Ofgallium(III)Complexeswhichareeitherimpermeabletothe 6 7Ga Samples of Known Activity

Home , Sodium nitrate

[CANCER RESEARCH 34, 2957â€”2960,November 1974J Effects ofBuffers and pH on in Vitro Binding of 67Gaby L1210 Leukemic Cells'

Jerry D. Glickson, John Webb,2 and Richard A. Gams Diviiion ofHematology, Department ofMedicine, and the Cancer Research and Training Center, University ofAlabama in Birmingham School of Medicine, Birmingham,Alabama 35294

SUMMARY MATERIALS AND METHODS

The effect of sodium nitrate and a series of buffers on in Buffer Inhibition. Stock solutions 6 â€˜Gacitrate and 67Ga vitro 67Ga binding to L1210 leukemic cells at pH 6.8 Â±0.2 nitrate (New England Nuclear, North Billirica, Mass.) and 37Â° at concentrations of iO@ to l02 M has been containing 2 mCi/ml at noon of the day of delivery were used investigated. The relative ability of these agents to inhibit within 2 weeks of purchase (tÂ½ 78.1 hr). Commercial cellular incorporation of 67Ga is ethylenediaminetetraacetate preparations of the citrate salt contained sodium citrate, 2 > nitrilotriacetic acid >> citrate >>> bicarbonate > mg/nil. The nitrate sample was approximately 0.5 M in nitrate. phosphate, lactate, Tris > morpholinopropane sulfonic acid Dilutions were made in 0.9% NaC1 solution and counted in a (MOPS), N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic Model 1185 automatic well scintillation counter (Nuclear @ acid, nitrate 0. Inhibition probably results from formation Chicago Corp., Des Planes, 111.),that had been calibrated with ofgallium(III)complexeswhichareeitherimpermeabletothe 6 7Ga samples of known activity. Buffers and salts used in this tumor membrane or which compete with intracellular receptor investigation included sodium bicarbonate (J. T. Baker complexes. However, direct interaction of buffers with the cell Chemical Co., Phfflipsburg, N. J.); potassium dihydrogen membrane or with gallium (III) receptors, as well as effects of phosphate (American Chemical Society certified; Fisher buffers on cellular metabolism, have not been excluded. A Scientific Co., Fair Lawn, N. J.); lactic acid (American monotonic decrease in the cellular incorporation of 67Ga Chemical Society reagent; Matheson, Coleman, and Bell, occursbetweenpH6.2and7.8inthepresenceoftheinert Norwood, Ohio); Tris (Fisher Scientific Co.; certified primary buffer, lO2 M morpholinopropane sulfonic acid. standard); morpholinopropane sulfonic acid (MOPS) (Sigma Chemical Co., St. Louis, Mo.); HEPES (Sigma); sodium nitrate (Baker; analyzed reagent); tnsodium citrate (Fisher; American INTRODUCTION Chemical Society certified), EDTA disodium salt (Cambridge Chemical Products, Inc., Detroit, Mich.), and NTA (Sigma). Preferential localization of 67Ga in a broad range of solid Except for modifications described below, the in vitro tumors and malignant lymphomas has been employed in the 6 7Ga-binding procedure described in detail elsewhere (8) was clinical detection and early staging of malignant disease by used. L12l0 cells were harvested from the peritoneal cavity of 6 7Ga scintigra@hy (4, 1 1). An understanding of the detailed female C57BL X DBA/2 F1 (hereafter called BD2F1 ) mice 6 mechanism of 7Ga localization in tumor cells may lead to the days after i.p. inoculation of i0@ leukemic cells. The cells were rational development of optimum methods for the detection washed 3 times with 0.9% NaCl solution, and a 4% packed-cell ofmalignantneoplasmsandmayalsorevealsomeunderlyingvolume (2.8 X l0@ cells/ml) suspension was prepared and differences between normal and transformed cells that stored in an ice bath. Samples of the buffer or salt (1 ml) determine their interaction with metal ions. To help attain adjusted to pH 6.8 Â±0.2 with HC1 and/or NaOH were these objectives, an in vitro technique for measuring binding of incubated in 12- x 75-mm Falcon 2052 counting tubes (Becton, Dickinson and Company, Oxnard, Calif.) for 1 hr at 6 7 Ga by dispersed cells was recently developed in this 0 . â€”8 . 6 laboratory (8). Preliminary experiments indicated that a 37 with 2.60 X 10 jimole (1.05 jiCi, 1.5 X 10 cpm) of number of buffers (bicarbonate, phosphate, Tris, barbital, 6 â€œGa(delivered in 100 jzl of0.9% NaC1 solution). At specified maleic acid, and citrate) inhibit in vitro uptake of 67Ga by timeintervals,250-jzlaliquotsofcellsuspensionwereaddedto Ll 2 10 leukemic cells. This study examines buffer inhibition duplicate tubes and the incubation was continued. The and pH effects in more detail. The need to study the effects of reaction was quenched by transferring the tubes to an ice bath counterions on the incorporation of 67Ga by isolated tumor and adding 2 ml of iced 0.9% NaCl solution to each tube. The cells is underscored by recent experiments by Konikowski et cells were washed 3 additional times with 0.9% NaCl solution aL (10), who report a significant difference in tumor (at 4Â°),and the residual radioactivity was determined. localization and clearance of the citrate, lactate, and chloride pH Studies. Cell-binding studies were performed as de-â€¢ @ salts of 67Ga as opposed to the DTPA salt. scribed above, with the use of an incubation medium consisting of 1 ml 10 2 M morpholinopropane sulfonic acid in 0.9% NaC1 solution adjusted to pH 6.2, 6.6, 7.0, 7.4, and 7.8. ReceivedMay 13, 1974; accepted July 16, 1974. The cell reaction was quenched after 2 hr of cell incubation. I This research was supported by Grant CA-13148 from the NIH. 2 Present address: Research School of Chemistry, Australian National University, P. 0. Box 4, Canberra, Australia 2600. JVâ€•-pentaceticacid; HEPES, N-2-hydroxyethylpiperazine N'-2-ethane 3The abbreviations used are: DTPA, diethylenetriamine-N,N,N',Nâ€•,sulfonic acid; NTA, nitriotriacetic acid.

NOVEMBER 1974 2957

5 60 , , I I 40 . BICARBONATE 20 0 0

z 0 o-o@! 0 -20 20 PHOSPHATE 0 0 0@â€” â€”0@0 Â° 0 -20 (I) @ w 2 20 LACTATE -J

0 z 0 2-20 I.â€” x I @2O TRIS ______,__@_____,0 zI 0 Iâ€”-20

z . MOPS0 w0 20 @00..-000 TIME (hr) @-2O Chart 1. Time course of uptake of 6â€˜7Gacitrate and 67Ga nitrate 20 HEPES.0.000 by 7.0 X 106 L1210 leukemic cells,at pH 6.8 Â±0.2and 37Â°.@ 0

RESULTS -20 .0o.. -40 Chart 1 summarized the kinetics of in vitro binding to 20 NITRATE.0 L1210 leukemic cells of 2 commercial preparations of 0 carrier-free 67Ga, i.e., the citrate and nitrate salts. The 67Ga -20 I0 II ?i concentration was 1.93 X 10-i â€˜M for both samples. Ap -7 -6 -5 -4 -3 -2 proximate concentrations of citrate and nitrate ions in the in log [ANIoN] @ vitro incubation mixture were 1.6 X 10 M and 1.2 X 10@@ Chart 2. Inhibition of 2-hr in vitro uptake of 67Ga by 7.0 x 106 M, respectively. Zinc, formed by radioactive decay of 67Ga, L1210 leukemic cells incubated with sodium nitrate or variousbuffers @@ was estimated at a level of about 6.65 X 10 M. It has at pH 6.8 Â±0.2 and 37Â°.Each point, average of duplicate previously been demonstrated that zinc at this concentration measurements. The percentage inhibition was (C â€”C0)/C0 x 100%, does not influence the extent of in vitro 6 7Ga binding by where C is the number of cpm measured for Li 210 cells incubated for L1210 leukemic cells (8). Incubation at 37Â°was limited to 2 2 hr with the specified buffer and C0 is the number of cpm measured @ hi, during which time the exclusion of the vital dye trypan for a control sample incubated with Ga containing no citrate blue suggests retention of membrane integrity of the L1210 other than that present in the commercial sample. A typical experiment cells (8). During this time interval, the cells responded in an measuring C0 appears in Chart 1.@MOPS, morpholinopropane sulfonic acid. identical manner to the citrate and nitrate salts of 6 7Ga (Chart 1). The inert buffer morpholinopropane sulfonic acid (10 2M) Chart 2 shows inhibition curves for nitrate, HEPES, was used to measure cellular 67Ga uptake between pH 6.2 and morpholinopropane sulfonic acid, Tris, lactate, phosphate, and 7.8 (Chart 4). A monotonic decrease in cellular localization of bicarbonate. Nitrate, HEPES, and morpholinopropane sulfonic the radioisotope is observed with increasing pH of the acid have no effect on in vitro 67Ga binding up to a incubation medium. concentration of 102 M. Tris, lactate, and phosphate inhibit @ slightly at concentrations above 10 M , whereas bicarbonate inhibits somewhat more. Citrate, NTA, and EDTA are much DISCUSSION more potent inhibitors (Chart 3). Since the pH remained at 6.8 Â± 0.2 after the 2-hr incubation, the inhibitory effects originate The aqueous chemistry of gallium (III) is complex and not from the specific buffers used in these experiments, rather completely characterized (2, 15). Salts such as the nitrate (18) than from pH changes. The relative order of inhibitory and perchlorate (5, 6) dissociate completely in strongly acidic capacity is EDTA > NTA > > citrate > > > bicarbonate > solution to yield Ga(H2O)6@'3 ions. However, the solubility @ phosphate, lactate, Tris > morpholinopropane sulfonic acid, product of Ga(OH)3 (about 5 X 10 7) (2) indicates that the @ HEPES, nitrate 0. maximum concentration of hydrated gallium (III) in neutral solution is about 5 X 10-i 6M. Consequently, gallium (III) is present in solution in the form of various complexes. The

4 Concentrations of gallium (III) have been calculated from the i@Ci perchiorate salt forms metastable polymers with maximum of 67Ga measured, assuming the radiochemical was carrier free ; 1.05 degrees of polymerization near pH 3 (3, 14). Citrate polymers @iCi(2.60x 108 mmoles) of 67Gawere administered. have also been detected in this laboratory by equilibrium

2958 CANCER RESEARCH VOL. 34-

I00 favor species with a greater or lesser permeability to tumor cell membranes or with a greater or lesser affinity for cellular z 90 receptors. The incorporation of buffers, particularly citrate, in 280 commercial preparations of 6 7Ga and the presence in the Iâ€” plasma of various potential complexing agents of both low and high molecular weight indicate a need to in'@stigate the effect @60 of counterions on tumor localization of this radioisotope. @50 Inhibition of 6 7Ga binding by Ll2lO leukemic cells @40 probably results from formation of 67Ga complexes that are either impermeable to the tumor membrane or that are more @30 stable than intracellular receptor complexes of the metal. This 0:: 20 theory is supported by the relative inhibitory capacity of the w a. 0 buffers, which is in the order anticipated for the stability of gallium (III) complexes, the chelating agents EDTA, NTA, and 0 citrate being the most potent inhibitors. Moreover, differential -@7 -6 -5 -4 -3 -2 thermal analysis (7) and ion-exchange chromatography (1) demonstrate the formation of gallium (III) citrate complexes. Log [Buffer] M EDTA complexes with gallium (III) are well known from Chart 3. Inhibition of 2-hr in vitro uptake of 67Ga by 7.0 x 106 titrimetric studies (13). The general inert response of L1210 leukemic cells incubated with sodium citrate (o), NTA (o), or morpholinopropane sulfonic acid and HEPES to polyvalent EDTA (V) at pH 6.8 Â±0.2 at 37Â°.Eachpoint, averageof duplicate cations is also well-documented (9). measurements.4 The shapes of the EDTA, NTA, and citrate inhibition curves (Chart 3) can readily be understood in terms of complex formation. These curves resemble citration curves for metal complex formation. At high buffer concentrations, all the available metal may be sequestered in complexes unavailable for tumor localization. These complexes, however, would a z dissociate at low buffer concentrations by a mass law effect, 0 thereby liberating 6 7Ga for tumor binding. Binding 6 7Ga to L12l0 cells may serve as a measure of the concentration of 0 (9 gallium (III) unassociated with buffer anions. Taken together, these observations suggest that the domi w(I) nant cause for inhibition of in vitro 6 7Ga binding by various -J buffers is the competitive formation of gallium (III) complexes 0 with these buffers. However, the possibility that buffers may interact in some manner with the cell to render it impermeable â€˜C to gallium (III) or to block receptor sites for this metal cannot 0 be totally discounted. Buffers such as citrate, phosphate, or bicarbonate may also affect cellular metabolism. The inhibitory effect of the buffers, at least that of the chelating agents, does not result solely from competition for cell surface-bound 67Ga. In the absence of buffer, only about 12% of the cell-bound 6 7Ga appears to be adsorbed on the pH cellular membrane (8), whereas the inhibitory capacities of Chart 4. The pH dependence of 67Ga bound by 7.0 x 106 L1210 EDTA, NTA, and citrate approach 100%. leukemic cellsincubated in vitro for 2 hr with 10 2 M morpholinopro Inhibitory effects such as those described in these in vitro pane sulfonic acid at 37Â°.@ experiments may be significant in the design and interpreta tion of in vivo studies of 67Ga localization. Average normal dialysis (unpublished data). By analogy with other metal ions, concentrations of citrate in serum are about 7.5 X i0@ M such as iron (III) (16, 17), which also undergo hydrolytic (19). This citrate concentration isjust below the level at which polymerization, one anticipates that the polymeric equilibrium significant inhibition of in vitro 6 7Ga uptake by Li 21 0 cells is probably shifts toward the formation of lower-molecular observed (Chart 3). The observation by Konikowski et a!. (10) weight complexes when buffer anions are present in great that under in vivo conditions citrate, lactate, and chloride salts excess (the condition under which the present study was of 67Ga localize to similar extents in tumors and are performed). In addition, various oxides of gallium (III) may be cleared at similar rates is consistent with the conclusion that present in neutral aqueous solution (15). Consequently, a citrate exerts a negligible inhibitory effect. However, these number of potential binders to tumor cells are present at authors did observe a significant diminution in the extent of neutral pH. Changes in concentrations of counterions and tumor localization of 67Ga when the DTPA salt was injected. changes in pH may influence the extent of in vitro binding of Spectrophotometric titration studies indicate that the stability . . â€”28 Â° 6 7Ga to Li 210 cells by shifting the molecular equilibrium to constant for gallium (III) DTPA is about 10 at 20 (12).

NOVEMBER 1974 2959

Such stable complexes may indeed inhibit tumor localization 5. Fiat, D., and Connick, R. E. Magnetic Resonance Studies of Ion Solvation. The Coordination Number of Gallium (III) Ions in of 67Ga. The effect of pH was studied in order to explore the Aqueous Solutions. J. Am. Chem. Soc., 88: 4754, 1966. 6. Fiat, D., and Connick, R. E. Oxygen-17 Magnetic Resonance molecular mechanism of cellular 6 7Ga incorporation. In the Studies of Ion Solvation. The Hydration of Aluminum (III) and limited pH range used in these studies, tumor uptake of the Gallium (III) Ions. J. Am. Chem. Soc., 90: 608â€”615,1968. radioisotope increased dramatically with decreasing pH. 7. Gallet, J. P.. and Paris, R. A. Etude Thermometrique de la Increasing uptake of the radioisotope in acidic media could be Formation des Complexes du Fer (III), de 1'Aluminum et du caused by : (a) stabilization of molecular states of 67Ga Gallium. Anal. Clam. Acts, 39: 341â€”348,1967. favorable for incorporation in the tumor, (b) titration of 8. Glickson, J. D., Ryel, R. B., Bordenca, M. M., Kim, K. H., and cellular receptors, or (c) decomposition of cellular membranes Gams, R. A. in vitro Binding of 67Ga to L1210 Cells. Cancer Res., in acidic media. A definitive choice between these mechanisms 33:2706â€”2713,1973. requires detailed information about the structure of the tumor 9. Good, N. E., Winget, G. D., Winter,W.,Connolly, T. W., Izawa, S., cell and the identification of the molecular species of gallium and Singh, R. M. M. Hydrogen Ion Buffers for Biological Research. (III) localized within tumor cells. If it is assumed that the first Biochemistry, 5: 467â€”477,1966. 10. Komkowski, T., Glenn, H. J., and Haynie, J. P. Kinetics of 67Ga effect,variationsintheconcentrationoftumorpermeable Compounds in Brain and Kidneys of Mice. J. Nucl. Med., 14: species of gallium (III), contributes, at least in part, to the 164â€”171,1973. observed variations of cellular uptake, the effect of pH could 11. Langhammer, H., Glaubitt, G., Grebe, S. F., Hampe, J. F., be used to help identify the species of this element which Haubold, V., Hor, G., Kaul, A., Koeppe, P., Koppenhagen, J., localize in Ll2lO cells. These would have to be species whose Roedler, H. D., and van der Schoot, J. B. 67-Gallium for Tumor concentration increases significantly between pH 8 and 6. Scanning. J. Nuci. Med., 13: 25â€”30,1971. Additional data on the aqueous chemistry of gallium (III) is 12. Nozaki, T., and Kasuga, K. Substitution Reactions of Some Iron (III)-Polyamine-N-polyacetate Complexes with Gallium (III) in required to identify these species. Aqueous Solution. J. Chem. Soc. Japan, 11: 2117â€”2122, 1973. 13. Ohnishi, H. Gallium, Indium, and Thallium. In: I. M. Kolthoff, P. T. Elving, and E. B. Sandell (eds), Treatise on Analytical ACKNOWLEDGMENTS Chemistry, Part 2, VoL 2, pp. 1â€”106. New York: Interscience Publishers, Inc., 1962. The authors are indebted to Dr. Paul Saltman, Dr. S. Y. Tyree, and 14. Ruff, J. K., and Tyree, S. Y. Light Scattering Studies on Aqueous Dr. Phillip Aisen for helpful discussion of the data, to Dr. K. H. Kim Gallium Perchlorate Solutions. J. Am. Chem. Soc., 20: and Mary Sabens for technical assistance, and to Dr. Frank M. Schabel 5654â€”5657, 1958. and Mary W. Trader for the generous provision of L1210 leukemic is. Sheka,I.A.,Chaus,I.S.,andMityu.reva,T.T.TheChemistryof BD2F, mice. Gallium, Chap. 3. Amsterdam: Elsevier Publishing Company, 1966. 16. Spiro, T. G., Bates, G., and Saltman, P. The Hydrolytic REFERENCES Polymerization of Ferric Citrate. II. The Influence of Excess Citrate. J. Am. Chem. Soc., 89: 5559â€”5562, 1967. I. Blanco, R. E., and Perkinson, J. U. Preparation ot Gallium Citrate 17. Spiro, T. G., Pape, L., and Saltman, P. The Hydrolytic by Ion Exchange. J. Am. Chem. Soc., 73:2696â€”2700, 1951. Polymerization of Ferric Citrate. I. The Chemistry of the Polymer. 2. Cotton, F. A., and Wilkinson, G. The Group III Elements: Al, Ga, J. Am. Chem. Soc., 89: 5555â€”5559, 1967. In, TI. In: Advanced Inorganic Chemistry, Ed. 3, pp. 260-282. New 18. Swift, T. J., Fritz, 0. G., and Stephenson, T. A. Determination of York: Interscience Publishers, Inc., 1972. the Hydration Number of Gaffium (III) in Aqueous Solution. J. 3. Craig, H. R., and Tyree, S. Y. Hydrolytic Behavior of Gallium (III) Chem. Phys., 46: 406â€”407,1967. Perchlorate. Inorg. Chem., 8: 591â€”594,1969. 19. Wolcott, G. H., and Boyer, P. 0. A Colorimetric Method for the 4. Edwards, D. L., and Hayes, R. L. Scanning malignant Neoplasms Determination of Citric Acid in Blood and Plasma. J. Biol. Chem., with Gaffium 67. J. Am. Med. Assoc., 212: 1182â€”1190, 1970. 172:729â€”736,1948.

2960 CANCER RESEARCH VOL. 34

Jerry D. Glickson, John Webb and Richard A. Gams

Cancer Res 1974;34:2957-2960.

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