: 1.8 g (49%). HPLC analysis showed a single peak at 305
nm with a retention time of 8.55 min., mp = 82 °C. Elemental analysis:
calcd/found C = 44.22/44.34%; H = 5.10/5.23%; N = 6.45/6.47%; S
= 29.51/28.52%. ESI-MS: m/z = 239.92 (M − Na)+.
1
MS, are summarized in Table 1. The H NMR spectra (300 MHz,
DMSO, 296 K) are provided in the Supporting Information.
The procedure described above for the synthesis of OPe-R121 was
used for the synthesis of OHe-Asp(OBzl)-Lys(Z)-OtBu (OHe-R121),
MeOHe-Asp(OBzl)-Lys(Z)-OtBu (MeOHe-R121), OPB-Asp(OBzl)-
Lys(Z)-OtBu (OPB-R121), and 6-(2-methyl-acryloylamino)-hexanoyl-
Asp(OBzl)-Lys(Z)-OtBu (Ma-Ahx-R121).
Synthesis of OHe-Asp(OBzl)-Lys(Z)-OtBu (OHe-R121). OHe-Asp-
(OBzl)-COOH was prepared by the reaction of H-Asp(OBzl)-OH
(0.32 g, 1.44 mmol) with OHe-TT (0.32 g, 1.44 mmol) in the
presence of NaHCO3 (0.12 g, 1.44 mmol). In the next step, OHe-
Asp(OBzl)-COOH (0.5 g, 1.44 mmol) was reacted with H-Lys(Z)-
OtBu·HCl (0.59 g, 1.59 mmol), HOBT (0.21 g, 1.59 mmol), DIPEA
(0.28 mL, 1.59 mmol), and EDC (0.37 g 1.91 mmol). HPLC analysis
showed a single peak at 220 nm with a retention time of 10.27 min.
Synthesis of MeOHe-Asp(OBzl)-Lys(Z)-OtBu (MeOHe-R121).
MeOHe-Asp(OBzl)-COOH was prepared by the reaction of H-
Asp(OBzl)-OH (0.5 g, 2.24 mmol) with MeOHe-TT (0.55 g, 2.24
mmol) in the presence of NaHCO3 (0.19 g, 2.24 mmol). In the next
step, MeOHe-Asp(OBzl)-COOH (0.78 g, 2.24 mmol) was reacted
with H-Lys(Z)-OtBu·HCl (0.92 g, 2.46 mmol), HOBT (0.33 g, 2.46
mmol), DIPEA (0.43 mL, 2.46 mmol), and EDC (0.52 g, 2.71 mmol).
The yield was 1.129 g (75.5%). HPLC analysis showed a single peak at
220 nm with a retention time of 10.58 min.
The procedure described for the synthesis of OPe-TT was used for
the synthesis of 1-(2-thioxo-thiazolidin-3-yl)-heptane-1,6-dione (OHe-
TT), 5-methyl-1-(2-thioxo-thiazolidin-3-yl)-hexane-1,4-dione
(MeOHe-TT), and 1-[4-(2-thioxo-thiazolidine-3-carbonyl)-phenyl]-
propan-2-one (OPB-TT).
Synthesis of 1-(2-Thioxo-thiazolidin-3-yl)-heptane-1,6-dione
(OHe-TT). The OHe-TT was prepared by the reaction of 6-
oxohexanoic acid (1.03 g, 7.14 mmol) with 4,5 dihydro-thiazole-2-
thiol (0.85 g, 7.14 mmol) in the presence of EDC (1.64 g, 8.57 mmol).
Yield: 1.4 g (37%). HPLC analysis showed a single peak at 305 nm
with a retention time of 9.12 min. Elemental analysis: calcd/found C =
48.95/49.01%; H = 6.16/6.14%; N = 5.71/5.66%; S = 26.14/25.84%.
ESI-MS: m/z = 268.00 (M − Na)+.
Synthesis of OPB-Asp(OBzl)-Lys(Z)-OtBu (OPB-R121). OPB-Asp-
(OBzl)-COOH was prepared by reaction of H-Asp(OBzl)-OH (0.6 g,
2.69 mmol) with OPB-TT (0.75 g, 2.69 mmol) in the presence of
NaHCO3 (0.23 g, 2.69 mmol). In the next step, OPB-Asp(OBzl)-
COOH (1.02 g, 2.66 mmol) was reacted with H-Lys(Z)-OtBu·HCl
Synthesis of 5-Methyl-1-(2-thioxo-thiazolidin-3-yl)-hexane-1,4-
dione (MeOHe-TT). 5-Methyl-4-oxohexanoic acid was synthesized
according Barberis and Per
́
ez-Prieto.36 The MeOHe-TT was prepared
by the reaction of 5-methyl-4-oxohexanoic acid (0.52 g; 3.58 mmol)
with 4,5 dihydro-thiazole-2-thiol (0.43 g, 3.58 mmol) in the presence
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Scheme 1. Synthesis of Derivatives of Reversin 121
Scheme 2. Synthesis of Derivatives of Ritonavir
(0.99 g, 2.66 mmol), HOBT (0.36 g, 2.66 mmol), DIPEA (0.46 mL,
2.66 mmol), and EDC (0.66 g, 3.46 mmol). The crude product was
purified on a silica gel column with a mobile phase of acetone/ethyl
acetate/hexane (1:1:1) and crystallized from a mixture of diethyl
ether/petrolether. The yield was 1.31 g (70.1%). HPLC analysis
showed a single peak at 220 nm with a retention time of 10.46 min.
Synthesis of 6-(2-Methyl-acryloylamino)-hexanoyl-Asp(OBzl)-
Lys(Z)-OtBu (Ma-Ahx-R121). Ma-Ahx-Asp(OBzl)-COOH was pre-
pared by reaction of H-Asp(OBzl)-OH (0.36 g, 1.63 mmol) with Ma-
Ahx-TT (0.49 g, 1.63 mmol) in the presence of NaHCO3 (0.14 g, 1.63
mmol). In the next step, Ma-Ahx-Asp(OBzl)-COOH (0.65 g, 1.61
mmol) was reacted with H-Lys(Z)-OtBu.HCl (0.60 g, 1.61 mmol),
HOBT (0.22 g, 1.61 mmol), DIPEA (0.28 mL, 1.61 mmol), and EDC
(0.46 g, 2.41 mmol). Ma-Ahx-R121 was isolated from the reaction
mixture by gel filtration on a silica gel column using a mobile phase of
ethyl acetate/methanol 90:10 (v/v) and crystallized from a mixture of
ethyl acetate/diethyl ether. The yield was 0.78 g (66.8%). The HPLC
analysis gave a single peak at 220 nm with a retention time of 9.67 min.
The synthetic scheme of the reversin 121 derivatives is shown in
Scheme 1, and the other characteristics, including melting point,
elemental analysis, and ESI-MS, of OPe-R121, OHe-R121, MeOHe-
R121, OPB-R121, and Ma-Ahx-R121 are summarized in Table 1. The
1H NMR spectra (300 MHz, DMSO, 296 K) are provided in the
Supporting Information.
Synthesis of 6-Oxoheptanoyl-ritonavir Ester (OHe-RIT). OHe-RIT
was prepared by esterification of ritonavir with 6-oxoheptanoic acid.
Ritonavir (0.1 g, 0.14 mmol) and 6-oxoheptanoic acid (0.026 g, 0.18
mmol) were dissolved in DCM (2 mL), and EDC (0.053 g, 0.276
mmol) was added. The reaction was catalyzed by DMAP. The reaction
mixture was stirred at room temperature for 20 h and then extracted
with distilled water (3 × 5 mL) and an aqueous solution of NaHCO3
(2 wt %). The organic layer was separated and dried over Na2SO4, and
the DCM was evaporated under reduced pressure. The oily crude
product was dissolved in a 60:40 mixture of acetonitrile/distilled water,
and the pure product was obtained by HPLC on a semipreparative
column (Chromolith SemiPrep Rp18e 100 × 10 mm) with UV
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Scheme 3. Synthesis of MeOHe-R205
detection at 236 nm. The fraction containing the pure product was
collected and evaporated under reduced pressure to dryness. The yield
was 0.055 g (47%). The HPLC analysis showed a single peak at 236
nm with a retention time of 9.81 min.
The synthetic scheme for the ritonavir esters is shown in Scheme 2,
and the other characteristics, including melting point, elemental
analysis, and ESI-MS, for all synthesized ritonavir esters (OPe-RIT,
OHe-RIT, MeOHe-RIT, OPB-RIT, and Ma-Ahx-RIT) are summar-
ized in Table 1. The 1H NMR spectra (300 MHz, MeOH, 296 K) are
provided in the Supporting Information.
HPLC analysis showed a single peak at 236 nm with a retention time
of 12.25 min.
Synthesis of 4-Oxopentanoyl-ritonavir Ester (OPe-RIT). OPe-RIT
was prepared by esterification of ritonavir (0.15 g, 0.208 mmol) with 4-
oxopentanoic acid (0.029 g, 0.25 mmol) using EDC (0.080 g, 0.416
mmol). The reaction was catalyzed by DMAP. The pure product was
obtained using the same method as described for OHe-RIT and OPB-
RIT. The yield was 0.12 g (70%). HPLC analysis showed a single peak
at 236 nm with a retention time of 9.25 min.
Synthesis of 6-(2-Methyl-acryloylamino)-hexanoyl-ritonavir Ester
(Ma-Ahx-RIT). Ma-Ahx-RIT was prepared by esterification of ritonavir
(0.2 g, 0.28 mmol) with Ma-Ahx-COOH (0.072 g, 0.36 mmol) using
EDC (0.106 g, 0.56 mmol) in DCM (4 mL). The reaction was
catalyzed by DMAP, and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was extracted with
distilled water (3 × 5 mL) and an aqueous NaHCO3 solution (2 wt
%). The organic layer was separated, dried over Na2SO4, and
evaporated under vacuum. MA-Ahx-RIT was obtained by trituration of
the residue with hexane. The yield was 0.12 g (48%). HPLC analysis
showed a single peak at 236 nm with a retention time of 10.55 min.
Synthesis of [Nα-MeOHe-Glu(OBzl), Nε-Boc-Glu(OBzl)]-Lys-OMe
(MeOHe-R205). The protected tripeptide was synthesized (Scheme 3)
through manual solid phase peptide synthesis, starting from the C-
terminus, using a standard Fmoc strategy with 4-Fmoc-hydrazino-
benzoyl AM Nova Gel (1.02 g; 0.56 mmol/g), PyBOP (3 equiv),
HOBt monohydrate (3 equiv), and DIPEA (6 equiv) in DMF. The
product was assembled by consecutive condensations of Fmoc-
Lys(ivDde)-OH (3 equiv), Fmoc-Glu(OBz)-OH (3 equiv), and 5-
methyl-4-oxohexanoic acid (3 equiv). After each condensation step,
the Fmoc protecting groups were removed using a 25% solution of
piperidine in DMF. Cleavage of the ivDde protecting group was
performed using a solution of 5% hydrazine monohydrate in DMF.
Finally, Boc-Glu(OBz)-OH (3 equiv) was coupled to the peptide-resin
as described above. The resin was removed by filtration, and the
amount of ivDde released into the filtrate was detected spectrophoto-
The procedure for the OHe-RIT synthesis described above was also
used for the synthesis of the other ritonavir esters, that is, 5-methyl-4-
oxohexanoyl-ritonavir ester (MeOHe-RIT), 5-oxohexanoyl-ritonavir
ester (OHe-RIT), 4-oxopentanoyl-ritonavir ester (OPe-RIT), 4-(2-
oxo-propyl)-benzoyl-ritonavir ester (OPB-RIT), and 6-(2-methyl-
acryloylamino)-hexanoyl-ritonavir ester (Ma-Ahx-RIT).
Synthesis of 5-Methyl-4-oxohexanoyl-ritonavir Ester (MeOHe-
RIT). MeOHe-RIT was prepared by esterification of ritonavir with 5-
methyl-4-oxohexanoic acid, catalyzed by DMAP. Ritonavir (0.1 g,
0.139 mmol), DMAP, and 5-methyl-4-oxohexanoic acid (0.026 g, 0.18
mmol) were dissolved in DCM (2 mL), and EDC (0.053 g, 0.276
mmol) was added. The reaction mixture was stirred at room
temperature for 5 h and then extracted with distilled water (3 × 5
mL) and an aqueous solution of NaHCO3 (2 wt %). The organic layer
was separated and dried over Na2SO4. The pure product was obtained
by chromatography on a silica gel column (mobile phase chloroform/
ethyl acetate/acetone 5:2:5) with UV detection at 236 nm. HPLC
analysis showed a single peak at 236 nm with a retention time of 10.78
min. The yield was 0.095 g (82%).
Synthesis of 4-(2-Oxopropyl)-benzoyl-ritonavir Ester (OPB-RIT).
OPB-RIT was prepared by esterification of ritonavir (0.15 g, 0.208
mmol) with 4-(2-oxopropyl)-benzoic acid (0.048 g, 0.27 mmol) in the
presence of EDC (0.080 g, 0.416 mmol), catalyzed by DMAP, using
the procedure described for MeOHe-RIT. The pure product was
obtained as described for OHe-RIT. The yield was 0.117 g (64%).
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Table 2. Characteristics of P(HPMA) Conjugates
P(HPMA) conjugate
Mw
Đ
inhibitor content [wt %]
Dox content [wt %]
P-Ahx-NH-NDox
27 000
33 100
33 100
33 100
29 600
33 100
27 700
33 100
30 700
30 700
30 700
30 700
30 700
31 900
31 900
1.8
9.8
P-Ahx-NH-NOPe-R121
P-Ahx-NH-NOHe-R121
P-Ahx-NH-NMeOHe-R121
P-Ahx-NH-NOPB-R121
P-Ahx-NH-NMeOHe-R121(Dox)
P-Ahx-R121
1.12
1.12
1.12
1.13
1.12
1.15
1.12
1.06
1.06
1.06
1.06
1.06
1.09
1.09
10.8
12.5
8.6
7.8
8.5
8.0
5.3
P-Ahx-NH-NH-MeOHe-R121
P-Ahx-NH-NMeOHe-R205
P-Ahx-NH-NOPe-RIT
9.2
7.0
12.6
18.3
8.3
P-Ahx-NH-NOHe-RIT
P-Ahx-NH-NMeOHe-RIT
P-Ahx-NH-NOPB-RIT
P-Ahx-NH-NMeOHe-RIT(Dox)
P-Ahx-NH-NH-MeOHe-RIT
18.8
7.3
9.4
7.3
metrically at 300 nm. Cleavage of the protected peptide methyl ester
from the resin was performed using Cu(OAc)2, pyridine and methanol
as the nucleophile.37 The product was triturated with diethyl ether and
dried. The crude peptide derivative was purified chromatographically
and characterized by HPLC and mass spectrometry.
The physicochemical characteristics of the derivatives of the ABC
transporter inhibitors reversin 121, reversin 205 and ritonavir esters
are summarized in Table 1.
Synthesis of P(HPMA) Precursor. The P(HPMA) precursors P-
Ahx-NH-NH-Boc (here and in the following text, P indicates the
HPMA copolymer backbone) with well-defined molecular weight and
low polydispersity Đ were prepared by reversible addition−
fragmentation chain transfer (RAFT) polymerization by using chain
transfer agent CTA and initiator AIBN. The ratio of monomer (M)/
CTA was 400:1, and the ratio of CTA/AIBN was 3:1. An example of
the polymerization follows: HPMA (1.0 g, 6.98 mmol), Ma-Ahx-NH-
NH-Boc (0.243 g, 0.78 mmol), CTA (12.9 mg, 5.82 × 10−2 mmol),
and AIBN (3.2 mg, 1.94 × 10−2 mmol) were dissolved in t-BuOH
(2.33 mL). The solution was inserted into an ampule and bubbled
with argon for 10 min. The ampule was sealed, and the polymerization
was performed at 70 °C for 16 h. The polymer was isolated by
precipitation into the mixture acetone/diethyl ether (3:1), filtered off,
and dried under vacuum. The yield was 0.85 g (68.0%). The terminal
dithio-benzoate group (DTB) was removed by the method described
by Perrier et al.38 The P(HPMA) precursor P-Ahx-NH-NH2 was
prepared by removing the Boc protecting group in the presence of
TFA.39
stirred for 6 h at room temperature. The conjugate P-Ahx-NH-NH-
MeOHe-R121 was purified on a PD-10 column with water as the
mobile phase and lyophilized. The yield was 47 mg.
The polymer conjugate P-Ahx-R121 was prepared by copolymeriza-
tion of HPMA (0.2 g) and Ma-Ahx-R121 (0.112 g) by RAFT
polymerization using the chain transfer agent CTA and the initiator
AIBN.
The conjugate P-Ahx-NH-NDox was prepared by the reaction of
the precursor P-Ahx-NH-NH2 with Dox·HCl in methanol in the dark
and was purified as described earlier.40 The characteristics of the
P(HPMA) conjugates are summarized in Table 2.
Synthesis of P(HPMA) Conjugates with Inhibitor Derivatives
and Doxorubicin. The conjugate P-Ahx-NH-NMeOHe-
R121(Dox) was prepared by reaction of the precursor P-Ahx-NH-
NH2 (50 mg) with MeOHe-R121 (10 mg) in dry methanol in the
presence of acetic acid (4 μL). The reaction was carried out for 16 h.
Dox·HCl (5 mg) was then added, and the reaction mixture was stirred
for 24 h. The reaction mixture was diluted with dry methanol (2 mL),
and the conjugate P-Ahx-NH-NMeOHe-R121(Dox) was purified
by column chromatography on a Sephadex LH-20 column with
methanol as the mobile phase. It was then precipitated into ethyl
acetate, isolated by filtration, and dried under vacuum. The yield was
48 mg of P-Ahx-NH-NMeOHe-R121(Dox).
Characterization of Monomers, P(HPMA) Precursor, and
P(HPMA) Conjugates. The monomers and inhibitor derivatives were
characterized using a HPLC Shimadzu system with a reverse-phase
column (Chromolith HighResulution RP-18e, 100 × 4.6 mm) (Merck,
Germany) equipped with a UV/vis photodiode array detector.
Gradient elution with 5−95% of acetonitrile for 15 min at a flow
rate of 1.0 mL/min was used. The molecular weights of the monomers
and inhibitors were determined using a mass spectrometer (MS LCQ
Fleet, Thermo Fisher Scientific). The structures were confirmed by 1H
NMR (300 MHz) using a Bruker DPX 300 spectrometer and by
elemental analysis.
Synthesis of P(HPMA) Conjugates with Inhibitor Deriva-
tives. The P(HPMA) conjugates with inhibitor derivatives bound
through pH-sensitive hydrazone bonds were prepared from the
polymer precursor P-Ahx-NH-NH2 (with reactive hydrazide groups in
its side chains) by reaction with OPe-R121, OHe-R121, MeOHe-
R121, OPB-R121, MeOHe-R205, OPe-RIT, OHe-RIT, MeOHe-RIT,
or OPB-RIT.
The number-average molecular weights (Mn), weight-average
molecular weights (Mw), and polydispersities (Đ) of the polymer
precursors and P(HPMA) conjugates were measured using size-
exclusion chromatography (SEC) on a HPLC Shimadzu system
equipped with a UV detector, an Optilab rEX differential
refractometer, and multiangle light scattering DAWN 8 (Wyatt
Technology) detector. For these experiments, a 20% 0.3 M acetate/
80% methanol (v/v) buffer and either a TSKgel G3000SW or a
TSKgel G4000SW column were used.
The content of the inhibitor in the polymer conjugates was
determined by amino acid analysis of the hydrolyzed P(HPMA)
conjugates (6 M HCl, 115 °C, 18 h in a sealed ampule) using a
reverse-phase column (Chromolith HighResulution RP-18e, 100 × 4.6
mm) (Merck, Germany) after precolumn derivatization with
phthalaldehyde (OPA) and 3-sulfanylpropanoic acid (excitation at
An example of the synthesis of the polymer conjugate P-Ahx-NH-
NMeOHe-R121 follows: P-Ahx-NH-NH2 (200 mg) was dissolved
in dry methanol (1.2 mL), and MeOHe-R121 (25 mg) was added,
followed by the addition of acetic acid (10 μL). The reaction mixture
was stirred for 16 h at room temperature. The reaction mixture was
then diluted with dry methanol (2 mL), and the conjugate P-Ahx-NH-
NMeOHe-R121 was purified by column chromatography on a
Sephadex LH-20 column in methanol with UV detection at 220 nm.
The compound was precipitated into ethyl acetate, isolated by
filtration, and dried under vacuum.
The polymer conjugate P-Ahx-NH-NH-MeOHe-R121 was pre-
pared by the reaction of P-Ahx-NH-NMeOHe-R121 with
cyanoborohydride. The conjugate P-Ahx-NH-NMeOHe-R121 (50
mg) was dissolved in PBS buffer. Cyanoborohydride (20 mg) was
added, and the pH was adjusted to 6.0. The reaction mixture was
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229 nm, emission at 450 nm). Gradient elution with 10−100% solvent
B for 35 min at a flow rate of 1.0 mL/min was used, where solvent A
was 0.05 M sodium acetate buffer, pH 6.5, and solvent B was 300 mL
of 0.17 M sodium acetate and 700 mL of methanol.
The Dox content was determined spectrophotometrically on a
Specord 205 (Jena Analytics) spectrophotometer (ε488 = 10 700 L·
mol−1·cm−1; methanol), and the content of the hydrazide groups was
determined by the TNBSA method (ε500 = 17 550 L·mol−1·cm−1;
borate buffer pH 9.3).40
105 cells per well (100 μL per well) in culture medium. The cells were
then incubated with the tested compounds or controls (in duplicates)
for 30 min. After this preincubation, calcein acetoxymethyl ester
(calcein-AM) was added (final concentration 0.1 μM), and the cells
were incubated for another 20 min in the dark. After this 20 min
incubation, the uptake of calcein-AM was terminated by centrifugation
(200g, 5 min, at 4 °C). The cell pellets were washed and centrifuged
twice with 200 μL and then resuspended in 100 μL of cold FACS
buffer (PBS with 2% FCS and 2 mmol EDTA). The flow cytometric
analysis was performed using an LSRII instrument (BD Biosciences),
and the data were analyzed using FlowJo software (Tree Star, Inc.).
Nonliving cells were detected and gated out using Hoechst 33258
staining (Sigma-Aldrich, Czech Republic). Each experiment was
performed at least three times.
In Vitro Release of the ABC Transporter Inhibitors and Dox
from the P(HPMA) Conjugates. The rate of release of the
derivatives of reversin 121, reversin 205, and ritonavir esters from
the P(HPMA) conjugates was investigated by incubation of the
conjugates in phosphate buffers at pH 5.0 or 7.4 (0.1 M phosphate
buffer with 0.05 M NaCl) at 37 °C. An example of a release
experiment follows: the conjugate P-Ahx-NH-NMeOHe-R121 (10.5
mg, 1.3 mM MeOHe-R121) was dissolved in phosphate buffers at
either pH 5.0 or pH 7.4. The stock solutions (0.3 mL) were placed in
Eppendorf vials and incubated at 37 °C. At predetermined time
intervals, chloroform (0.4 mL) was added to the vial and the mixture
was vortexed for 2 min. The chloroform layer (0.3 mL) was transferred
into a new vial. The residue, after evaporation of the chloroform, was
dissolved in methanol (0.1 mL), and the amount of released MeOHe-
R121 was determined by HPLC analysis performed on an HPLC
instrument (Shimadzu, Japan) using a reverse-phase column
(Chromolith Performance RP-18e; 100 × 4.6 mm) with UV detection
at 220 and 260 nm. The mobile phase was water−methanol with a
methanol gradient of 20−100 vol %, and the flow rate was 1.0 mL/
min. All release data are expressed as the percentage of the amount of
the free inhibitor derivative (drug) relative to the total inhibitor
derivative (drug) content in the polymer conjugate. All experiments
were carried out in triplicate.
Cell Lines and Cell Cultures. The murine monocytic leukemia
cell line P388 (parental cell line) and its doxorubicin-resistant subline
P388/MDR (a cell line that overexpresses P-gp) were obtained from
Prof. I. Lefkovits (Basel Institute for Immunology, Basel, Switzerland).
Both cell lines were propagated in RPMI-1640 medium supplemented
with heat-inactivated 10% fetal calf serum (FCS), 2 mM glutamine,
100 U/mL penicillin, 1 mM sodium pyruvate, 100 μg/mL
streptomycin, and 5 mL nonessential amino acids (Sigma-Aldrich,
Czech Republic). The P388/MDR cells were grown in the continuous
presence of 750 ng/mL doxorubicin to maintain the MDR-phenotype.
One day before each experiment, the cells were transferred to
doxorubicin-free culture medium. Both cell lines were propagated at
37 °C in a 5% CO2 atmosphere. The cell lines were tested for
mycoplasma infection (MycoAlert Mycoplasma Detection kit, Lonza,
Switzerland).
Cellular Drug Sensitivity Assay. To test the cytostatic effect of
the drugs in the presence or absence of chemosensitizers, cell growth
inhibition was determined using the [3H]-thymidine incorporation
assay. The cells (1 × 104 per well) were seeded into 96-well flat
bottom (FB) tissue culture plates (Nunc, Denmark). Various
concentrations of the samples were added to the wells to reach a
final volume of 250 μL. Triplicate wells were used for each test
condition. The plates were incubated in 5% CO2 at 37 °C for 72 h.
After this incubation, each well was pulsed with 1 μCi (37 kBq) of
[3H]-thymidine for 6 h. The cells were then collected on glass fiber
filters (Filtermat, Wallac, Finland) using a cell harvester (Tomtec), and
the radioactivity of the samples was measured in a scintillation counter
(1450 MicroBeta TriLux, Wallac, Finland). Cells cultivated in fresh
medium were used as controls. The inhibition of tumor cell growth is
expressed as the IC50, that is, the concentration of Dox (or equivalent)
that inhibited cell growth by 50%. All reported IC50 values are the
mean of at least three independent experiments. The resistance ratio
was calculated by dividing the IC50 value for the P388/MDR cell line
by the corresponding value in the P388 cells.
Statistical Analysis. The data from the various experiments are
reported as the means
standard deviation (SD). For statistical
analysis, Student’s t test for independent measurements was used. A
value of P < 0.05 was considered significant.
RESULTS AND DISCUSSION
■
Two cell lines, normal murine leukemia cell line P388 and its
Dox-resistant subline P388/MDR (for detailed characteristics,
see Materials and Methods), were used to study the biological
effects of the selected P-gp inhibitors, their derivatives, and
their P(HPMA) conjugates. P388 cells are sensitive to Dox, a
cytostatic drug used in our study, which shows an IC50 of
approximately 0.01 μM (Supporting Information Table 1). The
P388/MDR cell line is highly resistant to Dox, with an IC50 of
approximately 5.9 μM. Thus, the P388/MDR cells are
approximately 600 times less sensitive to Dox than the parental
P388 cell line. A similar situation is observed when the P-Ahx-
NH-NDox conjugate is used instead of free Dox: the IC50
concentrations for the P388 and P388/MDR cells are 0.024
and 92 μM, respectively. The calculated resistance ratio for P-
Ahx-NH-NDox is thus approximately 3800, even higher than
that for free Dox. The higher resistance ratio found for P-Ahx-
NH-NDox than for free Dox may be explained by lower
intracellular concentration of Dox released from P-Ahx-NH-
NDox entering the cell by slower endocytosis in comparison
with fast diffusion of free Dox which results in its more effective
efflux by P-gp and in this consequence also lower cytotoxicity of
the conjugate.
Synthesis of P(HPMA) Conjugates with Inhibitor
Derivatives and Doxorubicin. To increase the cytostatic
activity of free Dox and polymer conjugate P-Ahx-NH-NDox
in the Dox-resistant P388/MDR cell line, we synthesized novel
polymeric therapeutics based on HPMA copolymers bearing
either the anticancer drug, an inhibitor of ABC transporters or
both. The ABC transporter inhibitors selected for this study
were reversin 121, reversin 205 and ritonavir. Reversin 121
(Boc-Asp(OBzl)-Lys(Z)-OtBu) and reversin 205 (Nα,Nε-bis-
[Boc-Glu(OBzl)-Lys-OMe) exhibit good affinity and specificity
for P-gp,9,10,41 but these compounds do not contain any groups
suitable for covalent attachment to the HPMA copolymer. The
synthesis of derivatives of reversin 121 in which the Boc
protecting group was substituted with 4-oxopentanoate, 6-
oxoheptanoate, 5-methyl-4-oxohexanoate, and 4-(2-oxopropyl)-
benzoate enabled conjugation of these derivatives to the
P(HPMA) precursor bearing hydrazide groups via a pH-
sensitive hydrazone bond. This process is shown in Scheme 1.
The derivatives of ritonavir were prepared by esterification of
the ritonavir hydroxyl group with 4-oxopentanoic acid, 6-
oxoheptanoic acid, 5-methyl-4-oxohexanoic acid, and 4-(2-
oxopropyl)-benzoic acid as shown in Scheme 2. Reversin 205
Calcein Efflux Assay. The calcein assay was performed using 96-
well round-bottom (U-base) tissue culture plates (TPP, Switzerland).
All incubation steps were conducted in 5% CO2 at 37 °C. The drug-
sensitive and drug-resistant cells were seeded at a concentration of 1 ×
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derivatized with 5-methyl-4-oxohexanoic acid was prepared
using manual solid phase peptide synthesis, as shown in
Scheme 3. Substituents differing in their detailed structure were
selected to facilitate control of the rate of hydrolysis of the
hydrazone bond (the bond used for their attachment to the
polymer) and thus the rate of release of the derivatives of
inhibitors from the polymer carrier. The detailed procedures for
the synthesis of all inhibitor derivatives are described above and
the physicochemical characteristics of the derivatives of the
inhibitors are summarized in Table 1.
The P(HPMA) precursor containing hydrazide groups was
prepared by RAFT copolymerization of HPMA and Ma-Ahx-
NH-NH-Boc. The RAFT copolymerization enabled the
synthesis of polymer precursors with well-defined molecular
weights and low polydispersities (Đ). The terminal dithioben-
zoate group (DTB) was removed using the method described
by Perrier et al.38 The precursor P-Ahx-NH-NH2 was prepared
by removing the Boc protecting group from the copolymer
hydrazide groups in the presence of TFA.39
P(HPMA) conjugates containing derivatives of the inhibitors
were prepared by the reaction of the hydrazide groups of the
precursor P-Ahx-NH-NH2 with the carbonyl group of the
oxoacid-derivatized inhibitors or with Dox in methanol in the
presence of a trace of acetic acid. The content of the inhibitors
and Dox in the conjugates was controlled by the amount of
inhibitor derivative or Dox added to the reaction mixture and
by the amount of hydrazide groups in the precursor. In addition
to the conjugates described above, P(HPMA) conjugates
containing the inhibitors bound to the copolymer precursor
via nondegradable bonds were also prepared. The first type of
conjugates, P-Ahx-R121 and P-Ahx-RIT, was prepared by
copolymerization of HPMA with monomers Ma-Ahx-R121 or
Ma-Ahx-RIT, respectively, and the second type of conjugates,
P-Ahx-NH-NH-R121 and P-Ahx-NH-NH-RIT, was prepared
by reduction of the hydrazone bond with cyanoborohydride.
The characteristics of the P(HPMA) conjugates with all of the
inhibitors are summarized in Table 2.
the release rates of various drugs (dexamethasone, derivatives of
paclitaxel, docetaxel, or mitomycin C) from P(HPMA)
conjugates bearing the drugs bound via hydrazone
bonds.42−44 After 24 h of incubation of the P-Ahx-NH-N
MeOHe-R121 conjugate at pH 5.0, 90% of the MeOHe-R121
was released, while at pH 7.4 only 3% of the MeOHe-R121 was
released after the same incubation time.
The in vitro release experiments performed with the
conjugates P-Ahx-NH-NMeOHe-R121, P-Ahx-NH-N
OHe-R121, P-Ahx-NH-NOPe-R121, and P-Ahx-NH-N
OPB-R121 showed that the rate of release of MeOHe-R121,
OHe-R121, OPe-R121, and OPB-R121 is controlled by the
structure of the oxoacids used for the derivatization of reversin
121. After 24 h of incubation at pH 5.0, the amounts of the
reversin 121 derivatives released from the P(HPMA)
conjugates decrease in the order of MeOHe-R121 (90%),
OPe-R121 (59%), OHe-R121 (40%), and OPB-R121 (20%).
Additionally, the release profiles obtained for the polymer
conjugates P-Ahx-NH-NOHe-R121, P-Ahx-NH-NOPe-
R121, and P-Ahx-NH-NOPB-R121 showed that the rates
of release of the inhibitor derivatives at pH 7.4 are much lower
than those at pH 5.0. After 24 h of incubation at pH 7.4, only
3% of OPe-R121, 10% of OHe-R121, and 13% of OPB-R121
were released.
No release of R121 and MeOHe-R121 from the polymer
conjugates P-Ahx-R121, P-Ahx-NH-NH-MeOHe-R121 was
observed in buffer at pH 5.0 at 37 °C.
The in vitro release experiment carried out with the polymer
conjugate P-Ahx-NH-NMeOHe-R205 showed that, after 2 h
incubation at pH 5.0, 50% of the MeOHe-R205 was released.
After 24 h incubation at pH 5.0, 77% of the MeOHe-R205 was
released from the polymer conjugate, while at pH 7.4 only 13%
of the MeOHe-R205 was released.
The in vitro release profiles of the RIT esters from the
P(HPMA) conjugates at pH 5.0 and 7.4 (37 °C) are shown in
Figure 2. The release profiles obtained for the conjugates P-
In Vitro Release of Inhibitor Derivatives from
P(HPMA) Conjugates. The in vitro release profiles of the
derivatives of reversin 121 and reversin 205 from P(HPMA)
conjugates at pH 5.0 and 7.4 (37 °C) are shown in Figure 1.
The rates of release at pH 7.4 are much lower than those at pH
5.0. These results agree well with our earlier measurements of
Figure 2. Release of ritonavir esters from P(HPMA) conjugates. At
■
●
▲
pH 5.0: ( ) MeOHe-RIT; ( ) OPe-RIT; (∗) OHe-RIT; ( ) OPB-
○
△
RIT. At pH 7.4: ( ) MeOHe-RIT; ( ) OPe-RIT; (+) OHe-RIT;
□
( ) OPB-RIT.
Ahx-NH-NOPe-RIT, P-Ahx-NH-NMeOHe-RIT, P-Ahx-
NH-NOHe-RIT, and P-Ahx-NH-NOPB-RIT show that
the rates of release of the RIT esters at pH 7.4 are much lower
than those at pH 5.0. The rates of release of the RIT esters are
controlled by the structure of the oxoacids used for the
esterification of the RIT. After 24 h incubation at pH 5.0, the
amounts of RIT esters released decrease in the order of
Figure 1. Release of derivatives of reversin 121 and reversin 205 from
◆
■
polymer conjugates. At pH 5.0: ( ) MeOHe-R121; ( ) OHe-R121;
◇
●
) OPe-R121; (∗) OPB-R121; ( ) MeOHe-R205. At pH 7.4: ( )
▲
(
□
△
○
MeOHe-R121; ( ) OHe-R121; ( ) OPe-R121; (+) OPB-R121; ( )
MeOHe-R205.
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MeOHe-RIT (86%), OPe-RIT (83%), OHe-R121 (77%), and
OPB-R121 (68%).
Table 3. IC50’s of Different Inhibitors of P-gp Determined
after Culture with the Sensitive (P388) and Multidrug-
Resistant (P388/MDR) Cell Lines
The release profile obtained for the conjugate P-Ahx-NH-
NMeOHe-RIT shows that the rate of MeOHe-RIT release at
pH 7.4 is much lower than that at pH 5.0. After 24 h of
iincubation at pH 7.4, only 7% of MeOHe-RIT were released.
The rates of release of OPe-RIT, OHe-RIT, and OPB-RIT
from the conjugates P-Ahx-NH-NOPe-RIT, P-Ahx-NH-N
OHe-RIT, and P-Ahx-NH-NOPB-RIT were higher than
those for the release of MeOHe-RIT from the conjugate P-Ahx-
NH-NMeOHe-RIT at pH 7.4. After 24 h of incubation at
pH 7.4, 28% of OPe-RIT, 47% of OHe-RIT, and 25% of OPB-
R121 were released from their respective conjugates.
Hydrolysis of the ester bond and release of RIT from the
RIT esters were not detected after 24 h incubation at pH 7.4.
No release of RIT or MeOHe-RIT from the polymer
conjugates P-Ahx-RIT and P-Ahx-NH-NH-MeOHe-RIT was
observed after 24 h in a buffer at pH 5.0 at 37 °C.
a
P-gp inhibitor and derivatives P388 IC50 (μM)
P388/MDR IC50 (μM)
reversin 121
OPe-R121
OHe-R121
MeOHe-R121
OBP-R121
reversin 205
MeOHe-R205
ritonavir
12.1 1.45
>32
5.5 0.74
>32
11.9 1.65
10.6 1.75
23.0 0.50
3.9 0.25
13.2 0.60
7.2 0.72
11.1 0.76
9.1 0.82
11.5 1.50
21.5 1.00
15.3 2.66
14.1 2.03
22.7 0.20
34.3 0.80
24.9 1.02
14.2 0.56
12.4 1.40
43.0 1.00
OHe-RIT
MeOHe-RIT
OPB-RIT
a
IC50 (μM) values were calculated as the concentration of the tested
P-gp inhibitor or its derivative which inhibited the incorporation of
[3H]-thymidine into the exposed cells to 50% of the controls after 72 h
of culture (see Materials and Methods). The activity of control cells
was always greater than 50 000 cpm/well. The experiments were
repeated 2−6 times, and the average values SE are shown.
Prerequisites for effective intracellular delivery of derivatives
of MDR inhibitors are stability of their polymer conjugates
during transport in the blood circulation, as simulated here by
incubation in a phosphate buffer at pH 7.4, and rapid release at
the site of action as simulated by incubation in a phosphate
buffer at pH 5.0. The conjugates P-Ahx-NH-NMeOHe-R121
and P-Ahx-NH-NMeOHe-RIT fulfill these requirements
best, because they show the largest difference between their
good stability in buffer solution at pH 7.4 and rapid inhibitor
release at pH 5.0. For this reason, these P(HPMA) conjugates
were selected for further in vitro biological evaluation.
In Vitro Toxicity and Inhibitory Activity of Selected P-
gp Inhibitors and Their Derivatives. First, the in vitro
toxicities and activities of the original inhibitors, reversin 121,
reversin 205, and ritonavir, were evaluated and compared with
the activities of the derivatives of these agents suitable for
conjugation to the P(HPMA) carrier. The derivatives of the P-
gp inhibitors were intended to induce sensitization of the MDR
cancer cells to the cytostatic effect of Dox. However, it was
important that the concentration used did not affect the
proliferation of MDR cells per se. Thus, both P388 and P388/
MDR cells were incubated with titrated concentrations of the
tested compounds, and the IC50’s were determined by the
standard [3H]-thymidine incorporation assay. The results are
summarized in Table 3.
All reversin 121 derivatives except for OPe-R121, which
showed no toxicity in either cell line within the concentration
range used, exerted toxicity comparable to unmodified reversin
121 in the P388 cell line. However, MeOHe-R121, OHe-R121,
and OPB-R121 showed approximately 2.5−4 times lower
toxicity than reversin 121 in the P388/MDR cell line. Notably,
reversin 121 was more than 2-fold more toxic to the P388/
MDR cells than the P388 cells. Conversely, reversin 205 was
almost 6 times less toxic to the P388/MDR cells than to the
P388 cells. MeOHe-R205 was less toxic than parent compound
in both cell lines; however, the decrease in toxicity was much
more pronounced in the P388 cells (∼6 times versus 1.5
times). Ritonavir, the last P-gp inhibitor tested, was
approximately 3.5 times less toxic to the P388/MDR cells
than to the P388 cells. All ritonavir esters were shown to have
comparable or only slightly lower (up to 1.5 times) toxicity in
the P388 cell line. On the other hand, OHe-RIT and MeOHe-
RIT showed 2-fold higher toxicity and OPB-RIT showed 2-fold
lower toxicity than the parent compound in the P388/MDR
cell line.
The compounds described above were then tested for their
potential to inhibit the drug-efflux function of P-gp using the
calcein assay. Of the derivatives of reversin 121 tested, MeOHe-
R121 showed exceptionally high inhibitory activity (Figure 3A).
In fact, MeOHe-R121 was found to be even more potent as a
P-gp inhibitor than the unmodified reversin 121. Thus,
MeOHe-R121 was the first promising candidate compound
as a potent P-gp inhibitor, and it is possible to conjugate it to
the HPMA copolymer via hydrazone bonds. Moreover,
MeOHe-R121 is approximately 3 times less toxic than reversin
121 in the P388/MDR cells, and it shows very good P-gp
inhibitory activity at a concentration of 4 μM, which is far
below its IC50 in these cells. In contrast to the reversin 121
derivatives, MeOHe-R205 showed no P-gp inhibitory activity at
all (Figure 3B). MeOHe-RIT was identified to be a potent
inhibitor of P-gp function, while the other tested ritonavir esters
were significantly less potent (Figure 3C). As it was observed
for MeOHe-R121, MeOHe-RIT was also found to be even
more potent as a P-gp inhibitor than the unmodified ritonavir.
However, the toxicity of MeOHe-RIT is slightly higher and the
P-gp inhibitory activity is somewhat lower than that found for
MeOHe-R121. Thus, MeOHe-RIT was selected as another
candidate for further studies even though its expected activity is
slightly lower than that of MeOHe-R121. Interestingly, of the
nine derivatives of three different P-gp inhibitors, the two most
promising candidates were both obtained through modification
with 5-methyl-4-oxoheptanoic acid.
Sensitization of P388/MDR Cells to the Cytostatic
Activity of Dox by Selected P-gp Inhibitors and Their
Derivatives in Vitro. To evaluate the potential for the
selected P-gp inhibitors and their derivatives to overcome
multidrug resistance in the P-gp-overexpressing P388/MDR
cells as measured by the cytostatic effect of Dox, the cells were
incubated with titrated concentrations of the P-gp inhibitors or
their derivatives at a constant concentration of Dox (2 μM).
This concentration of Dox (∼3 times lower than IC50) has only
negligible cytostatic activity in the P388/MDR cells. MeOHe-
R121 showed the highest capacity to sensitize P388/MDR cells
to Dox among all of the derivatives of reversin 121 tested
(Figure 4A). The activity of MeOHe-R121 was comparable to
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Figure 4. Sensitization of P388/MDR cells to the cytostatic effect of
Dox by reversin 121 (A), reversin 205 (B), ritonavir (C), and their
derivatives. P388/MDR cells were incubated with titrated concen-
trations of P-gp inhibitors or their derivatives and a constant
concentration of Dox (2 μM) for 72 h under standard culture
conditions. The cell proliferation was assessed by [3H]-thymidine
incorporation by adding this tracer to the cultures for last 6 h of
incubation. The results are shown as the inhibition of the proliferation
of the exposed cells relative to the controls (cells incubated in medium
only). The activity of the control cells was always greater than 50 000
cpm/well. Each experimental point is the average of three experiments
SD.
Figure 3. Inhibition of P-gp function by reversin 121 (A), reversin 205
(B), and ritonavir (C) and their derivatives in P388/MDR cells. The
P388/MDR cells were incubated with titrated concentrations of the P-
gp inhibitors or their derivatives for 30 min under standard culture
conditions. Calcein-AM was then added and the cells were incubated
for another 20 min. The cells were analyzed by flow cytometry for
calcein fluorescence, and the dead cells were gated out using Hoechst
33258. P388/MDR cells incubated with 10 μM cyclosporine A (CsA)
and P388 cells incubated with calcein-AM only were used as positive
controls. The values shown are the averages of three to six experiments
SD.
the unmodified reversin 121 and it was evident at
concentrations of as low as 0.5 μM. Proliferation was
completely inhibited when the P388/MDR cells were exposed
to 1 μM MeOHe-R121. OPe-R121, OHe-R121, and OPB-R121
were much less potent, as demonstrated by the complete or
near complete inhibition of proliferation at concentrations as
high as 8 μM. Reversin 205 showed potency similar to that of
reversin 121 in sensitizing the P388/MDR cells to Dox.
However, its derivative, MeOHe-R205, was totally inactive
(Figure 4B). Ritonavir and its esters were generally much less
efficient in sensitizing the P388/MDR cells to Dox in
comparison to reversin 121 (Figure 4C). Nevertheless,
MeOHe-RIT showed reasonable activity, as demonstrated by
the observation that it caused nearly complete inhibition of the
proliferation at 5 μM. Of note, MeOHe-RIT was shown to be
significantly more potent (∼4 times) than the parent
compound ritonavir. The IC50’s for Dox in the presence of
titrated concentrations of reversin 121, reversin 205, ritonavir,
and their derivatives are summarized in Supporting Information
Tables 2−4. In conclusion, this study on the sensitization of the
P388/MDR cells to Dox treatment is in good agreement with
the results of the calcein assay experiments and identifies two
compounds, MeOHe-R121 and MeOHe-RIT, as promising
candidates for the subsequent study aimed at conjugation of
these inhibitors to the P(HPMA) carrier.
The effects of 1,7-substituted 3-n-propylxanthine derivatives
at 100 μM concentration on the influx and efflux of Dox in
P388 and P388/MDR leukemia cells were studied by Sadzuka
et al.45 The substituents in position 7, 3′-dimethylaminopropyl
and 3′-hydroxypropyl, significantly inhibited the Dox efflux
from P388 cells. In P388/MDR cells, most substituents in
positions 1 and 7 had no effect on Dox efflux except for the 3′-
dimethylaminopropyl derivative which facilitated influx of Dox
and inhibited its efflux in a dose-dependent manner.
Unfortunately, no experiments dealing with polymer conjugates
of these inhibitors were described.
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In Vitro Toxicity and P-gp Inhibitory Activity of the
P(HPMA) Conjugates P-Ahx-NH-NMeOHe-R121 and P-
Ahx-NH-NMeOHe-RIT. The polymer conjugates P-Ahx-
NH-NMeOHe-R121 and P-Ahx-NH-NMeOHe-RIT, con-
taining MeOHe-R121 or MeOHe-RIT, respectively, linked to
the carrier via a pH-sensitive hydrazone bond, were
synthesized. Their toxicity toward the P388/MDR cells was
tested in a manner similar to the method used for the selected
P-gp inhibitors and their derivatives. The P(HPMA) conjugate
P-Ahx-NH-NMeOHe-R121 showed no toxicity up to 24 μM
(the concentrations stated hereafter are those of the active
compound), and only modest toxicity was observed at 48 μM,
the highest concentration tested (Figure 5A). The P-Ahx-NH-
preincubated with the tested conjugates for 16 h, after which
the cells were used in the standard calcein assay. Both
conjugates, P-Ahx-NH-NMeOHe-R121 and P-Ahx-NH-
NMeOHe-RIT, showed very good P-gp inhibitory activity
at 12 μM, but lower concentrations were only partially effective
(Figure 6). None of the control P(HPMA) conjugates
described above (Figure 5) showed any activity.
Figure 6. Ability of the P(HPMA) conjugates P-Ahx-NH-N
MeOHe-R121, P-Ahx-NH-NH-MeOHe-R121, and P-Ahx-R121 (A),
and P-Ahx-NH-NMeOHe-RIT and P-Ahx-NH-NH-MeOHe-RIT
(B) to inhibit P-gp function in the P388/MDR cells. P388/MDR cells
were incubated with titrated concentrations of the P(HPMA)
conjugates P-Ahx-NH-NMeOHe-R121, P-Ahx-NH-NH-MeOHe-
R121, P-Ahx-R121, P-Ahx-NH-NMeOHe-RIT, and P-Ahx-NH-
NH-MeOHe-RIT for 16 h under standard culture conditions.
Calcein-AM was then added, and cells were incubated for another
30 min. The cells were analyzed by flow cytometry for calcein
fluorescence, and the dead cells were gated out using Hoechst 33258.
P-388/MDR cells incubated with 10 μM cyclosporine A (CsA) and
P388 cells incubated with calcein-AM only were used as positive
controls. The values shown are the averages of three experiments
SD.
Figure 5. Toxicity of P(HPMA) conjugates P-Ahx-NH-NMeOHe-
R121, P-Ahx-NH-NH-MeOHe-R121, and P-Ahx-R121 (A), and P-
Ahx-NH-NMeOHe-RIT and P-Ahx-NH-NH-MeOHe-RIT (B) as
indicated by inhibition of the proliferation of the P388/MDR cells.
The P388/MDR cells were incubated with titrated concentrations of
the P(HPMA) conjugates P-Ahx-NH-NMeOHe-R121, P-Ahx-NH-
NH-MeOHe-R121, P-Ahx-R121, P-Ahx-NH-NMeOHe-RIT, and P-
Ahx-NH-NH-MeOHe-RIT for 72 h under standard culture conditions.
Cell proliferation was assessed using [3H]-thymidine incorporation by
adding this tracer to the cultures for the last 6 h of incubation. The
results shown are the inhibition of the proliferation of the exposed
cells relative to the controls (cells incubated in medium only). The
activity of control cells was always greater than 50 000 cpm/well. Each
experimental point is the average of three experiments SD.
Sensitization of P388/MDR Cells to the Cytostatic
Activity of Dox by the P(HPMA) Conjugates P-Ahx-NH-
NMeOHe-R121 and P-Ahx-NH-NMeOHe-RIT in
vitro. To evaluate the potential for the polymer conjugates
P-Ahx-NH-NMeOHe-R121 and P-Ahx-NH-NMeOHe-
RIT to overcome multidrug resistance in the P-gp over-
expressing P388/MDR cells, as indicated by the cytostatic effect
of Dox, the cells were incubated with titrated concentrations of
Dox and with various concentrations of these conjugates. The
concentrations selected for conjugates P-Ahx-NH-N
MeOHe-R121 and P-Ahx-NH-NMeOHe-RIT were 24 and
12 μM, respectively, because higher concentrations were found
to be toxic.
NH-MeOHe-R121and P-Ahx-R121 conjugates containing
MeOHe-R121 or reversin 121, respectively, bound to the
P(HPMA) carrier via a nonbiodegradable bond were used as
controls and showed no toxicity. In contrast, the P-Ahx-NH-
NMeOHe-RIT polymer conjugate was significantly toxic at
48 and 24 μM. However, at concentrations up to 12 μM, this
conjugate showed no or negligible toxicity (Figure 5B). P-Ahx-
NH-NH-MeOHe-RIT and P-Ahx-RIT conjugates containing
MeOHe-RIT or RIT, respectively, bound to the P(HPMA)
carrier via nonbiodegradable bonds were used as controls and
showed no toxicity. Furthermore, the P-Ahx-NH-NMeOHe-
R121 and P-Ahx-NH-NMeOHe-RIT conjugates were tested
for their potential to inhibit the drug-efflux function of P-gp
using the calcein assay. The P388/MDR cells were
The P-Ahx-NH-NMeOHe-R121 conjugate showed a
gradually increasing capacity to sensitize the P388/MDR cells
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Figure 7. Sensitization of P388/MDR cells to the cytostatic effect of doxorubicin by the P(HPMA) conjugates P-Ahx-NH-NMeOHe-R121, P-
Ahx-NH-NH-MeOHe-R121, and P-Ahx-R121 (A), and P-Ahx-NH-NMeOHe-RIT and P-Ahx-NH-NH-MeOHe-RIT (B). P388/MDR cells were
incubated with titrated concentrations of the P(HPMA) conjugates P-Ahx-NH-NMeOHe-R121, P-Ahx-NH-NH-MeOHe-R121, P-Ahx-R121, P-
Ahx-NH-NMeOHe-RIT, or P-Ahx-NH-NH-MeOHe-RIT at the indicated concentration of Dox for 72 h under standard culture conditions. Cell
proliferation was assessed using [3H]-thymidine incorporation by adding this tracer to the cultures for the last 6 h of incubation. The results are
shown as the inhibition of the proliferation of the exposed cells relative to controls (cells incubated in medium only). The activity of the control cells
was always greater than 50 000 cpm/well. Each experimental point is the average of three experiments SD.
to Dox beginning at 1.5 μM, and approximately 50-fold
increase in the sensitization to Dox was achieved at 24 μM
(Figure 7A). The conjugates P-Ahx-NH-NH-MeOHe-R121
and P-Ahx-R121, used as controls, showed no activity. The P-
Ahx-NH-NMeOHe-RIT conjugate did not sensitize the cells
to Dox at concentrations up to 3 μM. This conjugate showed
moderate activity at 6 μM (∼10-fold increase in sensitization),
and a 50-fold increase in sensitization (comparable to conjugate
P-Ahx-NH-NMeOHe-R121 at 24 μM) was observed at 12
μM. However, the P-Ahx-NH-NMeOHe-RIT conjugate
showed some toxicity at 12 μM. Therefore, we cannot exclude
the possibility that the unexpectedly high sensitization activity
observed was due in part to its toxicity (Figure 7B). The P-Ahx-
NH-NH-MeOHe-RIT conjugate, used as a control, showed no
activity.
Cytostatic Activity of the P(HPMA) Conjugates P-Ahx-
NH-NMeOHe-R121(Dox) and P-Ahx-NH-NMeOHe-
RIT(Dox) in Vitro. Finally, the cytostatic activity of the
conjugate P-Ahx-NH-NMeOHe-R121(Dox) containing Dox
and the P-gp inhibitor MeOHe-R121, both bound via
hydrazone bonds to the P(HPMA) carrier, was determined
and compared to that of the mixture of the two conjugates, P-
Ahx-NH-NMeOHe-R121 and P-Ahx-NH-NDox. The P-
Ahx-NH-NMeOHe-R121(Dox) conjugate was far more
cytostatic than conjugate P-Ahx-NH-NDox (Figure 8A),
and the calculated IC50 was almost 30 times lower. This
conjugate was also slightly (less than twice) more cytostatic
than the comparable mixture of the conjugates P-Ahx-NH-N
MeOHe-R121 and P-Ahx-NH-NDox.
Polymer conjugate P-Ahx-NH-NMeOHe-RIT(Dox) ex-
erted almost 10 times higher cytostatic activity than P-Ahx-NH-
NDox; however, it had slightly lower cytostatic activity than
the mixture of P-Ahx-NH-NMeOHe-RIT and P-Ahx-NH-
NDox (Figure 8B).
Thus, we have demonstrated that simultaneous delivery of
cytostatic drug and P-gp inhibitor used either as a mixture of
single drug and single inhibitor-bearing P(HPMA) conjugates
or, better, as a combination of both molecules bound to the
same polymer carrier is a promising strategy to overcome
multidrug resistance in cancer cells.
CONCLUSIONS
■
The potential for novel polymeric therapeutics based on water-
soluble P(HPMA) conjugates bearing either the anticancer
drug doxorubicin, an inhibitor of ABC transporters or both to
increase the efficacy of Dox treatment of the Dox-resistant P-
gp-expressing P388/MDR leukemia cells was studied. Several
oxoacid analogues of the inhibitors of the ABC transporters
reversin 121, reversin 205, and ritonavir oxoacid esters were
synthesized, and their ability to inhibit P-gp mediated Dox
efflux in P388/MDR cells was evaluated. MeOHe-R121 and
MeOHe-RIT retained the inhibitory activity of the parent
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Figure 8. Overcoming multidrug resistance in P388/MDR cells in vitro by simultaneous delivery of the conjugates P-Ahx-NH-NDox and P-Ahx-
NH-NMeOHe-R121 in an equimolar drug/inhibitor ratio, or by the conjugate P-Ahx-NH-NMeOHe-R121(Dox) bearing both Dox and
MeOHe-R121 bound via hydrazone bonds (A) or by conjugates P-Ahx-NH-NDox and P-Ahx-NH-NMeOHe-RIT or P-Ahx-NH-NMeOHe-
RIT(Dox) containing Dox and P-gp inhibitor MeOHe-RIT (B). P388/MDR cells were incubated with titrated concentrations of P-Ahx-NH-N
Dox, with a mixture of P-Ahx-NH-NDox and P-Ahx-NH-NMeOHe-R121 or conjugate P-Ahx-NH-NMeOHe-R121(Dox) or with a mixture
of P-Ahx-NH-NDox and P-Ahx-NH-NMeOHe-RIT or conjugate P-Ahx-NH-NMeOHe-RIT(Dox) for 72 h at standard cultivation
conditions. The concentration of MeOHe-R121 or MeOHe-RIT was proportional to Dox concentration with the ratio 1:1 throughout the
experiment. Cell proliferation was assessed by [3H]-thymidine incorporation which was added to the cultures for the last 6 h of incubation. Results
are shown as inhibition of exposed cell proliferation relative to controls (cells incubated in medium only). The activity of control cells was always
higher than 50 000 cpm/well. Each experimental point is average of three experiments SD.
inhibitors, while MeOHe-R205 showed no P-gp inhibitory
activity.
The IC50 for the P-Ahx-NH-NMeOHe-R121(Dox)
conjugate, which contains both Dox and the P-gp inhibitor
MeOHe-R121 bound to the same carrier via hydrazone bonds,
was almost 30 times lower than that for the P-Ahx-NH-N
Dox conjugate. This IC50 was less than two times lower than
the IC50 for the comparable mixture of the two conjugates, P-
Ahx-NH-NMeOHe-R121 and P-Ahx-NH-NDox. Simi-
larly, P-Ahx-NH-NMeOHe-RIT(Dox) containing Dox and
MeOHe-RIT as a P-gp inhibitor both bound to the same carrier
was almost 10 times more cytostatic than P-Ahx-NH-NDox.
Contrary to the case of P-Ahx-NH-NMeOHe-R121(Dox),
the cytostatic activity of P-Ahx-NH-NMeOHe-RIT(Dox)
was slightly less cytostatic than the mixture of conjugates P-
Ahx-NH-NMeOHe-RIT and P-Ahx-NH-NDox.
The polymer conjugates P-Ahx-NH-NMeOHe-R121 and
P-Ahx-NH-NMeOHe-RIT, in which the MeOHe-R121 and
MeOHe-RIT, respectively, were bound to the water-soluble
polymer carrier via pH-senzitive hydrazone bonds, were
synthesized. P-Ahx-NH-NMeOHe-R121 showed no toxicity
up to 24 μM, and only modest toxicity was observed at 48 μM.
In contrast, the P-Ahx-NH-NMeOHe-RIT conjugate was
significantly toxic at concentrations of 24 and 48 μM, but little
or no toxicity was observed at concentrations below 12 μM.
Both conjugates, P-Ahx-NH-NMeOHe-R121 and P-Ahx-
NH-NMeOHe-RIT, showed very good P-gp inhibitory
activity. On the other hand, the conjugates P-Ahx-NH-NH-
MeOHe-R121, P-Ahx-R121, P-Ahx-NH-NH-MeOHe-RIT, and
P-Ahx-RIT, which contained MeOHe-R121, reversin 121,
MeOHe-RIT, and ritonavir, respectively, bound to the carrier
via nonbiodegradable bonds, showed no toxicity and no P-gp
inhibitory activity.
These results have confirmed that the use of water-soluble
P(HPMA) conjugates used to deliver a cytostatic drug and a P-
gp inhibitor simultaneously represents a promising strategy to
overcome multidrug resistance in cancer cells.
ASSOCIATED CONTENT
■
At 24 μM, the P-Ahx-NH-NMeOHe-R121 conjugate
increases the sensitivity of the P388/MDR cells to Dox by
approximately 50-fold. The P-Ahx-NH-NMeOHe-RIT con-
jugate showed moderate activity at 6 μM (∼10 times higher
sensitivity), and at 12 μM this conjugate increased the
sensitivity of the P388/MDR to Dox treatment by 50-fold.
S
* Supporting Information
1H NMR spectra of the synthesized compounds. Tables
containing the IC50 values determined in sensitive P388 and
multidrug-resistant P388/MDR cell lines for Dox and
P(HPMA) conjugate P-Ahx-NHNDox and IC50 values
determined in multidrug-resistant P388/MDR cell line for
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Biomacromolecules
Article
(21) Zhang, X.; Guo, S.; Fan, R.; Yu, M.; Li, F.; Zhu, C.; Gan, Y.
Biomaterials 2012, 33, 7103−7114.
doxorubicin in the presence of different P-gp inhibitors. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(22) Zhao, Y. Z.; Dai, D. D.; Lu, C. T.; Chen, L. J.; Lin, M.; Shen, X.
T.; Li, X. K.; Zhang, M.; Jiang, X.; Jin, R. R.; Li, X.; Lv, H. F.; Cai, L.;
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AUTHOR INFORMATION
(23) Prasad, P.; Cheng, J.; Shuhendler, A.; Rauth, A. M.; Wu, X. Y.
Drug Delivery Transl. Res. 2012, 2, 95−105.
(24) Shalviri, A.; Raval, G.; Prasad, P.; Chan, C.; Liu, Q.; Heerklotz,
H.; Rauth, A. M.; Wu, X. Y. Eur. J. Pharm. Biopharm. 2012, 82, 587−
597.
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Corresponding Author
*Tel.: +420-296809389. Fax: +420-296809410. E-mail: subr@
imc.cas.cz.
(25) Hu, C. M. J.; Zhang, L. Biochem. Pharmacol. 2012, 83, 1104−
Notes
1111.
The authors declare no competing financial interest.
(26) Minko, T.; Kopeck
59, 133−148.
(27) Minko, T.; Kopeck
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(28) Minko, T.; Kopeckova, P.; Kopece
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(29) Minko, T. Adv. Drug Delivery Rev. 2010, 62, 192−202.
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ACKNOWLEDGMENTS
̌
ova,
́ ̌
P.; Pozharov, V.; Kopecek, J. J.
■
The work was supported by the Grant Agency of the Czech
Republic (Grant No. P301/12/1254) and by the project
BIOCEV - Biotechnology and Biomedicine Centre of the
Academy of Sciences and Charles University (CZ.1.05/1.1.00/
02.0109) from the European Regional Development Fund. The
authors appreciate the excellent technical assistance of Helena
̌
́
̌
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D.; Etrych, T.; Ulbrich, K.; Ríhova,
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B. Eur.
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J. Cancer 2002, 38, 602−608.
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Misurcova and Pavlına Jungrova.
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