Small molecular weight PEGylation of diosgenin in an in vivo animal study for diabetic auditory impairment treatment

Article abstract of DOI:10.1016/j.bmcl.2012.05.094

Diosgenin was modified to control its in vivo bioavailability by conjugating a hydrophilic unit, tetraethylene glycol. The diosgenin- tetraethylene glycol conjugate (TE) was orally administered in streptozotocin induced diabetic mice for this auditory protection study. The bioactivity improvement of TE for in vivo diabetic auditory impairment treatment was clearly observed in three different auditory tests and compared with that of diosgenin. The improvement in in vivo efficacy suggests that the small molecular weight PEGylation of diosgenin is a synthetically robust and systematically applicable strategy to reform the poor pharmacokinetics of a hydrophobic aglycone.

Full text of DOI:10.1016/j.bmcl.2012.05.094

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4609–4612
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Small molecular weight PEGylation of diosgenin in an in vivo animal study
for diabetic auditory impairment treatment
a,
b,
a
b
a
b
Dong-Hwan Kim , Bin Na Hong , Hoa Thi Le , Ha Na Hong , Choon Woo Lim , Keun Ha Park ,
a,
⇑
b,
⇑
Tae Woo Kim , Tong Ho Kang
Graduate School of East–West Medical Science, Kyung Hee University, Gyeonggi-do 449-701, Republic of Korea
a
b
Department of Oriental Medicinal Materials & Processing, College of Life Sciences, Kyung Hee University, Gyeonggi-do 449-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Diosgenin was modified to control its in vivo bioavailability by conjugating a hydrophilic unit, tetraeth-
ylene glycol. The diosgenin–tetraethylene glycol conjugate (TE) was orally administered in streptozoto-
cin induced diabetic mice for this auditory protection study. The bioactivity improvement of TE for
in vivo diabetic auditory impairment treatment was clearly observed in three different auditory tests
and compared with that of diosgenin. The improvement in in vivo efficacy suggests that the small molec-
ular weight PEGylation of diosgenin is a synthetically robust and systematically applicable strategy to
reform the poor pharmacokinetics of a hydrophobic aglycone.
Received 24 February 2012
Revised 15 May 2012
Accepted 29 May 2012
Available online 5 June 2012
Keywords:
Diabetic auditory impairment
Diosgenin
Ó 2012 Elsevier Ltd. All rights reserved.
In vivo auditory nerve protection
Small molecular weight PEGylation
Diabetes mellitus (DM) is a common metabolic disease that
causes various impairments of body systems. The relationship be-
tween diabetes mellitus and hearing function has been studied for
a long time. Many clinical studies have shown a direct correlation
between hearing loss and diabetes.1 The effects of diabetes on
hearing have been investigated in animal models using diabeto-
genic drugs (streptozocin or alloxan) or genetic modifications.
Our previous study reported on the hearing threshold shift using
auditory brainstem responses (ABRs) and the auditory central dys-
auditory pathways in humans. Recently, Tauris et al. suggested
that NGF may play an important role in the response to noise-in-
duced injuries or ototoxic damage in mice.
1
7
Our cell-based, natural product screening for an NGF-inducer
provided us with hits that have broad molecular diversity (data
not shown). To validate their efficacy at animal level, screened nat-
ural products should satisfy two requirements: (1) minimum
water-solubility for precise administration and (2) gram-level
quantity supply. The second requirement is a practical bottle neck
of our diabetic auditory animal study because it demands long-
term monitoring (8 to ꢀ10 weeks).
–8
function using auditory middle latency responses (AMLRs) in
streptozotocin (STZ) induced mice.9
,10
Also, our previous study
suggested that diabetic mice may be a useful experimental animal
model for the evaluation of auditory neuropathy using ABR and
Diosgenin (DG, Fig. 1), a steroid sapogenin, exhibited notable
activity in our previous NGF-inducer screening. Fortunately DG is
commercially-available at a multi-gram scale and is supplied at
moderate prices. Researchers have reported the in vivo bioactivity
of DG including the suppression of cholesterol absorption in rats18
9
,11
AMLR tests.
Nerve growth factor (NGF) is one of the well characterized
members of the neurotrophin family and plays crucial roles in pro-
motion of the survival or differentiation of developing neurons.
Moreover, many previous studies have reported that NGF may
ameliorate toxin-induced neuropathy,1
2,13
diabetic neuropathy,
14
HO
OH
and HIV-associated sensory neuropathy.15 However, whether
NGF might prevent or treat diabetic auditory impairment is un-
known. Levels of NGF in patients with sensorineural hearing loss
are significantly lower.16 This suggests that NGF might be involved
in preventing damage and promoting recovery or degeneration of
O
HO
HO
O
O
O
O
HO
H
OH
O
HO
HO
H
H
DG
RO
O
⇑
Corresponding author. Tel.: +82 31 201 2391; fax: +82 31 622 9639.
E-mail addresses: tw1275@khu.ac.kr (T.W. Kim), panjae@khu.ac.kr (T.H. Kang).
These authors contributed equally to this work.
diosgenin : R = H
dioscin : R =
Figure 1. The molecular structures of diosgenin and dioscin.
0
960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.094

4
610
D.-H. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4609–4612
and the decrease of cholesterol plasma level in diet-induced hyper-
cholesterolemia rats. However, the pharmaceutical applications
Table 1
1
9
Body weights and blood glucose level measurements
of DG are very limited because of its poor pharmacokinetics (PK)
Weight (g)
Blood glucose level (mg/dl)
2
0
(
logP 5.7). On the other hand, its glycoside, dioscin (Fig. 1) has
an improved PK property because the hydrophilic sugar moiety
6-deoxy- -mannopyranosyl-(1-2)-[6-deoxy- -mannopyrano-
syl-(1-4)]-b- -glucopyranoside) balances the hydrophobicity of the
Initiala
Finalb
Initiala
Finalb
Normal
Control
DG10
DG20
G10
G20
TE10
TE20
32.25 ± 0.39
33.25 ± 0.75
32.83 ± 1.19
33.67 ± 1.12
33.17 ± 0.28
31.58 ± 0.86
30.50 ± 0.39
32.55 ± 1.13
39.67 ± 0.57
34.15 ± 1.16
38.33 ± 2.40
38.67 ± 1.33
33.39 ± 0.50
34.92 ± 1.23
33.72 ± 1.07
37.60 ± 1.57
165.30 ± 7.40
516.33 ± 46.73
P600.00
182.20 ± 16.18
529.90 ± 49.17
P600.00
(
a-
L
a-L
D
cholesterol backbone. Unfortunately, commercially-available dios-
cin is expensive and was not affordable for our diabetic auditory
animal study.
The PK control of the sugar moiety of glycoside is well estab-
lished. For example, the function of the sugar moiety of digoxin (a
cardiac glycoside, extracted from foxglove, Digitalis lanata; oral
administration marketed by GSK) has been investigated by in-depth
PK comparison studies between digoxin, its aglycone (digoxigenin),
574.17 ± 25.83
583.55 ± 16.26
571.75 ± 24.61
522.18 ± 26.01
582.80 ± 17.20
598.00 ± 2.00
P600.00
592.33 ± 7.67
565.56 ± 28.43
501.60 ± 68.80
Data are mean ± SEM.
a
One week after STZ injection.
Nine weeks after STZ injection.
b
and digoxigenin-mono(digitoxide) (2,6-dideoxy-b-
D
-ribo-hexopyr-
2
1–23
anosyloxyl digoxigenin).
A clinical study showed that an appro-
because it has only one free hydroxyl functional group. TE (dios-
genin–tetraethylene glycol conjugate) was prepared by the Wil-
priate sugar moiety is essential for the precise pharmacokinetic
control of digoxin. In addition, Veit and co-workers reported that
liamson ether synthesis between diosgenin and 11-methoxy-
0
0
31
the disposition of quercetin (5,7,3 ,4 -hydroxyflavanol, a polyphe-
3
,6,9-trioxaundecyl p-toluenesulfonate in a sodium hydride base
2
4
nol) in humans primarily depends on the sugar moiety.
(Scheme 1). For the control experiment, G (diosgenin–glucose con-
The molecular diversity of the sugar moiety of glycoside offers the
opportunity for glycoside-like drug development.25,26 However,
the natural complexity and heterogeneity of the sugar moiety of
glycoside have caused problems in the quality control and develop-
ment of glycosides.
jugate, trillin) was prepared by two-step reactions: the Koenigs–
Knorr reaction for b stereospecific glycosidic bond formation
32,33
and then conventional sodium methoxide deacetylation/proton-
34
1
13
ation.
NMR
The products were fully characterized by H,
C
35,36
and high resolution mass spectrometry and were scaled
Our molecular design started with the question: can we control
the PK of DG using synthetically robust and systematically applica-
ble modifications? PEGylation appeared to be a promising solution.
The covalent attachment of polyethylene glycol (PEG) to a drug im-
proves its pharmacokinetic, pharmacodynamic, and immunologi-
cal profiles, and thus enhances its therapeutic effect.2 In spite of
the success of protein PEGylation, the strategy of attaching a rela-
tively small PEG to a small molecule is still under debate because
small molecules have few sites to which PEGs can be attached
without compromising functionality.28 However, the recent clini-
cal development of PEG-naloxol (NKTR-118 from Nektar Therapeu-
37
up (Supplementary data A).
At one week and nine weeks after STZ injection, the blood glu-
cose levels of all groups were evaluated (see Supplementary data B
for details). Blood glucose levels of the control, DG20, G10, G20,
TE10, and normal groups increased slightly during the experimen-
tal periods. However blood glucose levels of the TE20 group de-
creased slightly during the experimental periods. In the DG10
and G10 groups, the mean final blood glucose levels was
P600 mg/dl, respectively, because the upper measurement limit
of the blood glucose analyzer was 600 mg/dl (Table 1). The initial
blood glucose level (one week after STZ injection) of each group
is quite different because of variation due to individual animal dif-
ferences after STZ injection. However, mice of all groups were
hyperglycemia. Our results show that the DG, G, and TE treatments
did not result in significantly decreased blood glucose level at nine
weeks after STZ injection. In other words, the treatments did not
improve severe hyperglycemias in a type 1 diabetic mice model.
To examine the protective effect against hearing loss due to DM,
auditory brainstem response (ABR) tests were performed using
7
tics, San Carlos, CA) spotlighted the benefits and challenges of
small molecular weight PEGylation of small molecules.2
9,30
Their
study implies that some natural products have ample space to con-
jugate low molecular weight PEG without a serious loss of bioactiv-
ity of the mother molecules.
DG may be an ideal natural product for application of the ‘small
molecular weight PEGylation of small molecules’ strategy. From
the viewpoint of pharmacokinetics, the PEGylation at 3 OH will en-
hance the water solubility of DG and improve its in vivo bioavail-
ability. DG can be easily modified by a tetraethylene glycol unit
4
kHz and 8 kHz TBs (tone bursts) at 8 weeks (Fig. 2). The hearing
threshold of the control group using ABR with 4 kHz and 8 kHz TBs
O
a
DG
O
H
H
H
TE
O
O
O
O
O
O
b, c
HO
HO
O
DG
H
HO
O
H
H
OH
O
G
Ò
Scheme 1. Synthesis. Reagents: (a) p-CH
3
(OCH
2
CH
2 4
) OTs, NaH, THF; (b) acetobromo-
a-
D
-glucose, Ag
2
CO
3
, CH
2
Cl
2
3
; (c) NaOCH , THF–water; DOWEX 50WX8.

D.-H. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4609–4612
4611
4
kHz TB threshold
8 kHz TB threshold
A
60
B
4
3
2
1
0
0
0
0
4
2
0
0
*
*
*
**
*
*
*
**
**
*
**
0
0
-10
Nor Con DG10DG20 G10 G20 TE10 TE20
Nor Con DG10 DG20
G10 G20
TE10 TE20
Figure 2. ABR Test. Results were obtained for the STZ-treated (Con), DG 10 mg/kg-treated (DG10), DG 20 mg/kg-treated (DG20), G 10 mg/kg-treated (G10), G 20 mg/kg-
treated (G20), TE 10 mg/kg-treated (TE10), TE 20 mg/kg-treated (TE20), and normal groups (Nor) with regard to ABR hearing thresholds at 4 kHz (A) and 8 kHz (B) TBs (tone
⁄
⁄⁄
⁄⁄⁄
bursts) stimulation at 8 weeks after sample treatments. Data are presented as mean ± SEM, N = 10 for each groups. p <0.05, p <0.01, p <0.001 versus control group.
increased significantly compared to that of the normal group (p
0.001). The G20, TE10, and TE20 groups significantly suppressed
These data indicate that high dosage G or TE might suppress cen-
tral auditory functional damage due to DM.
<
hearing threshold shifts compared to the control group (p <0.05
and p <0.01, respectively). These data indicate that high dosage G
or TE might suppress hearing threshold shifts in an STZ-induced
DM mouse model.
To investigate protective effects against abnormalities of central
auditory function caused by DM, auditory middle latency response
To assess the normality of the cochlear response in a DM mouse
model, transient evoked otoacoustic emission (TEOAE) tests were
performed at 8 weeks (Fig. 4). Signal intensity-to-noise (S/N) ratios
of the control group in response to TEOAEs at 2 kHz and 3 kHz de-
creased significantly compared to levels in the normal group (p
<0.05 and p <0.01); however, S/N ratios of the TE treatment groups
at 2 kHz were in the normal range. These results indicate that TE
might suppress functional damage to the outer hair cells in the or-
gan of Corti.
All three auditory tests show that TE significantly suppresses
damage and auditory impairment by STZ-induced DM compared
to those in the control and DG treatment groups. TE and high dose
G shows similar tendencies in ABR and AMLR, but TE has better
(
AMLR) tests were performed at 8 weeks (Fig. 3). The Pa (positive A
peak) latencies of the control group increased significantly and the
Na (negative A peak)-Pa amplitudes of the control group decreased
compared to the normal group (p <0.05). However, the G20, TE10,
and TE20 groups has significantly decreased Pa latency compared
to the control group (p <0.01). Also, G20, TE10, and TE20 groups
had increased Na-Pa amplitudes compared to the control group.
Pa latencies
Na-Pa amplitudes
A
60
B
2.5
2
1
1
0
.0
.5
.0
.5
u
4
2
0
0
*
*
*
*
0
0.0
Nor Con DG10 DG20 G10 G20
TE10 TE20
Nor Con DG10 DG20 G10 G20
TE10 TE20
Figure 3. AMLR Test. Results were obtained for the STZ-treated (Con), DG 10 mg/kg-treated (DG10), DG 20 mg/kg-treated (DG20), G 10 mg/kg-treated (G10), G 20 mg/kg-
treated (G20), TE 10 mg/kg-treated (TE10), TE 20 mg/kg-treated (TE20), and normal groups (Nor) with regard to Pa latencies (A) and Na-Pa amplitudes (B) of AMLR at 8 weeks
⁄
after sample treatments. Data are presented as the mean ± SEM, N = 10 for each group. p <0.05 versus control group.
2
kHz TEOAE
3 kHz TEOAE
A
15
B
8
6
4
2
0
*
*
* **
*
*
10
*
5
0
Nor Con DG10 DG20 G10 G20
TE10 TE20
Nor Con DG10 DG20 G10 G20
TE10 TE20
Figure 4. TEOAE Test. Results were obtained for the STZ-treated (Con), DG 10 mg/kg-treated (DG10), DG 20 mg/kg-treated (DG20), G 10 mg/kg-treated (G10), G 20 mg/kg-
treated (G20), TE 10 mg/kg-treated (TE10), TE 20 mg/kg-treated (TE20), and normal groups (Nor) with regard to signal-to-noise ratio (SNR) of TEOAE with 2 kHz (A) and 3 kHz
⁄ ⁄⁄
B) at 8 weeks after sample treatments. Data are presented as mean ± SEM, N = 10 for each group. p <0.05, p <0.01 versus control group.
(

4
612
D.-H. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4609–4612
efficacy than G in TEOAE. The blood glucose level of TE20 was de-
creased from 582–501 mg/dl, whereas that of TE10 was slightly in-
creased from 522–565 mg/dl. However, the auditory impairment
was improved in both TE10 and TE20 groups, regardless of glucose
levels. This data suggests that the improvement of the auditory
impairment in TE group is not related to glucose levels in diabetic
mice.
4. Frisina, S. T.; Mapes, F.; Kim, S.; Frisina, D. R.; Frisina, R. D. Hear. Res. 2006, 211,
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At a glance, these results conflict with the general viewpoint of
glycoside: inactive glycosides can be activated by enzyme hydro-
3
1
3
8
sis. From this point of view, the enzymatically/chemically inert
PEG conjugation of OH at aglycone will hamper the hydrolyzed
activation and the PEG-aglycone conjugate will not exhibit the ex-
pected in vivo bioactivity. However, a real metabolic activation of
glycoside is more complicated. For instance, a pharmacological
study on ginsenoside has shown that both deglycosylation by
intestinal bacteria and esterification with fatty acids are crucial
for the medical efficacy of ginseng.39 Our experiment implies that
some aglycones can be successfully conjugated by small molecular
weight PEG without compromising their functionality and even
improve their bioactivity.
1
1
1
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Dioscin has been reported to have antitumor, antifungal, and
antiviral bioactivity, but the absolute oral bioavailability of dioscin
1
4
0
is very low (0.2%). If we accept the structural simplicity and syn-
thetic availability of TE, small molecular weight PEGylated DG
derivatives can be proposed as potential congeners of dioscin.
In summary, our observations suggest that the auditory nerve
protection effect of TE in a diabetic mouse model results from
the appropriate PK adjustment of hydrophobic DG by hydrophilic
tetraethylene glycol conjugation. If we consider the synthetic reli-
ability of PEGylation and the natural complexity of glycosylation,
the replacement of the sugar moiety with small molecular weight
PEG might be a practical strategy to reform the poor PK of hydro-
phobic aglycones and to improve their in vivo bioavailability. To
our knowledge, our experiment is the first to apply this small
molecular weight PEGylation strategy to the PK control of aglycone
at an animal level.
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Acknowledgments
3
3
1
999, 37, 140.
7. Spectroscopic data for compound TE: 1H NMR (CDCl
1H), 3.63–3.50 (m, 16H), 3.40–3.51 (2H), 3.35 (s, 3H), 3.15 (m, 1H), 2.1–2.3
(2H), 2.0–1.0 (overlapping multiplets), 0.98 (3H), 0.95 (1H), 0.93 (3H), 0.76 (s,
This research was supported by the Basic Science Research
Program through the National Research Foundation of Korea
3
3
300 MHz): d 5.30 (1H), 4.38
(
(
(
NRF) funded by the Ministry of Education, Science and Technology
NRF-2009-0073817 and NRF-2010-0015906).
6
7
3
H); 13C NMR (CDCl
3
75 MHz): d 140.9, 121.2, 109.2, 80.7, 79.3, 71.8, 70.8, 70.5,
0.4, 67.2, 66.8, 62.0, 59.0, 56.4, 50.0, 41.5, 40.2, 39.7, 38.9, 37.1, 36.9, 32.0,
1.8, 31.3, 31.3, 30.2, 28.7, 28.2, 20.8, 19.3, 17.1, 16.2, 14.5; HRMS (FAB+) calcd
+
Supplementary data
mass 604.4339 for [C36
Spectroscopic data for compound G: H NMR (DMSO-d
H), 4.91–4.86 (m, 3H), 4.44 (app t, 1H, J = 5.7 Hz), 4.27–4.19 (m, 2H), 3.61
dd, 1H, J = 10.8, J = 5.6 Hz), 3.44–3.38 (m, 2H), 3.22–2.97 (m, 4H), 2.88 (m,
1H), 2.1–1.0 (overlapping multiplets), 0.95 (3H), 0.89 (4H), 0.72 (app s, 6H);
NMR (CD OD 75 MHz): d 141.9, 122.5, 110.5, 102.3, 82.2, 79.6, 78.0, 77.8, 75.0,
1.5, 67.8, 63.6, 62.7, 57.7, 51.6, 42.8, 41.4, 40.8, 39.6, 38.5, 38.0, 33.1, 32.7,
2.3, 31.4, 30.6, 29.8, 21.9, 19.8, 17.5, 16.7; HRMS (FAB+) calcd mass 576.3662
H
60
O
7
], found 605.4417 for [M+H] .
1
6
300 MHz): d 5.31 (br,
1
(
1
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05.094.
1
2
13
C
3
7
3
References and notes
+
52 8
for [C33H O ], found 577.3740 for [M+H] .
1
2
.
.
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1
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