Synthesis and Evaluation of Water-Soluble Polymeric Bone-Targeted

system for the treatment of bone diseases are obvious. ... Phone: + 801-581-4532. ..... A. Rodan, Eds.) pp 109-126, Academic Press, San Diego. (b). VÃ...
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Bioconjugate Chem. 2003, 14, 853−859

853

Synthesis and Evaluation of Water-Soluble Polymeric Bone-Targeted Drug Delivery Systems Dong Wang,† Scott Miller,§ Monika Sima,† Pavla Kopecˇkova´,† and Jindrˇich Kopecˇek*,†,‡ Department of Pharmaceutics and Pharmaceutical Chemistry/CCCD, Department of Bioengineering, Department of Radiobiology, University of Utah, Salt Lake City, Utah 84112, USA. Received May 23, 2003

Four polymeric bone-targeting conjugates were synthesized based on poly(ethylene glycol) (PEG, two conjugates) and poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA, two conjugates). The well-known bone-targeting compounds, alendronate and aspartic acid peptide, were used as bone-targeting moieties. Fluorescein isothiocyanate (FITC) was attached to the conjugates as a model drug for detection purposes. The bone-targeting potential of these conjugates was tested in vitro with hydroxyapatite (HA) and in mice. The data obtained indicated that these novel delivery systems could specifically accumulate in the bone tissue.

Bone is a highly specified form of connective tissue, which provides an internal support system in all vertebrates. It is also the major source of inorganic ions and actively participates in calcium homeostasis in the body (1). To maintain its normal function, bone is continuously being resorbed and rebuilt throughout the skeleton. In healthy individuals, bone resorption and formation are well balanced with the bone mass maintained in a steady state. Disturbances of this balance are characteristic of a number of bone diseases including osteoporosis, Paget’s disease, osteopetrosis, bone cancer, etc. (2). Over the past decade, our understanding of bone biology has improved dramatically (3, 4). Many molecules have been identified as new therapeutic agents for the treatment of bone diseases. Osteoprotegerin (OPG) (5), cathepsin K inhibitors (6), carbonic anhydrase II (CA2) inhibitors (7), Rvβ3 integrin antagonists (8), and Src (protein tyrosine kinase pp60c-Src) homology 2 inhibitors (9) have been studied for their antiresorptive activities. Prostaglandin E series (10), prostaglandin E EP4 receptor agonists (11), statins (12), parathyroid hormone (PTH) (13), and growth factors [including transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP)] (2) have all been considered for stimulation of bone growth. However, most of these therapeutic agents are not specific for bone, which can greatly hamper their clinical application to bone disease. The recent reports on the long-term effects of hormone replacement therapy (HRT) clearly demonstrated the adverse effects that can occur when bone therapeutic agents are not specifically targeted (14, 15). The advantages of a bone-targeted drug delivery system for the treatment of bone diseases are obvious. Such a system could easily impart osteotropicity to a * To whom correspondence should be addressed. University of Utah, Department of Pharmaceutics and Pharmaceutical Chemistry, 30 S 2000 E Rm. 301, Salt Lake City, UT 84112. Phone: + 801-581-4532. Fax: + 801-581-3674. E-mail: [email protected]. † Department of Pharmaceutics and Pharmaceutical Chemistry/ CCCD. ‡ Department of Bioengineering. § Department of Radiobiology.

variety of bone drugs and improve their therapeutic potential. A few attempts have been made previously to target drugs to hard tissue. Tetracycline and it analogues were linked to different drugs to increase their affinity to bone (16-18). Bisphosphonates were conjugated to different macromolecules (protein, PEG1) and low molecular weight compounds to render them osteotropic (19-21). Recently, glutamic acid and aspartic acid peptides were reported being used as bone-targeting moieties to deliver drugs to the bone (22). (Scheme 1, structures of molecules with strong affinity to bone). In the present study, we designed and synthesized water-soluble polymeric bone-targeting drug delivery systems based on PHPMA1 and PEG. These systems may be used as universal vehicles for the targeted delivery of bone therapeutics. It is hypothesized that this will enable a wide variety of bone therapeutics to be covalently loaded onto these delivery systems via acid- or enzymecleavable spacers. Other benefits derived from these conjugates may include improved pharmacokinetic parameters, such as area under the curve (AUC), and the increased water solubility of hydrophobic drugs. In some 1

Abbreviations: ACV, 4,4′-azobis(4-cyanopentanoic acid);

D-(Asp)8, octapeptide D-aspartic acid; D-(Asp-OtBu)8, octapeptide D-aspartic acid β-tert-butyl ester; FITC-PEG-alendronate, PEG

conjugate with one chain terminus linked to alendronate and the other linked to FITC; FITC-PEG-D-(Asp)8, PEG conjugate with one chain terminus linked to the N terminus of octapeptide D-aspartic acid and the other end linked to FITC; FITC-PEGNHS, heterobifunctional PEG with FITC at one terminus and N-hydroxysuccinimide ester at the other; HA, hydroxyapatite; MA-FITC, N-methacryloylaminopropyl fluorescein thiourea; MA-GG-D-(Asp)8, N-methacryloylglycylglycyl-D-(aspartic acid)8; P-alendronate-FITC, conjugate of P-GG-ONp-FITC with alendronate where alendronate was linked to the polymer via Gly-Gly spacer; P-D-(Asp)8-FITC, copolymer of HPMA, MAGG-D-(Asp)8, and MA-FITC; PEG, poly(ethylene glycol); PEGFITC, hydrolyzed from FITC-PEG-NHS; P-GG-ONp-FITC, FITC (fluorescein isothiocyanate)-labeled HPMA [N-(2-hydroxypropyl)methacrylamide] copolymer containing p-nitrophenyl ester, where P is the HPMA copolymer backbone; P-FITC, aminolyzed from P-GG-ONp-FITC with 2-amino propanol; PHPMA, poly[N-(2-hydroxypropyl)methacrylamide]; TFA, trifluoroacetic acid; Mw, weight average molecular weight;

10.1021/bc034090j CCC: $25.00 © 2003 American Chemical Society Published on Web 08/20/2003

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Scheme 1. Structures of Some Bone-Targeting Compounds

cases, these conjugates may also offer protection to therapeutic agents against degradation as they are transported to their target tissues (23). Alendronate and aspartic acid peptide are welldocumented bone-targeting compounds with strong boneaffinity and a primary amine useful for conjugation (1922). These were selected as bone-targeting moieties in this study. The conjugation of alendronate to P-GG-ONp-FITC1 could only be carried out in aqueous solution due to the poor solubility of alendronate in all organic solvents. The conjugation proceeded with the gradual addition of NaOH solution to a solution of alendronate and P-GG-ONpFITC at 25 °C. The conjugate was purified by column chromatography and dialysis, and then lyophilized.2 A similar procedure was used for the synthesis of FITCPEG-alendronate,1,3 where FITC-PEG-NHS1 (Nektar, formerly, Shearwater, Huntsville, AL) was used instead of P-GG-ONp-FITC. The content of alendronate in the conjugates was analyzed by HPLC, after hydrolysis with HCl (6 N) and a precolumn fluorochrome derivatization. L-Aspartic acid hexapeptide has been used previously as a bone-targeting moiety (22). To ensure proper in vivo stability, D-aspartic acid octapeptide was used in this study. A novel solid-phase synthesis (24) strategy was used for the synthesis of FITC-PEG-D-(Asp)8.1,4 FITC-PEGNHS was conjugated to the deprotected N-terminus of D-(Asp-OtBu)81 with the C-terminus still anchored to the surface of trityl chloride resin. This was followed by TFA1 cleavage to obtain the conjugate. With a similar strategy, 2 Synthesis of P-alendronate-FITC. Alendronate (100 mg, 3.08 × 10-4 mol) was added in water (1 mL). With vigorous stirring of the sample, P-GG-ONp-FITC (50 mg, ONp ) 2.75 × 10-5 mol, in 200 µL of DMF) was dropped into the aqueous solution. NaOH (0.2 M) was then dropped into the solution. Slowly, the pH value was increased to 7. Then, in 1 h, it was further increased to 8. Afterward, the pH value was quickly raised to 9 to finish the reaction. Free ONp and alendronate were removed with PD-10 columns (Amersham Pharmacia Biotech, Piscataway, NJ). The conjugate was then dialyzed against water (MWCO 6 ∼ 8 kDa). It was lyophilized to yield 36 mg of the titled product. 3 Synthesis of FITC-PEG-alendronate. Alendronate (150 mg, 4.6 × 10-4 mol) was added in water (1 mL). With vigorous stirring of the sample, FITC-PEG-NHS (50 mg, NHS ) 2. 5 × 10-5 mol, in 200 µL of DMF) was dropped into the aqueous solution. NaOH (0.2 M) was then dropped into the solution. Slowly, the pH value was increased to 7. Then, in 1 h, it was further increased to 8. Afterward, the pH value was quickly raised to 9 to finish the reaction. Free NHS and alendronate were removed with PD-10 columns (Amersham Pharmacia Biotech, Piscataway, NJ). The conjugate was then dialyzed against water (MWCO 2 kDa). It was lyophilized to yield 51.8 mg of the titled product, which was then analyzed with MALDITOF spectrometry (Supporting Information) and HPLC.

a polymerizable D-(Asp)81 derivative, MA-GG-D-(Asp)8,1 was synthesized5 and copolymerized with HPMA and MA-FITC1 to obtain P-D-(Asp)8-FITC.1,6 These conjugates were purified by column chromatography and dialysis, and then lyophilized. The D-(Asp)8 content in these conjugates was determined by HPLC after hydrolysis with HCl (6 N) and precolumn fluorochrome derivatization. The structures of FITC-PEG-D-(Asp)8 and MA-GG-D-(Asp)8 were also confirmed using MALDI-TOF mass spectrometry (Supporting Information). The characterization of all conjugates described above is summarized in Table 1. Their chemical structures are depicted in Schemes 2 and 3. As shown in Table 1, 90% of FITC-PEG-D-(Asp)8 chains have a D-(Asp)8 oligopeptide attached, whereas all FITC-PEG-alendronate chains have an alendronate appended to the chain terminus. The Mw1 of HPMA copolymer conjugates was determined with size exclusion chromatography (SEC) using PBS (pH ) 7.3) as eluent. 4 Synthesis of FITC-PEG-D-(Asp) . According to typical 8 solid-phase peptide synthesis procedure (24), Fmoc-D-(AspOtBu) (67 mg, 0.162 mmol) was loaded onto trityl chloride resin (300 mg, 0.324 mmol of -Cl, 50% loading). Stepwise growing procedure was followed until all eight Fmoc-D-(Asp-OtBu) had been connected. As the NH2 of the final Fmoc-D-Asp-OtBu was exposed by piperidine (20%), NHS-PEG-FITC (MW 2000, 400 mg, 0.2 mmol, in 2.5 mL DMF) and DIPEA (113 µL, 0.648 mmol) were added. The suspension was transferred into an ampule, purge with N2, and sealed by flame. It was agitated gently for 2 days. Then the resin was dried and cleaved with TFA. The raw product was dialyzed (MWCO 2000) and further purified on FPLC with Superdex 75 column. About 90 mg of FITCPEG-D-(Asp)8 was obtained. The structure and purity of the conjugate were confirmed with MALDI-TOF mass spectrometry (Supporting Information) and amino acid analysis. 5 Synthesis of MA-GG-D-(Asp) . D-(Asp-OtBu) protected pep8 8 tide was synthesized on solid phase as described in footnote 4. After the NH2 of the final Fmoc-D-Asp-OtBu was exposed with piperidine, MA-GG-ONp (260 mg. 0.810 mmol) and DIPEA (226 µL, 1.296 mmol) were added (in 1.5 mL DMF). It was transferred into a vial and rotated overnight. The resin was then washed thoroughly, and the product was cleaved off with TFA. The product was then purified with a Superdex 75 column to yield about 70 mg of MA-GG-D-(Asp)8. The m/z (MALDI-TOF, negative ion) of the product is 1119.24 (calculated 1119.89). 6 Synthesis of P-D-(Asp) -FITC. HPMA (50 mg, 3.5 × 10-4 8 mol) and MA-FITC (2.5 mg. 4.6 × 10-6 mol) were dissolved in DMSO (0.5 mL), and it was mixed with the aqueous solution (1 mL) of MA-GG-D-(Asp)8 (20 mg, 1.79 × 10-5 mol) and ACV (5.8 mg, 2.07 × 10-5 mol). The solution was then purged with N2 and sealed in an ampule for polymerization. It was polymerized at 50 °C for 18 h. The solution was then diluted and purified with PD-10 columns and dialyzed against water (MWCO 6∼8 kDa). The polymer was then further purified with FPLC (Superdex 75). The polymer fraction was dialyzed and finally obtained 44 mg of P-D-(Asp)8-FITC.

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Scheme 2. Structures of Bone-Targeting Poly(ethylene glycol) (PEG) Conjugates

Table 1. Characterization of Polymeric Bone-Targeting Conjugates bone-targeting moiety content conjugates PEG-FITCb FITC-PEG-alendronateb FITC-PEG-D-Asp8b P-FITCc P-alendronate-FITCc P-D-(Asp)8-FITCd

MWa (kDa) 2.4 2.6 3.4 17 35 58

mol/g

no./chaine

4.3 × 10-4 2.6 × 10-4

1 0.9

6.5 × 10-5 7.6 × 10-5

1.1 1-2

a Weight average molecular weights (M ) of PEG conjugates w were determined with MALDI-TOF mass spectrometry (see spectra in Supporting Information); Mw of HPMA copolymer conjugates were determined with SEC using PHPMA calibrations. Mw for P-alendronate-FITC and P-D-Asp8-FITC are apparent values, which are much higher due to the increase ionic osmotic pressures by introduction of electrolytes. b Synthesized from the same precursor of NHS-PEG-FITC (MW 2.4 kDa), PEG-FITC was hydrolyzed with H2O directly from the precursor. c Synthesized from the same precursor of P-ONp-FITC, P-FITC was directly aminolyzed with 1-amino-2-propanol from the precursor. The real Mw for P-alendronate-FITC is ∼17 kDa, similar to that of P-FITC. d Synthesized by copolymerization of HPMA, MA-FITC, and MA-GG-D-(Asp)8. Its estimated real Mw is 20-30 kDa. e Calculated according to the equation: no./chain ) bone-targeting moiety content (mol/g) × Mw (real).

The apparent increase in molecular weight of the conjugates when compared to unmodified macromolecules is likely an artifact and the result of increased hydrodynamic volumes of the modified polymers. This may be attributed to increased ionic osmotic pressures by the introduction of electrolytes [targeting moieties, alendronate and D-(Asp)8]. P-alendronate-FITC1 and P-FITC1 were prepared from the same polymer precursor, P-GGONp-FITC (Mw ) 17 kDa). Therefore, they have a similar real Mw (17 kDa). However, the apparent Mw of P-alendronate-FITC was almost doubled compared to the unmodified polymer. The HPLC analysis of the hydrolyzed P-alendronate-FITC showed that the alen-

Figure 1. The binding of polymeric bone-targeted conjugates to hydroxyapatite. Conjugates were dissolved in phosphate buffered saline (pH ) 7.4) with a concentration of 1 mg/mL. The conjugate solution (100 µL) and 100 µL of the same buffer was incubated with 5 mg of hydroxyapatite powder (HA, Bio-Gel HTP, DNA grade; BIO-RAD, Hercules, CA) in an eppendorf tube for 1 h at RT. Then the HA suspension was centrifuged. The UV absorbance at 490 nm of the supernatant was monitored with a microplate reader. Background correction was applied. Data are shown as the mean standard deviation from triplicate measurements.

dronate content in the conjugate was 4.3 × 10-4 mol/g. Conversion to units of number of alendronate molecule per chain produced a value of 1.1 [using the equation of no./chain ) bone-targeting moiety content (mol/g) × Mw (real)]. Due to the increased ionic osmotic pressure possibly caused by the introduction of D-(Asp)8, the real Mw of P-D-(Asp)8-FITC can be expected to be much lower than its apparent value of 58 kDa. Under similar

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Scheme 3. Structures of Bone-Targeting N-(2-Hydroxypropyl)methacrylamide (HPMA) Copolymer Conjugates

conditions, the copolymerization of HPMA and MA-FITC should yield a copolymer with an Mw of 30 kDa. Therefore, we estimate that the real Mw of P-D-(Asp)8-FITC was between 20 and 30 kDa. Since amino acid analysis of the conjugate revealed a D-(Asp)8 content of 2.6 × 10-4, the number of D-(Asp)8 per polymer chain was estimated to be between 1 and 2 [no./chain ) bone-targeting moiety content (mol/g) × Mw (real)]. All conjugates (Table 1) were screened in vitro for their bone-targeting capacity using HA1 powder as a model bone surface. Both bone-targeted PEG conjugates showed good binding to HA, whereas PEG-FITC1 (control polymer without targeting moiety) showed no binding to HA (Figure 1). About 90% of FITC-PEG-D-(Asp)8 was bound to HA, which correlated well with the fact that 90% of the FITC-PEG-D-(Asp)8 chain contains D-(Asp) 8 (Table 1). However, only 73% of FITC-PEG-alendronate bound to HA, though each polymer chain contains an alendronate moiety (Table 1). It has been proposed (22, 25) that the domain of -COOH groups in the aspartic acid peptide and the two neighboring phosphate groups in bisphosphonate are good ligands for chelation with calcium ions. Possibly, the structure of D-(Asp)8 may provide more

potential binding sites with HA surface calcium (multivalent binding) than alendronate, which contributed to its higher binding efficiency. Analogous to the PEG conjugates, bone-targeted HPMA copolymer conjugates also showed strong binding to HA, while the control polymer (P-FITC) yielded only nonspecific binding to HA (Figure 1). The higher HA binding efficiency of P-D-(Asp)8-FITC (80%) than that of P-alendronate-FITC (66%) may also be attributed to the stronger binding of D-(Asp)8 compared to alendronate. However, the contribution of multiple targeting moieties per chain in the case of P-D-(Asp)8-FITC is another possibility. The binding of the conjugates to the surface of HA was observed to occur very quickly. The result of the initial binding kinetic was shown in Figure 2. The binding of FITC-PEG-alendronate reached a plateau in 2-3 min with 69% of the conjugate bound to HA. Prolonged incubation of the conjugate with HA did not significantly improve binding efficiency (1 h, 73%, Figure 1). On the other hand, although FITC-PEG-D-(Asp)8 showed a similar pattern of HA binding in the first minute, its binding continued to climb to 80% at the end of 4 min.

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Figure 2. The initial binding kinetics of polymeric bonetargeted conjugates to hydroxyapatite. Conjugates were dissolved in phosphate buffered saline (pH ) 7.4) with a concentration of 1 mg/mL. The conjugate solution (100 µL) and 100 µL of the same buffer was incubated with 5 mg of hydroxyapatite powder (HA, Bio-Gel HTP, DNA grade; BIO-RAD, Hercules, CA) at RT. At selected time intervals, the incubation was stopped by centrifugation. The UV absorbance at 490 nm of the supernatant was monitored with a microplate reader. Background correction was applied. Data are shown as the mean standard deviation from triplicate measurements.

Prolonged incubation of the FITC-PEG-D-(Asp)8 with HA for 1 h led to an ultimate binding equilibrium of 90% bound. Further study is needed to understand the mechanism of these phenomena.

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The bone-targeting capacity of the conjugates was evaluated in vivo. Balb/c mice (∼20 g, male, Charles River Laboratories, Inc., Wilmington, MA) were injected i.v. (tail vein) with conjugates using an FITC dose of 1.84 × 10-5 mol/kg. All animals remained active with normal food and water consumption after the injection. Twentyfour hours later, the animals were sacrificed with a halothane overdose. Femurs and tibias were isolated, processed, and subjected to fluorescence microscopic analysis. No autofluorescence was observed in the animals injected with saline (Figure 3A). In those injected with nontargeted conjugates P-FITC and PEG-FITC (Figure 3B,C), no fluorescence labeling was observed either, which indicated the absence of the FITC-labeled polymers in the bone. In contrast, all bones of animals injected with FITC-labeled bone-targeting conjugates showed bright florescence. Both the epiphysis and diaphysis were labeled with fluorescence. A detailed examination indicated the strongest labeling around the epiphyseal plate and the diaphyseal funnel. In addition, both the endosteum and periosteum of the diaphyseal shaft were marked with clear lines of FITC label (Figure 3D-L). It appeared that the bone-targeting delivery systems preferred to accumulate in sites associated with high rates of bone turnover, perhaps in tissues where blood supplies are abundant. The in vivo binding of bone-targeting conjugates to hard tissue is much more complicated than the in vitro HA binding. Molecular weight and the negative charges of the conjugates have great influence on their clearance from the blood circulation via kidney glomerular filtration and liver uptake (negatively charged macromolecules can be recognized by scavenger receptor on nonparenchymal

Figure 3. The in vivo binding of polymeric bone-targeted conjugates to the bone. Bone samples were fixed with formalin, dehydrated with acetone, embedded in poly(methyl methacrylate), and sliced with a low speed diamond saw to the thickness of 100 µm and mounted onto a plastic cover slide for observation under a fluorescence microscope (Olympus BX41, Olympus America Corp., Mellville, NY.). All images were taken under similar microscope settings. Histological nomenclature for different parts of the long bone described below can be found in ref 33. (A) Saline injection; (B) P-FITC; (C) PEG-FITC; (D) FITC-PEG-alendronate, metaphyseal primary and secondary spongiosa labeled; (E) FITC-PEG-alendronate, endosteum of diaphyseal shaft labeled; (F) FITC-PEG-alendronate, endosteum and periosteum of diaphyseal shaft labeled; (G) FITC-PEG-D-(Asp)8, endosteum of diaphyseal shaft labeled; (H) FITCPEG-D-(Asp)8, metaphyseal spongiosa and cortex labeled; (I) P-alendronate-FITC, metaphyseal spongiosa and cortex labeled; (J) P-alendronate-FITC, endosteum and periosteum of diaphyseal shaft labeled; (K) P-D-(Asp)8-FITC, primary spongiosa and epiphysis labeled; (L) P-D-(Asp)8-FITC, endosteum of diaphyseal shaft labeled.

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cells) (26, 27). The saturated calcium ions in blood could be another important factor in the bone-binding process of these conjugates. It was reported that the addition of calcium ion would enhance the adsorption of polyphosphonate onto the surface of HA. Possibly, the calcium ion binding with the polyphosphonate would enhance the collapse of the macromolecule and consequently promote HA adsorption (28). Likely, this may also apply to FITCPEG-D-(Asp)8 and P-D-(Asp)8-FITC because of their multiple charges per chain. However, both alendronate conjugates may not benefit from this due to the existence of only one alendronate on each conjugate. Apparently, conjugates binding with free calcium ions in blood may still be able to bind to the HA surface in bone. Hypothetically, it could be an energy-favorable process, in which the phosphonate or carboxylic acid groups in the conjugates would release the bound calcium ion, exchange with HA-surface phosphate, and bind to surface calcium. It is also noteworthy that the vasculature in bone is unusually porous. Because of the fenestrated capillaries or sinusoids (29) in the bone with pore size of 80-100 nm, the extravasation of the bone-targeting conjugates described here (diameter < 10 nm) (30, 31) into the bone fluid should not be hampered. As shown in Figure 3G,H, the in vivo binding of FITCPEG-D-(Asp)8 to bone seems to be lower compared to the other conjugates. Most likely, this could be attributed to the high liver clearance of the conjugate from the blood circulation due to its highly negatively charged D-(Asp)8 and relatively low molecular weight of PEG (2.4 kDa), which direct the conjugate to scavenger receptor. In the case of P-D-(Asp)8-FITC, 1-2 D-(Asp)8 chains were introduced as side chains to the relatively high molecular weight HPMA copolymer (58 kDa). Therefore, the negative charges of D-(Asp)8 may be moderately shielded and the steric hindrance of HPMA copolymer could prevent its recognition by the scavenger receptors. It is also possible that the higher molecular weight of P-D-(Asp)8FITC could ensure a longer circulation time, which contributes to its better bone binding efficiency as well. Nevertheless, further biodistribution study is necessary to have a clear understanding of the bone-targeting efficiency of these conjugates. In summary, bone-targeting polymeric drug delivery systems based on PEG and HPMA copolymer were successfully synthesized. Alendronate and octapeptide D-aspartic acid were introduced into the delivery system as bone-targeting moieties by either direct conjugation or copolymerization. In vitro and in vivo studies indicated that alendronate- and D-(Asp)8-based conjugates were promising candidates for bone-targeted delivery of therapeutic agents. ACKNOWLEDGMENT

This work was supported in part by NIH Grant EB00251. Mass spectral data were acquired at the University of Utah Mass Spectrometry Facility, supported in part by NIH Grant P30 CA42014. Supporting Information Available: MALDI-TOF mass spectra of PEG conjugates. This material is available free of charge via Internet at http://pubs.acs.org/BC. LITERATURE CITED (1) Marks, S. C., Jr., and Odgren, P. R. (2002) Structure and Development of the Skeleton. Principles of Bone Biology, 2nd ed. (J. P. Bilezikian, L. G. Raisz, and G. A. Rodan, Eds.) pp 3-15, Academic Press, San Diego.

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