Prednisolone-glucose derivative conjugate: Synthesis, biodistribution and pharmacodynamics evaluation

Article abstract of DOI:10.1002/ardp.201200232

This study was aimed at synthesizing and evaluating a prednisolone-glucose derivative conjugate (PDG) that was expected to increase renal biodistribution without affecting pharmacological action and to decrease the systemic side effects of prednisolone. T

Full text of DOI:10.1002/ardp.201200232

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
1
Full Paper
Prednisolone-Glucose Derivative Conjugate: Synthesis,
Biodistribution and Pharmacodynamics Evaluation
Xing Liu1, Wenhao Li1, Zhen Liang1, Xuan Zhang2, Yangming Guo1, Tao Gong1, and Zhirong Zhang1
1 Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan
University, Chengdu, China
2 Shenzhen Salubris Pharmaceuticals Co., Ltd., Shenzhen, China
This study was aimed at synthesizing and evaluating a prednisolone-glucose derivative conjugate
(PDG) that was expected to increase renal biodistribution without affecting pharmacological action
and to decrease the systemic side effects of prednisolone. The PDG was designed and synthesized by
tethering 6-amino-6-deoxy-D-glucose (a D-glucose derivative) to prednisolone and its chemical structure
was confirmed by 1H NMR, 13C NMR, and LC-MS. This conjugate was then subjected to in vitro and in vivo
evaluation like stability studies, biological distribution, pharmacodynamics, and systemic side effects
studies. In these studies, PDG not only showed significant enhancement of renal target efficiency with
high values of relative uptake efficiency (RE, 24.1), concentration efficiency (CE, 8.6), and kidney targeting
index (KTI, 16.3), but retained the curative potency against minimal change nephrosis (MCN). In the
systemic side effects study, no osteoporosis was observed in rats after the administration of PDG for
20 days, which exhibited limited side effects. Conclusively, our findings showed a pharmacologically
active conjugate with the characteristics of renal targeting and limited systemic side effects. The
results implied the potential of PDG as a promising therapeutic in the treatment of renal diseases.
Keywords: D-Glucose derivative / Minimal change nephrosis (MCN) / Prednisolone / Renal targeting / Systemic side
effects
Received: May 26, 2012; Revised: July 18, 2012; Accepted: July 19, 2012
DOI 10.1002/ardp.201200232
Introduction
coids in the clinic as an immunosuppressive drug for kidney
transplants and nephritis treatment. Some renal target deliv-
ering systems of PD such as randomly 50% N-acetylated low
molecular weight chitosan (LMWC) [3] and prednisolone suc-
cinate-glucosamine conjugate (PSG) [4] have been developed
to improve its therapeutic efficacy and alleviate the systemic
side effects. Both LMWC and PSG have achieved specific renal
delivery and can be absorbed by renal epithelial cells.
However, the structure of LMWC is so complicated that
the precise quality control of this system is at present not
feasible. As for PSG, the instability of the ester linkage in vivo
limits its further application.
Most glucocorticoids, a type of highly potent anti-inflamma-
tory and immunosuppressive drugs, need to be adminis-
trated continuously and/or repeatedly in the clinic due to
their quick elimination. In addition, even their moderate
dose administration may cause many systemic side effects,
owing to their non-specific distribution to all organs,
multiple physiological effects, and pharmacological actions,
with the most serious being osteoporosis [1, 2]. Therefore, the
development of a drug delivery system that could transport
glucocorticoids to the desired site of action and reduce their
biodistribution in normal tissues would be of great value.
Prednisolone (PD) is one of the most widely used glucocorti-
D-Glucose, the main energy of cells, is delivered through
the bloodstream and transported to tissues by the glucose
transporter (GLUT) families. And so far, two types of GLUT
families in kidney have been identified [5–7]. One is the Naþ-
dependent glucose transporters (SGLTs), which mediate the
Naþ/glucose cotransport function in the kidney, the other is
the Naþ-independent GLUTs, which serve to facilitate diffu-
sion through the lipid bilayer [5]. In the mammalian kidney,
Correspondence: Zhirong Zhang and Tao Gong, Key Laboratory of Drug
Targeting and Drug Delivery Systems, West China School of Pharmacy,
Sichuan University, Sichuan 610041, China.
E-mail: zrzzl@vip.sina.com; gongtaoy@126.com
Fax: þ86 28 85501615
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X. Liu et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
Figure 1. The structure of prednisolone (PD) and prednisolone-glucose derivative conjugate (PDG).
SGLTs contribute to the reabsorption of glucose that is fil-
tered from the glomerulus in the process of urine formation.
Studies have been shown that the modification of the C-6
position or to tether a substituent at the C-6 position of
glucose has little influence on the affinity between the glu-
cose derivative and GLUTs [6]. Herein, we propose an inno-
vative covalent combination of PD and 6-amino-6-deoxy-D-
glucose (a D-glucose derivative, see Fig. 1) for a potential
SGLTs-mediated transportation. In addition, we also hope
that this targeted transportation could enhance patient com-
pliance and therapeutic efficacy of PD through the improved
pharmacokinetics and biodistribution [7, 8].
On the contrary, PD suffered quicker elimination in the
kidney, and was undetectable at 60 min. The results showed
that PDG could be specifically delivered to the kidney quickly,
with much lower concentration in organs (tissue) such as
blood, heart, spleen, and lung than that of PD.
Preliminary study on pharmacodynamics of PDG
At the beginning of the pharmacodynamics study, no signifi-
cant differences in the urinary protein content of rats were
observed among each group. After 14 days, the urinary
protein content of the normal group only slightly increased
(6.56 mg); however, in the control group, it increased about
nine-fold (78.31 mg; p < 0.05), which indicated a successful
establishment of the rat pathologic model of MCN. Although
the urinary protein content of the PD and PDG groups
increased about 3.5-fold (23.50 mg) after 14 days, they were
significantly lower than that of the control group (p < 0.05).
And no obvious difference was observed between the PD and
PDG groups on the 7th and 14th day, which suggested a
similar therapeutic effect against MCN (Fig. 4).
In our study, PD was adopted as a model drug and a novel
chemical conjugate (PDG) was synthesized and characterized.
Then its in vitro stability in rat plasma and liver, kidney
homogenate, biodistribution, systemic side effects, and cura-
tive potency against minimal change nephrosis (MCN) in rats
were evaluated.
Results
In vitro stability of PDG
PDG showed high enzymolysis resistance in vitro, as the con-
tent of PDG did not decrease and no released PD was detected
in both rat plasma and liver or kidney homogenate within
24 h (Fig. 2).
Biodistribution of PD and PDG in rat
The extraction recoveries of PDG in high, middle, and low
concentrations ranged from 77 to 95% in plasma and differ-
ent tissue homogenates, which were satisfying. PDG showed
excellent renal targeting efficiency with 12- to 28-fold con-
centrations in the kidney compared with PD at different time
points (Fig. 3). Its shorter elimination half-life in plasma led to
a shorter MRT of 14.1 min (Table 1), which would contribute
to decrease the non-targeted distribution of PDG. However,
PDG had a slower renal elimination rate, with an MRT of
2.7 h in the kidney (Table 2); as a result, it could still be
detected at a relatively high concentration at 2 h (51 nmol/g).
Figure 2. The in vitro stability of PDG in biological samples. PDG
was incubated with fresh rat plasma (50%), liver or kidney homo-
genate (33%) for 24 h. All the mixtures were incubated at 37 ꢀ 18C
in a shaking bath, and samples were taken at the predetermined
time points. Results are means ꢀ SD (n ¼ 3).
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
Prednisolone-Glucose Derivative Conjugate
3
Figure 3. Biodistribution of PD (A), PDG (B), and the concentrations of PD and PDG in rat kidneys and plasma versus time curves (C)
in Sprague–Dawley rats after intravenous injection (50 mmol/kg) within 2 h. Results are means ꢀ SD (n ¼ 5).
Table 1. Pharmacokinetic parameters of PD and PDG in rat plasma after intravenous injection of PD and PDG (50 mmol/kg).
T1/2 (min)
C
max (nmol/g)
MRT (min)
AUC0St (nmol/g h)
PD
PDG
22.2 ꢀ 3.3
5.3 ꢀ 1.9ꢁ
38.2 ꢀ 4.5
25.4 ꢀ 1.6
27.5 ꢀ 1.7
23.7 ꢀ 2.3
83.5 ꢀ 19.8ꢁ
14.1 ꢀ 1.38ꢁ
ꢁResults are means ꢀ SD (n ¼ 5).
p < 0.05 with respect to PD.
Table 2. Pharmacokinetic parameters of PD and PDG in rat kidneys after intravenous injection of PD and PDG (50 mmol/kg).
AUC0St (nmol/g h)
C
max (nmol/g)
MRT (min)
RE
CE
KTI
PD
PDG
10.6 ꢀ 2.0
20.0 ꢀ 3.1
11.4 ꢀ 0.6
31.2 ꢀ 1.2ꢁ
–
24.1
–
8.6
–
16.3
255.2 ꢀ 29.2ꢁ
572.2 ꢀ 53.1ꢁ
ꢁResults are means ꢀ SD (n ¼ 5).
p < 0.05 with respect to PD.
Serum analyses demonstrated that rats treated with PD
and PDG were in better condition than those in the control
group (see Fig. 5). Their biochemical indexes, including
creatinine (CERA) and triglyceride (Tg), recovered to normal
levels, which were similar with those in the normal group
but significantly lower than those in the control group
(p < 0.05). Blood urea nitrogen (BUN) content did not exhibit
much variation among the groups. Although the albumin
(ALB) levels of the rats in the PD and PDG groups did not
recover to normal state, they were similar to each other.
Conclusively, these data suggested that PDG kept the original
potency of PD.
Adverse effects of PDG in normal rats
Studies on glucocorticoid-induced toxicity of PD and PDG
were performed on male SD rats (120 ꢀ 10 g) during a period
of 20 days. The rats of the PD group grew much slower with
smaller body weight (212.3 ꢀ 10.2 g) and bone mineral
Figure 4. Urinary protein contents of rats in the various groups on
day 0, the 7th day and 14th day (ꢁp < 0.05 vs. the normal group;
#p < 0.05 vs. the control group). Results are means ꢀ SD (n ¼ 5).
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
Figure 5. The serum biochemical indexes of rats in the various groups (ꢁp < 0.05 vs. the normal group; ꢁꢁp < 0.05 vs. the control group).
Results are means ꢀ SD (n ¼ 5).
density (BMD, 0.210 ꢀ 0.003 g/cm2) than those of the normal Discussion
group (p < 0.05). In contrast, such influence on the rats of the
PDG group was not observed as there was no significant
difference in body weight and BMD between the PDG and
the normal groups. However, the body weight and BMD of the
rats in the PDG group were dramatically higher than those of
the PD group, which indicated the limited glucocorticoid-
induced side effects of PDG (p < 0.05; Fig. 6).
In order to improve the therapeutic effects and alleviate the
systemic side effects of some widely used drugs, researchers
have turned to chemical modification as an effective
approach to deliver them to the particular disease regions
and to improve their biodistribution [9]. Nowadays,
various transporter/receptor drug delivery systems have been
Figure 6. The BMD and body weight of the rats in various groups on the 21st day (ꢁp < 0.05 vs. the normal group; ꢁꢁp < 0.05 vs. the PD
group). Results are means ꢀ SD (n ¼ 5).
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
Prednisolone-Glucose Derivative Conjugate
5
developed, which are expected to achieve targeted drug
delivery, such as the glucosamine/megalin system [3, 10],
the D-glucose/GLUT1 system [11], the peptide transport sys-
tems, and the amino acid transporters [12]. Although most of
these systems could deliver drugs to the desired region, few
had achieved the fundamental purpose of reducing the
systemic side effects while maintaining the therapeutic
effects. This may be due to several reasons: (i) The release
mechanisms of drugs from macromolecular carriers are so
complicated, including swelling, hydrolysis, erosion, and
diffusion, as a result the drugs loaded on the carriers could
not be totally released; (ii) the linkages between drugs and
carriers are unstable in vivo, which results in their hydrolysis
in circulation and low target efficiency; (iii) due to the active
efflux systems, the released drug from the conjugates that do
reach the desired regions might be transported back to the
circulation and poor target efficiency happens; (iv) addition-
ally, some carriers might have toxicity in tissues, which
would notably limit their clinical application.
the molecular target for the treatment of type 2 diabetes
mellitus (T2DM) [15]. However, the renal-targeted delivery of
drugs by glucose reabsorption in the kidney has not been
reported until now, making the SGLT2 mediated renal target-
ing in this report a new method.
In biodistrubution study, PDG could still be detected at 2 h
in the kidney, whereas it was undetectable at 15 min in the
heart and the spleen, at 1 h in the lung and at 2 h in the
plasma after intravenous injection. The reason may be that
the reabsorbed PDG could not be released back into the
circulation through the same way as the reabsorbed glucose
released by the facilitative GLUTs [16]. This may be related
with the fact that the glucose C-6 position modification
prevented its transmembrane transport in cells [6]. Since
PDG could neither be hydrolyzed in vivo nor be released into
the circulation, it was ‘‘locked’’ in the kidney.
Minimal change nephrosis, originally named lipoid neph-
rosis, is mainly characterized by selective proteinuria, hypo-
albuminemia, and hyperlipidemia. Glucocorticoids have
been utilized for a long time in the treatment of MCN,
and PD was the most widely used one [19]. In our study,
MCN was induced in rats by a single intravenous adminis-
tration of daunomycin hydrochloride. The successful estab-
lishment of the MCN rat model was confirmed both by the
measurement of urinary protein contents and biochemical
indexes (CREA, BUN, Tg, and ALB) of the rats. Remission was
achieved after the treatment of PDG for 2 weeks with a
curative effect similar to that of PD. The fact that the ALB
level in both the PD and PDG groups did not recover to a
normal state in 2 weeks just shows why the low cure rate of
renal disease occurs in the clinic. Perhaps, the normalization
of serum albumin would happen after a longer period of
treatment [19].
Several renal targeting delivery systems of PD have been
developed [3, 4], but the issues mentioned above have not
been totally solved. In this study, a stable chemical conjugate
PDG was synthesized using 6-amino-6-deoxy-D-glucose as
a ligand, which was coupled to PD by a carbamate bond
[13, 14]. In contrast, two other 6-amino-6-deoxy-D-glucose-
PD conjugates via ester linkage (succinic acid or O-phthalic
acid) were hydrolyzed quickly in plasma in vitro and in vivo.
There was no conjugate, but only PD could be detected
within 3 min after intravenous administration, and their
renal distribution was similar to PD (data not shown).
Furthermore, the high stability of PDG could also avoid the
secondary distribution of the released PD from the kidney to
the blood circulation and accordingly evade the side effects.
The evaluation of PDG was performed at different aspects,
including performing a biodistribution study, analyzing
the pharmacodynamics against MCN, and studying its adverse
effects on normal rats. The results indicated a remarkable
renal target efficiency of PDG with the enhancement of kidney
distribution, accompanied by a decreased plasma distribution.
Furthermore, the PDG administration could alleviate the grav-
ity of MCN in rats with limited side effects.
In GC’s clinical application, osteoporosis is one of their
most common and serious side effects. The development of
glucocorticoids inducing osteoporosis is a multifactorial proc-
ess with complicated mechanisms [20, 21]. However, the non-
specific distribution of glucocorticoids might contribute to this
pathological condition. In PDG’s adverse effects study section,
the intravenous injection of PD resulted in osteoporosis and
growth inhibition in rats, whereas such side effects were not
observed in the PDG group. Therefore, PDG’s renal-targeted
delivery and scarce distribution in non-targeted tissues may
be the key issue of the limited side effects.
It is well known that glucose in the glomerular filtrate can
be efficiently reabsorbed by SGLT2 in the proximal convo-
luted tubule [15]. As a result, urine is essentially free from
glucose in healthy individuals [16]. In our study, PDG showed
a chemical structure with a glucose residue and a quick Conclusion
uptake process by the kidney. This phenomenon in exper-
iments might provide us with the evidence that PDG might
be an analogue of D-glucose. And the targeted accumulation
of PDG in the kidney could be the result of an active transport
process just like the reabsorption of the filtered glucose by
SGLT2 [17, 18]. Currently, SGLT2 has been widely studied as
In our study, the novel synthesized PDG exhibited an ideal
stability, excellent renal target efficiency, and the original
therapeutic efficiency of PD but limited side effects. Thus, we
conclude that PDG has the potential to be developed into a
novel renal target delivery system of glucocorticoids.
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X. Liu et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
primary silyl ether to provide 2b. The reaction of 2b with
diphenyl phosphorazidate (DPPA) and diisopropyl azodicarbox-
ylate (DIAD) afforded 3. To convert the azide into a primary
amine group, 3 was stirred at room temperature under H2 for
10 h which was catalyzed by Pd/C. PD and 4 were linked together
by another three steps. Firstly, 5 was obtained by conversion of
the prednisolone 21-hydroxyl group into 4-nitrophenyl carbon-
ate, and the amino group of 4 was then appended by a carbamate
bond to afford 6. Finally the chemical conjugate PDG was
obtained by deprotecting the silyl ether groups.
Experimental
Materials and methods
Prednisolone (99.5% purity) was purchased from Henan Lihua
Pharmaceutical CO., Ltd (Henan, China). All other chemicals and
reagents were obtained commercially and were of analytical
grade without further purification. Water was purified in an
Aquapro water purification system (Chongqing, China). TLC
(silica gel GF254) was used to detect spots by ultraviolet radi-
ation. The purification of intermediates and the final synthesized
compound were achieved by flash chromatography on silica gel.
1H NMR and 13C NMR analyses were performed using an AMX-400
Bruker spectrometer. Chemical shifts (d units) and coupling
constants (J values) are given in ppm and Hz, respectively.
LCMS spectroscopy was performed on an Agilent 6410 (Agilent
Technologies, Santa Clara, CA) utilizing electrospray ionization.
Chromatographic analysis was performed using an HPLC sys-
tem (Waters, Milford, MA) equipped with a Waters Delta 600
pump, a Waters 2487 Dual l absorbance detector, a Waters 600
controller, a model 100 column heater (CBL photoelectron tech-
nology) and a 20 mL injector loop. A Lichrospher column (C8,
250 mm ꢂ 4.6 mm) was used to separate the samples and was
protected by a Phenomenex guard column (4 mm ꢂ 3 mm, ODS).
All procedures involving animals were approved by the
Sichuan University animal ethical experimentation committee
according to the requirements of the National Act on the use of
experimental animals (People’s Republic of China). Sprague–
Dawley rats were provided by the Laboratory Animal Center of
Sichuan University and provided standard laboratory chow and
water ad libitum until the time of the experiment.
1,2,3,4,6-Penta-O-trimethylsilyl-D-glucose (2a)
D-Glucose (1, 360 mg, 2.00 mmol) was dissolved in anhydrous
pyridine (20 mL) and cooled to 08C. Hexamethyldisilazane
(HMDS, 0.94 mL, 4.50 mmol) and trimethylchlorosilane
(TMSCl, 1.89 mL, 14.95 mmol) were added sequentially. After
that, the mixture was warmed to 308C and stirred for another
10 h. Then the solvent was removed in vacuo, and white slurry
was obtained as the resulting crude product. The material was
suspended in petroleum ether (50 mL) and washed with water
(30 mL). The aqueous layer was extracted with petroleum ether
(3 ꢂ 20 mL). The organic layers were combined, washed with
brine (3 ꢂ 30 mL) and dried with Na2SO4. Solvent evaporation
provided viscous, colorless oil (98%) which was used without
further purification in the next step. Yield 1.06 g (98%). 1H
NMR (CDCl3, 400 MHz) d 5.00 (d, 1H, J ¼ 3.2 Hz, 1-H), 3.79–
3.64 (m, 4H, 4-H, 5-H, 6-H2), 3.40 (t, 1H, J ¼ 8.8 Hz, 3-H), 3.34–
3.32 (m, 1H, 2-H), 0.18–0.10 (m, 45H, 5 ꢂ Si(CH3)3). ESI–MS: m/z
[MþNa]þ: 563.1.
1,2,3,4-Tetra-O-trimethylsilyl-D-glucose (2b)
Chemicals
2a (957 mg, 1.70 mmol) was dissolved in dichlormethane (DCM,
12 mL) and cooled to 08C. Solution of AcOH (65 mL in 12 mL of
MeOH) was added dropwise over 30 min. The solution was stirred
for 8 h with the temperature below 58C and it was quenched
with saturated aqueous NaHCO3 (2 mL) and then washed with
water (15 mL), dried with Na2SO4 and concentrated under
vacuum. The resulting residue was purified by silica gel chroma-
The synthetic route of PDG is outlined in Scheme 1. PDG was
prepared by coupling prednisolone with the amino group of
6-amino-6-deoxy-D-glucose via a carbamate bond. 4 was obtained
in five steps from a commercially available D-glucose (1) [22]:
Per-TMS-protected D-glucose (2a) was prepared from 1 in a quan-
titative yield, and was selectively monodeprotected of the
Scheme 1. Conditions and reagents: (a) TMSCl, HMDS, Py, 08C to room temperature (rt); (b) AcOH, DCM/MeOH (1:1), 0–58C;
(c) DPPA, DIAD, PPh3, THF, 08C to rt; (d) H2, Pd/C, MeOH, rt; (e) Py, DCM; (f) Et3N, THF, rt; (g) TFA, DCM.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
Prednisolone-Glucose Derivative Conjugate
7
tography as colorless oil. Yield 0.60 g (75%). 1H NMR (CDCl3,
400 MHz) d 5.00 (d, 1H, J ¼ 3.2 Hz, 1-H), 3.81–3.65 (m, 4H, 4-H,
5-H, 6-H2), 3.45 (t, 1H, J ¼ 8.8 Hz, 3-H), 3.35–3.32 (m, 1H, 2-H),
0.18–0.10 (m, 36H, 4 ꢂ Si(CH3)3). ESI–MS: m/z [MþNa]þ: 491.1.
was introduced and the reaction was stirred overnight. Then
ethyl acetate was added and the mixture was washed with brine
(2 ꢂ 5 mL). The aqueous layers were re-extracted with ethyl
acetate (2 ꢂ 10 mL). The organic layers were combined and dried
with Na2SO4, followed by evaporation in vacuo. The crude product
of 6 was obtained as colorless oil which was used without puri-
fication. Then it was dissolved in DCM (6 mL), and trifluoroacetic
acid (TFA, 1.2 mL) was added dropwise into the reaction solution
slowly. The solution was stirred for 2 h at room temperature. It
was quenched with saturated aqueous NaHCO3 (5 mL) and then
extracted with DCM (3 ꢂ 10 mL). The organic layers were com-
bined, washed with brine (15 mL) and dried with Na2SO4. Solvent
was evaporated and the residue was purified with flash chroma-
tography on silica gel giving the pure product as a white powder.
6-Azido-6-deoxy-1,2,3,4-tetra-O-trimethylsilyl-D-glucose
(3)
Triphenylphosphine (PPh3, 1.17 g, 4.47 mmol), followed by DIAD
(0.90 g, 4.47 mmol) and then DPPA (1.23 g, 4.47 mmol) were
added sequentially to a solution of 2b (1.05 g, 2.24 mmol) in
THF (10 mL) at 08C. The mixture was allowed to warm to room
temperature and stirred for another 2 h. Then it was concen-
trated under reduced pressure, and purified by silica gel chroma-
tography. Yield 0.94 g (85%). 1H NMR (CDCl3, 400 MHz) d 5.01
(d, 0.6H, J ¼ 2.8 Hz, 1-H), 4.50 (d, 0.4H, J ¼ 7.2 Hz, 1-H), 3.90–3.25
(m, 6H, 2-H, 3-H, 4-H, 5-H, 6-H2), 0.18–0.14 (m, 36H, 4 ꢂ Si(CH3)3).
ESI–MS: m/z [MþNa]þ: 516.1.
1
Yield 0.20 g (62%). H NMR ([D6] DMSO, 400 MHz) d 7.32 (d, 1H,
J ¼ 10.0 Hz, prednisolone 2-H), 6.15 (d, 1H, J ¼ 10.4 Hz, predni-
solone 1-H), 5.91 (s, 1H, prednisolone 4-H), 4.97–4.87 (m, 2H,
prednisolone 21-H, 1-H), 4.69–4.64 (m, 1H, prednisolone 21-H),
4.28 (s, 1H, prednisolone 11-H), 3.63–3.58 (m, 1H, 6-H), 3.44–3.38
(m, 3H, prednisolone 6-H2, 6H), 3.13–2.87 (m, 4H, 2-H, 3-H, 4-H,
5-H), 2.30–2.27 (m, 2H, prednisolone 16-H2), 2.04–2.02 (m, 2H,
prednisolone 15-H2), 1.89–1.86 (m, 1H, prednisolone 9-H),
1.68–1.65 (m, 2H, prednisolone 12-H2), 1.44–1.40 (m, 2H, predni-
solone 7-H2), 1.39 (s, 3H, prednisolone 18-H3), 1.31–1.28 (m, 1H,
prednisolone 8-H), 1.03–0.97 (m, 1H, prednisolone 14-H), 0.80
(s, 3H, prednisolone 19-H3). 13C-NMR ([D6] DMSO, 400 MHz)
d 206.8 (C20), 185.4 (C3), 170.8 (C5), 157.0 (C1), 155.9 (OCONH),
127.2 (C2), 121.8 (C4), 97.2 (C17), 93.3 (C01), 76.0 (C03), 72.9 (C04), 72.5
(C02), 71.8 (C05), 70.2 (C11), 67.5 (C21), 55.6 (C9), 51.3 (C14), 47.2 (C13),
45.7 (C10), 44.0 (C12), 42.6 (C06), 34.2 (C8), 33.2 (C16), 31.5 (C6), 31.1
(C7), 23.7 (C15), 21.0 (C19), 16.7 (C18). ESI–MS: m/z [MþH]þ: 566.5.
6-Amino-6-deoxy-1,2,3,4-tetra-O-trimethylsilyl-D-glucose
(4)
10% Pd/C (20 mg) was added to a solution of 3 (211 mg,
0.43 mmol) in MeOH (5 mL). After having been stirred at room
temperature under H2 for 10 h, the reaction mixture was filtered
through Celite. The solvent was removed by concentration
in vacuo and then purified by silica gel chromatography. Yield
0.16 g. (79%). 1H NMR (CDCl3, 400 MHz) d 4.98 (d,1H, J ¼ 2.8 Hz,
1-H), 3.78 (t, 1H, J ¼ 8.8 Hz, 4-H), 3.66 (m, 1H, 5-H), 3.33 (m, 1H,
2-H), 3.27 (t, 1H, J ¼ 8.8 Hz, 3-H), 2.98 (dd, 1H, J1 ¼ 2.8 Hz,
J2 ¼ 13.2 Hz, 6-H), 2.64 (dd, 1H, J1 ¼ 7.6 Hz, J2 ¼ 13.2 Hz, 6-H),
0.17–0.13 (m, 36H, 4 ꢂ Si(CH3)3). ESI–MS: m/z [MþH]þ: 468.1.
In vitro stability of PDG
Prednisolone 21-(4-nitrophenyl carbonate) (5)
A working solution of PDG (10.55 mg/mL) was prepared by dis-
solving PDG (21.10 mg) in methanol (2.0 mL). Then a 20 mL
volume of the working solution was added to 5.0 mL of 50%
rat plasma (diluted by 0.05 M PBS, v/v), 33% liver or 33% kidney
homogenate (diluted by sterile saline, w/v) to investigate its
enzymatic hydrolysis. The mixture was incubated at 37 ꢀ 18C
under continuous shaking. Next, 200 mL of each incubated mix-
ture was sampled at the predetermined time points, mixed with
40 mL trichloroacetic acid (20%, g/mL). After vortexing (5 min), it
was centrifuged at 13,000 ꢂ g for 10 min. Aqueous supernatant
(20 mL) was injected into the HPLC system to determine the
concentration of PDG using the method described in the follow-
ing section.
Prednisolone (PD, 500 mg, 1.39 mmol) and 4-nitrophenyl chlor-
oformate (432 mg, 2.08 mmol) were dissolved in anhydrous DCM
(15 mL) and cooled to 08C. Pyridine (0.17 mL, 2.08 mmol) was
dropped in slowly, after that the mixture was allowed to warm
up and stirred for another 10 h at room temperature. Then DCM
(30 mL) was added and the mixture was washed with 1 M HCl
(3 ꢂ 30 mL) and brine (1 ꢂ 30 mL). The aqueous layer was re-
extracted with DCM (3 ꢂ 25 mL) and the organic phases were
combined and then dried with Na2SO4. Solvent was evaporated
and the residue was purified with flash chromatography on silica
gel giving the pure product as a white powder. Yield 0.61 g (84%).
1H NMR ([D6] DMSO, 400 MHz) d 8.34 (d, 2H, J ¼ 8.8 Hz, 20-H, 60-H),
7.56 (d, 2H, J ¼ 8.8 Hz, 30-H, 50-H), 7.31 (d, 1H, J ¼ 10.4 Hz, pred-
nisolone 2-H), 6.15 (d, 1H, J ¼ 10.4 Hz, prednisolone 1-H), 5.92 (s,
1H, prednisolone 4-H), 5.27 (d, 1H, J ¼ 18.0 Hz, prednisolone 21-
H), 4.94 (d, 1H, J ¼ 18.0 Hz, prednisolone 21-H), 4.28 (s, 1H,
prednisolone 11-H), 3.66–3.54 (m, 2H, prednisolone 6-H2),
2.30–2.25 (m, 2H, prednisolone 16-H2), 2.05–1.99 (m, 2H, predni-
solone 15-H2), 1.91–1.88 (m, 1H, prednisolone 9-H), 1.63–1.61 (m,
2H, prednisolone 12-H2), 1.51–1.47 (m, 2H, prednisolone 7-H2),
1.38 (s, 3H, prednisolone 18-H3), 1.19–1.16 (m, 1H, prednisolone
8-H), 1.03–1.00 (m, 1H, prednisolone 14-H), 0.80 (s, 3H, predniso-
lone 19-H3). ESI–MS: m/z [MþH]þ: 526.1.
HPLC analysis
HPLC assay method was used to monitor the concentrations of
PD and PDG in biological samples. A mixture of methanol/ace-
tonitrile/acetate buffer solution (adjusted by acetic acid to pH
4.0) at a ratio of 97:15:95 by volume was used as the mobile phase.
The flow rate was 1.0 mL/min, and the column effluent was
monitored at 254 nm. The total analytical time was 12 min
for the whole run. The obtained retention times were as follows:
PD, 10 min; PDG, 8.5 min, which, respectively, possessed the
limit of quantitation (LOQ) in biological samples of 0.02 and
0.03 mg/mL. Both PD and PDG in biological samples were com-
pletely separated under analytical conditions. And the standard
curves for PD ranging from 0.03 to 10.30 mg/mL were linear
(r2 > 0.99). As for PDG, its linearity range for kidney was
Prednisolone 21-(6-carbamate-D-glucose) PDG
Prednisolone 21-(4-nitrophenyl carbonate) (5, 270 mg,
0.58 mmol) was dissolved in anhydrous THF (6 mL) and
Et3N (0.6 mL) was added. After 5 min, 4 (609 mg, 1.30 mmol)
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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8
X. Liu et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
10.04–150.57 mg/mL and for plasma, heart, liver, spleen, lung it
was 0.20–40.17 mg/mL.
The biochemical indexes including ALB, BUN, CREA, and Tg in
the serum were monitored by a chemistry analyzer (Hitachi
7020, Japan).
Biodistribution of PD and PDG in rat
Adverse effects of PDG in rats
Fifty male Sprague–Dawley rats (190 ꢀ 10 g) were randomly
divided into two groups which were treated with PD and PDG,
respectively. For each preparation and sampling time point, five
rats were treated with a single dose of PD/PDG (50 mmol/kg)
through the caudal vein. The rats were killed at 5, 15, 30, 60,
and 120 min after injection.
At the predetermined time point, blood samples were
collected and placed into heparinized EP tubes. Rats were sacri-
ficed and tissue samples, including heart, liver, spleen, lung, and
kidney, were collected, flushed with cold saline and weighed
immediately, followed by homogenization with twofold volume
of 0.9% saline (g/mL). The plasma was separated by centrifugation
at 9200 ꢂ g for 1 min. All biological samples were stored at
ꢃ208C before assay. An 0.5 mL aliquot of plasma or tissue hom-
ogenate was mixed with 0.1 mL of methanol and then 0.1 mL of
trichloroacetic acid solution (20%, g/mL). After vortexing (5 min),
it was centrifuged at 13,000 ꢂ g for 10 min. The supernatant
(20 mL) was then injected into the HPLC system to determine the
concentrations of PD and PDG using the method described
above.
Tests for adverse effects were performed on 15 male Sprague–
Dawley rats (120 ꢀ 10 g), which were randomly divided into
three groups. In the normal group, rats were housed and fed
normally, whereas the rats of the PD group and PDG group were
treated with PD and PDG at a dose of 8.33 mmol kgꢃ1 dꢃ1 via
caudal vein for 20 days. All rats were weighed every 3 days after
the first administration, and the dosages were also changed
according to the variance of their body weights. On the 21st
day, rats were anesthetized with chloral hydrate (300 mg/kg,
i.p.), and their femoral densities were analyzed by an iDXA
instrument (GE Lunar, Madison, WI).
Statistical analysis
The area under the curve (AUC0ꢃt), maximal concentration
(Cmax), and mean retention time (MRT) in the biodistribution
study were calculated by Data and Statistics software (DAS 2.0,
Shanghai, China). The statistical analysis of the samples was
performed using a Student’s t-test with a p-value < 0.05 as the
minimal level of significance.
For the preparation of calibration curves of PD and PDG,
working solutions of PD (72.1 mg/mL) and PDG (1.05 mg/mL)
were prepared. Then they were diluted with methanol into other
six different concentration solutions, respectively, before use.
0.1 mL of each working solution was added to the blank bio-
logical samples, which was followed by biological sample pre-
treatment. Lastly, the concentration of PD or PDG was detected
by the HPLC. Their concentrations versus peak areas were
recorded and these calibrations were subjected to analytical
procedure to test the linearity of the method.
The relative uptake efficiency (RE), concentration efficiency
(CE), and kidney targeting index (KTI) were calculated to evaluate
the kidney target efficiency of PDG. Values of RE, CE, and KTI
were calculated as follows:
ðAUC0ꢃtÞPDG
RE ¼
ðAUC0ꢃtÞPD
ðCmaxÞPDG
CE ¼
ðCmaxÞPD
Preliminary study on pharmacodynamics of PDG
Minimal change nephrosis was employed as the pathological
model, which was established in rats by a single injection of
daunorubicin hydrochloride (12 mg/kg) through the caudal
vein [23].
Experiments were performed on 20 male Sprague–Dawley rats
(220 ꢀ 20 g), which were randomly divided into four groups. In
the normal group, rats were housed and fed without any treat-
ment. Rats in the control group received a single dose of daunor-
ubicin hydrochloride (12 mg/kg), whereas rats in the PD group
À
ꢀ
Á
AUCkidney AUCplasma
PDG
ꢀ
KTI ¼
ðAUCkidney AUCplasmaÞPD
This work was supported by the National Natural Science Foundation of
China (nos. 30430770 and 81130060). The authors would like to thank Rasa
Hamilton (H Lee Moffitt Cancer Center, FL) for editorial assistance.
The authors have declared no conflict of interest.
and PDG group were given PD and PDG at
a dose of
8.33 mmol kgꢃ1 dꢃ1 for 14 days separately after the adminis-
tration of daunorubicin hydrochloride (12 mg/kg).
References
Each rat was housed in a metabolism cage, and its urine voided
during 24 h was collected in tubes on the day before (day 0), the
7th day (day 7) after, and the 14th day (day 14) after the injection
of daunorubicin hydrochloride. The urine volume was recorded
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a urinary protein quantitation kit (Nanjing Jiancheng
Bioengineering Institute).
On the 15th day, blood samples were collected, placed into EP
tubes and left undisturbed at room temperature until clotted.
Samples were then centrifuged at 850 ꢂ g for 10 min, and the
serum obtained was immediately stored at ꢃ208C until assay.
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