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Osteotropic Peptide That Differentiates Functional Domains of the Skeleton Dong Wang,†,‡,⊥ Scott C. Miller,§,⊥ Luda S. Shlyakhtenko,† Alexander M. Portillo,† Xin-Ming Liu,† Kongnara Papangkorn,‡ Pavla Kopecˇkova´,‡ Yuri Lyubchenko,† William I. Higuchi,‡ and Jindrˇich Kopecˇek*,‡,| Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska 68198-6025, Department of Pharmaceutics and Pharmaceutical Chemistry and Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112, Department of Radiology/Radiobiology Division, University of Utah, Salt Lake City, Utah 84108. Received June 14, 2007; Revised Manuscript Received July 27, 2007
HPMA copolymer-D-aspartic acid octapeptide (D-Asp8) conjugates have been found to target the entire skeleton after systemic administration. In a recent study using the ovariectomized rat model of osteoporosis, we surprisingly discovered that D-Asp8 would favorably recognize resorption sites in skeletal tissues, while another bone-targeting moiety, alendronate (ALN), directs the delivery system to both formation and resorption sites. Atomic force microscopy (AFM) analyses reveal that ALN has a stronger binding force to hydroxyapatite (HA) than D-Asp8. In Vitro HA binding studies indicate that D-Asp8 is more sensitive to change of HA crystallinity than ALN. Because the bone apatite in the newly formed bone (formation sites) usually has lower crystallinity than the resorption sites (mainly mature bone), we believe that the favorable recognition of D-Asp8 to the bone resorption sites could be attributed to its relatively weak binding to apatite, when compared to bisphosphonates, and the different levels of crystallinity of bone apatite at different functional domains of the skeleton.
Bone is a specialized connective tissue, which provides mechanical support and participates in calcium homeostasis. It is continuously being resorbed and rebuilt to maintain its normal function. Disturbances of this resorption/formation balance are characteristic of most bone diseases (1-3). With ever-increasing understanding of bone biology, many new therapeutic agents have been identified for the treatment of bone diseases (415). However, most of them do not have tissue specificity to the skeleton (osteotropicity), which hampers their clinical application due to the side effects. In order to overcome this problem, we have developed bonetargeting water-soluble polymeric drug delivery systems. In the initial study, alendronate and D-Asp8 were used as the bonetargeting moieties and conjugated to fluorescein isothiocyanate (FITC)-labeled N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers (Figure 1A). The administration of these polymers to young growing balb/c mice led to their strong deposition to the skeleton, especially at high turnover sites in bone as determined histologically (16). A later pharmacokinetics and biodistribution study confirmed that HPMA copolymer using D-Asp8 as the bone-targeting moiety could recognize the skeleton, especially the high bone turnover sites, such as tibia and femur heads, lumbar vertebrae, and mandibular bone (17). To investigate whether the delivery system could deliver model drugs to the osteopenic skeleton, FITC-labeled HPMA copoly* Correspondence should be addressed to Jindrˇich Kopecˇek, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 30 S. 2000 E. Rm. 201, Salt Lake City, Utah 84112-5820, USA; Phone: (801) 581-7211; Fax: (801) 581-7848. E-mail:
[email protected]. † Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center. ‡ Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah. § Department of Radiology/Radiobiology Division, University of Utah. | Department of Bioengineering, University of Utah. ⊥ These authors contributed equally to this work.
mer-alendronate conjugate (P-ALN-FITC) and FITC-labeled HPMA copolymer-D-Asp8 conjugate (P-D-Asp8-FITC) were administered intravenously to ovariectomized (OVX) rats, a common model of postmenopausal osteoporosis. To allow the development of osteopenia, the OVX procedure was performed 3 months prior to the beginning of the study. To assist in the identification of different functional domains on bone surfaces, tetracycline (a fluorochrome marker of bone formation) (18) was administered intraperitoneally (20 mg/kg) 3 days prior to the administration of the conjugates. Twenty-four hours after administration of the conjugates, the OVX rats (three/group) were euthanized. Tibias, femurs, fifth lumbar vertebra, and mandibles were isolated, fixed, and processed for undecalcified histomorphometric analyses. As shown in Figure 1B and C, both conjugates were found to bind well to some bone surfaces in the OVX rats, which agrees with a previous study using young balb/c mice (16). The tetracycline marker labeled bone formation surfaces permitted easier identification of both bone formation and bone resorption sites. The presence of osteoclasts on the resorption surface and osteoblasts on the formation surfaces was confirmed by histology. Surprisingly, P-D-Asp8-FITC was found to preferentially bind to resorption surfaces, while P-ALN-FITC appeared to bind well at both formation and resorption surfaces. Such a result has never been reported before. As we examine the zone of dentine calcification, the binding of the P-ALN-FITC appeared to be stronger than P-D-Asp8-FITC (Figure 1D and E) in the continuously erupting incisor in the mandible. Because of this intriguing observation, we hypothesized that the binding strength of D-Asp8 to bone apatite (a poorly crystallized, CO3-containing, Ca-deficient hydroxyapatite analog) (19) may be weaker than that of alendronate. Therefore, it could be sensitive to the crystallinity of bone apatite. It is known that certain pathophysiological conditions such as Paget’s disease (20) and osteoporosis (21) can result in changes of the size of hydroxyapatite crystals. It is also understood that the crystallinity of newly formed bone mineral at active bone
10.1021/bc7002132 CCC: $37.00 © 2007 American Chemical Society Published on Web 08/17/2007
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Figure 2. AFM amplitude image of tooth enamel surface taken in PBS buffer using nonmodified tip. Bar ) 2 µm.
Figure 1. Different in ViVo bone-binding abilities of HPMA copolymers conjugated with alendronate (P-ALN-FITC) and D-aspartic acid octapeptide (P-D-Asp8-FITC). (A) Chemical structures of the osteotropic polymer conjugates. (B) Cancellous bone from an ovariectomized (OVX) rat injected with the bone formation marker tetracycline (TC) and the P-ALN-FITC. Both tetracycline (TC, yellow) and P-ALN-FITC (green) are seen on some surfaces (arrows) indicating uptake on boneforming surfaces. A selected area is magnified (×2, insert) to show the double label of TC and P-ALN-FITC. P-ALN-FITC is also evident on some resorption surfaces (double arrows). Bar ) 100 µm. (C) Cancellous bone from an OVX rat injected with TC and the P-D-Asp8FITC. Uptake of TC but little of P-D-Asp8-FITC is evident on bone formation surfaces (arrows). P-D-Asp8-FITC is evident on bone resorption surfaces (double arrows). Bar ) 100 µm. (D) Cross sections of the forming mandibular incisor illustrating the uptake of the P-ALNFITC. Bar ) 50 µm. (E) Cross sections of the forming mandibular incisor illustrating the uptake of the P-D-Asp8-FITC. Bar ) 50 µm. The TC label (yellow) is clearly evident in both sections (arrows). There is more apparent uptake of P-ALN-FITC (green label, double arrows) in the mineralizing dentin than uptake of P-D-Asp8-FITC.
formation sites is lower than that at the more mature sites where resorption occurs (20). This may help to explain the observed preferential binding of P-D-Asp8-FITC to resorption surfaces in skeletal tissues. To test this hypothesis, we employed force spectroscopy to directly measure the interaction of alendronate and D-Asp8 with hydroxyapatite. Since the AFM force measurement requires a rather flat substrate, we chose tooth enamel (Figure 2) as a model of hydroxyapatite surface. Alendronate or D-Asp8 was covalently attached to the AFM probes via glutaraldehyde crosslinks (22-24). Force-distance measurements were performed in PBS buffer at room temperature at identical experimental conditions. Force curves were taken at various random locations on the surface in order to account for any heterogeneity on the surface. Although both modified tips clearly indicated the tipsurface interaction (Figure 3A and B), the most frequent force-
distance curves were different for alendronate and D-Asp8. The force distance curves for the D-Asp8-modified tip more often showed a single adhesion peak, whereas the alendronatemodified tip often had two clear peaks, including adhesion. Each individual force plot was analyzed one at a time. The rupture force and the distance for each peak were also measured. The results obtained for a series of measurements of adhesion forces for the tips modified with alendronate and D-Asp8, respectively, are shown as histograms in Figure 3C and D. Both histograms demonstrate the interaction of modified tips with the tooth enamel surface, but they are clearly different. For alendronate, the distribution of forces is wide with majority of the forces recorded around 190 pN. For D-Asp8, the distribution is narrower with the majority of the forces recorded around 105 pN. In addition to the analyses of adhesion forces at zero distances, secondary peaks at distances away from the adhesion peak were also analyzed and shown in Figure 3E and F. Clearly, the data reveal a more pronounced difference in the interaction patterns for alendronate and D-Asp8. First, the number of secondary events was considerably larger for alendronate compared to D-Asp8. Second, the values for rupture forces were higher for alendronate than for D-Asp8. An additional difference was the position of the secondary peak. This effect is graphically illustrated in Figure 3G and H, on which the histograms for the distance distribution are plotted for alendronate and D-Asp8, respectively. The positions for the secondary rupture events are centered around 40 nm for alendronate, whereas the secondary peaks for D-Asp8 are scattered but with the majority of the events at distances of ∼10 nm. In spite of the heterogeneity in chemical composition and roughness of the enamel surface (Figure 2), which may widen the histograms for the interaction forces and distances, these AFM data strongly suggest that alendronate interacts more efficiently with the tooth enamel surface and has less sensitivity to the surface topography in comparison with D-Asp8. To further test the possibility that alendronate and D-Asp8 will bind differently to hydroxyapatite with different crystallinity, we synthesized two hydroxyapatites (HAa, high crystallinity, and HAb, low crystallinity; Table 1) (25, 26) for the in Vitro binding test. Solubility studies and X-ray diffraction evaluation of these hydroxyapatites indicate that the lattice disorder of the bulk mineral phase (which is reflected by the crystallite microstrain parameter value) correlates well with the lattice disorder at the crystal-solution interface (to be published). So, it would be reasonable to expect that the binding behavior of molecules to apatite surfaces can be dependent upon the lattice disorder of the bulk mineral phase. PBS solutions of P-ALN-FITC and P-D-Asp8-FITC were incubated with HAa and HAb. After removal of HA powders from the solutions, the percentage of the FITC-labeled conjugates bound to HAs was calculated from the decreased UV absorbance of the polymer solutions. As shown in Figure 4, the higher absolute binding
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Figure 3. AFM analyses of different interaction between osteotropic compounds (alendronate and D-Asp8) and tooth enamel surface. (A) Typical force-distance curve shows the interaction between alendronate modified tip and tooth enamel surface. (B) Typical force-distance curve shows the interaction between D-Asp8 modified tip and tooth enamel surface. (C) Histogram for rupture forces obtained for the force spectroscopy measurements with the tips functionalized with alendronate. (D) Histogram for rupture forces obtained for the force spectroscopy measurements with the tips functionalized with D-Asp8. (E) Histogram of the secondary rupture force events of Alendronate. (F) Histogram of the secondary rupture force events of D-Asp8. (G) Histogram of the rupture events for all distances except at zero for alendronate. (H) Histogram of the rupture events for all distances except at zero for D-Asp8. The most probable values obtained from the Gaussian fit for the histograms, which combine the analyses for more than 300 events are indicated in the inserts. No Gaussian fit could be obtained for (H). Table 1. Characterization of Synthetic Hydroxyapatite with Different Crystallinity crystallite specific CO32- PO43- Ca2+ Na+ microstrain surface sample (wt %) (wt %) (wt %) (wt %)a Ca/P parameter (X) area (m2/g) HAa HAb
0.5 8.7
53.9 45.4
38.1 37.3
0.23 0.58
1.68 1.95
0.17 0.61
26.7 30.0
a The remaining contents in HA and Ha are mainly water, hydroxide, a b and trace elements (27).
experiments were not performed on bone apatite, the data support the hypothesis that it is the weak binding ability of D-Asp8 to apatite that causes its in ViVo selectivity to the bone resorption surface (containing bone apatite with relatively higher crystallinity) over formation surfaces (mainly amorphous calcium phosphate) (19). Incorporation of molecular structures (e.g., tetracycline, D-Asp8) that could recognize different subtissue functional domains (osteolytic and osteoblastic) (18) into the new drug design and osteotropic drug delivery systems would greatly enhance the therapeutic index by delivering the drug directly to the desired cells and molecular targets.
ACKNOWLEDGMENT We acknowledge financial support from the College of Pharmacy, University of Nebraska Medical Center (D.W., L.S.S., A.M.P., X.M.L., and Y.L.) and NIH Grant GM069847 (S.C.M., P.K., and J.K.). Supporting Information Available: Detailed AFM procedures are described in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Figure 4. Binding ability of P-ALN-FITC and P-D-Asp8-FITC to hydroxyapatites with different crystallinity. [ALN] ) 3.2 × 10-5 mol/g for P-ALN-FITC; [D-Asp8] ) 7.0 × 10-5 mol/g for P-D-Asp8-FITC. HAa (9): microstrain ) 0.05 ( 0.03 (high crystallinity). HAb (0): microstrain ) 0.61 ( 0.03 (low crystallinity). The different crystallinity of hydroxyapatites caused 9% difference in binding for P-ALN-FITC and 22% difference for P-D-Asp8-FITC.
percentage of P-ALN-FITC to HAs, when compared to P-DAsp8-FITC, is probably confirming the stronger binding force of alendronate than D-Asp8. Nevertheless, the binding of P-ALN-FITC to HAs is less sensitive to the change of apatite crystallinity compared to P-D-Asp8-FITC. Evidently, the AFM study suggests that alendronate has stronger binding to hydroxyapatite than D-Asp8. Such differences in their ability to bind to hydroxyapatite coupled with their differences in binding with changes in apatite crystallinity would provide an explanation for the preferential binding of D-Asp8 to hydroxyapatite with higher crystallinity. Though these
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