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Biomedical subjects

Hasan Uludağ

Publications and source records attributed to Hasan Uludağ.

7 recordsLinked to original sources
PubMed2006

In vitro osteogenic response of rat bone marrow cells to bFGF and BMP-2 treatments.

Basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) are actively pursued for stimulation of bone formation. To assess their promise for systemic therapy of osteoporosis, we ascertained the effects of bFGF and BMP-2 on bone marrow cells in vitro. Bone marrow cells were obtained from young (8 weeks) and adult (32 weeks) rats by femoral aspiration and were exposed to osteogenic medium (ie, basal medium with 10 mM beta-glycerolphosphate and 100 nM dexamethasone) containing the growth factors. The cell viability in osteogenic medium was reduced after 3 weeks but not if the concentration of beta-glycerolphosphate/dexamethasone was reduced to 3 mM/30 nM. Unlike BMP-2, bFGF at 2-50 ng/mL was capable of enhancing long-term cell viability. Continuous treatment of bone marrow cells for 3 weeks resulted in dose-dependent stimulation of mineralization by BMP-2, but not by bFGF, whose activity was optimal at 2-10 ng/mL. To explore the effect of short-term exposure, bone marrow cells were treated with growth factors for 1 week and subsequent mineralization was investigated. BMP-2 exposure increased the extent of mineralization, but bFGF was not effective after the short exposure. We concluded bFGF was more potent (ie, required lower concentration) for stimulating osteogenic parameters, but BMP-2 effects were lasting on the bone marrow cells.

Animals↗
PubMed2005

A comparison of mineral affinity of bisphosphonate-protein conjugates constructed with disulfide and thioether linkages.

Chemical conjugation of bisphosphonates (BPs) to therapeutic proteins is an effective means to impart mineral affinity to proteins. Such conjugates can be implanted with mineral-based matrices to control the local delivery kinetics of the proteins. BPs linked to proteins with reversible (i.e., cleavable) linkages are desirable over conjugates with stable linkages to release the protein in free form. This study conducted a direct comparison of mineral affinity of BP-protein conjugates linked together with cleavable disulfide and non-cleavable thioether linkages. Bovine serum albumin (BSA) was used as a model protein and the desired conjugates were created with N-succinimidyl-3-(2-pyridyldithio)propionate (disulfide) and succinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate (thioether) linkers. The disulfide-linked conjugates were cleaved in the presence of a major thiol constituent of serum, cysteine. The imparted mineral affinity, as assessed by hydroxyapatite binding in vitro, was lost upon the cleavage of the disulfide-linked aminoBP. The presence of the serum did not accelerate the cleavage of disulfide-linked conjugates. The aminoBP-BSA conjugates formed with disulfide and thioether linkages were subcutaneously implanted in rats with two different mineral-based matrices to assess protein loss from the matrices. All conjugates exhibited a higher retention in mineral matrices as compared to unmodified BSA. However, no significant differences in in situ pharmacokinetics of the disulfide- and thioether-linked conjugates were observed. We conclude that disulfide-linked BP conjugates were readily cleavable by the amino acid cysteine in vitro, but in vivo cleavage of the disulfide-linked conjugates was not evident when the proteins were implanted adsorbed to mineral-based matrices. BP-protein conjugates with faster-cleaving tethers might be required to significantly influence the release of the BP conjugates from the mineral matrices.

Amides↗
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

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↗