Bifunctional bisphosphonate derivatives and platinum complexes with high affinity for bone hydroxyapatite.

A series of ethylenediamine/1,3-propanediamine derivatives containing bifunctional bisphosphonate substituents and their corresponding dichloroplatinum(II) complexes have been synthesized and characterized by elemental analysis, 1H NMR, 13C NMR, 31P NMR, and HRMS spectra. Based on WST-8 assay with CCK-8, in general, the newly synthesized dichloroplatinum complexes 1-6 showed higher in vitro antitumor activity than platinum-free compounds L1-L6 against three tumor cell lines (especially osteosarcoma MG-63). According to hydroxyapatite binding experiment, complexes 2, 3, and 6 showed much higher affinity (K'=3.7, 4.0, and 3.0, respectively) for bone hydroxyapatite than cisplatin (K'<0.1), comparable to zoledronate (K'=2.8). It can be found that representative complex 2 with high cytotoxicity and in vitro antiproliferative activity against osteosarcoma cell line, as well as promising hydroxyapatite binding ability has been screened as a potential bone-targeting antitumor agent for subsequent in vivo study. In addition, flow cytometry experiment was applied to investigate the mode of action of representative complex 2.

Inhibition of calcium hydroxyapatite formation by polyamines.

The lithogenic potential of bile depends not only on supersaturation of solutes but also on the presence of pro- and anti-nucleating factors. For example, glycine-conjugated dihydroxy bile salt dimers are potent inhibitors of calcium hydroxyapatite precipitation that function by "poisoning" the nascent crystal. Although most inhibitors of apatite formation are anions, theoretically polycations should also be effective, and because significant concentrations of polyamines are present in bile, we have investigated the ability of these molecules to inhibit apatite formation. In vitro, each polyamine (2-10 mmol/l) was able to inhibit apatite formation, and the inhibiting power was correlated with ionic charge. Thus putrescine (2+) was the weakest inhibitor and spermine (4+) was the strongest. Mixtures of polyamines were less effective than were the individual polyamines, except at higher concentrations. Although polyamines were effective over short periods of time (270 min), over longer times (3 days) spermine was unable to prevent apatite formation. Using infrared spectroscopy, we found no evidence for interaction between phosphate ions and spermine in solution. Taken together, these results suggest that polyamines are modest inhibitors of apatite formation that likely function by retarding the dissolution of the intermediate amorphous calcium phosphate phase.

Inhibition of the formation of oral calcium phosphate precipitates: beneficial effects of Chinese traditional (kampo) medicines.

Screening tests on the formation of calcium phosphate precipitates using 23 different kinds of Kampo medicines (Chinese traditional medicines) were carried out, at concentrations where the effects of chelation are not significant. Four of them, Hochu-ekki-to (TJ-41), Kyuki-kyogai-to (TJ-77), Oren-to (TJ-120) and Inchin-ko-to (TJ-135) showed an inhibitory effect on the formation of amorphous calcium phosphate (ACP). The inhibitory effect on the induction time and the rate of transformation to hydroxyapatite (HAP) varied greatly among the 23 Kampo medicines. We classified them according to their effects on increasing the induction time and/or decreasing the rate of HAP transformation. Ethane-1-hydroxy-1, 1-diphosphonate (EHDP) was used as the standard. This compound is a common toothpaste additive which decreases dental calculus formation. Two of the 23 Kampo medicines showed little or no inhibition either on the induction time or on the rate of HAP transformation. Twelve of them reduced the rate of HAP transformation by 20-40% and with 1.9- to 4.0-fold increases in the induction time. The remaining nine showed even greater activity. Keishi-ninjin-to (TJ-82), Dai-kenchu-to (TJ-100), Toki-to (TJ-102), Rikko-san (TJ-110) and San'o-shashin-to (TJ-113) showed the same inhibitory effect as EHDP. Shigyaku-san (TJ-35;5.2-fold), Dai-kanzo-to (TJ-84;4.9-fold), Oren-gedoku-to (TJ-15;12.7-fold) and Inchin-ko-to (TJ-135;9.5-fold) had a greater effect on the increase of induction time than EHDP and reduced the rate of HAP transformation by 50-60%. These results suggest that these nine kinds of Kampo medicines may have potential as anticalculus agents in toothpastes and mouthwashes.

Serum and plasma inhibit neutrophil stimulation by hydroxyapatite crystals. Evidence that serum alpha 2-HS glycoprotein is a potent and specific crystal-bound inhibitor.

Tissue deposits of basic calcium phosphate (BCP) crystals are associated with various clinical manifestations of inflammation. We addressed the possibility that native proteins modify the ability of hydroxyapatite (HA) crystals to stimulate human inflammatory cells. Neutrophil superoxide release and chemiluminescence in response to HA crystals (0.3-4.0 mg/ml) were blunted by serum and plasma. Inhibitory activity was progressively removed from serum by sequential adsorption with HA crystals, suggesting that the inhibitors were crystal-bound proteins. Thus, we characterized HA crystal-bound plasma proteins by O'Farrell gels: Fibronectin, transferrin, albumin, alpha 2-HS glycoprotein (AHSG), alpha 1-proteinase inhibitor, alpha 1-acid glycoprotein, Gc globulin, haptoglobin, and high density lipoprotein apolipoproteins were major bound species. Of these, AHSG was the most active inhibitor of HA-induced neutrophil superoxide release, and this glycoprotein partially (60%) restored inhibitory activity to HA-adsorbed serum. AHSG also bound in vitro to the related BCP crystal, octacalcium phosphate, but only minimally to calcium pyrophosphate dihydrate crystals and monosodium urate crystals. Suppressive effects on neutrophil stimulation exhibited by AHSG were also specific for BCP crystals. AHSG was present in noninflammatory synovial fluids bound to synthetic HA crystals in vitro, and AHSG could be detected on native synovial fluid HA crystals. We conclude that the binding of AHSG may modulate the inflammatory potential of BCP crystals.

Induction and inhibition of hydroxyapatite formation by rat dentine phosphoprotein in vitro.

Highly phosphorylated rat incisor phosphoprotein (PP-H) was purified and covalently attached to agarose beads. The beads were incubated for 24 h in solutions having an ionic strength of 0.165, a molar Ca/P ratio of 1.67, and a pH of 7.4. The calcium-phosphate concentration products [( Ca][P]) in the stable incubation solutions ranged from 1.0 to 1.8 mM2, from which no spontaneous precipitation occurred. In a timed series mineral formation was monitored by SEM and X-ray diffraction. The inhibitory capacity of PP-H, free solution, was also studied. The first mineral appeared after 10 min at a [Ca][P] product as low as 1.2 mM2; X-ray diffraction showed that the mineral was (hydroxy)apatite. Thus small amounts of PP-H attached to a surface are capable of inducing mineral formation in vitro at comparatively low supersaturation, whereas PP-H is a mineral inhibitor when free in solution.

Structural factors influencing the ability of compounds to inhibit hydroxyapatite formation.

For a compound to inhibit potently the transformation of amorphous calcium phosphate into hydroxyapatite, it is suggested that the minimum structural requirement is a phosphate group and, at some other position, either another phosphate group preferably or a carboxylic moiety. Primary amino groups abolish inhibitor potential. Inhibitor potency is modified by various secondary factors, including the number and proximity of active groups, their stereochemistry, steric factors, the lability of the molecule, and in special instances its lipophilicity. Parameters used to monitor the transformation indicate that inhibitors can be grouped into two classes, and it is suggested that this is because one class acts as a hydroxyapatite crystal growth inhibitor. The close proximity of two phosphate groups or of a phosphate and multiple carboxylic groups is proposed to determine in part whether or not a compound acts as a crystal growth inhibitor. Further, bulky side groups render a molecule inactive as a crystal growth poison, although it will still inhibit by other mechanisms.

Diphosphonates inhibit hydroxyapatite dissolution in vitro and bone resorption in tissue culture and in vivo.

Two diphosphonates containing the P-C-P bond, Cl(2)C(PO(3)HNa)(2), and H(2)C(PO(3)HNa)(2) retard the rate of dissolution of apatite crystals in vitro. They inhibit bone resorption induced by parathyroid extract in mouse calvaria in tissue culture and in thyroparathyroidectomized rats in vivo.