Content uploaded by Shohei Kasugai
Author content
All content in this area was uploaded by Shohei Kasugai on Jun 09, 2016
Content may be subject to copyright.
Selective Delivery of Estradiol to Bone by Aspartic Acid
Oligopeptide and Its Effects on Ovariectomized Mice
KOICHI YOKOGAWA, KAZUHIRO MIYA, TOHRU SEKIDO, YASUHIKO HIGASHI,
MASAAKI NOMURA, RYUICHI FUJISAWA, KEIKO MORITO, YUKITO MASAMUNE,
YOSHIHIRO WAKI, SHOHEI KASUGAI, AND KEN-ICHI MIYAMOTO
Department of Hospital Pharmacy, School of Medicine (K.Y., K.-i.M.), Kanazawa University,
Kanazawa 920-8641, Japan; Department of Clinical Pharmacy (K.Mi., T.S., Y.H., M.N.), Graduate
School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan; Department of
Biochemistry (R.F.), Faculty of Dentistry, Hokkaido University, Sapporo, Japan; Department of
Microbiology (K.Mo., Y.M.), Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa,
Japan; and Masticatory Function Control (Y.W., S.K.), Tokyo Medical and Dental University, Tokyo,
Japan
ABSTRACT
We have developed a novel osteotropic prodrug of estradiol (E
2
)
conjugated with L-Asp-hexapeptide (E
2
䡠3D
6
), which has very low af-
finity for estrogen receptors, and in this study, we examined its phar-
macokinetic behavior and pharmacological potential. After a single iv
injection of E
2
䡠3D
6
to mice, the half-time for elimination from plasma
was about 100 min; however, E
2
was selectively delivered to the bone
and eliminated very slowly, declining to the endogenous level at about
7 days. After a single iv injection of E
2
, the half-time in plasma was
about 70 min, whereas E
2
was highly distributed to the uterus, and
the bone concentration of E
2
was only slightly increased at 6 h. When
E
2
(0.37
mol/kg, sc, every third day) or E
2
䡠3D
6
(0.11 to 1.1
mol/kg,
sc, every seventh day) was administered to OVX mice for 4 weeks, E
2
increased the bone mineral density (BMD) together with weights of
liver and uterus, whereas E
2
䡠3D
6
increased only the BMD, in a dose-
dependent manner. E
2
䡠3D
6
enhanced the expression of messenger
RNAs of bone matrix proteins (osteopontin, bone sialoprotein, type I
collagen
␣
) of OVX mice at 4 h after administration, but E
2
did very
slightly. These results indicate that the E
2
prodrug was delivered to
the bone, where it gradually released E
2
, thereby ameliorating bone
loss. This acidic oligopeptide appears to be a good candidate for se-
lective drug delivery to bone. (Endocrinology 142: 1228–1233, 2001)
OSTEOPOROSIS is a serious problem for postmeno-
pausal and aged women, because estrogen deficiency
plays a causative role in the development of osteoporosis.
Estrogen can act directly or indirectly on osteoblasts and
osteoclasts through estrogen receptor-mediated mechanisms
(1–5), such as inhibition of production of bone-resorptive
cytokines from bone marrow stromal cells (6), direct inhibi-
tion of activity of osteoclasts (7, 8), enhancement of osteoblast
proliferation, and stimulation of secretion of bone matrix
proteins from osteoblasts (9, 10), resulting anabolic effect on
bone formation in estrogen-deficient animal models (11, 12).
Consequently, estrogen replacement therapy is an effective
treatment in postmenopausal women to prevent reduction of
the bone mineral density (13). However, prolonged therapy
may increase the risks of endometritis, breast cancer, and
uterus cancer (14, 15). Then, a selective drug delivery system
(DDS) to bone is desirable for osteoporosis therapy without
adverse reactions.
Oldberg et al. (16, 17). and Butler (18) demonstrated that
several bone noncollagenous proteins in bone matrix have
repeating sequences of acidic amino acids (Asp or Glu). We
considered that the repetitive acidic amino acid sequences
may be binding sites to the hydroxyapatite (HA) component
of bone, and we attempted to use acidic oligopeptides for
drug delivery to the bone. We have shown that small acidic
peptides conjugated with fluorescein isothiocyanate, as a
detection marker, are adsorbed preferentially on the surface
of HA in vitro (19, 20), and are selectively delivered to the
bone after systemic administration to mice (21). In this study,
to confirm the usefulness of acidic oligopeptide for bone
targeting, we synthesized a conjugate of 17

-estradiol (E
2
)
linked at position 3 with l-Asp-hexapeptide via succinate
and examined its pharmacokinetics and pharmacodynamics
in ovariectomized (OVX) mice.
Materials and Methods
Materials
17

-Estradiol (E
2
) was purchased from Wako Pure Chemicals Co.
(Osaka, Japan). 17

-Estradiol-3-succinate-(l-Asp)
6
(E
2
䡠3D
6
) was synthe-
sized in Peptide Institute, Inc. Ltd. (Osaka, Japan) and its molecular
structure is presented in Fig. 1. 17

-Estradiol Correlate-CLIA Kit was
purchased from Assay Designs Inc. (MI). [
3
H]-17

-E
2
was purchased
from Amersham Pharmacia Biotech (Buckinghamshire, UK).
Animal experiments
The animal studies were performed according to the regulations
approved by the institutional animal care committee. For the pharma-
cokinetic study, E
2
solution in 30% ethanol or E
2
䡠3D
6
in saline was
injected via the jugular vein into female ddY mice (8 weeks old, Nippon
SLC, Inc., Hamamatsu, Japan) at a dose of 3.7
mol/kg in a volume of
50
l. The blood samples were collected from the intraorbital venous
plexus using heparinized capillary tubes under light diethyl ether an-
esthesia at designated time intervals. The plasma was separated by
Received May 30, 2000.
Address all correspondence and requests for reprints to: Ken-ichi
Miyamoto, Ph.D., Prof., Department of Hospital Pharmacy, School of
Medicine, Kanazawa University, 13–1 Takara-machi, Kanazawa 920-
8641, Japan. E-mail: miyaken@kenroku.ipc.kanazawa-u.ac.jp.
0013-7227/01/$03.00/0 Vol. 142, No. 3
Endocrinology Printed in U.S.A.
Copyright © 001 by The Endocrine Society
1228
centrifugation and stored at ⫺30 C until assay. Mice were killed by
decapitation, then tissues were quickly excised, rinsed well with ice-cold
saline, blotted dry, and weighed.
Female ddY mice (9 weeks old) were ovariectomized under pento-
barbital anesthesia and divided into six groups; sham-operated control,
untreated control, E
2
(0.37
mol/kg), E
2
䡠3D
6
(0.11, 0.37, 1.1
mol/kg),
six in a group. From 8 weeks after the operation, E
2
and E
2
䡠3D
6
were sc
injected at the hind back every third day and every seventh day, re-
spectively. At 28 days after the first administration, mice were killed and
the major organs were weighed. The femur was removed from the leg
and stored in ethanol. The bone mineral density (BMD) was measured
by using a dual x-ray absorptiometer (DCS-600R, Aloka Corp., Tokyo,
Japan).
Analysis for E
2
and E
2
䡠3D
6
E
2
in plasma was assayed with a 17

-Estradiol Correlate-CLIA Kit
according to the manufacturer’s instructions. After administration of E
2
and E
2
䡠3D
6
, tissues were digested in 1 nNaOH at 60 C for1hand
neutralized with 1 nHCl, then E
2
was extracted with diethyl ether. Bone
was incubated in 6 nHCl at 60 C for 1 h and neutralized with 6 nNaOH,
then E
2
was extracted with diethyl ether three times. The diethyl ether
extracts of tissue and bone were dried under nitrogen stream and the
residue was dissolved in ethanol. Fifty microliters of ethanol solution
was mixed with 250
l of the assay buffer and E
2
was assayed with the
17

-Estradiol Correlate-CLIA Kit.
Before assay, we confirmed, using HPLC, that E
2
䡠3D
6
was stable in
blood, whereas in 1 nNaOH and 6 nHCl solution at 60 C it was
completely hydrolyzed to release free E
2
within 1 h.
Binding competition experiments to human recombinant
estrogen receptors
Estrogen receptor ER
␣
cDNA isolated from pBacPAK9-HEGO by
digestion with BamHI and XhoI was kindly provided by Kato et al. (22).
ER

cDNA was isolated from pGEX-4T-2ER

by digestion with BamHI
and XhoI. These fragments were ligated into the BamHI/XhoI sites of the
baculovirus donor vector pFastBac1 (Life Technologies, Inc., Gaithers-
burg, MD). Recombinant baculoviruses were generated using the BAC-
TO-BAC expression system (Life Technologies, Inc.) in accordance with
the manufacturer’s instructions. The isolated Bacmid DNA was trans-
fected into Sf21 cells, which were then cultured at 28 C for 72 h. The
obtained viruses were amplified and used to infect Sf21 cells. Infected
cells were harvested 72 h post infection, suspended in 10% glycerin, and
disrupted by ultrasonication. After the centrifugation, the obtained su-
pernatant was used as human recombinant ER
␣
or ER

in estrogen
receptor binding competition experiments.
A solution of ER
␣
or ER

(10
l, 40
g protein) containing [
3
H]-17

-
E
2
(2.5 pmol) was supplemented with various concentrations of E
2
or
E
2
䡠3D
6
in a total volume of 250
l. The mixture was incubated at 0 C for
15 h, then an equal volume of a suspension of activated charcoal powder
was added and the mixture was kept at 0 C for 10 min. After the
centrifugation, the radioactivity of the supernatant was counted in a
liquid scintillation counter (Aloka LSC-5100).
Measurement of estrogen-sensitive transcripts in OVX mice
Female 8-week-old ddY mice were ovariectomized under pentobar-
bital anesthesia at 14 days before use. E
2
and E
2
䡠3D
6
were sc injected at
a dose of 0.37
mol/kg. After designated times, uterus and femur were
removed and frozen immediately in liquid nitrogen. Frozen tissues were
crushed and RNA was prepared by using ISOGEN (Nippon Gene Co.,
Tokyo, Japan) according to the manufacturer’s instructions. Then, RT
reaction was performed in 75 mmKCl, 50 mmTris-HCl (pH 8.3), 6 mm
MgCl
2
,10mmdithiothreitol, 0.6 mmeach dATP, dTTP, dGTP, dCTP
mixture, 10 U of RNase inhibitor (Promega Corp., Madison, WI), 100
pmol of random hexamer, 600 U of M-MLV reverse transcriptase (Life
Technologies, Inc., Berlin, Germany) and 10
g of RNA in a final volume
of 50
l at 37 C for 1 h. PCR was conducted in final volume of 20
l
containing 1
l of RT mixture, 50 mmKCl, 20 mmTris-HCl (pH 8.3), 1.75
mmMgCl
2
, 0.25 mmeach dATP, dTTP, dGTP, dCTP mixture, 1
m
specific oligonucleotide primers, and 1.5 U of Taq DNA polymerase (Life
Technologies, Inc.). Osteopontin, bone sialoprotein, and type I collagen
␣
2 were amplified by 20 cycles consisting of 94 C for 30 sec, 52 C for 60
sec, and 72 C for 60 sec. Oligonucleotide primers were obtained from
Takara (Ohtsu, Japan). The sequences of primers for osteopontin were;
sense: 5⬘-CAT TGC CTC CTC CCT CCC GGT G-3⬘and antisense: 5⬘-ATC
ACC TCG GCC GTT GGG G-3⬘. Predicted fragment size was 402 bp. The
sequences of primers for bone sialoprotein were: sense: 5⬘-GAG CCA
GGA CTG CCG AAA GGA A-3⬘and antisense: 5⬘-CCG TTG TCT CCT
CCGCTG CTG C-3⬘. Predicted fragment size was 652 bp. The sequences
of primers for type I collagen
␣
2 were; sense: 5⬘-TGG TCC TCT GGG
CAT CTC AGG C-3⬘and antisense: 5⬘-GGT GAA CCT GCT GTT GCC
CTC A-3⬘. Predicted fragment size was 1248 bp.

-Actin primers and the
thermal cycling procedure were described in our previous paper (23),
and the predicted fragment size was 456 bp. Amplified fragments were
analyzed by agarose gel electrophoresis.
Data analysis
The pharmacokinetic parameters were estimated by means of model-
independent moment analysis as described by Yamaoka et al. (24). The
FIG. 1. Structure of 3

-estradiol-3-succinate-(L-Asp)
6
(E
2
䡠3D
6
).
FIG. 2. Time courses of plasma (A) and
femur (B) concentration of E
2
after a
single iv administration of E
2
(E) and
E
2
䡠3D
6
(F) (3.7
mol/kg) to normal mice.
Each point with bar represents the
mean ⫾SE of three animals.
EFFECTS OF ESTRADIOL-OLIGOPEPTIDE CONJUGATE ON OSTEOPOROSIS 1229
data were analyzed using Student’s ttest to compare the unpaired mean
values of two sets of data. The number of determinations is noted in each
table and figure. A value of P⬍0.05 was taken to indicate a significant
difference between sets of data.
Results
Disposition pharmacokinetics of E
2
䡠3D
6
in mice
Figure 2 shows the plasma concentration-time courses af-
ter a single iv injection of E
2
or E
2
䡠3D
6
(3.7
mol/kg). The
behaviors of E
2
and E
2
䡠3D
6
were biphasic, with half-times for
the elimination phase of about 70 and 100 min, respectively.
After injection of E
2
, the plasma concentration of E
2
de-
creased to the endogenous E
2
level (7.6 ⫾1.9 pmol/ml,
mean ⫾se,n⫽8) by 360 min. After injection of E
2
䡠3D
6
, it was
confirmed by using a combination of HPLC and the 17

-
estradiol enzyme-immunoassay system that no degradation
products were detectable in plasma, and the plasma E
2
level
was unchanged up to 360 min (data not shown). As shown
in Fig. 2 and Table 1, unchanged E
2
䡠3D
6
slowly decreased.
The value of the area under the plasma concentration-time
curve (AUC) was significantly higher than that of E
2
and the
value of the total clearance (CLtot) was lower than that of E
2
.
The distribution volume at the steady-state (Vdss) of E
2
䡠3D
6
was a little lower than that of E
2
.
Figure 2 also shows the bone concentration-time course of
E
2
after injection of E
2
䡠3D
6
. The bone concentration decreased
very slowly, falling to the endogenous level (10.5 ⫾3.3
pmol/ml, mean ⫾se,n⫽8) by 7 days, whereas after E
2
injection the bone concentration of E
2
increased a little within
360 min, then declined to within the basal range.
Figure 3 shows the apparent tissue-to-plasma concentra-
tion ratio (Kp,app) at 360 min after a single injection of E
2
or
E
2
䡠3D
6
.E
2
was distributed most highly to the uterus among
all the organs examined. After E
2
䡠3D
6
injection, E
2
concen-
tration in the bone was the highest, although the concentra-
tions of E
2
in other organs tended to be lower than those
after E
2
.
Binding affinity of E
2
䡠3D
6
to human estrogen receptors
Figure 4 shows the profiles of inhibition by E
2
and E
2
䡠3D
6
of [
3
H]E
2
binding to ER
␣
and ER

. These results indicate that
the binding affinities of E
2
䡠3D
6
to these receptors were about
1/100 of those of E
2
.
Effects of E
2
䡠3D
6
on OVX mice
To clarify the bone-selective effect of E
2
䡠3D
6
, OVX mice
were treated with 0.11, 0.37, or 1.1
mol/kg of E
2
䡠3D
6
every
seventh day or 0.37
mol/kg of E
2
every third day for 28
days. There were no large changes of body and major organs
FIG. 3. Apparent tissue-to-plasma concentration ratios (Kp,app) of E
2
at
360 min after a single iv administration of E
2
(䡺)orE
2
䡠3D
6
(f)(3.7
mol/kg) to normal mice. Each column with bar represents the mean ⫾
SE of three animals. *, Significantly different from E
2
at P⬍0.01.
TABLE 1. Pharmacokinetic parameters of E
2
and E
2
䡠3D
6
after a
single intravenous injection at a dose of 3.7
mol/kg in mice
Parameter E
2
E
2
䡠3D
6
AUC
0-360
(nmol䡠min/ml) 196 ⫾12 513 ⫾23
a
MRT (min) 23.5 ⫾2.9 48.1 ⫾5.0
a
Vd
ss
(ml/kg) 443 ⫾73 347 ⫾45
b
CL
tot
(ml/min/kg) 18.9 ⫾1.2 7.2 ⫾0.3
a
Pharmacokinetic parameters were estimated by means of model-
independent moment analysis. Each value represents the mean ⫾SD
of five mice. AUC
0-360
; the area under the plasma concentration-time
curve from 0 to 360 min, MRT; the mean residence time, Vd
ss
; the
distribution volume at the steady-state, CL
tot
; the total body clear-
ance.
a,b
Significantly different from estradiol at P⬍0.01 and 0.05,
respectively.
FIG. 4. Binding affinity of E
2
and
E
2
䡠3D
6
to human estrogen receptors,
ER
␣
(A) and ER

(B). Data indicate
binding % of [
3
H]E
2
(2.5 pmol) to each
receptor in the presence of various con-
centrations of E
2
(E)orE
2
䡠3D
6
(F).
1230 EFFECTS OF ESTRADIOL-OLIGOPEPTIDE CONJUGATE ON OSTEOPOROSIS Endo •2001
Vol. 142 •No. 3
weights, except for the liver and uterus, among the sham-
operated group, OVX-untreated control and treated groups.
The liver weight of OVX mice treated with E
2
was signifi-
cantly higher than that of the E
2
䡠3D
6
group, whereas the liver
weight was hardly changed after OVX (Fig. 5). The treatment
with E
2
induced a vacuolar degeneration histologically re-
sembling fatty liver.
Figure 6 shows the uterine weight in each group. The
uterine weight of the OVX-untreated control mice was sig-
nificantly decreased compared with that of sham-operated
mice. E
2
treatment almost completely restored the uterine
weight, whereas E
2
䡠3D
6
treatment tended to increase the
uterine weight, though the change was not significant com-
pared with that of the untreated-control group. Although the
BMD of femurs from untreated control mice was signifi-
FIG. 5. Effects of E
2
and E
2
䡠3D
6
on the liver weight of OVX mice. OVX
mice were treated with the indicated dose of E
2
(every third day) or
E
2
䡠3D
6
(every seventh day) for 28 days. Sham, Sham-operated group;
Control, untreated control group. Each column with bar represents
the mean ⫾SD of six animals. *, Significantly different from control
mice at P⬍0.01.
FIG. 6. Effects of E
2
and E
2
䡠3D
6
on the uterine weight of OVX mice.
OVX mice were treated with the indicated dose of E
2
(every third day)
or E
2
䡠3D
6
(every seventh day) for 28 days. Sham, Sham-operated
group; Control, untreated control group. Each column with bar rep-
resents the mean ⫾SD of six animals. *, Significantly different from
control mice at P⬍0.01.
FIG. 7. Effects of E
2
and E
2
䡠3D
6
on the femoral BMD of OVX mice.
OVX mice were treated with the indicated dose of E
2
(every third day)
or E
2
䡠3D
6
(every seventh day) for 28 days. Sham, Sham-operated
group; Control, untreated control group. Each column with bar rep-
resents the mean ⫾SD of six animals. *, **, Significantly different
from control mice at P⬍0.05 and 0.01, respectively.
FIG. 8. Microscopic observations of the soft tissue-free femur of OVX
mice. OVX mice were treated with the indicated dose of E
2
(every third
day) or E
2
䡠3D
6
(every seventh day) for 28 days, after treatment femur
was removed and incubated in 1 NNaOH over night, and then pho-
tographed under a microscope. A, Sham-operated control; B, un-
treated control, C, E
2
(0.37
mol/kg); D, E
2
䡠3D
6
(0.37
mol/kg).
EFFECTS OF ESTRADIOL-OLIGOPEPTIDE CONJUGATE ON OSTEOPOROSIS 1231
cantly decreased compared with that of the sham-operated
mice, the treatment with E
2
almost completely inhibited the
decrease of BMD. Similarly, E
2
䡠3D
6
significantly inhibited the
decrease of femur BMD of OVX mice in a dose-dependent
manner (Fig. 7). Figure 8 shows that E
2
䡠3D
6
(0.37
mol/kg,
every third day) inhibited the loss of trabecular bone of OVX
mice, as well as E
2
䡠3D
6
(0.37
mol/kg, every seventh day)
.
Effects of E
2
and E
2
䡠3D
6
on expression of bone-matrix
protein mRNAs
To confirm the bone-selective effect of E
2
䡠3D
6
, expression
of bone-matrix protein mRNAs in bone was examined using
RT-PCR method 4 h after iv injection of E
2
or E
2
䡠3D
6
(0.37
mol/kg) into OVX mice. The predicted sizes of the PCR
products were detected, and the sequences also agreed with
the respective sequences reported elsewhere. After injection
of E
2
, the expression of mRNAs of bone matrix proteins,
osteopontin, type I collagen
␣
, and bone sialoprotein, in the
femur was changed slightly, whereas the expression of these
all mRNAs was increased about 3-fold in the bone of OVX
mice treated with E
2
䡠3D
6
(Fig. 9).
Discussion
In this study, we examined the pharmacokinetics and
pharmacodynamics of a new type of E
2
prodrug, which con-
jugated with E
2
and an Asp-oligopeptide carrier. We found
that it showed a potent antiosteoporotic effect with reduced
side effects and could be administered less frequently than
E
2
in OVX mice.
After iv administration at a dose of 3.7
mol/kg, E
2
䡠3D
6
was not detectably degraded in blood within 360 min, and
its AUC was larger and its clearance was slower than those
of E
2
, but its distribution to various tissues except for bone
was smaller than that of E
2
(Figs. 2 and 3). These results
suggest that E
2
䡠3D
6
was taken up less into the soft tissues than
E
2
, presumably because of increased hydrophilicity due to
the acidic peptide moiety in the molecule. On the other hand,
after injection of E
2
䡠3D
6
,E
2
was highly distributed to the bone
and its level decreased very slowly to the endogenous level
at 7 days (Fig. 2), indicating that the acidic oligopeptide acts
as a bone-selective DDS.
Then, we examined whether the effects of E
2
䡠3D
6
are se-
lective for bone, compared with those of E
2
, using OVX mice.
The OVX mice exhibited severe uterus atrophy and bone loss
by 12 weeks. When E
2
was administered at a dose of 0.37
mol/kg every third day for the last 4 weeks, the changes of
uterine weight and BMD in OVX mice were almost com-
pletely inhibited, and the liver weight was significantly in-
creased (Figs. 5–7). On the other hand, E
2
䡠3D
6
affected only
the bone, exhibiting inhibition of the decreases in the BMD
(Fig. 7) and the number of trabecular bones (Fig. 8), when
administered every seventh day. Thus, E
2
conjugated with
Asp-hexapeptide selectively and continuously acted on the
bone and prevented bone loss in OVX mice.
It is well known that hepatic hypertrophy with nonalco-
holic steatohepatitis is induced by massive doses of synthetic
estrogen (25), and the uterus weight increase is estrogen
dependent (26). In this study, we observed that E
2
enlarged
the liver, which histopathologically showed fatty degener-
ation, and restored the uterine weight of OVX mice to the
level of sham-operated mice, whereas E
2
䡠3D
6
did not affect
these organs. One reason might be the smaller number of
injections and longer intervals of treatment with E
2
䡠3D
6
than
with E
2
. However, after a single injection of E
2
䡠3D
6
, the dis-
tribution of E
2
in these organs was similar to that of E
2
after
injection of E
2
(Fig. 3). There are at least two estrogen re-
ceptors, ER
␣
and ER

(27–29). We confirmed that the affin-
ities of E
2
䡠3D
6
for human ER
␣
and ER

were very much lower
than those of E
2
(Fig. 4). From these results, it is suggested
FIG. 9. RT-PCR of mRNAs of bone matrix proteins in femur of animals treated with E
2
or E
2
䡠3D
6
. OVX mice were sc injected with E
2
or E
2
䡠3D
6
at a dose of 0.37
mol/kg. A, Typical electrophoresis. Lane 1, untreated control; lane 2, E
2
4 h; lane 3, E
2
䡠3D
6
4 h. B, Intensity ratio of each
band vs.

-actin analyzed by using NIH Image. Data are the mean ⫾SD done in three experiments. Open column, type I collagen; dotted column,
osteopontin; filled column, bone sialoprotein. *, Significantly different from control mice at P⬍0.05.
1232 EFFECTS OF ESTRADIOL-OLIGOPEPTIDE CONJUGATE ON OSTEOPOROSIS Endo •2001
Vol. 142 •No. 3
that the conjugated E
2
may not cause the systemic adverse
reactions of E
2
, which include carcinogenesis (30, 31).
On the other hand, estrogen acts directly on osteoblasts
through estrogen receptor-mediated mechanisms and stim-
ulates secretion of bone-matrix proteins (9, 10). In this study,
high expression of mRNAs of osteopontin, type I collagen
␣
and bone sialoprotein was observed in bone at 4 h after
E
2
䡠3D
6
, but not after E
2
(Fig. 9). This indicates that E
2
䡠3D
6
acted on the osteogenic cells for longer time than E
2
. Al-
though we measured E
2
䡠3D
6
in tissues as E
2
after complete
hydrolysis, E
2
retained in the bone (over 100-fold more than
E
2
after E
2
administration) (Fig. 2B) was considered to be
present as the unchanged form of E
2
䡠3D
6
, bound to HA,
because of its prolonged residence time in the bone. It is
unlikely that E
2
䡠3D
6
directly acted on the estrogen receptors,
because the compound binds tightly to HA via the peptide
moiety (20) and is hardly transported into bone cells due to
its hydrophilicity. Even if it were taken up into the cells, its
affinity for the estrogen receptors is very low (about 100-fold
less than that of E
2
) (Fig. 4). Consequently, we speculate that
E
2
䡠3D
6
distributed to bone was gradually hydrolyzed at the
bone surface, possibly by peptidases and/or acid secreted
from osteoclasts, releasing E
2
, which was transported into the
bone cells and acted on the estrogen receptor(s).
In conclusion, this study indicated that our E
2
-prodrug,
consisting of E
2
conjugated with a novel acidic oligopeptide
carrier, is a promising candidate as an osteotropic drug, for
estrogen-replacement therapy of postmenopausal osteopo-
rosis, because of its selective and long-term action on bone
without the adverse side effects of E
2
. The use of E
2
䡠3D
6
would extend medication intervals, resulting in an improved
quality of life for patients. Selective delivery to bone using an
acidic oligpeptide carrier may also be applicable to other
osteotropic drugs.
References
1. Eriksen EF, Colvard DS, Berg NJ, Graham ML, Mann KG, Spelsberg TC,
Riggs BL 1988 Evidence of estrogen receptors in normal human osteoblast-like
cells. Science 241:84–86
2. Komm BS, Terpening CM, Benz DJ, Graeme KA, Gallegos A, Korc M,
Greene GL, O’Malley BW, Haussler MR 1988 Estrogen binding, receptor
mRNA, and biologic response in osteoblast-like osteosarcoma cells. Science
241:81–84
3. Oursler MJ, Osdoby P, Pyfferoen J, Riggs BL, Spelsberg TC 1991 Avian
osteoclasts as estrogen target cells. Proc Natl Acad Sci USA 88:6613–6617
4. Onoe Y, Miyaura C, Ohta H, Nozawa S, Suda T 1997 Expression of estrogen
receptor beta in rat bone. Endocrinology 138:4509–4512
5. Arts J, Kuiper GG, Janssen JM, Gustafsson JA, Lowik CW, Pols HA, van
Leeuwen JP 1997 Differential expression of estrogen receptors alpha and beta
mRNA during differentiation of human osteoblast SV-HFO cells. Endocrinol-
ogy 138:5067–5070
6. Girasol G, Jilka RL, Passeri G, Boswell S, Boder G, Williams DC, Manolagas
SC 1992 17

-estradiol inhibits interleukin-6 production by bone marrow-
derived stromal cells and osteoblasts in vitro: a potential mechanism for the
antiosteoporotic effect of estrogens. J Clin Invest 89:883–891
7. Oursler MJ, Pederson L, Fitzpatrick L, Riggs BL, Spelsberg T 1994 Human
giant cell tumors of the bone (osteoclastomas) are estrogen target cells. Proc
Natl Acad Sci USA 91:5227–5231
8. Kameda T, Mano H, Yuasa T, Mori Y, Miyazawa K, Shiokawa M, Nakamaru
Y, Hiroi E, Hiura K, Kameda A, Yang NN, Hakeda Y, Kumegawa M 1997
Estrogen inhibits bone resorption by directly inducing apoptosis of the bone-
resorbing osteoclasts. J Exp Med 186:489–495
9. Craig AM, Denhardt DT 1991 The murine gene encoding secreted phospho-
protein 1 (osteopontin): promoter structure, activity, and induction in vivo by
estrogen and progesterone. Gene 100:163–171
10. Mathieu E, Merregaert J 1994 Characterization of the stromal osteogenic cell
line MN7: mRNA steady-state level of selected osteogenic markers depends on
cell density and is influenced by 17 beta-estradiol. J Bone Miner Res 9:183–192
11. Bain SD, Bailey MC, Edwards MW 1992 The anabolic effect of estrogen on
endosteal bone formation in the mouse is attenuated by ovariohysterectomy:
a role for the uterus in the skeletal response to estrogen? Calcif Tissue Int
51:223–228
12. Wronski TJ, Yen CF, Qi H, Dann LM 1993 Parathyroid hormone is more
effective than estrogen or bisphosphonates for restoration of lost bone mass in
ovariectomized rats. Endocrinology 132:823–831
13. Lindsay R, Tohme JF 1990 Estrogen treatment of patients with established
postmenopausal osteoporosis. Obstet Gynecol 76:290–295
14. Astedt B 1981 On the role of estrogens in endometrial carcinogenesis. Acta
Obstet Gynecol Scand Suppl 106:33–35
15. Gambrell RD Jr 1982 The menopause: benefits and risks of estrogen-proges-
togen replacement therapy. Fertil Steril 37:457–474
16. Oldberg A, Franzen A, Heinegard D 1986 Cloning and sequence analysis of
rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding
sequence. Proc Natl Acad Sci USA 83:8819–8823
17. Oldberg A, Franzen A, Heinegard D 1988 The primary structure of a cell-
binding bone sialoprotein. J Biol Chem 263:19430–19432
18. Butler WT 1989 The nature and significance of osteopontin. Connect Tissue Res
23:123–136
19. Fujisawa R, Kuboki Y 1991 Preferential adsorption of dentin and bone acidic
proteins on the (100) face of hydroxyapatite crystals. Biochim Biophys Acta
1075:56–60
20. Fujisawa R, Wada Y, Nodasaka Y, Kuboki Y 1996 Acidic amino acid-rich
sequence as binding sites of osteonectin to hydroxyapatite crystals. Biochim
Biophys Acta 1292:53–60
21. Kasugai S, Fujisawa R, Waki Y, Miyamoto K, Ohya K 2000 Selective drug
delivery system to bone: small peptide (Asp)6 conjugation. J Bone Miner Res
15:936–943
22. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige
S, Gotoh Y, Nishida E 1995 Activation of the estrogen receptor through
phosphorylation by mitogen-activated protein kinase. Science 270:1491–1494
23. Waki Y, Miyamoto K, Kasugai S, Ohya K 1995 Osteoporosis-like changes in
Walker carcinoma 256-bearing rats, not accompanied with hypercalcemia or
parathyroid hormone-related protein production. Jpn J Cancer Res 86:470 –476
24. Yamaoka K, Nakagawa T, Uno T 1978 Statistical moments in pharmacoki-
netics. J Pharmacokinet Biopharm 6:547–558
25. Seki K, Minami Y, Nishikawa M, Kawata S, Miyoshi S, Imai Y, Tarui S 1983
“Nonalcoholic steatohepatitis” induced by massive doses of synthetic estro-
gen. Gastroenterol Jpn 18:197–203
26. Williams CL, Stancel GM 1996 Estrogens and progestins. In: Hardman JG,
Limbird LE (eds) The Pharmacological Basis of Therapeutics. McGraw-Hill,
New York, pp 1411–1440
27. Kuiper GG, Enmark E, Pelt-Huikko M, Nilsson S, Gustafsson JA 1996
Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc
Natl Acad Sci USA 93:5925–5930
28. Mosselman S, Polman J, Dijkema R 1996 ER beta: identification and char-
acterization of a novel human estrogen receptor. FEBS Lett 392:49–53
29. Pettersson K, Grandien K, Kuiper GG, Gustafsson JA 1997 Mouse estrogen
receptor beta forms estrogen response element-binding heterodimers with
estrogen receptor alpha. Mol Endocrinol 11:1486–1496
30. Loose-Mitchell DS, Chiappetta C, Stancel GM 1988 Estrogen regulation of
c-fos messenger ribonucleic acid. Mol Endocrinol 2:946–951
31. Morishita S, Niwa K, Ichigo S, Hori M, Murase T, Fujimoto J, Tamaya T 1995
Overexpressions of c-fos/jun mRNA and their oncoproteins (Fos/Jun) in the
mouse uterus treated with three natural estrogens. Cancer Lett 97:225–231
EFFECTS OF ESTRADIOL-OLIGOPEPTIDE CONJUGATE ON OSTEOPOROSIS 1233
