Complexes of DOTA− Bisphosphonate Conjugates: Probes for

1952

Langmuir 2008, 24, 1952-1958

Complexes of DOTA-Bisphosphonate Conjugates: Probes for Determination of Adsorption Capacity and Affinity Constants of Hydroxyapatite Toma´sˇ Vitha,†,‡ Vojteˇch Kubı´cˇek,*,†,‡ Petr Hermann,‡ Zvonimir I. Kolar,*,§ Hubert Th. Wolterbeek,§ Joop A. Peters,† and Ivan Lukesˇ‡ Biocatalysis and Organic Chemistry, Department of Biotechnology, Delft UniVersity of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands, Department of Inorganic Chemistry, Faculty of Science, Charles UniVersity in Prague, HlaVoVa 8, 128 40 Prague, The Czech Republic, and Department of Radiation, Radionuclides & Reactors, Faculty of Applied Sciences, Delft UniVersity of Technology, 2629 JB Delft, The Netherlands ReceiVed September 5, 2007. In Final Form: NoVember 5, 2007 The adsorption on hydroxyapatite of three conjugates of a bisphosphonate and a macrocycle having C1, C2, and C3 spacers and their terbium complexes was studied by the radiotracer method using 160Tb as the label. The radiotracercontaining complex of the conjugate with the C3 spacer was used as a probe for the determination of the adsorption parameters of other bisphosphonates that lack a DOTA unit. A physicochemical model describing the competitive adsorption was successfully applied in the fitting of the obtained data. The maximum adsorption capacity of bisphosphonates containing bulky substituents is determined mainly by their size. For bisphosphonates having no DOTA moiety, the maximum adsorption capacity is determined by the electrostatic repulsion between negatively charged bisphosphonate groups. Compounds with a hydroxy or amino group attached to the R-carbon atom show higher affinities. Macrocyclic compounds containing a short spacer between the different bisphosphonic acid groups and the macrocyclic unit exhibit high affinities, indicating a synergic effect of the bisphosphonic and the macrocyclic groups during adsorption. The competition method described uses a well-characterized complex and allows a simple evaluation of the adsorption behavior of bisphosphonates. The application of the macrocycle-bisphosphonate conjugates allows easy radiolabeling via complexation of a suitable metal isotope.

Introduction Phosphonates are known to exhibit a high affinity for the surface of many inorganic oxides and salts.1-5 If more than one phosphonic acid group is present in a molecule, the adsorption is usually stronger, and then the adsorption abilities correlate with the number of carbon atoms separating the phosphonic acid groups.6,7 For example, geminal bisphosphonates show the highest affinities for the surface of hydroxyapatite (HA), which is the main inorganic component of bones.6,7 This class of bisphosphonates has found important applications in the treatment of diseases connected with disorder of calcium metabolism, including osteoporosis and Paget’s disease.1 Complexes of radioactive metal ions with bisphosphonates have been applied in bone cancer radiotherapy and in palliation of pain associated with metastatic bone tumors.1,8,9 During the past decade, the * To whom correspondence should be addressed. E-mail: [email protected] (V.K.); [email protected] (Z.I.K.). † Department of Biotechnology, Delft University of Technology. ‡ Charles University in Prague. § Department of Radiation, Radionuclides & Reactors, Delft University of Technology. (1) Fleisch, H. Bisphosphonates in Bone Disease, 4th ed.; Academic Press: London, 2000. (2) Fleish, H. Endocr. ReV. 1998, 19, 80-100. (3) Vioux, A.; Le Bideau, J.; Mutin, P. H.; Leclerq, D. Top. Curr. Chem. 2004, 232, 145-174. (4) Mingalyov, P. G.; Lisichkin, G. V. Russ. Chem. ReV. 2006, 75, 541-557. (5) Mutin, P. H.; Guerrero, G.; Vioux, A. J. Mater. Chem. 2005, 15, 37613768. (6) Quin, L. D. A Guide to Organophosphorus Chemistry; Wiley & Sons: New York, 2000. (7) Hilderbrand, R. L. The Role of Phosphonates in LiVing Systems; CRC Press: Boca Raton, FL, 1983. (8) Deutsch, E.; Libson, K.; Jurisson, S.; Lindoy, L. F. Prog. Inorg. Chem. 1983, 30, 75-139.

bisphosphonic acid group has been suggested as a bone-targeting vector in various tissue-specific contrast agents.10-13 The high affinity of bisphosphonates for calcified tissues has been explained by their strong adsorption on the surface of HA. Recently, we have reported on a ligand, BPAMD (see Figure 1), which combines a bisphosphonic acid targeting function with a DOTA core.12 The latter group can, after complexation with an appropriate metal ion or radiotracer, function as a reporter group in various molecular imaging techniques including MRI, SPECT, and SPECT-CT. It should be noted that lanthanide complexes of the DOTA ligand are thermodynamically and kinetically more stable than those of bisphosphonates, and therefore, the bisphosphonate group in Ln-BPAMD remains free for binding to HA.12 Alternatively, compounds of this class have potential as therapeutic agents in molecular therapy. The promising results of the previous study12 prompted us to synthesize two other ligands, BPAPD and BPPED (see Figure 1), containing spacers of different lengths between the DOTA and the bisphosphonic acid moieties. To get more insight into the adsorption behavior of this type of compound, we studied the adsorption of radiolabeled Tb complexes of the three ligands mentioned. The 160Tb radionuclide was selected because it is suitable for radiochemical work due to 100% natural abundance of its precursor, 159Tb, which in addition has a large cross-section (9) Neville-Webbe, H. L.; Holen, I.; Coleman, R. E. Cancer Treat. ReV. 2002, 28, 305-319. (10) Adzamli, I. K.; Johnson, D.; Blau, M. InVest. Radiol. 1991, 26, 143-148. (11) Adzamli, I. K.; Blau, M.; Pfeffer, M. A.; Davis, M. A. Magn. Reson. Med. 1993, 29, 505-511. (12) Kubı´cˇek, V.; Rudovsky´, J.; Kotek, J.; Hermann, P.; Vander Elst, L.; Muller, R. N.; Kolar, Z. I.; Wolterbeek, H. T.; Peters, J. A.; Lukesˇ, I. J. Am. Chem. Soc. 2005, 127, 16477-16485. (13) Zhang, S.; Gangal, G.; Uludag, H. Chem. Soc. ReV. 2007, 36, 507-531.

10.1021/la702753j CCC: $40.75 © 2008 American Chemical Society Published on Web 01/29/2008

Complexes of DOTA-Bisphosphonate Conjugates

Figure 1. Discussed compounds.

for neutron capture. Furthermore, the 160Tb half-life of 72 days allows comfortable handling of the complexes. To evaluate the effect of the macrocyclic moiety in these ligands on the affinity for HA, a comparison was made with a series of bisphosphonates lacking the DOTA moiety - AMP2, AcMP2, APP2, PAM, and HEDP (Figure 1). The Tb-BPAPD complex was selected to probe the interactions between these compounds and HA via competitive adsorption experiments. A physicochemical model based on the Langmuir adsorption isotherm was evaluated to describe competitive adsorption of two bisphosphonates. The presented method of competitive adsorption with a kinetically inert complex as a probe enables quantitative evaluation of adsorption abilities of different substrates (i.e., bisphospohonates). Experimental Section Materials. Compounds BPAMD,12 PAM,14,15 AMP2,14,16 APP2,14,17 and AcMP214 were prepared according to the published procedures. The syntheses of BPAPD and BPPED are described in the Supporting Information. Other chemicals were purchased from commercial sources and used without further purification. The radionuclide 160Tb was prepared by thermal neutron irradiation of Tb(NO ) ‚ 3 3 6H2O (20.8 mg, 46 µmol) purchased from Aldrich in a nuclear reactor (thermal neutron flux 4.49 × 1012 cm-2 s-1, irradiation time 1.5 h). The material obtained (specific activity A ) 65 GBq mol-1) was used without further purification. An aqueous solution of 14Clabeled HEDP was obtained from NEN Life Sciences Products, Boston (4.33 MBq mL-1, specific activity A ) 68.8 GBq mol-1). Hydroxyapatite was purchased from Fluka (catalog number 55496); two batches were used with specific surface areas of 63 and 73 m2 g-1, respectively, as determined by N2 adsorption by means of a (14) Kubı´cˇek, V.; Kotek, J.; Hermann, P.; Lukesˇ, I. Eur. J. Inorg. Chem. 2007, 333-344. (15) Kieczykowski, G. R.; Jobson, D. F.; Melillo, D. G.; Reinhold, D. F.; Grenda, V. J.; Shinkai, I. J. Org. Chem. 1995, 60, 8310-8312. (16) Kantoci, D.; Denike, J. K.; Wechter, W. J. Synth. Commun. 1996, 26, 2037-2043. (17) Winckler, W.; Pieper, T.; Keppler, B. K. Phosphorus, Sulfur Silicon Relat. Elem. 1996, 112, 137-141.

Langmuir, Vol. 24, No. 5, 2008 1953 Quantachrome Autosorb-6B apparatus. Surface analysis of both batches shows the same Ca:P ratio, 5:4, as determined on an Omicron ESCAProbeP apparatus. The ζ potential, -18 mV at pH 7.5 (for both batches), was determined on a Brookhaven BI-Zeta PALS apparatus. Preparation of Solutions of 160Tb-Containing Tb Complexes. (Materials containing 14C and 160Tb isotopes and their solutions are denoted as “labeled” in the Experimental Section; other materials are denoted as “nonlabeled”.) The appropriate macrocyclic ligand (260 µmol) was suspended in H2O (3 mL). An aqueous solution of NaOH (10%) was added slowly until all ligand was dissolved. The resulting solution was dropped slowly into a solution of irradiated (11 mg, 24 µmol) and nonirradiated (102 mg, 225 µmol) Tb(NO3)3‚ 6H2O in water (2 mL). After adjustment of the pH of the solution to 9 using 10% aqueous NaOH, it was kept at 80 °C (BPAMD, BPAPD) or at room temperature (BPPED) for 12 h. Then, the pH was readjusted to the desired value (7.5) with aqueous HCl. In a 25 mL volumetric flask, the solution of the complex obtained was mixed with a Tris‚HCl buffer solution (1 mol L-1, 2.5 mL) and diluted with water to a final complex concentration of 0.010 mol L-1 (A ) 60 MBq L-1) in Tris‚HCl buffer solution (0.1 mol L-1, pH 7.5). Stock Solution of HEDP Containing Traces of 14C-Labeled HEDP. Nonlabeled HEDP (100 mg, 0.49 mmol) was dissolved in H2O (10 mL). A solution of 14C-labeled HEDP (0.2 mL, 3 mg, 0.013 mmol), was added and the pH was adjusted to the desired value (7.5) with aqueous NaOH (10%). The solution obtained was transferred into a 50 mL volumetric flask and diluted to a final concentration of 0.010 mol L-1 (A ) 17.3 MBq L-1) using Tris‚HCl buffer solution (0.10 mol L-1, pH 7.5). Stock Solutions of Nonlabeled Bisphosphonates. The nonlabeled bisphosphonate (250 µmol) was dissolved in H2O (5 mL). pH was adjusted to the desired value (7.5) with aqueous NaOH (10%). In a 25 mL volumetric flask, the solution of the compound obtained was mixed with a Tris‚HCl buffer solution (1 mol L-1, 2.5 mL) and diluted with water to a final bisphosphonate concentration of 0.010 mol L-1 in Tris‚HCl buffer solution (0.1 mol L-1, pH 7.5). Adsorption Experiments: General Procedure. Unless stated otherwise, the following procedure was applied. In a 5 mL vial, HA was suspended in a Tris‚HCl buffer solution (0.10 mol L-1, pH 7.5), and a solution of the compound under study was added to get a final volume of 3.0 mL. The suspension was gently shaken for 72 h and then filtered through a Millipore filter (0.22 µm). The radioactivity in 1.0 mL of the filtrate was determined by measuring the intensity of emitted γ rays using a NaI(Tl) scintillation detector based γ ray counter (Wallac). The radioactivity in samples containing 14C-labeled HEDP was measured in a liquid scintillation counter (Tricarb Spectrometer, Packard, Meriden, CT). The adsorbed amounts of the bisphosphonates were calculated from the observed counting rates of 160Tb or 14C in filtered solutions by comparison with the counting rate of a mixture of a stock solution of the labeled compound under study (0.1 mL) and a Tris‚HCl buffer solution (0.10 mol L-1, 0.9 mL). The experimental data were fitted by means of a least-squares fitting procedure using the Micromath Scientist program, version 2.0 (Salt Lake City, UT). Estimation of the Time Required To Establish Thermodynamic Equilibrium. Solutions of labeled Tb complex with total concentrations of the complex under study of 0.33, 0.66, and 1.00 mmol L-1 were incubated with HA (50 mg) for 1, 3, 5, 7, 24, 48, or 72 h and processed as described above. Reversibility of the Adsorption. A solution of labeled Tb complex was incubated with HA (50 mg) for 72 h. Then a solution of the nonlabeled complex was added. The total concentration of labeled Tb in the samples was 0.33 or 0.67 mmol L-1, whereas the total concentration of nonlabeled Tb was 1.50 mmol L-1. The resulting mixture was shaken for another 72 h and processed as described above. For comparison, the labeled and the nonlabeled Tb complex solutions of the same concentrations were mixed prior to incubation. The mixture was incubated with HA (50 mg) for 72 h and processed as described above.

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Adsorption Isotherm of the Tb Complexes. A solution of the labeled Tb complex under study (the total concentration of labeled Tb in the samples was 0.17-2.00 mmol L-1) was incubated with HA (50 mg) and processed as described above. Adsorption Isotherm of HEDP. A stock solution of labeled HEDP (the total concentration of labeled HEDP in the sample was 0.3-7.3 mmol L-1) was incubated with HA (50 mg) and processed as described above. Competitive Adsorption Experiments. A solution of labeled Tb-BPAPD complex and a solution of the competing bisphosphonate were incubated with HA (50 mg) and processed as described above. Five sets of experiments were performed: (experiment 1) a series in which the concentration of the labeled Tb-BPAPD complex was constant (0.33 mmol L-1), whereas the concentration of the competing bisphosphonate was varied between 0.17 and 3.00 mmol L-1; (experiment 2) a series with constant concentration of labeled TbBPAPD (0.67 mmol L-1) and variation of the concentration of the competing bisphosphonate between 0.17 and 3.00 mmol L-1; (experiment 3) a series with equimolar concentrations of labeled Tb-BPAPD and competing bisphosphonate, varying between 0.08 and 1.00 mmol L-1; (experment 4) a series with a molar concentration ratio of labeled Tb-BPAPD and competing bisphosphonate of 1:3; the concentration of the bisphosphonate was varied from 0.08 to 1.50 mmol L-1; (experiment 5) a series with a molar concentration ratio of labeled Tb-BPAPD and competing bisphosphonate of 1:6; the concentration of the bisphosphonate was varied from 0.20 to 2.00 mmol L-1. Competitive Adsorption Experiments with Tb Complexes and Their Corresponding Free Ligands. Solutions of the free ligand and its labeled Tb complex were incubated with HA under the same conditions as described in the previous section (“Competitive Adsorption Experiments”) and processed as described above.

Results Adsorption Isotherms of the Conjugates of DOTA and Bisphosphonates. The adsorption isotherms of the three Tb complexes of the DOTA derivatives with a bisphosphonatecontaining pendant arm (Tb-BPAMD, Tb-BPAPD, TbBPPED) on HA were determined using 160Tb as a radiolabel. Many models to describe adsorption isotherms have been reported in the literature.18,19 The Langmuir (eq 1) and the Langmuir-Freundlich (eq 1) isotherms represent the most

(Kc)n X ) Xm 1 + (Kc)n

(1)

commonly applied ones. Here, K is the affinity constant (L mol-1), Xm the maximum adsorption capacity (mol m-2), X the specific adsorbed amount (mol m-2), c the equilibrium concentration in the solution (mol L-1), and n a coefficient describing the adsorption energy distribution (Langmuir, n ) 1; LangmuirFreundlich, 0 < n < 1). The Langmuir model describes the system as a surface showing homogeneous distribution of the places available for adsorption without taking into account any interaction between adsorbed molecules, whereas the LangmuirFreundlich model assumes the adsorption energies to have an exponential distribution. This could be caused by nonhomogeneity of the sorbent surface and/or interactions between sorbate molecules as a result of, for example, Coulomb interactions. All three complexes (Tb-BPAMD, Tb-BPAPD, and TbBPPED) showed fast adsorption; most of each complex (>90%) was adsorbed during the first hour, and the equilibrium was reached within 24 h for all tested initial concentrations. The reversibility of the adsorptions was demonstrated by adding (18) Marczewski, A. W.; Jaroniec, M. Monatsh. Chem. 1983, 114, 711-715. (19) Jaroniec, M.; Derylo, A.; Marczewski, A. W. Monatsh. Chem. 1983, 114, 393-397.

nonlabeled Tb complex to the suspensions obtained. After 72 h, the equilibrium activity was the same as that of a mixture of labeled/nonlabeled complex of the same concentrations in the presence of fresh HA. The experimental adsorption isotherms were fitted with both the Langmuir and the Langmuir-Freundlich models. For the Tb complexes of BPAMD and BPAPD, excellent fits were obtained with the Langmuir model (see Figure 2A,B), whereas the Langmuir-Freundlich model resulted in a best fit for n values of about 1. For the Tb-BPPED complex a significantly better fit was obtained with the Langmuir-Freundlich model (Figure 2C) and n ) 0.46. The best-fit parameters obtained are summarized in Table 1. Competition Experiments. To evaluate the binding characteristics of the free ligands BPAMD, BPAPD, and BPPED and of a series of bisphosphonate derivatives lacking a macrocyclic moiety (see Figure 1), competition experiments were performed on a mixture containing the bisphosphonate in question and the labeled Tb complex as a probe. Models for simultaneous adsorption of two compounds have been described in the literature.20-22 Unfortunately, all these models require knowledge of the adsorption parameters of the individual compounds in the mixture and do not provide the possibility to use one of the compounds as a probe for the study of the second one. Competitive adsorption of bisphosphonates on HA and their effect on HA crystal growth have been reported,23-25 but the information about the adsorption parameters of the studied compounds obtained from those experiments is limited. Therefore, a new mathematical model for determination of Xm and K constants for multiadsorbate solid-solution systems has been developed. The Langmuir and the Langmuir-Freundlich equations can be written as in eq 2. Here, the term Xm - X represents the free surface capacity. For simultaneous adsorption of two or more compounds on a surface, the free capacity of each individual compound is efficiently decreased by the adsorption of the others. Therefore, the term Xm - X can be substituted by the term Xf representing the effective free capacity (eq 3). This term can be expressed in the free fraction of the HA surface (Sf) and the total surface of HA (S) (eq 4). Together with the equations relating Xi to Si, the HA surface fractions occupied by each compound (eq 5), the HA surface balance (eq 6), and the mass balance of the adsorbates (eq 7; mtot is the total molar amount of the compound, mads the adsorbed molar amount, and msol the molar amount remaining in the solution), a set of equations is obtained that describes the multicomponent adsorption process.

Kn )

X c (Xm - X)

(2)

X c Xf

(3)

n

Kn )

n

Sf X f ) Xm S Si Xi ) Xm S S)

∑Si + Sf

mtot ) mads + msol

(4) (5) (6) (7)

The competition experiments were performed under five different conditions: two series with different but constant concentrations of the Tb complex (the probe) and varying

Complexes of DOTA-Bisphosphonate Conjugates

Langmuir, Vol. 24, No. 5, 2008 1955

Figure 2. Adsorption isotherms of the Tb-BPAMD (A),12 Tb-BPAPD (B), and Tb-BPPED (C) complexes and HEDP (D) on HA at 25 °C. The curves represent the results of fittings of the experimental data with the Langmuir model (solid line). In (C) and (D) the dashed curve gives the result of a fitting with the Langmuir-Freundlich model. Table 1. Adsorption Parameters of the Tb Complexes TbBPAMDa,b K/103 (L mol-1) Xm/10-6 (mol m-2) n a

TbBPAPDa

TbBPPEDa

TbBPPEDc

196 ( 11

14.1 ( 2

249 ( 44

129 ( 21

0.621 ( 6

0.722 ( 10

0.652 ( 18

0.778 ( 18 0.46 ( 3

Langmuir model. b Reference 12. c Langmuir-Freundlich model.

concentrations of the second compound (experiments 1 and 2) and three different series of experiments with constant probe complex:bisphosphonate ratios (experiment 3, 1:1; experiment 4, 1:3; experiment 5, 1:6). The results of all five experiments were processed simultaneously. The results of fittings according to the Langmuir-Freundlich model showed strong correlation between the parameters n and Xm, and therefore, all fittings of the competition experiments were only performed according to the Langmuir model. Very good fits were obtained, which are given as curves in Figure 3, and the best-fit parameters are summarized in Tables 2 (free ligands) and 3 (“simple” bisphosphonates). To prove the validity of the competition method, the adsorption parameters of HEDP were determined from the adsorption isotherm of the 14C-labeled HEDP (Figure 2D, Table 4). Similar (20) Rush, U.; Borkovec, M.; Daicic, J.; van Riemsdijk, W. H. J. Colloid Interface Sci. 1997, 191, 247-255. (21) Derylo, A.; Jaroniec, M. Chem. Scr. 1982, 19, 108-115. (22) Jaroniec, M.; Derylo, A.; Marczewski, A. W. Chem. Eng. Sci. 1983, 38, 307-311. (23) van Beek, E. R.; Lowik, C. W. G. M.; Ebetino, F. H.; Papapoulos, S. E. Bone 1998, 23, 437-442. (24) Leu, C.; Luegmayr, E.; Freedman, L. P.; Rodan, G. A.; Reska, A. A. Bone 2006, 38, 628-636. (25) Nancollas, G. H.; Tang, R.; Phipps, R. J.; Henneman, Z.; Gulde, S.; Wu, W.; Mangood, A.; Russell, R. G. G.; Ebetino, F. H. Bone 2006, 38, 617-627.

to the results on the Tb-BPPED complex, a much better fit was obtained with the Langmuir-Freundlich than with the Langmuir model. The results obtained are in good agreement with those obtained from the competition experiments (see Table 3).

Discussion As HA is an ionic compound, Ca2+, PO43-, and OH- ions can be present on its surface. Bisphosphonates show a high affinity for the Ca2+ ion. High stability constants14,27 and several crystal structures28,29 of their calcium complexes have been reported. Thus, coordination of the bisphosphonates on the Ca2+ ions on the surface and/or substitution of the surface phosphate anions are believed to be the main mechanism of the adsorption.7,30 The adsorption is then described as a bidentate coordination via two phosphonate oxygen atoms or tridentate coordination via two phosphonate oxygen atoms and a hydroxy or amino group bound at the R-carbon atom.13 In complexes of simple bisphosphonates, the metal ion is coordinated by phosphonate oxygen atoms, which are also responsible for the adsorption on the surface of HA. Those complexes are not kinetically inert. Therefore, the adsorption is a complicated process including dissociation/formation of the complexes and adsorption of all the complex species as well as its parts (i.e., ligand and metal ion) separately.26 The complexes of the studied macrocycle-bisphosphonate conjugates show a (26) Claessens, R.A.M.J.; Kolar, Z.I. Langmuir 2000, 16, 1360-1367. (27) (a) Martell, A. E.; Smith, R. M. Critical Stability Constants; Plenum Press: New York, 1974-1989; Vols. 1-6. (b) NIST Standard Reference Database 46 (Critically Selected Stability Constants of Metal Complexes), version 7.0; National Institute of Standards and Technology: Gaithersburg, MD, 2003. (28) Fernandez, D.; Vega, D.; Goeta, A. Acta Crystallogr., Sect. C: Cryst. Struct. Commun. 2002, 58, m494-m497. (29) Fernandez, D.; Vega, D.; Goeta, A. Acta Crystallogr., Sect. C: Cryst. Struct. Commun. 2003, 59, m543-m545. (30) Haque, S.; Rehman, I.; Darr, J. A. Langmuir 2007, 23, 6671-6676.

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Figure 3. Competitive adsorption experiments of AcMP2-(Tb-BPAPD) (A, B) and APP2-(Tb-BPAPD) (C, D). The total amount of simple bisphosphonate, mtot(bisphosphonate), is on the x axis, and the adsorbed amount of Tb-BPAPD, mads(probe), is on the y axis. Experiment: 1 ([), 2 (2), 3 (b), 4 (9), 5 (O). The results of the best fits are represented by the curves (solid lines). Table 2. Adsorption Parameters of the Free Ligands K/103 (L mol-1) Xm/10-6 (mol m-2)

BPAMD

BPAPD

BPPED

236 ( 39 0.609 ( 8

29 ( 7 0.687 ( 63

285 ( 135 0.693 ( 36

Table 3. Adsorption Parameters of the Simple Bisphosphonates Determined from Competitive Sorption Experiments AcMP2

AMP2

APP2

PAM

HEDP

46 ( 5 59 ( 13 16 ( 2 44 ( 10 53 ( 9 (L mol-1) Xm/10-6 1.19 ( 3 2.00 ( 7 1.50 ( 6 1.82 ( 7 1.58 ( 5 (mol m-2) K/103

Table 4. Adsorption Parameters of HEDP Langmuir model K/103 (L mol-1) Xm/10-6 (mol m-2) n

62 ( 12 1.89 ( 7

LangmuirFreundlich model 34 ( 9 2.17 ( 12 0.58 ( 7

ref 26 3.3 2.9

completely different coordination mode. Complexes of the DOTA-like ligands with trivalent lanthanide ions show high thermodynamic and kinetic stability and, therefore, are resistant to decomplexation, transmetalation, and/or ligand exchange.31 The structural characterizations of the lanthanide complexes of the studied macrocycle-bisphosphonate conjugates have shown that Tb ion is bound exclusively in the macrocyclic cavity, whereas the bisphosphonate group is not coordinated to the Tb ion.12 Thus, Tb ion cannot be involved directly in any adsorption process. Further, the described procedure12 yields exclusively complexes with a 1:1 metal:ligand ratio. Therefore, the adsorption can be (31) Merbach, A. E., To´th, E., Eds. The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging; John Wiley and Sons: Chichester, U.K., 2001.

considered as an interaction between the HA surface and a free bisphosphonate attached to a bulky substituent (the macrocyclic complex). The adsorption isotherms for the Tb-BPAMD and TbBPAPD complexes could be described by the Langmuir model, whereas for Tb-BPPED the Langmuir-Freundlich model gave a considerably better fit. This can be ascribed to a difference in charge of the macrocyclic part of the complexes. Whereas the amide complexes (Tb-BPAMD and Tb-BPAPD) are electroneutral, the phosphinate-containing one (Tb-BPPED) bears a negative charge, and as a result repulsion between the adsorbed molecules has an effect on the adsorption behavior. All complexes showed comparable maximum adsorption capacities. However, the affinity constants differ over 1 order of magnitude. The adsorption parameters of the corresponding free ligands are generally comparable with those of their Tb complexes, showing that the presence of a Tb ion in the macrocyclic cavity does not have much influence on the adsorption behavior. The results of the adsorption experiments show that maximum adsorption capacities of the studied compounds correlate mainly with the size of the molecules (Figure 4). The bisphosphonates bearing the bulky DOTA macrocycle exhibit a 2-3 times lower maximum adsorption capacity than the simple bisphosphonates. However, from the values of Xm and the total surface, it can be calculated that the surface area occupied by one molecule for the macrocyclic derivatives (∼200 Å2) is in good agreement with that area as determined by computer modeling32 (taking into account free rotation of the macrocycle moiety around the anchored bisphosphonic acid group), whereas for the simple bisphosphonates the values obtained from the experiment (∼80100 Å2) and from the modeling (∼20-30 Å2) are very different. (32) HyperChem Release 7.5 for Windows, Molecular Modeling System, Hypercube, Inc., Ontario, Canada, 2002.

Complexes of DOTA-Bisphosphonate Conjugates

Figure 4. Comparison of maximum adsorption capacities of the studied compounds.

Figure 5. Comparison of affinity constants of the studied compounds.

This can be explained by the negative charge of the bisphosphonate group and/or the positive charge of protonated amino groups of the ligands AMP2, APP2, and PAM,14 resulting in a strong electrostatic interaction between molecules adsorbed on the HA surface. Therefore, the HA surface cannot be fully covered with the latter compounds. It may be concluded that, in the case of the macrocycle-containing bisphosphonates, the area occupied by one molecule is determined by the size of the molecule, whereas for the simple bisphosphonates, the shortest distance between two adsorbed molecules is determined mainly by their charge. It is generally believed that the presence of a hydroxy or amino group attached to the R-carbon atom of bisphosphonates is essential for their efficient adsorption on HA.7 This is usually explained with a tridentate adsorption (two phosphonate oxygen atoms and the R-heteroatom interacting with HA).13 The present results confirm this hypothesis. The values of the affinity constants of APP2, BPAPD, and Tb-BPAPD are significantly lower than those of the other compounds (Figure 5). In the case of BPPED and the Tb-BPPED complex, which do not contain an R-heteroatom, the high value of the affinity constant can be explained by a synergic effect of the negatively charged phosphinic acid group in the β-position. Similarly, an increase of the HA affinity with incorporation of a sulfonate group at the β-position of a bisphosphonate has been reported.33 (33) Lisic, E. C.; Phillips, M.; Ensor, D.; Nash, K. L.; Beets, A.; Knapp, F. F. Nucl. Med. Biol. 2001, 28, 419-424.

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Very high affinities were found for BPAMD and its Tb complex. These compounds have affinity constants that are higher than that of the analogue without a macrocyclic moiety, AcMP2. Although a negligible adsorption of the polyazapolycarboxylates on HA has been reported previously,34 the relatively high affinity of BPAMD and its Tb complex should be ascribed to a synergic effect of the macrocycle and the bisphosphonic acid groups, which are in close proximity in this case. A possible explanation may be sterical hindrance of the bisphosphonic acid group by the DOTA moiety. Similarly, it has been shown that, for bisphosphonate-peptide conjugates, a longer spacer between both parts of the conjugate results in a lower affinity for HA.35 We assume that an increase in HA affinity may originate from a limited excess of water molecules to the adsorbed bisphosphonate moiety due to the presence of a bulky substituent and/or hydrophobic group. This suggests an important role of water molecules in the adsorption/desorption mechanism. There are two possible mechanisms: (i) The “free” surface of the adsorbent is solvated (i.e., covered with a layer of water molecules). The adsorption equilibrium can then be described as an exchange between adsorbed/free water and bisphosphonate molecules. The decrease of the local water concentration could “push” the equilibrium into the direction of bisphosphonate adsorption. (ii) The desorption mechanism could involve protonation of the phosphonate group (analogous to the dissociation of a metal ion from phosphonate complexes in homogeneous solution) and/or protonation of the HA surface. The protonation is dependent on the availability of H3O+ ions. A decrease of the local water (and so H3O+) concentration may result in a lower desorption rate and, consequently, in a higher HA affinity for the bisphosphonate. The previously reported studies on adsorptions of bisphosphonates were performed using mainly 14C or 32P labeling,13 which are not easily accessible, and their applications are complicated with storage of the wastes due to the long half-life of the carbon isotope. Determination of the bisphosphonate affinities is important for characterization of the newly developed pharmaceuticals and contrast agents.12,13,36 The competition method described in this paper has several advantages. With one radiolabeled complex, many different nonlabeled bisphosphonates can be studied via the competition experiments in a direct comparison. The application of the bisphosphonate-macrocycle conjugates (ligands BPAMD, BPAPD, and BPPED) allows easy radiolabeling via complexation of a radioactive metal ion by the DOTA moiety. As DOTA-like ligands can be used for complexation of a large group of metal ions (all lanthanide as well as many transition- or main-group-metal ions), the properties of the probe complex can be easily tuned. Different types and energies of the radiation as well as half-lives can be obtained by selection of the applied metal isotope. The determination of surface affinities of bisphosphonates is not complicated by solution speciation of bisphosphonate complexes as the probe macrocycles form 1:1 ligand-metal complexes with the metal ions.

Conclusions The adsorption behavior of three macrocyclic ligands containing a bisphosphonic acid group in a side chain and their Tb complexes on hydroxyapatite was studied by a radiotracer method making use of 160Tb as the label. The adsorption of the ligands (34) Li, W. P.; Ma, D. S.; Higginbotham, C.; Hoffman, T.; Ketring, A. R.; Cutler, C. S.; Jurisson, S. S. Nucl. Med. Biol. 2001, 28, 145-154. (35) Gittens, S. A.; Kitov, P. I.; Matyas, J. R.; Lobenberg, R.; Uludag, H. Pharm. Res. 2004, 21, 608-616. (36) Palma, E.; Oliveira, B. L.; Correia, J. D. G.; Gano, L.; Maria, L.; Santos, I. C.; Santos, I. J. Biol. Inorg. Chem. 2007, 12, 667-679.

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and their complexes is fast and reversible. One of the complexes was used to probe the adsorption affinity of nonlabeled compounds. A physicochemical model describing the competitive adsorption was evaluated and successfully applied in the calculation of adsorption parameters for a group of bisphosphonates. The maximum adsorption capacity of bulky bisphosphonates is determined mainly by their size. For simple bisphosphonates, the maximum adsorption capacity is determined by the electrostatic repulsion between negatively charged bisphosphonate groups. The values of the affinity constants depend on the structures of the bisphosphonic acid group. The compounds containing a hydroxy or amino group attached to the R-carbon atom show higher affinities. Macrocyclic compounds containing a short spacer between the disphosphonic acid group and the macrocycle unit exhibit high affinities, indicating a synergic effect of the bisphosphonic and the macrocyclic groups during adsorption. The competition method proposed here expands the possibilities for studying the sorption behavior of bisphosphonates and other compounds. The application of the macrocycle-bisphosphonate

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conjugates allows easy radiolabeling and provides the possibility of tuning of probe complex properties. The method described can be generally used for a comparative study of other groups of compounds and sorbents. Acknowledgment. Thanks are due to the EU for financial support via a Marie Curie training site host fellowship (MESTCT-2004-7442). Support from the Grant Agency of the Czech Republic (Grant No. 203/06/0467), Grant Agency of the Academy of Science of the Czech Republic (Grant No. KAN201110651), and Long Term Research Plan of the Ministry of Education of the Czech Republic (Grant No. MSM0021620857) is acknowledged. The work was carried out in the frame of COST D38 and the EU-supported NoE projects EMIL (Grant No. LSHC-2004503569) and DiMI (Grant No. LSHB-2005-512146). Supporting Information Available: Description of the syntheses of compounds BPAPD and BPPED. This material is available free of charge via the Internet at http://pubs.acs.org. LA702753J