Multifunctional Tumor-Targeting Cathepsin B-Sensitive Gemcitabine Prodrug Covalently Targets Albumin in Situ and Improves Cancer Therapy

Article abstract of DOI:10.1021/acs.bioconjchem.8b00223

We report a new type of amide bond-bearing cathepsin B-sensitive gemcitabine (GEM) prodrugs, capable of in situ covalently targeting circulating albumin and then making a hitchhike to the tumor. Specially, less plasma-enzyme deactivation, long plasma half-life, independence on nucleoside transporters, outstanding tumor targeting, and site-specific drug release are achieved, and as such these multifunctional advantages contribute to the dramatically increased in vivo antitumor efficacy.

Full text of DOI:10.1021/acs.bioconjchem.8b00223

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Communication
Multifunctional tumor-targeting cathepsin B-sensitive gemcitabine
prodrug covalently targets albumin in situ and improves cancer therapy
Huicong Zhang, Zhisu Sun, Kuanglei Wang, Na Li, Hongxiang
Chen, Xiao Tan, Lingxiao Li, Zhonggui He, and Jin Sun
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00223 • Publication Date (Web): 18 May 2018
Downloaded from http://pubs.acs.org on May 19, 2018
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GAMOriginal Research Communication
Title:
Multifunctional
Tumor-Targeting
Cathepsin
B-Sensitive
Gemcitabine Prodrug Covalently Targets Albumin In Situ and
Improves Cancer Therapy
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a
a
b,c
d
Authors: Huicong Zhang , Zhisu Sun , Kuanglei Wang , Na Li , Hongxiang
e
f
g,h
a,*
a,*
Chen , Xiao Tan , Lingxiao Li , Zhonggui He , Jin Sun
Affiliations:
a
Department of Pharmaceutics, Wuya College of Innovation, Shenyang Pharmaceutical
University, Shenyang, Liaoning, 110016, P. R. China
b
Wuyi University, Jiangmen, Guangdong, 529020, P. R. China
c
International Healthcare Innovation Institute (Jiangmen), Jiangmen, Guangdong, 529080, P. R.
China
d
Clinical Pharmacy, Wuya College of Innovation, Shenyang Pharmaceutical University,
Shenyang, Liaoning, 110016, P. R. China
e
Center for Drug Evaluation, China Food and Drug Administration, Beijing, 100022, P. R.
China
f
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R.
China
g
School of Pharmacy, Queen’s University of Belfast, 97 Lisburn Road, Belfast BT9 7BL,
Northern Ireland, UK
h
School of Pharmacy, China Medical University, Shenyang, Liaoning, 110013, P. R. China
*Corresponding author:
Prof. Zhonggui He, Ph.D and Prof. Jin Sun, Ph.D
No. 59 Mailbox, Department of Pharmaceutics, Wuya College of Innovation, Shenyang
Pharmaceutical University, NO. 103 Wenhua Road, Shenyang 110016, China
Tel: +86ꢀ024ꢀ23986321; Fax: +86ꢀ024ꢀ23986321
Eꢀmail address: hezhonggui@vip.163.com; sunjin@syphu.edu.cn
ABSTRACT
We report a new type of amide bondꢀbearing cathepsin Bꢀsensitive gemcitabine (GEM)
prodrugs, capable of in situ covalently targeting circulating albumin and then making a hitchhike
to the tumor. Specially, less plasmaꢀenzyme deactivation, long plasma halfꢀlife, independence on
nucleoside transporters, outstanding tumor targeting and siteꢀspecific drug release are achieved
and such these multifunctional advantages contribute to the dramatically increased in vivo
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antitumor efficacy.
Key words: Gemcitabine prodrug, Circulating albumin, Hitchhike, Tumor targeting, Cathepsin
Bꢀsensitive.
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Many factors limit the clinical use of GEM: (a) very short plasma halfꢀlife resulting from rapid
deamination into inactive 2′ꢀdeoxyꢀ2′, 2′ꢀdifluorouridine (dFdU) by ubiquitous cytidine deaminase
1
ꢀ2
(
CDA) in the bloodstream ; (b) acquired drug resistance (ADR) caused by reduced expression of
nucleoside transporters, especially human equilibrative nucleoside transporter (hENT) required for
3ꢀ4
GEM uptake by cancer cells ; (c) lack of tumorꢀspecific delivery. Up until now, there have been
two major solutions to address the above problems, nanoparticulate delivery systems and
5
ꢀ6
lipophilic gemcitabine prodrugs , but the two strategies have only succeeded in one or two
aspects aforementioned. Therefore a novel method to win in all aspects and to show great potential
5
, 7
towards clinical translation is still in an urgent need . In vivo macromolecular prodrug strategy
8ꢀ9
may serve as a promising alternative . The strategy applied here employs rapid covalently
binding of endogenous circulating albumin with maleimideꢀcontaining prodrugs. INNOꢀ206 is a
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0ꢀ11
successful example
.
With approximately 19 days plasma halfꢀlife and excellent accumulation capacity in solid
1
2ꢀ13
tumors, albumin becomes an ideal drug carrier for chemotherapeutic drugs
. In vivo albuminꢀ
binding prodrugs would have several advantages: (a) enhanced stability against enzyme
deactivation in systemic circulation; (b) prolonged retention time in the bloodstream; (c) possible
change in cell entry pathway to circumvent special transporters required for hydrophilic drugs; (d)
tumorꢀspecific drug delivery; (e) avoiding the use of exogenous albumin which may be expensive
and even immunogenic or pathogenic; (f) cheap and simple manufacture as well as quality control,
just like other smallꢀmolecule drugs.
To achieve siteꢀspecific drug release, the enzymeꢀbased approach represents an elegant
19
biocompatible method of both high sensitivity and selectivity , when compared with other
delivery approaches utilizing certain internal or external stimuli, such as pH, temperature, and
light. Cathepsin B is exactly a lysosomal cysteine proteinase only overexpressed in tumors and has
14ꢀ16
therefore been an ideal target for enzymeꢀtriggered, tumorꢀtargeting delivery
. Moreover,
albuminꢀprodrug conjugates allow for the albuminꢀmediated cellular entry into cells in a hENTꢀ
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independent manner of GEM, leading to circumvention of corresponding ADR. So we incorporate
a cathepsin Bꢀsensitive linker into the GEM prodrug to obtain hyperꢀselective tumor bioactivation,
lower offꢀtarget toxicity and even bypass ADR.
Herein, we synthesized a novel prodrug (GAM) of GEM, equipping GEM with maleimide
group through an amide bondꢀbearing cathepsin Bꢀsensitive linker (Figure 1). Another similar
prodrug (GCM) with a carbamate bondꢀbearing nonꢀcathepsin Bꢀsensitive linker was used as a
control (Figure 2B).
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Figure 1. Schematic diagram to describe the whole process of GAM covalently targeting albumin in situ,
accumulating at the tumor target and then cathepsin Bꢀmediated parent drug release after intravenous
administration.
First, GAM was synthesized according to the synthetic routes shown in Figure 2A. Maleic
anhydride (3) and 6ꢀaminocaproic acid were refluxed in glacial acetic acid to obtain 6ꢀ
maleimidocaproic acid (4, EMC). The two hydroxyl groups of GEM were masked by TBS groups
to furnish compound 2. Then compounds 2 and 4 were reacted in the presence of HATU to afford
5
. Finally, tetrabutylammonium fluoride (TBAF) unveiled the protective groups of 5 to give target
1
compound GAM. The identity of GAM was confirmed by H NMR (Figure S1) and MS.
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a
Figure 2. (A) Synthesis of GAM . (B) structures of GAM and GCM.
a
Reagents and Reactants: (a) 6ꢀaminocaproic acid, glacial acetic acid. (b) TBSCl, imidazole, dichloromethane.
(
c) HATU, 4ꢀMethylmorpholine, dichloromethane. (d) TBAF, THF.
To investigate the binding ability of GAM with albumin, an in vitro binding test was carried out
using an HPLC method and the binding ability was measured by the decrease in the prodrug peak
intensity. As shown in Figure 3A, the majority (ca. 90%) of GAM came into conjugation with
BSA within the first 1 min and the binding process almost completed after 30 min incubation.
However, when free Cysꢀ34 of BSA was blocked by excessive 6ꢀmaleimidocaproic acid (EMC)
added in advance, the peak of GAM showed only marginal decrease. The above result validated
the specificity of GAM conjugation with albumin through the ꢀSH group of Cysꢀ34.
Another binding test, by in vitro incubation with fresh rat blood, came to the same conclusion.
Nearly all GAM disappeared after incubation for 1 min (Figure 3B), demonstrating a quick
albuminꢀbinding rate in blood. By contrast, GAM exhibited no obvious change after incubation
for 1 min with blood preincubated with excessive EMC.
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Figure 3. The binding abilities of GAM with albumin at 37°C. (A) Chromatograms. (B) Incubation study of
GAM with fresh rat blood (n=3).
GAM existed mainly in the form of albuminꢀGAM conjugates after intravenous administration,
so we prepared BSAꢀGAM in advance to investigate the enzymeꢀsensitivity of GAM in vitro.
Experiments were conducted under different pH values with or without cathepsin B. At pH 7.4,
less than 3% of GEM was released from BSAꢀGAM within 72 h (Figure 4), illustrating that the
albuminꢀGAM conjugates could retain good stability under physiological conditions (i.e., in the
blood circulation). In contrast, the BSAꢀGAM showed dramatically accelerated GEM release in
the presence of cathepsin B at pH 5.4 (simulated conditions in endosomes and lysosomes) due to
the degradation of the amide bondꢀbearing linker between GEM and albumin. However, the nonꢀ
enzyme sensitive control, BSAꢀGCM, released less GEM either in the presence or absence of
cathepsin B at corresponding pH.
Figure 4. Release of GEM from BSAꢀGCM and BSAꢀGAM with cathepsin B at pH 5.4 or without cathepsin B
at pH 7.4 (n=3).
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To test the stability of BSAꢀGAM conjugates against metabolism by CDA, GEM, BSAꢀGAM
and BSAꢀGCM were incubated with fresh rat plasma at an equivalent GEM concentration of 16
ꢁg/mL at 37°C for 12 h, respectively. Both the free GEM and the degraded dFdU product were
detected. GEM release rate in the plasma for BSAꢀGAM and BSAꢀGCM was comparable to that
in buffer at pH 7.4. As shown in Figure 5A, no more than 5% of free GEM was released from
either BSAꢀGAM or BSAꢀGCM group. In Figure 5B, the area under the curve (AUC) of the
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dFdU from BSAꢀGAM group (AUC: 160 ng·mL ·h) was 17.5 times lower than that from the
ꢀ1
GEM group (AUC: 2960 ng·mL ·h). These data suggested that CDAꢀdependent degradation only
occurred in the free form of GEM and the albuminꢀconjugated form could provide a good
shielding effect from albumin.
Figure 5. Metabolic stability of GEM and two BSAꢀprodrug conjugates against CDA in fresh rat blood. (A)
Concentration change of free GEM. (B) Concentration change of dFdU (n=3).
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Next, Cy5ꢀlabelled BSAꢀGAM conjugates were synthesized to investigate the cellular uptake
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. BSAꢀGAM and Cy5ꢀBSAꢀGAM showed similar circular dichroism (CD) spectra to that of
BSA (Figure 6A), implying that the synthesis process had a negligible influence on the secondary
structure of BSA. 4T1 tumor cells were incubated with Cy5ꢀBSA, Cy5ꢀBSAꢀGAM and Cy5ꢀBSAꢀ
GCM for different time and temperature. As shown in Figure 6B, cellular uptake of BSA, BSAꢀ
GCM and BSAꢀGAM depended on both time and temperature and demonstrated no significant
difference quantitatively. It suggested that smallꢀmolecule bioconjugation did not affect the
albumin cellular uptake performance.
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Figure 6. (A) CD spectra of BSA, BSAꢀGAM, Cy5ꢀBSA and Cy5ꢀBSAꢀGAM. (B) Cellular uptake of Cy5ꢀ
BSA, Cy5ꢀBSAꢀGCM, Cy5ꢀBSAꢀGAM after incubation with cells at 37°C for 0.5 or 2 h, or at 4°C for 2 h, *P <
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.05,**P < 0.01, ***P < 0.001 (n=3).
To determine whether GEM could be released from albuminꢀGAM conjugates after cellular
uptake, BSAꢀGAM and BSAꢀGCM were incubated with 4T1 tumor cells for 6, 12 and 18 h. BSAꢀ
GAM demonstrated significantly accelerated intracellular GEM release when compared with the
nonꢀstimuliꢀsensitive control BSAꢀGCM (Figure 7).
Figure 7. Intracellular GEM release from BSAꢀGAM and BSAꢀGCM inside 4T1 cells (n=3).
The cytotoxicity results (Table S1) were in a good agreement with the intracellular GEM
release experiments. GEM was a potent growth inhibitor for 4T1 cells with IC50 value being 9.7 ±
1.2 nM. However, given the delayed release of GEM, both free GAM and BSAꢀGAM
demonstrated less cytotoxicity but still exhibited IC50 values in the low micromolar range (0.2ꢀ0.5
µM). In contrast, both free GCM and BSAꢀGCM showed very poor cytotoxicity. Additionally,
cytotoxicity of different prodrugs against A549 cells was listed in Table S2.
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In order to investigate the sensitivity of nucleoside transporterꢀdependent membrane permeation
of BSAꢀGAM, dipyridamole (DP) was used as an inhibitor of hENT during cytotoxicity
evaluation (Table S1). In 4T1 cells, DP significantly undermined GEM cytotoxicity by 34 times,
while didn’t affect the cytotoxicity of BSAꢀGAM. The results suggested that BSAꢀGAM entered
into tumor cells in a nucleoside transporterꢀindependent but albumin receptorꢀdependent manner,
probably circumventing ADR mediated by decreased expression of hENT.
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Pharmacokinetic profile was studied in male SpragueꢀDawley (SD) rats. GEM, GCM and GAM
were intravenously administrated at an equivalent GEM dose of 1 mg/kg. As shown in Figure 8A
and Table S3, the AUC of free GEM from the GA
M group was less than the half of the GEM group, while t1/2 value of free GEM from the GAM
group was 5.5 times longer than that of the GEM group. Moreover, the dFdU generated by the
GEM group was 6.6 times higher than that of the GAM group (Figure 8B). The above data
suggested the role of albuminꢀGAM as a GEM reservoir and protector, leading to less free GEM
exposure to plasmaꢀrich CDA.
Figure 8. Pharmacokinetic profiles of GEM, GAM and GCM in SD rats. (A) Plasma concentration–time curves
of free GEM. (B) Plasma concentration–time curves of dFdU (n=4).
The in vivo and ex vivo distribution of albumin and albuminꢀprodrug conjugates was studied in
mice bearing 4T1 tumor. Free Cy5 was eliminated quickly from the body and did not manifest
obvious accumulation in special organs, while Cy5ꢀBSA, Cy5ꢀBSAꢀGAM and Cy5ꢀBSAꢀGCM
showed strongest fluorescence of tumor sites either in in vivo imaging (Figure 9A) or among the
dissected organs (Figure 9C). Surprisingly, both BSA and BSAꢀprodrug conjugates could bypass
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renal elimination and mononuclear phagocytic system deeply plaguing nanoparticulate drug
delivery systems, which was validated by the weaker fluorescence intensity of the liver, spleen
and kidney than that of the tumor. These data highlighted the outstanding performance of albuminꢀ
prodrug conjugates in tumor targeting and sparing much less distribution in normal tissues and
organs.
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Encouraged by these results, we determined tumor freeꢀGEM accumulation in 4T1 tumorꢀ
bearing mice. As shown in Figure 10A and 10B, the tumor freeꢀGEM accumulation of the GAM
group was 2.6 times greater than that of GEM solution group. However, GCM showed poor tumor
freeꢀGEM accumulation which was even a little less than that of GEM. It was speculated that lack
of an enzymeꢀsensitive linker significantly hindered the release of free GEM from albuminꢀGCM
conjugates and directly limited the accumulation of free GEM in the tumor.
Figure 9. The in vivo and ex vivo fluorescence imaging of 4T1 tumorꢀbearing mice after intravenously injection
of Cy5, Cy5ꢀBSA, Cy5ꢀBSAꢀGAM and Cy5ꢀBSAꢀGCM. (A) Wholeꢀbody distribution for 12 and 24 h. (B)
Quantitative analysis of relative tumor accumulation in a live body. (C) Relative dissected major organs harvested
at 24 h postꢀadministration. (D) Quantitative analysis of relative dissected major organs (n=3).
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Figure 10. Quantification of tumor freeꢀGEM accumulation for GEM and two prodrugs. (A) Tumor freeꢀGEM
concentrationꢀtime curves. (B) AUC of free GEM in the tumor, ns: no significance versus GEM, ***P < 0.001
versus GEM (n=3).
To investigate the antitumor efficacy of GAM in vivo, 4T1 tumorꢀbearing mice were randomly
divided into four groups and received five injections of vehicle, GEM, GCM and GAM
respectively at an equivalent GEM dose of 8 mg/kg per injection. As shown in Figure 11A, 11B,
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the vehicle group showed a rapid increase in tumor volume (1602 mm at day 22), and GEM was
3
able to somewhat suppress tumor growth but the tumor volume still reached to 595 mm at day 22.
In consistent with the in vivo tumor freeꢀGEM accumulation result, GCM manifested poor tumor
inhibition efficacy, but GAM demonstrated much better antitumor efficacy when compared with
3
GEM. GAM dramatically delayed tumor progression with the final tumor volume of 241 mm .
The potent antitumor efficacy may be due to: (a) rapid covalently binding of endogenous albumin,
(b) good stability in systemic circulation against CDA, (c) long circulation ability, (d) outstanding
performance of tumor targeting, (e) independence on hENT, (f) cathepsin Bꢀsensitive GEM
release inside tumor cells. All these advantages made GAM a promising candidate for GEM
delivery. In addition, GAM demonstrated similar safety profile to GEM confirmed by body weight
change (Figure 11C), hematological parameters (Figure 11D), and H&E stained major organs
(
Figure S2). These suggested that GAM with potent antitumor activity elicited no significant offꢀ
target toxicities to major organs.
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Figure 11. Anticancer efficiency and safety profiles of two prodrugs in vivo in a 4T1 tumor xenograft model.
(A) Tumor growth curves. (B) Excised tumor weight after last treatment. (C) The weight growth curves of mice,
ns: no significance, *P < 0.05, ***P < 0.001 (n=4). (D) Hematological parameters after last treatment (n=3).
In summary, GAM was synthesized for the first time by linking GEM with a maleimide through
an amide bondꢀbearing cathepsin Bꢀsensitive linker. GCM with a carbamate bondꢀbearing linker
was used as a nonꢀenzymeꢀsensitive control. As a result of covalently targeting circulating
albumin and tumorꢀstimuliꢀtriggered drug release, GAM exhibited distinct superiority over both
GEM and GCM in terms of significantly delayed tumor progression and favorable safety profile.
The present study successfully exhibited how to exploit endogenous albumin as a carrier to
circumvent as many obstacles as possible in GEM delivery through the combination of in vivo
macromolecular prodrug strategy and ingenious selection of siteꢀspecific drug release.
ASSOCIATED CONTENT
Supplementary Information
The supporting information is available free of charge on the ACS Publication website at DOI:
1
0.1021/acs.bioconjchem.xxxxxxx.
AUTHOR INFORMATION
Corresponding Authors
*Eꢀmail address: hezhonggui@vip.163.com;
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*
Eꢀmail address: sunjin@syphu.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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This work was financially supported by National Natural Science Foundation of China (NO.
8
1473164, 81573371, 81773656).
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Table of Contents graphic
Maleimideꢀfunctionalised cathepsin Bꢀsensitive linkerꢀbearing gemcitabine prodrug
dramatically improves pharmacokinetic profiles, tumor cell targetingꢀbioactivation, and
overcomes acquired drug resistance.
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Figure 1. Schematic diagram to describe the whole process of GAM covalently targeting albumin in situ,
accumulating at the tumor target and then cathepsin B-mediated parent drug release after intravenous
administration.
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Figure 2. (A) Synthesis of GAM. (B) structures of GAM and GCM.
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Figure 3. The binding abilities of GAM with albumin at 37°C. (A) Chromatograms. (B) Incubation study of
GAM with fresh rat blood (n=3).
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Figure 6. (A) CD spectra of BSA, BSA-GAM, Cy5-BSA and Cy5-BSA-GAM. (B) Cellular uptake of Cy5-BSA,
Cy5-BSA-GCM, Cy5-BSA-GAM after incubation with cells at 37°C for 0.5 or 2 h, or at 4°C for 2 h
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Figure 9. The in vivo and ex vivo fluorescence imaging of 4T1 tumor-bearing mice after intravenously
injection of Cy5, Cy5-BSA, Cy5-BSA-GAM and Cy5-BSA-GCM. (A) Whole-body distribution for 12 and 24 h.
(B) Quantitative analysis of relative tumor accumulation in a live body. (C) Relative dissected major organs
harvested at 24 h post-administration. (D) Quantitative analysis of relative dissected major organs (n=3).
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Figure 11. Anticancer efficiency and safety profiles of two prodrugs in vivo in a 4T1 tumor xenograft model.
(
A) Tumor growth curves. (B) Excised tumor weight after last treatment. (C) The weight growth curves of
mice, ns: no significance, *P < 0.05, ***P < 0.001 (n=4). (D) Hematological parameters after last
treatment (n=3).
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