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.

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.

Cleavage of disulfide-linked fetuin-bisphosphonate conjugates with three physiological thiols.

An effective therapeutic agent for treatment of bone diseases is expected to exhibit a high affinity to bone. Conjugating proteins to bisphosphonates (BPs), a class of molecules with an exceptional affinity to bone mineral hydroxyapatite (HA), is a feasible means to impart such a bone affinity. Protein-BP conjugates with cleavable linkages, which allow protein release from the mineral, are preferable over conjugates with stable linkages. To this end, 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (thiolBP) was conjugated onto fetuin, a model protein, using N-succinimidyl-3-(2-pyridyldithio)propionate to create disulfide-linked conjugates. Although the fetuin-thiolBP conjugates were stable under aqueous conditions, the disulfide linkage was readily cleaved in the presence of the physiological thiols l-cysteine, dl-homocysteine, and l-glutathione. dl-Homocysteine exhibited the highest cleavage of the disulfide linkage among these thiols. The imparted bone affinity as a result of thiolBP conjugation, as assessed by HA binding in vitro, was eliminated upon cleavage of the disulfide linkage. The cleavage of the conjugates bound to HA was as effective as the conjugate cleavage in solution, and even more so at high concentrations of l-glutathione. In conclusion, disulfide-linked fetuin-thiolBP conjugates exhibited a high affinity to HA, which was readily lost upon cleavage with thiols found in physiological milieu.

Imparting mineral affinity to proteins with thiol-labile disulfide linkages.

Chemical conjugation of bisphosphonates (BPs) to proteins is an effective means to enhance binding of proteins to mineral-containing biomaterials. BPs linked to proteins with reversible (i.e., cleavable) linkages were considered desirable over the conjugates linked with stable linkages because cleavable linkages allow protein release in free form from the mineral-containing biomaterials. To explore the feasibility of creating cleavable BP-protein conjugates, an amine- and a thiol-containing BP were conjugated to the model protein Bovine Serum Albumin (BSA) with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), which resulted in disulfide-linked BP-BSA conjugates. Although disulfide-linked conjugates were stable under aqueous conditions, the conjugates in solution were readily cleaved in the presence of physiological concentrations (approximately 0.3 mM) of the thiol compound, cysteine. The imparted mineral affinity as a result of BP conjugation, as assessed by hydroxyapatite (HA) binding in vitro, was lost upon cleavage of the disulfide-linked BP. The conjugates bound to HA were also cleavable with cysteine, but their cleavage rate was significantly reduced as compared to the conjugates in solution. In conclusion, disulfide-linked BP conjugates were shown to be readily cleavable by the amino acid cysteine and this resulted in the loss of imparted mineral affinity of the proteins. The proposed approach will be useful for modulating in vivo delivery of proteins implanted with mineral-containing biomaterials.

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.

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.