Inhibition of hydroxyapatite formation in aqueous solutions by hafnocene dichlorides S. Koutsopoulos, E. Dalas,* N. Tzavellas and N. Klouras Dept. of Chemistry, University of Patras GR-26500, Patras, Greece The kinetics of crystal growth of hydroxyapatite HAP], the thermodynamically most stable calcium phosphate [Ca 5 (PO 4 ) 3 OH, phase at 37 ¡C, pH 7.40 and ionic strength 0.15 M in the presence of hafnocene dichlorides has been investigated. The kinetic results showed a surface di†usion controlled mechanism. The presence of hafnocene dichlorides used as drugs for therapeutic applications inhibited the crystal growth of HAP, possibly through adsorption onto the active growth sites for crystal growth. The kinetic results favored a Langmuir type isotherm, suggesting a high affinity of hafnocene dichloride for HAP. Introduction Metallocene dihalides are drugs of proven therapeutic impor- tance and have been used against various tumors, e.g. Ehrlich ascites tumor, sarcoma 180, B16 melanoma, lymphoid leuke- mia L1210, lymphocytic leukemia P388, colon 38 carcin- oma.1h3 These are organometallic complexes (X: Cp 2 MX 2 , halogen) where the cyclopentadienyl ring ligands (Cp : C 5 H 5 , etc.) are n-bonded to the central atom M (Ti, Zr, MeC 5 H 4 , Hf, V, Nb, Mo) (Fig. 1). Recently the antiarthritic activity of titanocene and zirconocene dihalides has been measured.4 h 6 These complexes along with gold(I) complexes (auranoÐn) are clinically well approved therapeutic drugs against arthritis4 and they are characterized be pronounced antiinÑammatory properties, giving relief at optimum doses of 20È100 mg kg~1. The typical plasma levels after administration was reported to be approximately 1 mM of However, when applied Cp 2 MX 2 .7 in dimethylformamide or dimethyl sulfoxide to the skin, they manifested both antiinÑammatory and antiarthritic activity without much local skin damage. They might therefore have the potential of being useful drugs, especially if released slowly.1 Despite the rather widespread use of metallocene dichlo- rides as drugs, very little is known concerning their inÑuence on the formation of hard tissues of humans, composed mainly of basic calcium phosphate. Hydroxyapatite [Ca 5 (PO 4 ) 3 OH, HAP] is the model compound for the inorganic component of bones and teeth and was observed in pathological calciÐcation of the articular cartilage.8h10 In the present work we have investigated the e†ect of various hafnocene dichlorides, on the crystal growth of HAP by the constant composition technique.11,12 Experimental All experiments were performed at 37 ^ 0.1 ¡C in a ther- mostated double walled, water jacketed Pyrex vessel, volume Fig. 1 Hafnocene dichloride, a complex with a tilted sandwich struc- ture totalling 0.250 dm~3. Solid reagent-grade (Merck) calcium chloride, potassium dihydrogen phosphate, sodium chloride and triply distilled water were used in the prep- CO 2 -free aration of the solutions. Potassium hydroxide solutions were prepared from concentrated standards (Merck, Titrisol). The standardization of the stock solutions is described in detail elsewhere.11h13 The supersaturated solution were prepared in the thermostated vessel by mixing the appropriate volumes of calcium chloride and potassium dihydrogen phosphate. The ionic strength of the solutions was adjusted to 0.15 mol dm~3. by the addition of sodium chloride. The solution pH was mea- sured by a glass/saturated calomel pair of electrodes (Metrohm, 6.0101.100 and 6.0726.100, respectively) stan- dardized before and after each experiment by NBS bu†er solu- tions (pH \ 6.841 and 7.386 at 37 ¡C).14 Following pH adjustment, by the addition of dilute potassium hydroxide M), the crystal growth process was initiated by the addition (C K of 20 mg of well characterized HAP seed crystals prepared by a method described elsewhere.15 The speciÐc surface area of the seed crystals, as determined by a multiple-point BET method (Perkin-Elmer Sorptometer 212 D), was found to be 34.6 m2 g~1. The solid precipitates were analyzed by IR spectroscopy (KBr pellet method, FT-IR Perkin-Elmer 16-PC) and by powder X-ray di†raction (Philips PW 1830/1840) using aluminium as an internal standard and elemental analysis. The synthetic crystals displayed the char- acteristic powder X-ray di†raction pattern16 and the IR spec- trum of stoichiometric HAP13 and the experimentally determined stoichiometric ratio Ca : P was 1.67 ^ 0.01. The hafnocene dichlorides Hf(g5- (HC) and the C 5 H 5 ) 2 Cl 2 1,1@-disubstituted hafnocene dichlorides Hf(g5-C 5 H 4 Me) 2 Cl 2 (MHC) and Hf(g5- (SMHC) were prepared C 5 H 4 SiMe 3 ) 2 Cl 2 and puriÐed according to literature methods.17h19 In the HAP growth experiments in the presence of hafno- cene complexes, the latter were added in the phosphate solu- tions along with the seed crystals, 30 min before the experiment started by the addition of calcium chloride. When following the above procedure, adsorption phenomena did not interfere with the kinetic measurements. Throughout the course of the crystallization process water-saturated puriÐed nitrogen was bubbled through the solution in order to pre- clude atmospheric carbon dioxide from dissolving into the solution. During HAP formation, protons are released in the solu- tion, thus o†ering a very sensitive means of monitoring its formation. A pH-meter (Metrohm 691) was used for measur- ing pH. Connection of the pH-meter to pH-stat (Metrohm 614 Impulsomat with 654 Dosigraph) which was modiÐed so as to accommodate two burettes, mechanically coupled and J. Chem. Soc., Faraday T rans., 1997, 93(23), 4183È4186 4183