Langmuir 1992,8, 1003-1006
Inhibition of Hydroxyapatite Formation by Titanocenes E. Dalas,* N. Klouras, and C. Maniatis Department of Chemistry, University of Patras, GR-26110, Patras, Greece Received June 6, 1991. I n Final Form: December 17,1991 The effects of eight titanocenes, (v5-C5H5)2TiX2and (v5-C5H&H3)2TiXz(X = C1, F, Br, I),used as drugs for various therapeutic applications, on the crystal growth of hydroxyapatite were investigated a t sustained supersaturation. The rates of crystal growth measured in the presence of titanocenes a t concentrations as low as 1.6 to 20.1 p M were drastically reduced, possibly through adsorption onto the active sites for crystal growth. Kinetics analysis suggested Langmuir-type adsorption of titanocene complexes on the hydroxyapatite surface.
Introduction Metallocene dihalides showing tumor-inhibiting efficacy are mainly represented by complexes of type RzMXz (X = F, C1, Br, J). These are organometallic complexes, t h e cyclopentadienyl ring ligands (R = C5H5, CH3C5Hd) being bound t o the central atom M (M = Ti, V, Nb, Mo) by carbon-metal bonds.' Titanocene complexes have shown antiproliferative activity against various experimental tumors, e.g., Ehrich ascites tumor, sarcoma 180, B16 melanoma, colon 38 carcinoma, and Lewis lung carcinoma, and against xenografted human carcinomas,lp2and gold(1) complexes like auranofin are clinically well-approved therapeutic drugs against rheumatoid a r t h r i t i ~ .Recently ~ t h e anti-arthritic activity of titanocene complexes has been m e a ~ u r e d . ~ These complexes are characterized by pronounced tumorinhibiting properties causing cures with optimum doses of 40-100 mg/kg. Typical plasma levels after administration have reported t o be approximately 1 mM of R2-
MX2.5 Despite the rather widespread use of titanocene dihalides as drugs, very little is known concerning their influence on the formation of t h e hard tissues of humans, composed mainly of basic calcium phosphate. Hydroxyapatite (CadP04)30H, HAP) is the model compound for the inorganic component of bones and teeth and was observed in pathological calcifications of the articular cartilage.6* In t h e present work we have investigated the effect of various titanocene dihalides which are listed in Table I. Their effect on the crystal growth of HAP was studied by t h e constant composition a p p r o a ~ h . ~ , ' ~
Experimental Section The experiments were done at 37 f 0.1 O C in a thermostated double-waled,water-JacketedPyrex vessel, volume totaling 0.200 dm3. Solid reagent-grade calcium chloride, potassium dihydro(1) Kopf-Maier, P.; Hesse, B.; Voigtlander, R.; Kopf, H. J.Cancer Res. Clin. Oncol. 1980, 97, 31. (2) Kopf-Maier, P.; Preiss, F.; Marx, T.; Klapotke, T.; Kopf, H. Anticancer Res. 1986, 6, 33. (3) Simon, T. M.; Kunishima, D. H.; Vibert, G. J.; Lorber, A. Cancer Res. 1981, 41, 94. (4) Fairlie, D. P.; Whitehouse, M. W.; Broomhead, J . A. Chem.-Biol. Interact. 1987, 61, 277. (5) Toney, J. H.; Marks, T. J. J. A m . Chem. SOC.1985, 107,947. (6) Gordon, G. V.; Villanueva,T.; Shumacher, H. R.; Gohel, V. J.Rheumatol. 1984, 11,861. (7) Gibilisco, P. A.; Schumacher, H. R., Jr.: Hollander, J. L.: SoDer, K. A. Arthritis Rheum. 1985, 28, 511. (8) Boskey, A. L.; Bullough, P. G. Scanning Electron Microsc. 1984, I I , 943. (9) Koutsoukos, P. G.; Amjad, Z.; Tomson, M. B.; Nancollas, G. H. J. Am. Chem. Soc. 1980,102, 1553. (IO)Tomson, M. B.; Nancollas, G. H. Science 1978,200, 1059.
Table I. Titanocenes, Studied with Respect to Their Effect on the Crystal Growth of Hydroxyapatite titanocene structure TF (w5-CsHdzTiFz TC TB TI MTF MTC MTB MTI gen phosphate, sodium chloride (Merck), and triply distilled, COz-free water were used in the preparation of the solutions. Potassium hydroxide solutions were prepared from concentrated standards (Merck, Titrisol). The standardization of the stock solutions prepared is described in detail elsewhere.gJ0 The supersaturated solutions were prepared in a thermostated vessel by mixing equal volumes 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 measured by a glass/saturated calomel pair of electrodes (Radiometer, C202G and K402, respectively), standardized before and after each experiment by NBS buffer solutions.ll Following pH adjustment, by the addition of dilute potassium hydroxide the crystal growth process was initiated by the addition of known quantities of well-characterizedHAP seed crystalsprepared by a method described elsewhere.12 The specific surface area of the seed crystals, as determined by a multiplepoint BET method (Perkin-Elmersorptometer 212D),was found to be 34.6 m2g-l. The solid precipitates were analyzed by infrared spectroscopy and by powder X-ray diffraction using aluminum as an internal standard. The synthetic crystals displayed the characteristic powder X-ray diffraction patternl3 and the infrared spectrum (KBr pellet method, Perkin-Elmer 577 infrared spectrometer) of stoichiometric HAP14and the stoichiometric ratio Ca:P experimentally determined was 1.67 0.01. ($-CsH&TiC12I5was purchased from Alfa Chemical Co. and recrystallized from toluene. The 1,l'-disubstituted titanocene dichloride ($C5H4CH3)2TiC12l6 was prepared and purified according to a published procedure.15 The remaining titanocene complexes (v5C5H5)zTiXzand (v5-C5H4CH3)2TiXz(X = F, Br, I) were obtained from chloridetitanocene complexes by halide-exchangereactions and purified according to literature methods.1e1* All compounds gave correct elemental analyses (C,H, X) and were characterized by lH NMR and infrared spectroscopy.
(11) Bates, R. G. In Determination of pH; Wiley: New York, 1973. (12) Koutsoukos, P. G.; Amjad, Z.; Nancollas,G. H. J. Colloid Interface Sci. 1981, 83, 599. (13) ASTM file Card No. 9-432. (14) Koutsoukos, P. C. Ph.D. Thesis, SUNYAB,1980. (15) Reynolds, L.; Wilkinson, G. J. Inorg. Nucl. Chem. 1959, 9, 86. (16) Wilkinson, G.; Birmingham, J. M. J. Am. Chem. SOC.1954, 76, 4281. (17) Samuel, E. Bull. SOC.Chim. Fr. 1966, 11, 3548. (18) Kopf, H.; Klouras, N. Chem. Scr. 1982, 19, 122.
0743-746319212408-1003$03.00/0 0 1992 American Chemical Society
Dalae et al.
1004 Langmuir, Vol. 8, No. 3, 1992
Table 11. Crystallization of HAP on HAP Seed Crystals in the Presence of Titanocenes: pH 7.40; 37 OC; 0.16 M NaC1; Total Calcium (Cat):Total Phosphate (Pt) = 1.67 titanwenel lo-5 AGIkJ mol-’ exDtl no. CadlO-4mol dm-3 mol dm-3 HAP TCP OCP DCPD R110-8 mol min-1 m-2 -3.5 -1.2 0.1 3.4 4.01 TF10.93 5 1 -3.5 -1.2 0.1 3.4 5.15 MTFl0.82 5 2 -3.5 -1.2 0.1 3.4 TCl0.80 3.15 5 3 MTC10.72 -3.5 -1.2 0.1 3.4 3.86 5 4 TBl0.59 -3.5 -1.2 0.1 3.4 4.01 5 5 MTBl0.56 -3.5 -1.2 0.1 3.4 1.00 5 6 TIl0.46 -3.5 -1.2 0.1 3.4 5 2.86 7 MTIl0.43 -3.5 -1.2 0.1 3.4 1.14 5 8 TCl0.16 -3.5 -1.2 3.4 5.15 0.1 5 9 TCl0.32 -3.5 3.4 4.01 -1.2 0.1 5 10 -1.2 0.1 TCl1.07 -3.5 3.4 2.58 5 11 -3.5 3.4 -1.2 0.1 TC/2.01 2.29 5 12 -3.5 -1.2 0.1 TCl4.00 3.4 1.72 5 13 -3.0 -0.6 0.6 TCl0.80 4 3.9 1.37 14 -2.7 4.2 -0.3 0.9 TCl0.80 1.36 3.5 15 -2.0 5.1 0.5 1.6 0.86 TCl0.80 3 16 -1.5 1.1 2.0 0.57 5.6 TCl0.80 2.5 17 -3.5 -1.2 0.1 9.72 3.4 5 18 -3.0 3.9 -0.6 0.6 5.38 4 19 -2.7 4.2 -0.3 0.9 3.85 3.5 20 -2.0 5.1 0.5 1.6 3.04 3 21 -1.5 1.68 5.6 1.1 2.0 2.5 22 ~
In the HAP crystal growth experiments in the presence of titanocene complexes, the latter were dissolved in the supersaturated solutions. Throughout the course of the crystallization process water-saturated, purified nitrogen was bubbled through the solution in order to preclude atmospheric carbon dioxide from dissolving into the solution. The precipitation reaction in ah cases started immediately following the introduction of the seed crystals in the crystallization medium. During HAP formation protons are released in the solution, thus offering a very sensitive means of monitoring its formation. A pH meter (Metrohm 632) was used for measuring pH. Connection of the pH meter to a pH-stat (Metrohm 614 Impulsomat with 654 dosigraph) which was modified so as to accommodate two burets, mechanically coupled and mounted onto the shaft of the piston buret, allowed us, through the simultaneous addition of exactly equal volumes of reagents, to achieve invariability of all species in solution. This may be done by adding simultaneously calcium chloride-sodium chloride and potassium hydrogen phosphatepotassium hydroxide in a way such that the stoichiometry of the precipitating phase is preserved.10 At plethostatic conditions therefore, the recorded motion of the buret piston can be easily translated into moles of HAP formed per unit time and unit area of the introduced seed crystale. Experiments done both in the presence and in the absence of titanocene drugs at different seed concentrations showed that crystallization took place exclusively on the surface of the introduced seed crystale. The reproducibility of the measured rates was better than 5% (a mean of five experiments). During the crystallization process, samples were withdrawn and filtered through membrane filters(Millipore,0.22 Fm). The fiitrates were analyzed for calcium by atomic absorption and for phosphate by spectrophotometric means.lg
Results and Discussion The experimental conditions and the kinetic results obtained are summarized in Table 11. The rate of crystal growth was reduced upon increasing the solution concentration of titanocene dichloride. Potentiometric titrations of the chloride titanocene complexes at the conditions of the experiments (0.15 M NaCl), both in the presence and in the absence of total calcium, 5 X M, did not show any appreciable complexation, thus suggesting that the observed inhibitions are not due to a decrease in solution supersaturation because of the Ca2+ sequestration by the titanocenes. Thus the inhibition (19)Reynolds, L.;Wilkinson, G.J . Inorg. Nuol. Chem. 1959, 9, 86.
Figure 1. Kinetics of HAP crystal growth in the presence of various concentrations of (v5-C5H&TiC12,according to the Langmuir kinetic model: pH 7.40; 37 OC; 0.15 mol dm-a NaCl.
observed may be ascribed to further blocking of the active growth sites of the seed crystals. This assumption was tested by fitting the kinetics results in a Langmuir-type isotherm. It should be noted however that the basic assumptions on which the kinetic isotherm is based are that adsorption free energy is constant over the entire adsorbent surface and that there are no lateral interactions between the adsorbed molecules. The rates of crystal growth in the absence, Ro,and in the presence, Ri,of the inhibitors may be related to their concentrations in the supersaturated solutions, Ci,according to
In eq 1 k, and k d are the specific rate constants for the adsorption and desorption, respectively. The ratio (k$ kd),defined as the “affinity constant” may be determined from linear plots according to eq 1,like the plot shown in Figure 1. From the slope of the straight line, a value of 68.88 X lo4mol dm-3 was obtained for the affinity constant of titanocene, TC. For comparison only,valuesof “affinity constants” for other inhibitors for the crystal growth of
Langmuir, Vol. 8, No. 3, 1992 1005
Inhibition of Hydroxyapatite Formation Table 111. Affinity Constants for Various Inhibitors of HAP Crystal Growth inhibitor ~~
phytic acid citric acid Mg2+ aminotris(methy1enephosphonic acid) acid ethane- I-hydroxy-1,l-diphosphonic melitic acid glucose bis(su1fonamides) titanocenes (q5-C5H&Ti(H20)~2+ [email protected]
(ka/kd)/ moldm-3 8.4 1.5 1.54 62 208 160 10.22 3.51 68.88
20 21 22 21 20 21 23 24 a
HAP are given in Table 111. The higher value of the "affinity" constant points to the stronger adsorption of the inhibitor on the HAP surface. The driving force for the formation of a crystalline phase, M"+,+ X2-+,-(u = u+ + u-) is the average change in Gibbs free energy, per ion, AG, for the transition from the supersaturated solution to equilibrium and is given by
where the parentheses denote ionic activities, T is the absolute temperature, R, is the gas constant, Q is the supersaturation ratio, and KO,is the thermodynamic solubility product of the precipitating solid phase. A number of calcium phosphates may be formed in the supersaturated calcium phosphate solutions in the order of increasing solubility: hydroxyapatite [HAP], tricalcium phosphate [Ca3(PO4)2, TCPI, octacalcium phosphate [CadH(P04)[email protected]
, OCPI, and dicalcium phosphate dihydrate [CaHP04.2H20, DCPD]. The following values were used for the thermodynamic solubility products of the various calcium phosphates: for HAP, KO, = 2.35 X 10-59;25for TCP, KO,= 2.83 X 10-30;26for OCP, KO,= 5.01 X 10-50;27 for DCPD, KO, = 1.87 X The computation of the activities of the free ions M" and Xz-wasdone as previously de~cribed.~JO In all cases, the measured crystal growth rates, R, were proportional to the relative solution supersaturation, u, with respect to hydroxyapatite 0
R = kSa'
where k is the precipitation rate constant, S a function of the active growth sites on the seed crystals, and 7 the apparent order of reaction. Kinetics plots according to eq 4 gave a satisfactory fit, as may be seen in Figure 2. From the linear plots, a value of 7 = 2 f 0.2 was obtained for ~
(20)Koutsoukce, P. G.;Amjad, Z.; Nancollas, G. H. J.Colloidlnterface Sci. 1981, 83, 599. (21)Amjad, Z . Langmuir 1987, 3, 1063. (22)Amjad,Z.; Koutsoukos,P. G.;Nancollas, G. H.J. ColloidInterface Sci. 1984, 101, 250. (23)Dalas,E.;Koutsoukos, P. G. J. Chem. SOC.,Faraday Trans. 1 1989,85, 2465. (24)Maniatis, Ch.; Dalas, E.; Zafiropoulos, Th. F.; Koutsoukos, P. G. Langmuir 1991, 7, 1542. (25)McDowel, H.; Gregory, T. M.; Brown, W. E. J. Res. Natl. Bur. Stds. 1977,81, 273. (26)Gregory, T. M.; Moreno, E. C.; Patel, J. M.; Brown, W. E. J.Res. Natl. Bur. Stand. (US.) 1974, 78, 667. (27)Shyn, L.J.; Perez, L.; Zawacki, S. J.; Heughebaert, J. C.; Nancollas, G. H. J.Dent. Res. 1983, 62, 398. (28)Marchall, R. Ph.D.Thesis,State Universityof New Yorkat Buffalo, Buffalo, NY, 1970.
Figure 2. Kinetics of H A P crystallization on H A P seed crystals both in the presence ( 0 )and in t h e absence ( 0 )of 8 X lo4 mol dm-3 ($426Hs)zTiC12: pH 7.40; 37 "C;0.15 mol dm-3 NaC1.
the crystallization of HAP both in the presence and in the absence of chloride titanocenes, which is higher than the value reported for the crystallization in low ionic strength media.12.14.23 The cyclopentadienyl hydrolysis, initially follows approximate first-order kinetics, with half-life t1p = 114 f 11h at 37 "C and 0.103 M NaCl.5 The free cyclopentadiene in the working solution (3-5 h after titanocene introduction in the working solution the crystal growth rate was measured) was in insignificant amounts. On the other hand chloride hydrolysis resulted in a rapid increase of the hydrolysis products in the working solution ($-C,H,),TiCl,
+ H20 e (~5-C2H5)2Ti(H20)C1+ + C1- (5)
despite the presence of 0.15 M NaCl., The half-life for the first hydrolysis step of the TC is very short (cannot be measured by chloride potentiometry) and the half-life for eq 6 is 49.5 min. The creation therefore of more positively charged centers via chloride hydrolysis is expected to lead to stronger interactions (electrostatic) between the negatively charged HAP and the positive sites of (q5-C5H&Ti(H20)C1+and (~+C5H&Ti(Hz0)2~+ complexes. The hydrolysis of fluoride, bromide, and iodine titanocenes produces additional anions of F-, Br-, and I-, respectively, that probably interfere in the inhibition procedure. It is well-known that fluoride ions F- promote calcium phosphate cryst a l l i z a t i ~ n .Consequently ~~ only the "affinity" constant for the titanocene dichloride was measured. The relative reduction of HAP crystal growth rates in the presence of titanocene dichlorides was found to follow the trend blank > MTC > TC. This may be due to the fact that the presence of the additional -CH3 group decreases the positive charge of the second chloride hydrolysis product. Finally if the crystallization started 24 h after titanocene chloride introduction the rate measured was 2.29 x 10-8 mol min-l m-2, leading to the conclusion that there was an additional affect of the cyclopentadienyl hydrolysis on the inhibition of crystal growth. (29)Koutsoukos, P. G.; Nancollas, G. H. J.Cryst. Growth 1981,53,10.
1006 Langmuir, Vol. 8,No. 3, 1992
Conclusions In the present work, the effect of titanocene complexes on the crystallization of HAP,in the concentration range 1.6 x 104 to 2.01 X 10-6 mol dm-3 was investigated in solutions supersaturated only with respect to HAP,at constant solution composition. The titanocene dihalides investigated reduced the rates of crystal growth of HAP by 47-90 % . This inhibitory effect may be explained by
Dalas et al.
the adsorption and subsequent blocking of the active growth sites. The adsorption assumption may be justified through the satisfactory fit of the results to a kinetics, Langmuir-type isotherm. Registry NO. TF, 309-89-7;TC, 1271-19-8; TB, 1293-73-8; TI, 12152-92-0;MTF, 38498-31-6;MTC, 1282-40-2; MTB, 7262232-3;MTI, 72622-33-4; hydroxyapatita, 1306-06-5.