Prostate-Specific Membrane Antigen Targeted Therapy of Prostate Cancer Using a DUPA-Paclitaxel Conjugate

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00026

Prostate cancer (PCa) is the most prevalent cancer among men in the United States and remains the second-leading cause of cancer mortality in men. Paclitaxel (PTX) is the first line chemotherapy for PCa treatment, but its therapeutic efficacy is greatly restricted by the nonspecific distribution in vivo. Prostate-specific membrane antigen (PSMA) is overexpressed on the surface of most PCa cells, and its expression level increases with cancer aggressiveness, while being present at low levels in normal cells. The high expression level of PSMA in PCa cells offers an opportunity for target delivery of nonspecific cytotoxic drugs to PCa cells, thus improving therapeutic efficacy and reducing toxicity. PSMA has high affinity for DUPA, a glutamate urea ligand. Herein, a novel DUPA-PTX conjugate is developed using DUPA as the targeting ligand to deliver PTX specifically for treatment of PSMA expressing PCa. The targeting ligand DUPA enhances the transport capability and selectivity of PTX to tumor cells via PSMA mediated endocytosis. Besides, DUPA is conjugated with PTX via a disulfide bond, which facilitates the rapid and differential drug release in tumor cells. The DUPA-PTX conjugate exhibits potent cytotoxicity in PSMA expressing cell lines and induces a complete cessation of tumor growth with no obvious toxicity. Our findings give new insight into the PSMA-targeted delivery of chemotherapeutics and provide an opportunity for the development of novel active targeting drug delivery systems for PCa therapy.

Full text of DOI:10.1021/acs.molpharmaceut.8b00026

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Prostate-Speci#c Membrane Antigen Targeted Therapy
of Prostate Cancer Using a DUPA–Paclitaxel Conjugate
Qingzhi Lv, Jincheng Yang, Ruoshi Zhang, Zimeng Yang,
Zhengtao Yang, Yongjun Wang, Youjun Xu, and Zhonggui He
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00026 • Publication Date (Web): 02 Apr 2018
Downloaded from http://pubs.acs.org on April 3, 2018
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ProstateꢀSpecific Membrane Antigen Targeted
Therapy of Prostate Cancer Using a DUPA–
Paclitaxel Conjugate
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†‡
§
†
†
§
Qingzhi Lv, Jincheng Yang, Ruoshi Zhang, Zimeng Yang, Zhengtao Yang,
†
§
†
Yongjun Wang,* Youjun Xu,* Zhonggui He*
†
Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua
Road, Shenyang, 110016, China
‡
School of Pharmacy, Binzhou Medical University, 346 Guanhai Road, Yantai
264003, China
§
Key Laboratory of StructureꢀBased Drug Design & Discovery of Ministry of
Education, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang
110016, China
Key words: Paclitaxel; Prostateꢀspecific membrane antigen; DUPA; Prostate cancer;
Disulfide bond
ABSTRACT
Prostate cancer (PCa) is the most prevalent cancer among men in the United States
and remains the secondꢀleading cause of cancer mortality in men. Paclitaxel (PTX) is
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the first line chemotherapy for PCa treatment, but its therapeutic efficacy is greatly
restricted by the nonꢀspecific distribution in vivo. Prostateꢀspecific membrane antigen
(PSMA) is overexpressed on the surface of most PCa cells and its expression level
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increases with cancer aggressiveness, while being present at low levels in normal
cells. The high expression level of PSMA in PCa cells offers an opportunity for target
delivery of nonspecific cytotoxic drugs to PCa cells, thus improving therapeutic
efficacy and reducing toxicity. PSMA has high affinity for DUPA, a glutamate urea
ligand. Herein, a novel DUPA−PTX conjugate is developed using DUPA as the
targeting ligand to deliver PTX specifically for treatment of PSMA expressing PCa.
The targeting ligand DUPA enhances the transport capability and selectivity of PTX
to tumor cells via PSMA mediated endocytosis. Besides, DUPA is conjugated with
PTX via a disulfide bond, which facilitates the rapid and differential drug release in
tumor cells. The DUPA−PTX conjugate exhibits potent cytotoxicity in PSMA
expressing cell lines and induces a complete cessation of tumor growth with no
obvious toxicity. Our findings give a new insight into the PSMAꢀtargeted delivery of
chemotherapeutics, and provide an opportunity for the development of novel active
targeting drug delivery systems for PCa therapy.
INTRODUCTION
Prostate cancer (PCa) is the most prevalent cancer and remains the secondꢀleading
cause of cancer mortality and death affecting American males, in spite of recent
1
ꢀ3
advances in early diagnosis and surgical intervention. Current standard therapies
include radical prostatectomy, radiation, and adjuvant hormonal therapy. However,
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these therapies are effective only in curing localized, lowꢀgrade PCa, the majority of
4
ꢀ8
patients initially diagnosed with localized PCa ultimately relapse. Chemotherapy is
the most widely used approach to combat advanced PCa, but its therapeutic effect is
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often unsatisfactory due to lack of specificity and toxicity. Paclitaxel (PTX), a mitotic
inhibitor, is the first line chemotherapy for PCa treatment. However, the clinical
efficiency of PTX was greatly restricted by the significant side effects, which would
9
be attributed to the nonꢀspecific biodistribution in the body. Therefore, there is a
critical need for target delivery of PCa therapeutic agents.
Prostateꢀspecific membrane antigen (PSMA), also known as glutamate
10
carboxypeptidase II, is a 100 KDa type II transmembrane glycosylated protein.
PSMA is overexpressed on the surface of nearly all PCa cells and the neovasculature
11
of most solid malignant tumors. More importantly, its expression level increases
1
2
with cancer aggressiveness, recurrence and metastasis. Although PSMA is
expressed in some normal tissues, such as prostate, brain, small intestine and
13, 14
kidney,
its expression level is 100 to 1000 fold higher in PCa cells compared to
1
5, 16
normal cells.
The exceptionally high expression level of PSMA in PCa cells
makes it becomes an excellent active target for the delivery of PCa therapeutic and
imaging agents. Additionally, PSMA has internalization function via clathrinꢀcoated
pits and endocytosis, and this transport capability could be increased three fold upon
1
7, 18
ligand binding.
DUPA belongs to a class of glutamate ureas and has high binding
19ꢀ21
affinity with PSMA (K
i
=8 nM).
After binding to DUPA, PSMA undergoes
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internalization, releases DUPA into cytoplasm, and then returns to the cell
2
1, 22
membrane.
Based on the above considerations, we utilized DUPA as the targeting ligand to
deliver PTX actively for treatment of PSMA expressing PCa. We hypothesized that
conjugation of the targeting moiety DUPA will enhance the transport capability and
selectivity of PTX to tumor cells via PSMA mediated endocytosis. Therefore,
PTXꢀSSꢀDUPA was developed by conjugating DUPA with PTX, using a
reductionꢀsensitive disulfide bond as a linkage to facilitate a rapid release of PTX in
tumor cells (Scheme 1). An ester prodrug (PTXꢀDUPA) was synthesized as the
nonꢀsensitive conjugate. The tumorꢀtargeting drug delivery and tumorꢀspecific drug
release would lead to improved antitumor efficacy and reduced toxicity.
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Scheme 1. Redoxꢀsensitive drug release mechanism of PTXꢀSSꢀDUPA triggered by
GSH.
MATERIALS AND METHODS
Materials. PTX was obtained from Jingzhu Bioꢀtechnology Co. Ltd. (Nanjing,
China). diꢀtertꢀbutyl Lꢀglutamate hydrochloride, DMAP and HATU were purchased
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from Aladdin (Shanghai, China). F12K, 1640, DMEM media and fetal bovine serum
(FBS) were obtained from Gibco BRL (Gaithersburg, MD, USA). Thiazolyl blue
tetrazoliumbromide (MTT) and trypsinꢀEDTA were purchased from SigmaꢀAldrich
(St. Louis, MO, USA). Alexa Fluor® 488ꢀlabled antiꢀPSMA antibody were purchased
from Abcam (Cambridge, MA, USA). DAPI was purchased from Beyotime
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(Shanghai, China). All other chemicals and reagents applied in this work were
analytical or HPLC grade.
The PCꢀ3, LNCaP, HKꢀ2 and 22RV1 cell lines were obtained from the Cell Bank of
the Chinese Academy of Sciences (Shanghai, China). 22RV1 and LNCaP human PCa
cells were cultured in 1640 media, PCꢀ3 and HKꢀ2 cells were cultured in F12K and
ꢀ1
DMEM media respectively supplemented with 10% FBS, 100U mL penicillin, and
ꢀ1
1
00 ꢁg mL streptomycin. Cells were grown in a humidified 5% CO
2
atmosphere at
37 °C.
Synthesis of DUPA-conjugates.
-benzyl
-(tert-butyl)-(((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-glutama
5
1
te (1). To a solution of triphosgene (163 mg, 0.55 mmol) in DCM at ꢀ78 °C under
argon, diꢀtertꢀbutyl Lꢀglutamate hydrochloride (488 mg, 1.65 mmol) and
triethylamine (TEA, 0.5 mL) in DCM was added. After stirring for 2 h at ꢀ78 °C, a
solution of 5ꢀbenzyl 1ꢀ(tertꢀbutyl) Lꢀglutamate hydrochloride (600 mg, 1.82 mmol)
and TEA (0.3 mL) in DCM was added. The resulting mixture was stirred for 2 h at the
same temperature and allowed to come to room temperature (rt). The reaction mixture
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was stirred for additional 12 h and then quenched with 1 M HCl (30 mL), the organic
phase was washed with brine, dried over Na
2 4
SO , and concentrated. The residual oil
was purified by silica column chromatography to yield 1 (778.68 mg, 81.55%). MS
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+
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(ESI) m/z: 601.3 [M+Na] . H NMR (600MHz, DMSOꢀd ) δ 7.39 – 7.28 (m, 5H),
6
6.30 (m, J = 11.8, 8.3 Hz, 2H), 5.08 (s, 2H), 4.03 (qd, J = 8.7, 5.3 Hz, 2H), 2.46 –
2.31 (m, 2H), 2.28 – 2.14 (m, 2H), 1.99 – 1.81 (m, 2H), 1.70 (m, J = 51.2, 14.4, 8.7,
6.0 Hz, 2H), 1.41 – 1.27 (m, 27H).
(S)-5-(tert-butoxy)-4-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-5-o
t
xopentanoic acid (2, DUPA(O Bu)-OH). To a stirred solution of compound 1 (300
mg, 0.52 mmol) in DCM, 10% Pd/C was added. The reaction mixture was vigorously
stirred under a hydrogen atmosphere for 36 h at rt. The Pd/C was filtered off and wash
with DCM, the organic phase was combined and evaporated under vacuum. The
residue was purified using silica column chromatography to yield 2 (216.82 mg,
+
1
8
1
5.39%). MS (ESI) m/z: 511.3 [M+Na] . H NMR (600MHz, DMSOꢀd ) δ 12.13 (s,
6
H), 6.29 (dd, J = 8.4, 3.6 Hz, 2H), 4.02 (m, J = 11.9, 8.6, 5.3 Hz, 2H), 2.31 – 2.15
(m, 4H), 1.87 (m, J = 15.2, 8.8, 6.1 Hz, 2H), 1.68 (m, J = 19.6, 14.4, 8.7, 6.1 Hz, 2H),
1.41 – 1.37 (m, 27H).
di-tert-butyl
(((S)-5-((2-aminoethyl)amino)-1-(tert-butoxy)-1,5-dioxopentan-2-yl)carbamoyl)-L
-glutamate (3). To a stirred solution of ethylenediamine (82.14 ꢁL, 1.2 mmol) and
DIPEA (143.2ꢁL, 0.8mmol) in DCM under argon, a mixture of compound 2 (200 mg,
0.41 mmol) and HATU (187.07 mg, 0.48 mmol) in DCM was added. The resulting
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mixture was stirred overnight. After completion of the reaction (monitored by TLC),
the reaction mixture was concentrated and purified by silica column chromatography
+
1
to yield 3 (171.87 mg, 63.29%). MS (ESI) m/z: 531.5 [M+H] . H NMR (400MHz,
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DMSOꢀd ) δ 8.03 (t, J = 5.6 Hz, 1H), 6.37 (dd, J = 8.4, 6.2 Hz, 2H), 4.01 (m, J =
6
3
4.1, 8.4, 5.2 Hz, 2H), 3.23 – 3.10 (m, 2H), 2.71 (t, J = 6.3 Hz, 2H), 2.32 – 2.15 (m,
H), 2.13 (d, J = 7.9 Hz, 2H), 1.95 – 1.81 (m, 3H), 1.69 (m, J = 17.3, 14.7, 8.7, 6.5
2
Hz, 2H), 1.40 (d, J = 5.9 Hz, 27H), 1.33 – 1.22 (m, 1H).
-((2-(((1 ,2 )-1-benzamido-3-(((2a ,4 ,4a ,6 ,9
diacetoxy-12-(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4
a,5,6,9,10,11,12,12a,12b-dodecahydro-1 -7,11-methanocyclodeca[3,4]benzo[1,2-
2
S
R
R
S
S R S,11S,12S,12aR,12bS)-6,12b-
H
b]oxet-9-yl)oxy)-3-oxo-1-phenylpropan-2-yl)oxy)-2-oxoethyl)disulfanyl)acetic
acid (4). A solution of 2,2'ꢀdisulfanediyldiacetic acid (366 mg, 2 mmol) in acetic
anhydride (6 mL) was stirred at rt for 2 h under argon. Excessive acetic anhydride was
removed with toluene under high vacuum at rt. The residue was reacted with PTX
(1.71 g, 2 mmol) and DMAP (73.32 mg, 0.6 mmol) in DCM. The reaction mixture
was stirred for 12 h under argon, and then concentrated. The residue was purified by
－
silica gel chromatography to afford 4 (1.60 g, 78.66%). MS (ESI) m/z: 1016.5 [MꢀH]
1
.
H NMR (600MHz, Chloroformꢀd) δ 8.14 – 8.06 (m, 2H), 7.81 – 7.76 (m, 2H), 7.66
7.59 (m, 1H), 7.55 – 7.46 (m, 3H), 7.41 (dq, J = 20.7, 7.7, 7.3 Hz, 7H), 7.33 – 7.27
–
(m, 1H), 6.27 (s, 1H), 6.19 – 6.08 (m, 1H), 5.96 (td, J = 8.5, 8.0, 4.5 Hz, 1H), 5.68 –
.46 (m, 2H), 4.96 (dd, J = 9.5, 2.2 Hz, 1H), 4.39 (dd, J = 10.9, 6.7 Hz, 1H), 4.29 (dd,
5
J = 8.5, 4.6 Hz, 1H), 4.16 (dd, J = 15.8, 8.6 Hz, 1H), 3.75 (d, J = 7.0 Hz, 1H), 3.71 –
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.59 (m, 2H), 3.55 – 3.47 (m, 2H), 2.58 – 2.49 (m, 1H), 2.43 (s, 2H), 2.41 (d, J = 23.6
Hz, 3H), 2.24 – 2.17 (m, 3H), 1.94 – 1.89 (m, 1H), 1.89 – 1.83 (m, 3H), 1.79 (s, 1H),
.66 (s, 1H), 1.25 (s, 3H), 1.21 (s, 2H), 1.18 (s, 1H), 1.12 (d, J = 5.9 Hz, 3H).
1-((1 ,2 )-1-benzamido-3-(((2a ,4 ,4a ,6 ,9 ,11 ,12 ,12a ,12b )-6,12b-diace
toxy-12-(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,
,10,11,12,12a,12b-dodecahydro-1 -7,11-methanocyclodeca[3,4]benzo[1,2-b]oxet
9-yl)oxy)-3-oxo-1-phenylpropan-2-yl) 13,17,19-tri-tert-butyl
13 ,17 )-5,10,15-trioxo-2,3-dithia-6,9,14,16-tetraazanonadecane-1,13,17,19-tetra
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S
R
R
S
S
R
S
S
S
R
S
9
H
-
(
S
S
carboxylate (5). To a stirred mixture of compound 4 (509 mg, 0.5 mmol) and HATU
456mg, 1.2 mmol) in DCM under argon, compound 3 (270 mg, 0.5 mmol) and
DIPEA (350 ꢁL, 2 mmol) in DCM was added. The resulting mixture was stirred for
8 h. Upon completion (monitored by TLC), the reaction mixture was washed with
brine (50 mL), dried over Na SO , and concentrated. The crude oil was purified by
silica gel chromatography obtain 5 (469.97 mg, 61.45%). MS (ESI) m/z: 1553.1
(
4
2
4
+
1
[
M+Na] . H NMR (600MHz, Chloroformꢀd) δ 8.31 (d, J = 8.4 Hz, 1H), 8.14 – 8.07
(m, 2H), 7.85 (dt, J = 7.2, 1.3 Hz, 2H), 7.66 – 7.59 (m, 2H), 7.56 – 7.48 (m, 4H), 7.48
–
7.41 (m, 1H), 7.37 (dt, J = 19.2, 7.7 Hz, 4H), 7.23 – 7.17 (m, 1H), 6.89 (t, J = 5.6
Hz, 1H), 6.28 (s, 1H), 6.16 – 6.06 (m, 1H), 5.86 (dd, J = 8.4, 6.2 Hz, 1H), 5.62 (t, J =
.9 Hz, 3H), 5.47 (d, J = 6.2 Hz, 1H), 4.97 – 4.92 (m, 1H), 4.40 (dt, J = 11.8, 6.2 Hz,
1H), 4.27 (d, J = 8.5 Hz, 1H), 4.23 (td, J = 9.2, 8.3, 5.3 Hz, 2H), 4.16 (d, J = 8.5 Hz,
H), 3.75 (d, J = 7.0 Hz, 1H), 3.71 – 3.53 (m, 2H), 3.45 – 3.34 (m, 3H), 3.30 (d, J =
6
1
14.2 Hz, 1H), 3.24 – 3.17 (m, 1H), 3.08 (s, 1H), 2.98 (d, J = 5.1 Hz, 1H), 2.55 – 2.46
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(
m, 1H), 2.39 (s, 3H), 2.31 (ddd, J = 11.4, 8.5, 5.0 Hz, 3H), 2.25 – 2.14 (m, 7H), 2.12
– 2.04 (m, 3H), 1.94 (s, 1H), 1.91 – 1.85 (m, 5H), 1.81 (dq, J = 17.5, 8.6, 8.0 Hz, 1H),
1
.43 (d, J = 10.5 Hz, 27H), 1.38 (dd, J = 10.7, 5.3 Hz, 2H), 1.19 (s, 3H), 1.10 (s, 3H).
(3 ,4 ,19 ,23 )-4-((((2a ,4 ,4a ,6 ,9 ,11 ,12 ,12a ,12b )-6,12b-diacetoxy-1
-(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11
-7,11-methanocyclodeca[3,4]benzo[1,2-b]oxet-9-yl)o
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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0
1
2
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5
6
7
8
9
0
S
R
S
S
R
S
S
R
S
S
S
R
S
2
,
12,12a,12b-dodecahydro-1
H
xy)carbonyl)-1,6,11,16,21-pentaoxo-1,3-diphenyl-5-oxa-8,9-dithia-2,12,15,20,22-p
entaazapentacosane-19,23,25-tricarboxylic acid (6, PTX-SS-DUPA). To a stirred
solution of compound 5 (300 mg, 0.2 mmol) in DCM under argon, TFA was added
dropwise. The resulting reaction was stirred at rt for 1.5 h. The mixture was diluted in
acetonitrile (ACN, 30 mL) and the solvent was evaporated under vacuum. The residue
was purified by preparative reverse phase HPLC [solvent gradient: 20% A to 80% A
in 50 min; A ACN; B 0.1% TFA; λ=227 nm]. The pure fractions were lyophilized for
+
2
4 h to yield PTXꢀSSꢀDUPA (108.51 mg, 40.66%). MS (ESI) m/z: 1362.4 [M+H] .
1
4
H NMR (600MHz, Methanolꢀd ) δ 8.12 (d, J = 7.7 Hz, 1H), 8.01 (dd, J = 17.4, 7.8
Hz, 2H), 7.88 (dd, J = 9.3, 7.7 Hz, 2H), 7.78 (d, J = 7.7 Hz, 1H), 7.63 (m, 1H), 7.60 –
7
.38 (m, 10H), 7.37 – 7.26 (m, 2H), 6.51 (d, J = 14.6 Hz, 1H), 5.93 (d, J = 4.9 Hz,
H), 5.75 (d, J = 7.0 Hz, 1H), 5.60 (d, J = 5.0 Hz, 1H), 5.56 – 5.48 (m, 2H), 5.43 (dd,
1
J = 13.8, 7.2 Hz, 1H), 5.36 (d, J = 6.8 Hz, 1H), 5.32 – 5.27 (m, 1H), 4.73 (d, J = 5.8
Hz, 1H), 4.43 – 4.37 (m, 1H), 4.35 – 4.28 (m, 3H), 4.16 (d, J = 12.1 Hz, 1H), 3.70 (d,
J = 11.5 Hz, 1H), 3.67 – 3.62 (m, 1H), 3.41 (d, J = 3.5 Hz, 3H), 3.35 (s, 2H),
3.22ꢀ3.18 (m, 1H), 2.57 – 2.50 (m, 1H), 2.48 – 2.39 (m, 4H), 2.36 – 2.27 (m, 3H),
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6
2
.24 (s, 1H), 2.18 (s, 2H), 2.15 (d, J = 14.1 Hz, 1H), 2.13 ꢀ2.10 (m, 5H), 2.00 – 1.94
(m, 1H), 1.92 (m, 1H), 1.85 – 1.81 (m, 2H), 1.74 (s, 2H), 1.72 (d, J = 3.6 Hz, 3H),
1.33 (s, 2H), 1.29 (t, J = 7 Hz, 3H).
0
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9
0
HPLC purity: 98.03%. [solvent gradient: 20% A to 80% A in 25 min; A ACN; B 10
mM NH
-((1
toxy-12-(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,
,10,11,12,12a,12b-dodecahydro-1 -7,11-methanocyclodeca[3,4]benzo[1,2- ]oxet
4
OAc; λ=227 nm].
5
S
,2 )-1-benzamido-3-(((2a
R
R,4S,4aS,6R,9S,11S,12S,12aR,12bS)-6,12b-diace
9
H
b
-9-yl)oxy)-3-oxo-1-phenylpropan-2-yl)
1-(tert-butyl)
(((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-
L-glutamate (7). To a
stirred solution of compound 2 (244.2 mg, 0.5 mmol), DCC (206.2 mg, 1 mmol) and
DMAP (12.22 mg, 0.1 mmol) in DCM under argon, PTX (427.0 mg, 0.5 mmol) in
DCM was added dropwise. The resulting mixture was stirred at rt for 48 h. Upon
completion (monitored by TLC), the reaction mixture was filtered and concentrated.
The crude oil was purified by silica gel column chromatography to obtain 7 (469.97
+
1
mg, 61.45%). MS (ESI) m/z: 1325.3 [M+H] . H NMR (600MHz, Methanolꢀd
4
) δ
8
2
1
.15 – 8.10 (m, 2H), 7.86 – 7.81 (m, 2H), 7.68 (t, J = 7.4 Hz, 1H), 7.59 (t, J = 7.7 Hz,
H), 7.56 – 7.41 (m, 8H), 6.46 (s, 1H), 6.10 (t, J = 9.3 Hz, 1H), 5.87 (d, J = 5.9 Hz,
H), 5.65 (d, J = 7.1 Hz, 1H), 5.47 (d, J = 6.0 Hz, 1H), 4.59 (s, 4H), 4.40 – 4.32 (m,
2H), 4.21 – 4.15 (m, 3H), 4.10 (q, J = 7.1 Hz, 2H), 3.83 (d, J = 7.2 Hz, 1H), 2.61 –
.54 (m, 1H), 2.49 (dt, J = 15.6, 7.2 Hz, 2H), 2.43 (s, 3H), 2.29 (q, J = 8.2 Hz, 2H),
2.26 – 2.17 (m, 2H), 2.17 (s, 3H), 2.15 – 2.08 (m, 1H), 2.01 (s, 4H), 1.95 (d, J = 1.5
2
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Hz, 3H), 1.93 – 1.75 (m, 4H), 1.71 (d, J = 13.6 Hz, 1H), 1.66 (s, 3H), 1.60 (s, 2H),
1.45 (d, J = 2.1 Hz, 16H), 1.41 (s, 8H), 1.24ꢀ1.14 (t, J = 7.1 Hz, 4H).
(3S R,9S,13S)-4-((((2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-6,12b-diacetoxy-12-
,4
(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,1
,12a,12b-dodecahydro-1 -7,11-methanocyclodeca[3,4]benzo[1,2- ]oxet-9-yl)oxy
carbonyl)-1,6,11-trioxo-1,3-diphenyl-5-oxa-2,10,12-triazapentadecane-9,13,15-tr
icarboxylic acid (8, PTX-DUPA). PTXꢀDUPA was obtained as described for
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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6
0
1
2
3
4
5
6
7
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0
1
2
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0
1
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0
1
2
3
4
5
6
7
8
9
0
2
H
b
)
+
1
PTXꢀSSꢀDUPA starting from compound 7. MS (ESI) m/z: 1156.4 [M+H] . H NMR
(
600MHz, Methanolꢀd ) δ 7.99 – 7.94 (m, 2H), 7.90 – 7.86 (m, 2H), 7.61 – 7.54 (m,
4
1
H), 7.54 – 7.39 (m, 9H), 7.36 (d, J = 32.6 Hz, 1H), 7.29 (t, J = 7.4 Hz, 1H), 6.56 –
6.50 (m, 1H), 5.94 (d, J = 4.5 Hz, 1H), 5.59 – 5.50 (m, 2H), 5.36 (d, J = 6.7 Hz, 1H),
.78 (s, 1H), 4.67 (s, 1H), 4.16 (d, J = 12.1 Hz, 1H), 3.91 – 3.81 (m, 2H), 3.45 (d, J =
6.8 Hz, 1H), 2.64 – 2.53 (m, 2H), 2.53 – 2.45 (m, 2H), 2.42 – 2.32 (m, 2H), 2.25 –
4
2
.03 (m, 8H), 1.98 – 1.81 (m, 3H), 1.78 (s, 1H), 1.74 (d, J = 11.8 Hz, 6H), 1.62 (s,
H), 1.34 (s, 3H), 1.30 (d, J = 10.2 Hz, 1H).
3
HPLC purity: 98.92%. [solvent gradient: 20% A to 80% A in 25 min; A ACN; B 10
4
mM NH OAc; λ=227 nm].
In vitro release of PTX from DUPA-drug Conjugates.
The release of PTX from PTXꢀSSꢀDUPA and PTXꢀDUPA were studied in PBS (pH
7.4) containing 20% ethanol with 0 ꢁM, 20 ꢁM, 1 mM or 10 mM dithiothreitol
(DTT), respectively. Briefly, DUPA conjugates (6 mg PTX equivalent) were
incubated in 30 mL of release medium at 37 °C. At timed intervals, 0.2 mL of sample
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was withdrawn, and the content of PTX, PTXꢀSSꢀDUPA and PTXꢀDUPA was
determined by HPLC (Waters e2695 Separations Module and Waters 2489 UVꢀvis
Detector on a reverse ODS Shiseido ꢀC18 column (250 mm × 4.6 mm, 5 ꢁm) with
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solvent gradient: 20% A to 80% A in 12 min; A ACN; B 10 mM NH OAc) at a flow
4
ꢀ1
rate of 1.0 mL min . The UV detector was kept at 227 nm. Each experiment was
repeated in triplicate, and the results were expressed by mean ± SD.
To investigate the release mechanism of PTXꢀSSꢀDUPA, a solution of
ꢀ1
PTXꢀSSꢀDUPA (200 ꢁg mL ) in PBS (pH 7.4) containing 20% ethanol was
incubated with 10 mM DTT. The efficiency of free PTX release from PTXꢀSSꢀDUPA
was monitored by HPLC and Bruker microTOFꢀQ timeꢀofꢀflight mass spectrometer.
In vitro cytotoxicity of DUPA-drug Conjugates.
The in vitro cytotoxicity of PTX, PTXꢀSSꢀDUPA and PTXꢀDUPA was evaluated in
PSMAꢀpositive cell lines (22RV1 and LNCaP), a PSMAꢀnegative cell line (PCꢀ3) and
a
3
2
2
human
proximal
tubular
cell
line
(HKꢀ2)
by
the
ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyl tetrazolium bromide (MTT) assay. Briefly,
2RV1 cells were seeded in 96ꢀwell plates at a density of 8000 cells per well. After
4 h incubation, cells were exposed to a series of PTX or its DUPA conjugates
concentrations ranging from 2 to 500 nM at 37 °C for 48 h with or without 100ꢀfold
excess PMPA (a competitive inhibitor of PSMA). PTX, PTXꢀDUPA and
PTXꢀSSꢀDUPA were dissolved in DMSO. The concentration of DMSO was no more
ꢀ1
than 0.05%. To assess cell viability, 20 ꢁL of MTT (5 mg mL ) solution was added
and incubated for 4 h at 37 °C. Then, the media was replaced with 200 ꢁL of DMSO
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to dissolve the formazan crystals formed by living cells. The absorbance was
measured at 570 nm using a microplate reader (Varioskan Flash, Thermo Scientific,
Waltham, MA, USA).
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PSMA receptor expression and cellular uptake of PTX in PCa cells.
PSMA expression levels were determined by combined immunocytostaining and
flow cytometry. For immunocytostaining analysis, cells were planted on sterile cover
slips placed in a 24ꢀwell plate at a density of 50,000 cells per well, and cells were
allowed to attach for 24 h. The cells were then washed with PBS thrice, followed by
fixation with 4% paraformaldehyde for 20 min. Fixed cells were stained using Alexa
Fluor 488 labeled monoclonal antiꢀPSMA antibody (1:100 dilution; Abcam,
Cambridge, MA, USA) overnight at 4 °C. Cells were counterꢀstained with DAPI for
nuclear recognition prior to imaging on the confocal laser scanning microscopy
(C2SI, Nikon Corp., Tokyo, Japan). For flow cytometry analysis, 100,000 cells were
ꢀ1
incubated with Alexa Fluor 488 labeled monoclonal antiꢀPSMA antibody (6 ꢁg mL )
for 15 min at 25 °C. Following the incubation, the cells were washed three times with
PBS, gently dissociated from the wells with trypsin, and reꢀsuspended in 0.4 mL PBS.
The fluorescence intensity was analyzed on a flow cytometer (Becton Dickinson
Immunocytochemistry Systems, San Jose, CA, USA).
For cellular uptake studies, 22RV1 cells were grown on 12ꢀwell plate at a density of
150,000 cells per well and cultured for 24 h. Cells were exposed to PTX or its DUPA
conjugates solution (1 ꢁM, dissolved in 0.05% DMSO) at 37 °C for 1, 4 and 8 h with
or without 100ꢀfold excess PMPA. The samples were collected and subjected to
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ultrasonication. PTX concentration was determined by the UPLCꢀMS/MS method. A
BCA protein assay kit was used to determine the protein content in cell lysates, and
the cellular uptake of PTX was corrected to per gram protein. Each experiment was
repeated three times.
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Animals and tumor xenograft model.
Athymic nude BALB/c male mice (23ꢀ26 g) were purchased from Vital River
Laboratory Animal Technology Co. Ltd. (Beijing, China). All animal studies were
carried out under Institutional Animal Care and Use Committeeꢀapproved protocols of
Shenyang Pharmaceutical University. The tumor xenograft models were established
6
by injecting 5×10 PSMAꢀexpressing 22RV1 cells in 50% Matrigel (BD Biosciences,
SanJose, CA) into the armpit region of mice.
Tissue distribution study
To determine the tissue distribution of PTX and PTXꢀSSꢀDUPA, we used mouse
3
xenografts bearing 22RV1 tumors. Mice with subcutaneous tumors of ~100ꢀ150 mm
were administered through tail vein injection of PTX, PTXꢀSSꢀDUPA or PTXꢀDUPA
ꢀ1
solution at an equivalent PTX dose of 10 mg kg . At 24 h postꢀtreatment, tumor,
heart, liver, spleen, lung and kidney were harvested. UPLCꢀMS/MS was used to
measure the concentration of PTX in tissue samples.
In vivo anticancer efficacy of DUPA-drug Conjugates
Mice bearing 22RV1 tumors were utilized to investigate the in vivo antitumor
activity of PTX and its DUPA conjugates. When the average tumor volume reached
3
~100 mm , the mice were randomly assigned to 4 groups (n=5 per group): (1) saline
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(
the control group), (2) free PTX solution, (3) PTXꢀSSꢀDUPA solution, (4)
PTXꢀDUPA solution. These preparations were administrated through tail veins every
ꢀ1
other day for five times at an equivalent PTX dose of 10 mg kg . Tumor volume and
1
1
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1
1
1
1
1
1
1
2
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body weight were recorded. Mice were sacrificed 24 h after the final injection, and
blood was collected from the retroꢀorbital sinus. The serum levels of blood urea
nitrogen (BUN), creatinine, aspartate transaminase (AST), alanine transaminase
(ALT) and uric acid were determined to assess hepatic and renal function. The serum
prostate specific antigen (PSA) levels were determined by an ELISA kit (Cusabio
Biotech, Wuhan, China) in accordance with the manufacturer’s protocols.
The tumors and major organs (heart, liver, spleen, lung, kidney) were harvested and
sectioned for further pathological assessment. Tumor cell apoptosis was investigated
by the terminal deoxynucleotidyl transferaseꢀmediated dUTP nick endꢀlabeling
(TUNEL) assay using an apoptosis detection kit (KeyGen Biotech, Nanjing, China)
according to the manufacturer’s instructions. The nuclei of tumor slices were stained
by DAPI. All stained samples were observed with a confocal laser scanning
microscopy (C2SI, Nikon Corp., Tokyo, Japan). The formalinꢀembedded tissues were
stained with hematoxylin and eosin (H&E) to assay the systematic toxicity.
Statistical analysis.
Data are expressed as mean ± SD. A oneꢀway ANOVA was used to determine the
significance in the experiments. Statistical differences were significant at P < 0.05 and
highly significant at P < 0.01.
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RESULTS AND DISCUSSIONS
Design and synthesis of DUPA-conjugates.
t
The synthesis of tertꢀbutyl protected DUPA (2, DUPA(O Bu)ꢀOH) was shown in
Scheme 2. Commercially available γꢀbenzoylated glutamic acid was treated with
triphosgene in DCM containing TEA at 78 °C for 2 h followed by addition of
tertꢀbutylꢀprotected glutamic acid to obtain 1. Deprotection of the benzyl group of 1
0
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obtained DUPA(O Bu)ꢀOH (2). The structure of compound 1 and 2 was confirmed by
1
H NMR (Supporting Information, Figure S1, S2).
Scheme 2
The synthesis of the desired PTXꢀSSꢀDUPA conjugate 6 was shown in Scheme 3.
The carboxylic acid 2 was coupled with ethylenediamine to obtain 3 in the presence
of HATU and DIPEA in DCM. Dithiodiglycolic acid was converted to the
corresponding anhydride using acetic anhydride as dehydration agent. The residue
was reacted with PTX, using DMAP as catalyst, to obtain 4. The amine 3 was coupled
with carboxylic acid 4 in the presence of HATU and DIPEA in DCM to produce 5.
1
The H NMR spectrums of compound 3ꢀ5 were illustrated in Figure S3ꢀ5, the mass
spectrum of 5 was illustrated in Figure S6. Deprotection of the tertꢀbutyl group of 5
obtained the target compound PTXꢀSSꢀDUPA (6). The final product was purified
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using preparative reverse phase HPLC and characterized by H NMR and HRMS
(Figure S7, 8).
Scheme 4 shows the synthesis of ester prodrug PTXꢀDUPA (8). The carboxylic acid
2 was coupled with PTX in the presence of DCC and DMAP to produce 7 which was
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confirmed by mass spectrometry and H NMR (Figure S9, S10). Deprotection of the
tertꢀbutyl group of 7 obtained the ester prodrug PTXꢀDUPA (8). The final product
1
was purified using preparative reverse phase HPLC and characterized by H NMR and
HRMS (Figure S11, 12). The octanolꢀwater distribution coefficients (log P) of
DUPAꢀSSꢀPTX and PTXꢀDUPA was 1.199 and 2.674, respectively. The
hydrophilicity of DUPAꢀSSꢀPTX and PTXꢀDUPA was slightly increased due to the
introduction of DUPA moiety.
Scheme 3
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Scheme 4
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In vitro release of PTX from DUPA-drug Conjugates.
The in vitro drug release profiles of PTXꢀDUPA and PTXꢀSSꢀDUPA were
2
3, 24
investigated using DTT (a prevailing GSH simulant) containing release media
.
As shown in Figure 1 A, less than 20% of PTX was released from PTXꢀSSꢀDUPA
within 24 h in blank PBS (pH 7.4) and PBS containing 20 ꢁM DTT, and about 80%
of PTX was released within 4 h in the presence of 1 mM and 10 mM DTT. By
contrast, there was negligible PTX (<2.1%) released from PTXꢀDUPA after 24 h
incubation in the presence or absence of 10 mM DTT (Figure 1 B). These results
suggested that the drug release from ester prodrug (PTXꢀDUPA) was extremely slow,
and PTXꢀSSꢀDUPA had a remarkable redoxꢀresponsive drug release. In addition, the
results indicated that PTXꢀSSꢀDUPA conjugate would remain stable in circulation but
25
release PTX following disulfide reduction in tumor cell. The rapid and differential
drug release within tumor cells would lead to enhanced antitumor efficacy and
2
4
reduced toxicity.
To investigate the mechanism of DTTꢀtriggered drug release, the change of
molecule weight was analyzed by HPLC and HRMS. Figure 1 C indicated that all
PTXꢀSSꢀDUPA prodrugs were reduced within 2 h after addition of DTT. The
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breakage of the disulfide bond generated a thiolꢀcontaining intermediate (PTXꢀSH,
confirmed by HRMS) which would eventually release free PTX (Scheme 1).
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Figure 1. In vitro release of PTX from prodrugs. (A) Release profiles of PTX from
PTXꢀSSꢀDUPA with 0 ꢁM, 20 ꢁM, 1 mM or 10 mM DTT in PBS (pH 7.4) (n = 3). (B)
Release profiles of PTX from PTXꢀDUPA and the content of PTXꢀDUPA in release
medium. (C) HPLC spectra of PTXꢀSSꢀDUPA with 10 mM DTT at 0 and 2 h. HRMS
spectra of PTXꢀSSꢀDUPA, PTXꢀSH, and PTX.
In vitro cytotoxicity of DUPA-drug Conjugates.
The in vitro cytotoxicity of PTXꢀDUPA and PTXꢀSSꢀDUPA was evaluated in
PSMAꢀpositive cell lines (22RV1 and LNCaP), a PSMAꢀnegative cell line (PCꢀ3) and
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a human proximal tubular cell line (HKꢀ2) using MTT assay. As shown in Figure 2 A
and S13 A, cell viability decreased with increasing concentrations of PTX and
PTXꢀSSꢀDUPA in PSMA expressing 22RV1 and LNCaP cells. PTXꢀSSꢀDUPA
(IC = 121.1 nM) was less potent than PTX (IC =14.25 nM) in 22RV1 cells, possibly
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because of the delayed release of PTX (Figure 2 C). The high efficacy of
PTXꢀSSꢀDUPA was quantitatively inhibited by excess PMPA, which suggested that
21
cytotoxicity required PSMA receptors. By contrast, PTXꢀDUPA (IC50=563.3 nM)
exhibited poor cytotoxicity within the studied range of drug concentrations, due to the
extremely slow hydrolysis rate of PTX. In addition, PMPA alone cause no
cytotoxicity even at the concentration of 30 ꢁM (cell viability=96.69%), suggesting
PMPA did not affect the cell function. As shown in Figure 2 B and S13 B,
PTXꢀSSꢀDUPA exhibited inferior cytotoxicity in PSMA negative PCꢀ3 and HKꢀ2
cells, showing no significant difference compared with PTXꢀDUPA at the
concentration of 500 nM. The results further suggested cell killing required PSMA
receptors and rapid release of active PTX moiety within tumor cells.
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Figure 2. In vitro cytotoxicity of PTX and its DUPA conjugates in 22RV1 and PCꢀ3
cells. (A) Cytotoxicity of PTX, PTXꢀSSꢀDUPA, PTXꢀDUPA, PMPA (100ꢀfold
excess), and cytotoxicity of DUPAꢀdrug conjugates in the presence of 100ꢀfold excess
PMPA in 22RV1 cells. (B) Cytotoxicity of PTX, PTXꢀSSꢀDUPA and PTXꢀDUPA in
PCꢀ3 cells. (C) IC50 of PTX, PTXꢀSSꢀDUPA and PTXꢀDUPA in PCa cells. The data
are presented as means ± SD, P values: *P < 0.05, **P < 0.01.
PSMA expression and cellular uptake of PTX in PCa cells.
PSMA receptor expression of human PCa cell lines 22RV1 and PCꢀ3 was
determined by combined immunocytostaining and flow cytometry. As shown in
Figure 3 A, PSMA receptor was robustly expressed by 22RV1 cells and complete
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absence of staining observed in PCꢀ3 cells (green, PSMA receptor; blue, nucleus). For
flow cytometry, human PCa cell lines 22RV1 and PCꢀ3 were stained with Alexa Fluor
488 labeled monoclonal antiꢀPSMA antibody. As shown in Figure 3 B, expression of
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a high level of PSMA was observed in the 22RV1 cells, whereas PCꢀ3 cells lacked
PSMA expression (Cyan, unstained cells; red, cells with antibody).
To confirm the contribution of PSMA to the transport of the targeted DUPAꢀdrug
conjugates, PSMAꢀpositive 22RV1 cells were used to evaluate the cellular uptake
efficiency. PTX concentration in PCa cells was determined by UPLCꢀMS/MS. As
shown in Figure 3 C, the intracellular concentration of PTX increased with time after
incubation with free PTX and its DUPA conjugates in 22RV1 cells. The PTX
concentration in cells derived from PTXꢀSSꢀDUPA and free PTX solution was
markedly higher than PTXꢀDUPA. Besides, the cellular uptake of PTXꢀSSꢀDUPA
was quantitatively inhibited by excess PMPA, indicating PMPA effectively prevented
PTXꢀSSꢀDUPA from binding to PSMA, thereby blocking the cellular uptake of
PTXꢀSSꢀDUPA. In the same time, the higher intracellular concentration of PTX
derived from PTXꢀSSꢀDUPA suggested it could release active PTX molecules rapidly
following internalization into the tumor cells.
By contrast, the PTX concentration in 22RV1 cells of free PTX solution did not
change in the absence of excess PMPA, indicating that the PTX cellular uptake occur
through passive diffusion into the cell but not PSMA mediated internalization. The
PTX concentration in 22RV1 cells of PTXꢀDUPA was poor because of slow drug
release from ester prodrug, which may lead to poor antitumor efficiency.
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Figure 3. Expression of PSMA and cellular uptake of PTX in 22RV1 and PCꢀ3 cells.
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(
A) Expression of PSMA demonstrated by confocal fluorescent microscopy in 22RV1
and PCꢀ3 cells (green, PSMA receptor; blue, nucleus). (B) Quantitative expression of
PSMA in 22RV1 cells investigated by flow cytometry after incubation of cells with
antiꢀPSMAꢀAlexa Fluor 488 conjugate (Cyan, unstained cells, control; red, cells with
antibody, PSMA). (C) Cellular uptake of PTX in 22RV1 cells (n=3). The data are
presented as means ± SD, P values: **P < 0.01.
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Tissue distribution study
In view of the favorable cellular uptake of PTXꢀSSꢀDUPA in PCa cells, we further
investigated the tissue distribution of PTXꢀSSꢀDUPA and PTXꢀDUPA in mice
xenograft models bearing 22RV1 tumors in comparison with PTX solution. The
content of PTX in major organs was determined using UPLCꢀMS/MS, and illustrated
in Figure 4 A and B. At 24 h after injection, the accumulation of PTX in tumor of
PTXꢀSSꢀDUPA group was less than that of PTX solution. However, PTX solution did
ꢀ1
not show tumor targeting with high drug contents in lung (6.35 ꢁg g ), which will
lead to toxicity in normal organs. The content of PTX in tumors of PTXꢀSSꢀDUPA
group was higher than that in other organs. The potential value of cancer targeting by
PTXꢀSSꢀDUPA would be derived from its lack of toxicity in other organs, which
might make it a less toxic anticancer drug than PTX. There was negligible PTX (0.01
ꢀ1
ꢁg g ) detected in tumors of PTXꢀDUPA group due to extremely slow release rate of
PTX. The PSMAꢀtargeting moiety DUPA significantly changed the biodistribution
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and leaded to much higher accumulation of drug in tumor compared with other major
organs due to the selective uptake by PCa cells.
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Figure 4. The content of PTX in major organs and tumor of PTX and its DUPA
conjugate. (A) The content of PTX in major organs and tumor of PTX,
PTXꢀSSꢀDUPA and PTXꢀDUPA group on 22RV1 tumor xenografts. (B) PTX content
in major tissues of PTX, PTXꢀSSꢀDUPA and PTXꢀDUPA group determined by
UPLCꢀMS/MS.
In vivo anticancer efficacy of DUPA-drug Conjugates.
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Based on the favorable antiꢀtumor effect of PTXꢀSSꢀDUPA in vitro, we further
investigated the in vivo antiꢀtumor activities of PTXꢀSSꢀDUPA in mice xenograft
models bearing 22RV1 tumors. PTX, PTXꢀSSꢀDUPA or PTXꢀDUPA were injected
intravenously into mice every 2 days, with a total of 5 injections. As shown in Figure
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A and B, PTXꢀDUPA exhibited a moderate antiꢀtumor effect as compared to the
PTX group and PTXꢀSSꢀDUPA group, which may due to its slow release rate of
active PTX molecular and low intracellular PTX concentration. In contrast, the mice
treated with PTXꢀSSꢀDUPA displayed a significant decrease in tumor volume,
showing no significant difference compared with PTX. (Figure 5 B, D). PSA levels in
mice were determined on day 10, and PTXꢀSSꢀDUPA treated mice had a significant
reduction in PSA levels (Figure 5 C). The potent antitumor efficacy of
PTXꢀSSꢀDUPA could be attributed to the efficient cellular uptake mediated by PSMA
and onꢀdemand release of active PTX molecules in tumor cells, which will result in
potent chemotherapeutic efficacy with decreased offꢀtarget toxicities compared with
2
1
PTX.
The TUNEL assay was used to evaluate if apoptosis is induced in tumor cells by
PTX, PTXꢀSSꢀDUPA and PTXꢀDUPA treatment (Figure 5 E). A robust and
significant increase in cell apoptosis was observed in tumor samples treated with PTX
and PTXꢀSSꢀDUPA, as compared to both saline and PTXꢀDUPA group. Additionally,
there was no significant change observed in body weight or in the hematological
parameters between treated and control groups (Figure 6 A, B). Furthermore, no
noticeable histological abnormalities were observed in H&E staining of major organ
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sections, indicating that the DUPA conjugates treatments were well tolerated. This
confirms that the PTXꢀSSꢀDUPA conjugate has potent antitumor activity and no
treatmentꢀinduced toxicity (Figure 7). The superior safety of PTXꢀSSꢀDUPA could be
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attributed to that the targeting moiety DUPA enhances the transport capability and
8, 20, 26, 27
selectivity of PTX to tumor cells via PSMA mediated endocytosis.
Besides,
DUPA were conjugated with PTX via a disulfide bond, which facilitates the rapid and
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differential drug release in tumor cells.
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Figure 5. In vivo antitumor efficiency of PTX, PTXꢀSSꢀDUPA and PTXꢀDUPA on
22RV1 tumor xenografts. (A) The tumor volume after intravenous administration of
saline, PTX, PTXꢀSSꢀDUPA or PTXꢀDUPA (n=5). (B) The morphology of dissected
tumors from 22RV1 tumorꢀbearing mice of different groups at the end of tests (n=5).
29
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