Prednisolone succinate-glucosamine conjugate: Synthesis, characterization and in vitro cellular uptake by kidney cell lines

Article abstract of DOI:10.1016/j.cclet.2011.07.023

Prednisolone succinate-glucosamine (PSG) conjugate, a prodrug for prednisolone, was synthesized and confirmed by NMR and MS spectrum. The stabilities of the prodrug in PBS (pH 2.50, 5.00, 7.20, and 7.89) were studied. Cytotoxicity and uptake assay of the prodrug were perfomed on HK-2 and MDCK cell lines. The results showed that compared with prednisolone, the PSG not only did not increase the cytotoxicity but also improved the uptake to 2.2 times of prednisolone by the cells. Thus, it indicated that glucosamine might be a potential carrier for kidney-targeting delivery of prednisolone.

Full text of DOI:10.1016/j.cclet.2011.07.023

Available online at www.sciencedirect.com
Chinese Chemical Letters 23 (2012) 25–28
www.elsevier.com/locate/cclet
Prednisolone succinate–glucosamine conjugate: Synthesis,
characterization and in vitro cellular uptake by kidney cell lines
*
Yan Lin, Xun Sun, Tao Gong, Zhi Rong Zhang
Key Laboratory of Drug Targeting of Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
Received 27 May 2011
Available online 8 November 2011
Abstract
Prednisolone succinate–glucosamine (PSG) conjugate, a prodrug for prednisolone, was synthesized and confirmed by NMR and
MS spectrum. The stabilities of the prodrug in PBS (pH 2.50, 5.00, 7.20, and 7.89) were studied. Cytotoxicity and uptake assay of
the prodrug were perfomed on HK-2 and MDCK cell lines. The results showed that compared with prednisolone, the PSG not only
did not increase the cytotoxicity but also improved the uptake to 2.2 times of prednisolone by the cells. Thus, it indicated that
glucosamine might be a potential carrier for kidney-targeting delivery of prednisolone.
# 2011 Zhi Rong Zhang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Glucosamine; Prednisolone; Prodrug; Cytotoxicity; Uptake
Glucocorticoid such as prednisolone has been generally used for the therapy of nephrotic syndrome. However, the
long-term administration of this kind of drug is always accompanied by systemic toxicities and side effects, including
osteoporosis, amyotrophy, and central obesity, etc. Therefore, it is necessary to develop a novel kidney-targeted drug
delivery system to increase the therapeutic effect and reduce the systemic toxicities. Several renal drug delivery
systems have been previously developed, such as poly (vinylpyrrolidone-co-dimethyl maleic acid) (PVD) [1], low
molecular weight proteins [2], sugar-modified low-molecular-weight peptides prodrugs, and randomly 50% N-
acetylated low molecular weight chitosan (LMWC) [3]. However, their complex structures hinder their further
development for clinical applications. Thus, our aim is to synthesize a kidney-targeted prodrug with a simple chemical
structure. As the constitutional unit of chitosan, glucosamine was chosen to synthesize the prodrug.
In the current research, the following studies have been investigated: (1) synthesizing the prednisolone succinate–
glucosamine conjugate (PSG); (2) the in vitro stabilities of the PSG; (3) cytotoxicity assay and uptake assay of the PSG
on kidney cell lines.
The synthetic routes of the intermediate 10 and PSG were presented as Scheme 1 [4,5] and Scheme 2. The
1
structures of the intermediate 10 and PSG were characterized by H NMR, 13C NMR and ESI-MS [6].
The stability of PSG was investigated in PBS (pH 2.50, 5.00, 7.20 and 7.89) at 37 Æ 1 8C. The percentages of the
residual PSG were determined by HPLC. In Fig. 1, it could be known that the prodrug was relatively stable in acidic
environment (pH 2.50, 5.00), while in slightly basic environment (pH 7.20, 7.89), most of the PSG were hydrolyzed
within 9 and 24 h, respectively.
* Corresponding author.
1001-8417/$ – see front matter # 2011 Zhi Rong Zhang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2011.07.023

OH
OAc
OH
OAc
O
O
O
a
b
c
O
HO
HO
AcO
AcO
HO
HO
AcO
AcO
OH
NH .HCl
OAc
OH
OAc
N
N
2
NH .HCl
2
Glucosamine
4
Ph
Ph
3
2
OAc
OAc
OAc
O
e
g
d
O
f
O
AcO
AcO
AcO
AcO
AcO
OAc
NHCOCCl
O
AcO
O
N
3
3
Br
NHCOCCl
3
NHCOCCl
3
5
6
7
OH
O
OH
OH
h
O
i
HO
HO
O
HO
HO
O
HO
HO
O
O
NH .HCl
N
3
N
3
NHCOCCl
3
NH .HCl
2
NH
2
2
8
9
10
Scheme 1. Synthesis of 2-amino-1-(2-aminoethoxy)-glycose. Reagents and conditions: (a) benzaldehyde in NaOH aqueous solution; (b) Ac2O, Py; (c) HCl; (d) Cl3CCOCl, Et3N; (e) 2-
bromoethanol, BF3ÁEt2O; (f) NaN3, DMF; (g) CH3ONa; (h) 0.3 mol/L NaOH; and (i) Pd/C (10%), H2 in MeOH.

Y. Lin et al. / Chinese Chemical Letters 23 (2012) 25–28
27
O
OH
OH
OH
OH
O
O
O
OH
OH
HCl.H2N
O
O
O
OH
HO
HO
H
N
O
O
O
OH
a
b
O
HO
10
O
O
11
prednisolone
O
1
Scheme 2. Synthesis of prednisolone succinate–glucosamine conjugate (PSG). Reagents and conditions: (a) succinic anhydride; and (b) DCC/
DMAP.
100
80
60
40
20
0
0
5
10
15
20
25
Time(h)
pH5.00
pH2.50
pH7.20
pH7.89
Fig. 1. Stability of PSG in PBS buffers at 37 Æ 1 8C. Error bars represent standard deviation of the mean (n = 3).
The cytotoxicity of PSG and prednisolone were both determined on Madin-Darby Canine kidney (MDCK) cell
lines and human proximal renal tubular epithelial (HK-2) cells after 24 h incubation at 37 8C using the MTT test. The
results, reported in Fig. 2, showed the cell viability of both prednisolone and PSG higher than 90% when the
concentration was lower than 0.28 mmol/mL. Compared with prednisolone, PSG showed no differences in
cytotoxicity (P > 0.05).
Uptake experiments were also performed at 37 8C on MDCK and HK-2 cell lines. Prednisolone and PSG were
respectively dissolved in the serum-free medium to a final concentration of 0.28 mmol/mL. Afterwards, they were
incubated with the two cell lines for 2 h. Then, MDCK and HK-2 cells were collected for the determination of the drug
contents. Compared with the prednisolone group, 2.2 times of the intracellular content of prednisolone in the PSG
group was certified by the HPLC method (P < 0.01) (Fig. 3).
In conclusion, Prednisolone succinate–glucosamine conjugate was synthesized successfully. The cytotoxicity and
uptake assay of PSG on MDCK and HK-2 cell lines showed that PSG did not increase the cytotoxicity while it could
MDCK
HK-2
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Prednisolone
PSG
Prednisolone
PSG
0.056 0.112 0.168 0.224 0.280
Concentration (μmol/L)
0.056 0.112 0.168 0.224 0.280
Concentration (μmol/L)
Fig. 2. Effect of prednisolone (white columns) and PSG (black columns) concentration on MDCK and HK-2 cell viability as determined by the MTT
assay after 24 h incubation. Mean Æ SD (n = 3).

28
Y. Lin et al. / Chinese Chemical Letters 23 (2012) 25–28
2
*
*
1.8
1.6
1.4
1.2
1
Prednisolone
PSG
0.8
0.6
0.4
0.2
0
MDCK
HK-2
Fig. 3. In vitro uptake assay of prednisolone (white) and PSG (black) on MDCK and HK-2 cells. Mean Æ SD (n = 3), (*P < 0.01).
improve the uptake 2.2 times of prednisolone by the cells. Therefore, the conjugate could be used as a potential kidney-
targeted delivery system for prednisolone. Further studies of this conjugate are ongoing in our laboratory.
Acknowledgment
The present study is supported by the National Science & Technology Major Project of China (No. 2009ZX09310-
002).
References
[1] H. Kodaira, Y. Tsutsumi, Y. Yoshioka, Biomaterials 25 (2004) 4309.
[2] T. Maaek, V. Johnson, S.T. Kau, Kidney Int. 16 (1979) 251.
[3] Z.X. Yuan, Z.R. Zhang, Mol. Pharm. 6 (2009) 305.
[4] Z.X. Guo, X.Q. Xiong, Chin. J. Org. Chem. 25 (2005) 1437.
[5] W. Dullenkopf, J.C. Castro-Palomino, L. Manzoai, Carbohydr. Res. 296 (1996) 135.
[6] Analytical data for the intermediate 10. yeild: 87%; 1H NMR (400 MHz, D2O): d 4.71 (d, 1H, J = 18.8 Hz), 4.20 (m, 1H), 4.01 (m, 2H), 3.81 (d,
1H, J = 5.6 Hz), 3.79 (d, 1H, J = 5.6 Hz), 3.53 (m, 3H), 3.32 (m, 2H); 13C NMR (200 MHz, D2O): d 36.8, 53.5, 57.9, 63.3, 67.0, 71.3, 73.5, 98.2;
ESI-MS (m/z): 245.0 [M+Na]+. Compound 1. Yield: 57%; 1H NMR (400 MHz, DMSO-d6): d 7.33 (d, 1H, J = 10.4 Hz), 6.16 (d, 1H,
J = 10.4 Hz), 5.92 (s, 1H), 5.06 (d, 1H, J = 17.2 Hz), 4.76 (d, 1H, J = 17.6 Hz), 4.29 (d, 1H, J = 3.2 Hz), 4.07 (m, 2H), 3.48 (m, 4H), 3.15 (m,
2H), 3.04 (m, 4H), 2.60 (m, 3H), 2.42 (m, 3H), 2.31 (m, 2H), 2.02 (m, 2H), 1.64 (m, 3H), 1.38 (s, 3H), 1.25 (m, 2H), 1.02 (dd, 1H, J = 13.2 and
4 Hz), 0.89 (dd, 1H, J = 10.8 and 3.2 Hz), 0.77 (s, 3H); 13C NMR (200 MHz, DMSO-d6): d 16.8, 21.1, 22.8, 23.7, 28.4, 28.9, 29.9, 31.1, 31.8,
34.2, 44.1, 47.3, 51.3, 55.6, 57.2, 61.2, 66.0, 68.1, 68.3, 70.2, 76.2, 77.4, 88. 8, 103.9, 115.9, 121.8, 127.3, 157.1, 172.2, 173.4, 174.3, 185.5,
205.6; ESI-MS (m/z): 665.4 [M+H]+.