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Endocrinology Vol. 142, No. 3 1228-1233
Copyright © 2001 by The Endocrine Society

ARTICLES

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 {alpha}) 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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{beta}-estradiol (E2) linked at position 3 with L-Asp-hexapeptide via succinate and examined its pharmacokinetics and pharmacodynamics in ovariectomized (OVX) mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
17{beta}-Estradiol (E2) was purchased from Wako Pure Chemicals Co. (Osaka, Japan). 17{beta}-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. 1Go. 17{beta}-Estradiol Correlate-CLIA Kit was purchased from Assay Designs Inc. (MI). [3H]-17 {beta}- E2 was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK).



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  		Figure 1. Structure of 3{beta}-estradiol-3-succinate-(L-Asp)6 (E2·3D6).

 
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{beta}-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{beta}-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{alpha} cDNA isolated from pBacPAK9-HEGO by digestion with BamHI and XhoI was kindly provided by Kato et al. (22). ER{beta} cDNA was isolated from pGEX-4T-2ER{beta} 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{alpha} or ER{beta} in estrogen receptor binding competition experiments.

A solution of ER{alpha} or ER{beta} (10 µl, 40 µg protein) containing [3H]-17 {beta}- 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 {alpha}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 {alpha}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. {beta}-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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Disposition pharmacokinetics of E2·3D6 in mice
Figure 2Go 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{beta}-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. 2Go and Table 1Go, 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 ({circ}) 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

 
Figure 2Go 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 3Go 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.



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  		Figure 3. Apparent tissue-to-plasma concentration ratios (Kp,app) of E2 at 360 min after a single iv administration of E2 ({square}) or E2·3D6 ({blacksquare})(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.

 
Binding affinity of E2·3D6 to human estrogen receptors
Figure 4Go shows the profiles of inhibition by E2 and E2·3D6 of [3H]E2 binding to ER{alpha} and ER{beta}. These results indicate that the binding affinities of E2·3D6 to these receptors were about 1/100 of those of E2.



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  		Figure 4. Binding affinity of E2 and E2·3D6 to human estrogen receptors, ER{alpha} (A) and ER{beta} (B). Data indicate binding % of [3H]E2 (2.5 pmol) to each receptor in the presence of various concentrations of E2 ({circ}) or E2·3D6 (•).

 
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. 5Go). 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.

 
Figure 6Go 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. 7Go). Figure 8Go 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).

 
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 {alpha}, 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. 9Go).



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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. {beta}-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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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. 2Go and 3Go). 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. 2Go), 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. 5–7GoGoGo). On the other hand, E2·3D6 affected only the bone, exhibiting inhibition of the decreases in the BMD (Fig. 7Go) and the number of trabecular bones (Fig. 8Go), 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. 3Go). There are at least two estrogen receptors, ER{alpha} and ER{beta} (27, 28, 29). We confirmed that the affinities of E2·3D6 for human ER{alpha} and ER{beta} were very much lower than those of E2 (Fig. 4Go). 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 {alpha} and bone sialoprotein was observed in bone at 4 h after E2·3D6, but not after E2 (Fig. 9Go). 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. 2BGo) 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. 4Go). 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.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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E. Bonnelye, V. Kung, C. Laplace, D. L. Galson, and J. E. Aubin
Estrogen Receptor-Related Receptor {alpha} Impinges on the Estrogen Axis in Bone: Potential Function in Osteoporosis
Endocrinology, September 1, 2002; 143(9): 3658 - 3670.
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