Endocrinology Vol. 142, No. 3 1228-1233
Copyright © 2001 by The Endocrine Society
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
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{at}kenroku.ipc.kanazawa-u.ac.jp
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Abstract
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We have developed a novel osteotropic prodrug of estradiol
(E2) conjugated with L-Asp-hexapeptide
(E2·3D6), which has very low affinity for
estrogen receptors, and in this study, we examined its pharmacokinetic
behavior and pharmacological potential. After a single iv injection of
E2·3D6 to mice, the half-time for elimination
from plasma was about 100 min; however, E2 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
E2, the half-time in plasma was about 70 min, whereas
E2 was highly distributed to the uterus, and the bone
concentration of E2 was only slightly increased at 6
h. When E2 (0.37 µmol/kg, sc, every third day) or
E2·3D6 (0.11 to 1.1 µmol/kg, sc, every
seventh day) was administered to OVX mice for 4 weeks, E2
increased the bone mineral density (BMD) together with weights of liver
and uterus, whereas E2·3D6 increased only the
BMD, in a dose-dependent manner. E2·3D6
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 E2 did very slightly.
These results indicate that the E2 prodrug was delivered to
the bone, where it gradually released E2, thereby
ameliorating bone loss. This acidic oligopeptide appears to be a good
candidate for selective drug delivery to bone.
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Introduction
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OSTEOPOROSIS is a serious problem for
postmenopausal 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, 2, 3, 4, 5), such as inhibition of
production of bone-resorptive cytokines from bone marrow stromal cells
(6), direct inhibition 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 (E2) linked at
position 3 with L-Asp-hexapeptide via succinate
and examined its pharmacokinetics and pharmacodynamics in
ovariectomized (OVX) mice.
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Materials and Methods
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Materials
17-Estradiol (E2) was purchased from
Wako Pure Chemicals Co. (Osaka, Japan).
17-Estradiol-3-succinate-(L-Asp)6
(E2·3D6) was synthesized
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).
[3H]-17 - E2 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
pharmacokinetic study, E2 solution in 30%
ethanol or E2·3D6 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 µmol/kg in
a volume of 50 µl. The blood samples were collected from the
intraorbital venous plexus using heparinized capillary tubes under
light diethyl ether anesthesia at designated time intervals. The plasma
was separated by 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 pentobarbital
anesthesia and divided into six groups; sham-operated control,
untreated control, E2 (0.37 µmol/kg),
E2·3D6 (0.11, 0.37, 1.1
µmol/kg), six in a group. From 8 weeks after the operation,
E2 and
E2·3D6 were sc injected
at the hind back every third day and every seventh day, respectively.
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 E2 and
E2·3D6
E2 in plasma was assayed with a
17-Estradiol Correlate-CLIA Kit according to the manufacturer’s
instructions. After administration of E2 and
E2·3D6, tissues were
digested in 1 N NaOH at 60 C for 1 h and neutralized
with 1 N HCl, then E2 was extracted
with diethyl ether. Bone was incubated in 6 N HCl at 60 C
for 1 h and neutralized with 6 N NaOH, then
E2 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 µl of the assay buffer and
E2 was assayed with the 17-Estradiol
Correlate-CLIA Kit.
Before assay, we confirmed, using HPLC, that
E2·3D6 was stable in
blood, whereas in 1 N NaOH and 6 N HCl solution
at 60 C it was completely hydrolyzed to release free
E2 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., Gaithersburg, 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
transfected 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 supernatant was used as human recombinant
ER or ER in estrogen receptor binding competition
experiments.
A solution of ER or ER (10 µl, 40 µg protein) containing
[3H]-17 - E2 (2.5
pmol) was supplemented with various concentrations of
E2 or
E2·3D6 in a total volume
of 250 µl. 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
pentobarbital anesthesia at 14 days before use.
E2 and
E2·3D6 were sc injected
at a dose of 0.37 µmol/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 mM KCl, 50 mM
Tris-HCl (pH 8.3), 6 mM MgCl2, 10
mM dithiothreitol, 0.6 mM each 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 µg of RNA in a final volume of 50 µl at 37 C for
1 h. PCR was conducted in final volume of 20 µl containing 1
µl of RT mixture, 50 mM KCl, 20 mM Tris-HCl
(pH 8.3), 1.75 mM MgCl2, 0.25
mM each dATP, dTTP, dGTP, dCTP mixture, 1 µM
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 data were analyzed using Student’s
t test 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.
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Results
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Disposition pharmacokinetics of E2·3D6 in
mice
Figure 2 shows the plasma
concentration-time courses after a single iv injection of
E2 or
E2·3D6 (3.7 µmol/kg).
The behaviors of E2 and
E2·3D6 were biphasic,
with half-times for the elimination phase of about 70 and 100 min,
respectively. After injection of E2, the plasma
concentration of E2 decreased to the endogenous
E2 level (7.6 ± 1.9 pmol/ml, mean ±
SE, n = 8) by 360 min. After injection of
E2·3D6, 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 E2 level was unchanged
up to 360 min (data not shown). As shown in Fig. 2 and Table 1, unchanged
E2·3D6 slowly decreased.
The value of the area under the plasma concentration-time curve (AUC)
was significantly higher than that of E2 and the
value of the total clearance (CLtot) was lower than that of
E2. The distribution volume at the steady-state
(Vdss) of E2·3D6 was a
little lower than that of E2.
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Figure 2. Time courses of plasma (A) and femur (B)
concentration of E2 after a single iv administration of
E2 ( ) and E2·3D6 (•) (3.7
µmol/kg) to normal mice. Each point with bar
represents the mean ± SE of three animals.
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Table 1. Pharmacokinetic parameters of E2 and
E2 · 3D6 after a single intravenous injection
at a dose of 3.7 µmol/kg in mice
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Figure 2
also shows the bone concentration-time course of
E2 after injection of
E2·3D6. 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 E2 injection the bone
concentration of E2 increased a little within 360
min, then declined to within the basal range.
Figure 3 shows the apparent
tissue-to-plasma concentration ratio (Kp,app) at 360 min after a single
injection of E2 or
E2·3D6.
E2 was distributed most highly to the uterus
among all the organs examined. After
E2·3D6 injection,
E2 concentration in the bone was the highest,
although the concentrations of E2 in other organs
tended to be lower than those after E2.
Binding affinity of E2·3D6 to human
estrogen receptors
Figure 4 shows the profiles of
inhibition by E2 and
E2·3D6 of
[3H]E2 binding to ER
and ER. These results indicate that the binding affinities of
E2·3D6 to these receptors
were about 1/100 of those of E2.
Effects of E2·3D6 on OVX mice
To clarify the bone-selective effect of
E2·3D6, OVX mice were
treated with 0.11, 0.37, or 1.1 µmol/kg of
E2·3D6 every seventh day
or 0.37 µmol/kg of E2 every third day for 28
days. There were no large changes of body and major organs 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 E2 was significantly higher than
that of the E2·3D6 group,
whereas the liver weight was hardly changed after OVX (Fig. 5). The treatment with
E2 induced a vacuolar degeneration histologically
resembling fatty liver.
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Figure 5. Effects of E2 and
E2·3D6 on the liver weight of OVX mice. OVX
mice were treated with the indicated dose of E2 (every
third day) or E2·3D6 (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.
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Figure 6 shows the uterine weight in each
group. The uterine weight of the OVX-untreated control mice was
significantly decreased compared with that of sham-operated mice.
E2 treatment almost completely restored the
uterine weight, whereas
E2·3D6 treatment tended
to increase the uterine weight, though the change was not significant
compared with that of the untreated-control group. Although the BMD of
femurs from untreated control mice was significantly decreased compared
with that of the sham-operated mice, the treatment with
E2 almost completely inhibited the decrease of
BMD. Similarly, E2·3D6
significantly inhibited the decrease of femur BMD of OVX mice in a
dose-dependent manner (Fig. 7). Figure 8 shows that
E2·3D6 (0.37 µmol/kg,
every third day) inhibited the loss of trabecular bone of OVX mice, as
well as E2·3D6 (0.37
µmol/kg, every seventh day).
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Figure 6. Effects of E2 and
E2·3D6 on the uterine weight of OVX mice. OVX
mice were treated with the indicated dose of E2 (every
third day) or E2·3D6 (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.
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Figure 7. Effects of E2 and
E2·3D6 on the femoral BMD of OVX mice. OVX
mice were treated with the indicated dose of E2 (every
third day) or E2·3D6 (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 < 0.05 and 0.01, respectively.
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Figure 8. Microscopic observations of the soft tissue-free
femur of OVX mice. OVX mice were treated with the indicated dose of
E2 (every third day) or E2·3D6
(every seventh day) for 28 days, after treatment femur was removed and
incubated in 1 N NaOH over night, and then photographed
under a microscope. A, Sham-operated control; B, untreated control, C,
E2 (0.37 µmol/kg); D, E2·3D6
(0.37 µmol/kg).
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Effects of E2 and E2·3D6 on
expression of bone-matrix protein mRNAs
To confirm the bone-selective effect of
E2·3D6, expression of
bone-matrix protein mRNAs in bone was examined using RT-PCR method
4 h after iv injection of E2 or
E2·3D6 (0.37 µmol/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 E2, 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
E2·3D6 (Fig. 9).
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Figure 9. RT-PCR of mRNAs of bone matrix proteins in femur
of animals treated with E2 or
E2·3D6. OVX mice were sc injected with
E2 or E2·3D6 at a dose of 0.37
µmol/kg. A, Typical electrophoresis. Lane 1, untreated control; lane
2, E2 4 h; lane 3, E2·3D6
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.
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Discussion
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In this study, we examined the pharmacokinetics and
pharmacodynamics of a new type of E2 prodrug,
which conjugated with E2 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
E2 in OVX mice.
After iv administration at a dose of 3.7 µmol/kg,
E2·3D6 was not detectably
degraded in blood within 360 min, and its AUC was larger and its
clearance was slower than those of E2, but its
distribution to various tissues except for bone was smaller than that
of E2 (Figs. 2 and 3). These results suggest that
E2·3D6 was taken up less
into the soft tissues than E2, presumably because
of increased hydrophilicity due to the acidic peptide moiety in the
molecule. On the other hand, after injection of
E2·3D6,
E2 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
E2·3D6 are selective for
bone, compared with those of E2, using OVX mice.
The OVX mice exhibited severe uterus atrophy and bone loss by 12 weeks.
When E2 was administered at a dose of 0.37
µmol/kg every third day for the last 4 weeks, the changes of uterine
weight and BMD in OVX mice were almost completely inhibited, and the
liver weight was significantly increased (
Figs. 5–7). On the other
hand, E2·3D6 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, E2 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 nonalcoholic
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
E2 enlarged the liver, which histopathologically
showed fatty degeneration, and restored the uterine weight of OVX mice
to the level of sham-operated mice, whereas
E2·3D6 did not affect
these organs. One reason might be the smaller number of injections and
longer intervals of treatment with
E2·3D6 than with
E2. However, after a single injection of
E2·3D6, the distribution
of E2 in these organs was similar to that of
E2 after injection of E2
(Fig. 3). There are at least two estrogen receptors, ER and ER
(27, 28, 29). We confirmed that the affinities of
E2·3D6 for human ER
and ER were very much lower than those of E2
(Fig. 4). From these results, it is suggested that the conjugated
E2 may not cause the systemic adverse reactions
of E2, which include carcinogenesis (30, 31).
On the other hand, estrogen acts directly on osteoblasts through
estrogen receptor-mediated mechanisms and stimulates 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
E2·3D6, but not after
E2 (Fig. 9). This indicates that
E2·3D6 acted on the
osteogenic cells for longer time than E2.
Although we measured
E2·3D6 in tissues as
E2 after complete hydrolysis,
E2 retained in the bone (over 100-fold more than
E2 after E2 administration)
(Fig. 2B) was considered to be present as the unchanged form of
E2·3D6, bound to HA,
because of its prolonged residence time in the bone. It is unlikely
that E2·3D6 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 E2) (Fig. 4).
Consequently, we speculate that
E2·3D6 distributed to
bone was gradually hydrolyzed at the bone surface, possibly by
peptidases and/or acid secreted from osteoclasts, releasing
E2, which was transported into the bone cells and
acted on the estrogen receptor(s).
In conclusion, this study indicated that our
E2-prodrug, consisting of
E2 conjugated with a novel acidic oligopeptide
carrier, is a promising candidate as an osteotropic drug, for
estrogen-replacement therapy of postmenopausal osteoporosis, because of
its selective and long-term action on bone without the adverse side
effects of E2. The use of
E2·3D6 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.
Received May 30, 2000.
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Abstract
Introduction
) or
E2·3D6 (
)(3.7 µmol/kg) to normal mice.
Each column with bar represents the mean ±
SE of three animals. *, Significantly different from
E2 at P < 0.01.