Biomedical subjects

Sébastien A Gittens

Publications and source records attributed to Sébastien A Gittens.

7 recordsLinked to original sources

PubMed2005

Designing proteins for bone targeting.

Protein-based therapeutic agents intended for bone diseases should ideally exhibit a high affinity to bone tissue, so that their systemic administration will result in specific delivery to bone with minimal distribution to extra-skeletal sites. This was shown possible in the authors' lab by modifying a desired protein with bisphosphonates (BPs) that exhibit an exceptionally high affinity to the bone-mineral hydroxyapatite. In this review, we explore the potential applications of that concept by summarizing the bone diseases and candidate proteins that will benefit from the proposed bone delivery approach. A selective synopsis of BP synthesis is presented to highlight the synthesis of functional BPs suitable for covalent attachment to proteins. Finally, we present a summary of recent research results from the authors' laboratory emphasizing factors influencing bone affinity of the conjugates. We conclude with future research avenues that are considered critical for clinical entry of the BP-targeted therapeutic agents.

Animals↗

PubMed2004

Imparting bone mineral affinity to osteogenic proteins through heparin-bisphosphonate conjugates.

Although numerous growth factors can promote the regeneration of bone upon parenteral administration, all exhibit undesirable side-effects that prevent their clinical utility. These side-effects arise due to the growth factors' inherent lack of bone affinity. The goal of this study was to develop a means to enhance the bone mineral affinity of osteogenic growth factors so as to minimize their extra-skeletal distribution. Heparin, a glycosaminoglycan that exhibits a high affinity to numerous growth factors, was modified with bisphosphonates to enhance its affinity to bone mineral (i.e. hydroxyapatite, HA). To this end, conjugation of 1-amino-1,1-diphosphonate methane (aminoBP) onto periodate-oxidized heparin using 4-(maleimidomethyl)cyclohexane-1-carboxyl-hydrazide was attempted. Using this chemistry, the number of aminoBPs conjugated onto heparin was modulated by varying the reacting reagent concentrations (to a maximum of 7 aminoBPs per heparin). Increasing the number of aminoBPs resulted in a more than twofold increase in heparin's affinity for HA in vitro. Subsequently, the ability of aminoBP-heparin conjugates to enhance the mineral affinity of basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) was explored. The results revealed that the complexation between the conjugates and growth factors had occurred and that the conjugates increased the bone mineral affinity of bFGF and BMP-2 in an aminoBP-dependent manner. In conclusion, the conjugation of aminoBP onto heparin is a feasible approach to enhance the affinity of heparin-binding, osteogenic growth factors to HA.

Bone Morphogenetic Protein 2↗

PubMed2004

Systemic bone formation with weekly PTH administration in ovariectomized rats.

PURPOSE: Weekly subcutaneous administration of 0 (vehicle), 10 and 80 microg/kg doses of human parathyroid hormone (1-34) [PTH (1-34)] were compared based on their capacity to induce systemic formation of bone in 9 month-old ovariectomized (OVX) Sprague-Dawley rats. METHODS: Changes elicited at bone tissue after 4 weeks of treatment were assessed using dual x-ray absorptiometry, micro-computed tomography (microCT), and ashing. RESULTS: The 10 microg/kg dose led to a significant increase (p<0.025) in femoral bone mineral density (BMD) over vehicle- and 80 microg/kg-treated groups. Similarly, structural analysis of the femoral neck trabecular bone by microCT revealed increases in bone volume fraction and trabecular thickness over the pre-treatment baseline, and vehicle- and 80 microg/kg-treated groups. CONCLUSIONS: The data suggest that the weekly administration of 10 microg/kg of PTH (1-34) was sufficient to significantly promote the bone mineral density systemically. The weekly administration of 10 microg/kg over a 4-week treatment period is, to our knowledge, one of the lowest reported total dose of PTH (1-34) shown to induce a net anabolic effect on skeletal tissue in OVX rats.

Animals↗

PubMed2004

A di(bisphosphonic acid) for protein coupling and targeting to bone.

Proteins intended for treatment of bone diseases should ideally exhibit a high bone affinity, so that they are preferentially deposited to bones after systemic administration. This can be achieved by combining molecules having a high affinity to bone with the proteins. Bisphosphonates (BPs) are chemical analogs of pyrophosphate that possess exceptional bone mineral affinity. To this end, we synthesized a novel BP, 3,5-di(ethylamino-2,2-bisphosphono)benzoic acid (6), which contains two BP moieties on a single molecule, unlike conventional BPs that contain one BP moiety per molecule. 6 was then conjugated to two model proteins, bovine serum albumin and nonspecific bovine immunoglobulin G by the carbodiimide chemistry. By varying the reagent concentrations, the conjugation efficiency (i.e., number of 6 per protein) was readily controlled under the experimental conditions. The protein-6 conjugates exhibited an in vitro mineral affinity that was proportional to the number of conjugated 6. The 6-conjugates of both bovine serum albumin and immunoglobulin G were found to be bone seeking in rats, based on the increased concentration of 6-conjugated proteins in bone tissue after intravenous administration. We conclude that the novel BP synthesized (6) can serve as a carrier for bone delivery while reducing the extent of protein modification necessary for bone targeting.

Animals↗

PubMed2004

Impact of tether length on bone mineral affinity of protein-bisphosphonate conjugates.

PURPOSE: To determine the effect of tether length on bone mineral affinity of fetuin-bisphosphonate conjugates. METHODS: 1-Amino-1,1-diphosphonate methane (aminoBP) was conjugated onto the lysine residues of fetuin by using five different crosslinkers that varied in length. Both the conjugation efficiency (i.e., the number of aminoBPs per protein) as well as molecular dynamics modeling of the resulting conjugates were assessed. Furthermore, the in vitro and in vivo bone mineral affinity of the conjugates were compared to one another. RESULTS: The tethers, whose extended lengths varied from 5.7 to approximately 136 A, were effective in conjugating aminoBP onto fetuin. Molecular dynamics modeling revealed an inverse relationship between tether length and the maximal radial density of the pendent ligand. The capacity of the conjugates to bind to various bone matrices in vitro differed significantly, as aminoBPs tethered onto fetuin via shorter cross-linkers afforded a superior affinity for various mineral matrices than those tethered via longer cross-linkers. Results from the in vivo mineral implantation studies corroborated the in vitro findings, as higher binding was achieved with the shorter conjugates. Thus, the binding capacities of the conjugates paralleled the maximal radial densities of the pendent ligands. CONCLUSIONS: The use of any of the chosen cross-linkers was feasible in conjugating aminoBP onto fetuin. Maximal mineral binding of the fetuin-aminoBP conjugates, however, was typically achieved using the shorter cross-linkers.

Animals↗

PubMed2003

Imparting bone affinity to glycoproteins through the conjugation of bisphosphonates.

PURPOSE: To develop a novel means of conjugating bisphosphonates onto the carbohydrate moieties of glycoproteins to enhance protein affinity to bone. METHODS: 1-Amino-1,1-diphosphonate methane (aminoBP) was conjugated onto the carbohydrate moietites of oxidized fetuin by using 4-(maleimidomethyl)cyclohexane-1-carboxyl-hydrazide (MMCCH). Bone affinity of the resulting conjugates was compared to proteins obtained from another means of conjugation, whereby aminoBP was conjugated onto fetuin's lysine moieties by using succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). RESULTS: The use of the MMCCH resulted in the conjugation of up to seven aminoBPs per molecule of fetuin. These conjugates gave a 2.6-, 2.0-, 30.5-. and 1.84-fold increased affinity for untreated, ashed, demineralized bone and hydroxyapatite, respectively, as compared to conjugates from the SMCC reaction. Both conjugates exhibited a pH-independent, equally slow degradation in adult bovine serum-containing media. CONCLUSION: The use of the MMCCH chemistry to conjugate aminoBP onto fetuin was feasible. Furthermore, the described processes of conjugation resulted in amino-BP-dependent increase in the glycoprotein's affinity to various bone matrices in a manner that exceeds the affinity produced by the previously established method, which used SMCC.

Animals↗

PubMedDate not supplied

Imparting mineral affinity to fetuin by bisphosphonate conjugation: a comparison of three bisphosphonate conjugation schemes.

Protein conjugation to bisphosphonic acids (BPs), such as 1-amino-1,1-diphosphonate methane (aminoBP) and 3,5-di(ethylamino-2,2-bisphosphono)benzoic acid (diBP), was proposed as a foundation for bone-specific delivery of protein therapeutics. This study was performed to directly compare the mineral affinity of protein-BP conjugates prepared by three different approaches. Fetuin, serving as a model protein, was derivatized with BPs by the following approaches: (i) by attaching the aminoBPs onto protein lysines using succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC); (ii) by attaching the aminoBPs onto protein carbohydrates using 4-(maleimidomethyl)-cyclohexane-1-carboxyl-hydrazide (MMCCH), and (iii) by conjugating diBP to protein lysines using the carbodiimide chemistry. The results indicated that conjugation of aminoBP and diBP to fetuin by all three means unequivocally enhanced the protein's affinity for hydroxyapatite in vitro. Similarly, conjugation of aminoBP and diBP onto fetuin increased the protein's retention in a mineral-containing matrix (Pro-Osteon) when the proteins were implanted in a rat subcutaneous model. Upon parenteral administration, however, no discernible differences were found between the SMCC- or MMCCH-linked conjugates and unmodified fetuin to target to bony tissues. DiBP-fetuin conjugates, however, led to successful bone targeting after intravenous injection in rats. We conclude that all three conjugation schemes were equally effective in imparting an affinity to the proteins toward mineral-containing matrices. Bone targeting, however, was achieved only with diBP conjugation to fetuin, supportive of the superior ability of this BP with a higher density of bisphosphonic acid groups.

Animals↗