Appl. Radiat. lsot. Vol. 42, No. 8, pp. 707-712, 1991 Int. J. Radiat. Appl. Instrum. Part A
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Potential Radiopharmaceuticals Labeled with Titanium-45* K I I C H I I S H I W A T A J t , T A T S U O I D O l, M I N O R U M O N M A l, M A T S U T A R O M U R A K A M P , H I R O S H I F U K U D A 2, M O T O N O B U K A M E Y A M A 3, K E N J I Y A M A D A 2, S A T O S H I E N D O 2, S E I R O Y O S H I O K A 2, T A C H I O S A T O 2 a n d T A I J U M A T S U Z A W A z ~Division of Radiopharmaceutical Chemistry, Cyclotron and Radioisotope Center, 2Department of Radiology and Nuclear Medicine, The Research Institute for Tuberculosis and Cancer and 3Division of Neurosurgery, Institute of Brain Diseases, School of Medicine, Tohoku University, Sendai 980, Japan (Received 15 October 1990; in revised form 17January 1991)
The potential of some compounds labeled with cyclotron-produced titanium-45 (45Ti) as radiopharmaceuticals was studied. Properties of colloid formation of 45TIOC12 or 45TiO-phytate in vivo resulted in the highest radioactivity uptake in the rat liver, followed by the spleen, suggesting potential for imaging the reticuloendothelial system. Three 45TiO-complexeswith diethylenetriaminepentaacetic acid, citric acid and human serum albumin showed the highest radioactivity levels in the blood over 6 h. The binding of the 45Ti with plasma transferrin in vitro and in vivo suggested that these compounds can be used for estimating the blood volume. Also, potential as an indicator representing the breakdown of the blood-brain barrier in the rat was demonstrated by autoradiography.
Introduction Titanium-45 (45Ti) has a half-life of 3.08 and a high positron branching ratio (85%), and can be produced using even small cyclotrons in positron emission tomography (PET) centers. It is uncertain whether Ti is an essential trace element in human body; however, the formation of complexes with chelating agents or organometallic compounds (Clark, 1968, 1975) could suggest the usefulness of radiolabeled Ti in nuclear medicine as potential radiopharmaceuticals, as well as in biological and environmental studies (Merrill et al., 1978). Preliminary works aimed at the application of 45Ti-labeled compounds to PET and biological studies were carried out (Ishiwata et al., 1982; Kameyama et al., 1983; Kawamura et al., 1986). We now report our results in the preparation of some 45Ti-labeled compounds and their animal studies using a tissue sampling method and in vivo imaging.
Materials and Methods Production a n d separation o f 4STi
Preparation of 45Ti was carried out according to the method of Merrill et al. (1978) with some modifica*Presented in part at the Fourth International Symposium on Radiopharmaceutical Chemistry, Jiilich, Germany, August 1982. 1"Author for correspondence.
tions. The 45Ti was produced by the proton irradiation of 65-130 mg of scandium foil (0.127 or 0.254 mm thickness, 99.9% purity, Alfa Division, Ventron Corporation, Danvers, U.S.A.) at 11.5 MeV via the 45Sc(p, n)45Ti reaction using the AVF cyclotron at Tohoku University. Analysis of radionuclidic purity by a gamma-ray spectrometer using a Ge(pure) detector showed only 45Ti as the radioactive nuclide. The 45Ti was obtained with yields of 25 _ 2 and 48 +_ 0.5 mCi/pA saturation using 0.127 and 0.254 mm thick foils, respectively. The 45Ti was separated by the method of Nelson et al. (1964). Following the irradiation, the scandium foil was dissolved in 2 mL of 6 M HCI to give 45TIC13, and a drop of conc. HNO3 was added to oxidize the 45Ti(III) to the + 4 state. The solution was evaporated to dryness and dissolved in 6 m HC1 again. This step was repeated three times to remove HNO3 completely. The 45Ti dissolved in 2 mL of 6 M HCI was loaded on an A G 50W x 8 column (100-200mesh, Bio-Rad, 1.9cm i.d. x 13cm) prewashed with 6 M HCI. The 45Ti was eluted with 6 M HC1. The scandium was eluted with 4 M HC1 containing 0.1 M HF. After drying of the 45Ti fraction, the 45Ti was dissolved in 1-2 mL of 1 M HCI. This 45Ti(IV) preparation is probably an oxotitanium species, 45TIOC12. Separation was done within 2 h of the end of irradiation with decay-corrected radiochemical yields of 75-90%.
707
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KIICHI ISHIWATA et al.
Table 1. Relative migrating distances of the 4STi-labeled complexes on the paper electrophoresis 4~i preparation
Ligand
pH 6.5*
pH 3.6*
*~Ti(IV) 45Ti(III) 4~Ti(1V) 4~i(IV)
DTPA DTPA CA Fluoride
1.0 0.87 0 (leading) 0 (leading)
1.0 0.63 0.48 (tailing) 0.48 (tailing)
*Migrating distances of the DTPA complex to the cathode were 5.2 cm at pH 6.5 (at 5 mA and 500 V for 30 min) and 4.8 cm at pH 3.6 (at 20 mA and 700 V for 20 rain).
Preparation of 45Ti compounds A small amount ( < 50 # L) of 45TIOC12solution was added to 0.2 mL of the chelating reagent solution in saline: 0.6 mg/mL sodium phytate (Thechne Phytate Kit, Daichi Radioisotope Lab., Tokyo), 5 mg/mL human serum albumin (HSA), 10 mg/mL diethylenetriaminepentaacetic acid (DPTA) and 21 mg/mL citric acid (CA). The solutions were adjusted to pH 4-6 with 0.1 M NaOH and passed a through 0.45 #m filter. HSA and DPPA were kindly supplied by Daichi Radioisotope Lab., Tokyo. Because the 45TIOC12solution produced the precipitate, 45TIO2 (Merrill et al. 1978) or 45Ti(OH)4, in neutral pH conditions, was used for animal studies, along with the 45TIOC12 solution adjusted approximately to pH 4-5 with NaOH. A 45Ti(III)-DPTA complex was also prepared by mixing a 45Ti(III) solution with DPTA, and a fluoride complex was prepared from a 45TIOC12 solution and KF. 45Ti-complexes were analyzed by paper electrophoresis (PE) which was performed using two solutions of pyridine, acetic acid and water in different amounts: at pH 6.5 (100:4:900, v/v) or at pH 3.6 (1:10:89, v/v). Table 1 shows the results of the analyses. By thin-layer chromatography (TLC) on silica gel with a solvent of acetone and water (1 : 1, v/v), both 45TiO-DTPA and 45TiO-CA showed a spot (Rf value = 0.60) with tailing. 45TiO-phytate and 45TIOC12preparations did not migrate in either analysis. By gel-filtration chromatography as described below, about 30% each of 45TiO-DTPA and 45TiO-HSA was recovered in the low molecular region and in the protein fraction, respectively. Tissue distribution studies Male Donryu rats weighing 140-160 g were injected i.v. with 0.37MBq 45Ti-labeled compounds. Two groups of rats implanted with AHI09A hepatoma on their back were injected with 45TiO-DTPA or 45TiOCA. The rats were killed by dislocation at various time intervals. Tissues were dissected, counted for 45Ti and weighed. The tissue uptake was expressed as the differential absorption ratio (DAR). The DAR stands for (counts of tissue/total injected counts) x (g body wt/g tissue). In vitro plasma binding assay 45TiO-DTPA (0.37 MBq/20 #L) was incubated with 80 #L of rat plasma at 37°C for 10 min. The solution was applied to the HPLC analyses. A gel filtration column, TSKgel G3000SW (7.5mm i.d. x 60cm,
Tosoh, Tokyo), was used with 20 mM sodium acetate buffer, pH 5.9, containing 0.15 M NaCI at a flow rate of 1.0 mL/min. An anion-exchange TSKgel D E A E 5PW column (7.5 mm i.d. x 7.5 cm, Tosoh) was used with Tris buffer with a gradient modifier with a flow rate of 0.5 mL/min. For the first 2 min, 20 mM TrisHC1, pH 8.0, was used for elution, after which a solvent with a linear gradient of 0-0.3 M CH3 COONa for 35 min, was eluted. For the last 30 min, Tris buffer containing 0.3 M CH3COONa was eluted. In vivo imaging Whole-body imaging of rats given 19 MBq of 45TiO-phytate or 45TiO-DTPA was performed using a gamma-camera (Pho/Gamma HP, Ohio Nuclear) equipped with a pin-hole collimator for 60 min after injection. Rats in which a small part of the brain was frozen by liquid nitrogen were injected i.v. with 80-150MBq of 45TiO-DTPA and 0.2mL of 2% Evan's Blue, and decapitated 15 min later. The frozen brain was cut into sections of 20 #m thickness in a cryostat. The sections were exposed to Kodak NMC-I film, and the image of 45Ti was obtained.
Results and Discussion Chemical and solution state of the 4STi compounds 45Ti(IV) easily takes a colloidal form under neutral conditions. When 45TIOC12 in the acidic condition 10
T
T - -
A,T
CA1
o
~o (B) 15
A 10
~
5
: .,,.. ..:'"
0 0
.... [ " . . . . . . . . 10
"""'" 20
/ 30
..., - '~ '
~ 40
50
60
Retention Time (rain)
Fig. I. HPLC profiles of 45TiO-DTPA incubated in vitro with rat plasma on a gel filtration column (A) and an anion-exchange column (B). Percentages of radioactivity in each fraction for the total radioactivity applied on the columns were calculated. Dotted lines represent the elution profiles of plasma proteins measured using a u.v. detector at 280 nm. Arrows show elution times of 45TiO-DTPA, transferrin (T) and albumin (A).
Fig. 2. Single-photon scintigrams of 45TiO-phytate (A) and 45TiO-DTPA (B). Whole-body images were obtained 10 min after injection of the tracers. Head, upper side; tail, lower side.
Fig. 3. Autoradiogram of coronal brain sections of the rat given 45TiO-DTPA and Evan's Blue. The region of the brain, indicated by the arrow, was damaged by freezing with liquid nitrogen.
709
Ti-45 labeled compounds
711
Table 2. Tissue distribution of radioactivity after i.v. injection of 45TiOC12 or 4~l'iO-phytate in rats 45TiOC12 30 min Blood Brain Heart Lung Liver Spleen Kidney Muscle
0.89 + 0.08 0.040 + 0.007 0.23 ± 0.03 1.36 __.0.48 15.49 + 1.69 3.60 _.+1.53 0.31 _+0.04 0.059 + 0.021
45TiO-phytate
60 min 0.68 + 0.026 + 0.24 + 1.26 + 14.83 + 4.87 + 0.24 + 0.072 +
0.12 0.002 0.06 0.06 1.25 2.02 0.02 0.009
Uptake, DAR* 180 min
30 rain
0.52 + 0.07 0.028 + 0.005 0.19 ± 0.01 0.81 + 0.47 17.70 + 4.02 4.53 + 1.49 0.30 5- 0.05 0.057 + 0.021
0.12 + 0.05 0.012 __.0.003 0.041 __.0.011 0.45 -[- 0.13 19.93 + 2.29 2.90 + 0.50 0.083 + 0.015 0.026 _+0.007
*Mean + SD (n = 3--4).
w a s neutralized w i t h N a O H , the colloidal precipitate, p r o b a b l y 45TiO2 o r 45Ti(OH)4 (Merrill et al. 1978), a p p e a r e d . Also, n e i t h e r the 45TiOC12 a d j u s t e d to p H 4 - 5 n o r 45TiO-phytate m i g r a t e d o n P E a n d
Tissue distribution I n rats given 45TiOC12, the h i g h e s t radioactivity
T L C analyses, s u g g e s t i n g the colloid f o r m a t i o n o f the 45Ti.
level w a s f o u n d in the liver, followed by t h a t in the spleen a n d l u n g ( T a b l e 2). U p t a k e in the b r a i n a n d m u s c l e w a s very low. 45TiO-phytate s h o w e d distribu-
45Ti-complexes m i g r a t e d to the c a t h o d e by PE in the
tion similar to 45TiOC12 30 m i n postinjection, w h i c h is
order of Ti(IV)-DTPA, Ti(III)-DTPA and Ti(IV)C A = T i ( I V ) - f l u o r i d e (Table 1). T h e 4 5 T i ( I V ) - D T P A m i g r a t e d faster t h a n the 4 5 T i ( I I I ) - D T P A u n d e r b o t h
a characteristic o f the colloid f o r m a t i o n . 45TiO--DTPA, 45TiO-CA a n d 45TiO-HSA s h o w e d
p H c o n d i t i o n s . T h e g r e a t e r negative c h a r g e o f the T i ( I V ) - D T P A c o m p l e x explains the fact t h a t D T P A
radioactivity decreased very slowly. B l o o d - t o - t i s s u e r a t i o s o f all tissues were nearly c o n s t a n t f o r the
with five c a r b o x y l g r o u p s c o o r d i n a t e s w i t h a n o x o t i t a n i u m species. T h e c o m p l e x is p r o b a b l y e x p r e s s e d as 4 5 T i O - D T P A , w i t h an electric c h a r g e o f - 3 . T h e 4~Ti(III) p r o b a b l y f o r m s an o c t a h e d r a l c o m p l e x o f 4 5 T i - D T P A w i t h - 2 electric charge. C o m p l e x e s with
first 6 0 m i n , after w h i c h the ratios for the liver, spleen, lung, kidney a n d m u s c l e increased slightly. I n A H l 0 9 A - b e a r i n g r a t s given 4 5 T i O - D T P A o r 45TiO-CA, the t u m o r u p t a k e levels 3 h p o s t i n j e c t i o n
C A a n d fluoride s e e m e d to have l o w e r c h a r g e s a n d to be less stable t h a n the D T P A c o m p l e x e s . T h e C A c o m p l e x is p r o b a b l y expressed as T i O ( O H ) ~ C A o r T i O 4 S A , a n d the fluoride c o m p l e x as TiOF42- .
similar tissue distributions (Table 3). T h e highest b l o o d
were t w o times h i g h e r t h a n t h o s e m e a s u r e d at 10 m i n b u t were l o w e r t h a n the levels in the blood. T h e results suggest t h a t ligands o f the 45TiO-complexes were displaced by p l a s m a p r o t e i n s o r t h a t the c o m p l e x e s b o u n d t h e m s e l v e s w i t h the p l a s m a proteins.
Table 3. Tissue distribution of radioactivity in rats after i.v. injection of 45TiO-DTPA, 45TiO-CA or 'STiO-HSA Uptake, DAR* Blood Brain Heart Lung Liver Spleen Kidney Muscle AH109A~"
45TiO-DTPA 45TiO-CA 45TiO-HSA 45TiO-DTPA 45TiO-CA 45TiO-HSA 45TiO-DTPA 45TiO-CA 45TiO-HSA 45TiO-DTPA 4~TiO42A 45TiO-HSA 45TiO-DTPA 45TiO-CA 45TiO-HSA 45TiO-DTPA 45TiO4~A 45TiO-HSA 45TiO-DTPA 45TiO-CA 45TiO-HSA 45TiO-DTPA 45TiO-CA 45TiO--HSA 4~I'iO--DTPA 45TiO-CA
10 min
30 min
60 min
3h
6h
12.63 + 0.78 10.97 + 0.98 14.91 4- 2.06 0.50 + 0.17 0.46 _+0.13 0.47 + 0.22 3.02 __.0.28 2.33 + 0.30 2.39 + 0.57 5.29 + 0.17 3.17+0.18 5.50 __. 1.61 2.80 + 0.21 1.87 + 0.20 2.62 + 0.53 2.05 + 0.92 1.94 __.0.28 2.61 __.0.84 3.94 + 0.19 2.25 + 0.14 3.47 __.0.80 0.71 _+0.10 0.23 + 0.02 0.31 + 0.07 0.72 + 0.14 0.51 + 0.14
10.73 + 0.53 10.08 + 0.60
9.11 __.0.83 8.82 ___0.44 10.12 _+0.85 0.26 + 0.02 0.30 __.0.13 0.36 + 0.05 2.45 __.0.38 2.56 _+0.30 2.58 + 0.63 4.26 _+0.95 3.42+0.93 3.32 __.0.67 2.50 + 0.90 1.58 ___0.18 1.89 + 0.15 1.85 + 0.15 1.82 + 0.22 1.92 + 0.28 2.55 + 0.42 1.82 + 0.22 2.50 ___0.42 0.48 __.0.04 0.30 +_0.05 0.25 ± 0.01
6.44 __. 1.39 5.06 + 1.24 5.71 _+ 1.52 0.21 + 0.08 0.23 __.0.06 0.14 + 0.02 1.84 __.0.45 1.51 + 0.44 1.39 + 0.26 2.59 + 0.48 2.00+0.51 2.72 + 0.73 2.62 ___0.84 2.24 ___0.76 1.35 _+0.33 1.71 + 0.38 1.47 ___0.29 1.36 + 0.26 1.98 + 0.42 1.50 + 0.37 1.43 ___0.33 0.43 __.0.11 0.26 + 0.08 0.26 __.0.06 1.81 + 0.48 1.31 ___0.30
3.76 + 0.88 3.00 -t- 0.31 3.44 __0.52 0.14 + 0.03 0.17 ___0.03 0.11 + 0.04 1.32 + 0.29 0.84 + 0.07 1.06 -t- 0.29 1.83 + 0.34 1.37+0.26 1.86 + 0.64 2.49 + 0.31 1.96 + 0.39 1.37 __.0.42 2.07 __.0.50 1.28 + 0.24 1.50 + 0.52 1.72 + 0.09 1.16 _.+0.53 1.62 + 0.44 0.43 + 0.03 0.31 + 0.06 0.27 __.0.12 1.61 + 0.22 1.23 __.0.21
*Mean _+SD (n = 3~,). tDifferent groups'of rats were used.
0.52 + 0.02 0.38 + 0.05 3.65 _.+0.18 2.67 + 0.53 6.20 + 1.12 3.77+0.89 2.88 + 0.28 1.68 __.0.21 2.15 _+0.17 1.69 + 0.10 3.85 + 0.34 2.08 + 0.36 0.88 + 0.04 0.22 + 0.03
712
KIICHI ISHIWATAet al.
Plasma protein binding o f ~STiO-DTPA
The 45TiO-DTPA preparation was not eluted from a DEAE-5PW column, and 23% of the total radioactivity was eluted from a G3000SW column. The in vitro sample showed very similar elution profiles of radioactivity and plasma proteins on the G3000SW column [Fig. I(A)]. Main radioactivity was eluted in an albumin fraction that included transferrin, and 2.4% of the total was recovered in the retention volume of the 45TiO-DTPA; however, the in vivo plasma sample gave a radioactive peak corresponding to the albumin fraction. On the DEAE-5PW column, both the in vitro and in vivo samples showed a radioactive peak in the same retention time as that of the transferrin [Fig. I(B)]. The recovery yields of in vivo and in vitro samples using two columns were 22-26% for the G3000SW and 29-33% for the DEAE-5PW. The results indicate that transferrin is a primary plasma binding protein with 45Ti, and the affinity of the 45Ti to other proteins seems to be very weak in vivo. It is suggested that the weak affinity may be mediated via DTPA and that the DTPA is displaced with transferrin to give a 45TiO-transferrin complex. Although DTPA is a strong chelating reagent, the analyses of 45TiO-DTPA on TLC and on a G3000SW column suggest partial dissociation of the complex. Low recovery yields of the in vivo and in vitro samples on both columns may be explained by the affinity of 45TiO-DTPA and 45Ti-plasma protein complex to the columns or partial dissociation of the complex in the analytical conditions. The dissociated 45Ti was absorbed on the columns. In vivo imaging
In a gamma scintigraphy using rats, radioactivity of 45TiO-phytate was highly concentrated in the liver [Fig. 2(A)], and diffused slightly 60 min later. The image probably represents the reticuloendothelial system (RES) as 99mTc-phytate (Subramanian et al., 1973). Some 45TiO-phytate preparation formed large colloids, most of which did not pass through a 0.45 #m membrane filter. Such preparations showed the highest uptake by the lung (Ishiwata et al., 1982). The colloid formation with different sizes may be due to small amounts of contaminants in the 45Ti preparation or to the preparation conditions such as the acidity. The size of colloid particles should be controlled to perform the optimal imaging of the RES, because the uptake by the RES is mainly dependent on the properties and sizes of the colloid particles (Kuperus, 1979). The 45TiO-DTPA was uniformly distributed in a body without any changes for 60 min [Fig. 2(B)]. The binding of the 45Ti with plasma transferrin indicates that the tracer has potential for measuring the circulating plasma volume or blood pools. Of the three complexes, a relatively stable 45TiO-DTPA may be a better tracer. Miura et al. (1987) used the 45Ti-DTPA
for evaluating the blood pool in the left ventricular cavity of the dog heart. In another application, the 45TiO-DTPA is a possible tracer as a blood-brain barrier (BBB) indicator. An autoradiographic study using rats given 45TiO-DTPA showed an image of the breakage of the BBB (Fig. 3). The image of the 45Ti was coincident with that of the Evan's Blue. The radioactivity level of the damaged region was 8.6 times higher than that of the normal brain region. The tracer will be useful for animal studies, especially those using the double radionuclide autoradiographic technique (Kameyama et al., 1983). In conclusion, 45Ti was produced by the proton irradiation to develop a new type of positron-emitting metallic radiopharmaceutical. 45TiO-phytate has potential for the imaging of RES, and 45TiO-DTPA will be useful for measuring the blood volume or as a BBB indicator. The significance of the titanium in the biological processes has not been ascertained. On the other hand, because the chemistry has been well investigated (Clark, 1968, 1975), it is hoped that several types of 45Ti-labeled compounds will be developed for biological studies. Acknowledgement--The authors thank the staff at the
Cyclotron and Radioisotope Center for their cooperation. References
Clark R. J. H. (1968) The Chemistry of Titanium and Vanadium. Elsevier, Amsterdam. Clark R. J. H. (1975) Titanium. In Comprehensive Inorganic Chemistry 3, p. 355. Pergamon Press, New York. Ishiwata K., Ido T., Monma M., Murakami M., Kameyama M., Fukuda H. and Matsuzawa T. (1982) Preparation of 45Ti-labeled compounds and their medical application. J. Label. Compds Radiopharm. 19, 1539 (abstract). Kameyama M., Ogawa A., Shirane R., Tsurumi Y., Suzuki J., Takahashi T., Ishiwata K. and Ido T. (1983) New approach to autoradiography using positron emitting radionuclide. J. Cereb. Blood Flow Metab. 3(suppl.l), S109 (abstract). Kawamura M., Ido T., Ishiwata K., Inoue K., Kimura S., Matsuda K., Kawashima K. and Kameyama M. (1986) Metabolism of 45Ti-labeled compounds: effect of ascorbic acid. J. Label. Compds Radiopharm. 23, 1360 (abstract). Kuperus J. H. (1979) The role of phagocytosis and pinocytosis in the localization of radiotracers. In Principles of Radiopharmacology, Vol. III, (Colombetti L. G., ed), p. 267. CRC Press, Boca Raton, FL. Merrill J. C., Lambrecht R. M. and Wolf A. P. (1978) Cyclotron isotopes and radiopharmaceuticals XXIV. Titanium-45. Int. J. Appl. Radiat. Isot. 29, 115. Miura Y., Kagaya Y., Nozaki E., Ishide N., Maruyama Y., Takishima T., Takahashi T., Ishiwata K., Watanuki S. and Ido T. (1987) Myocardial imaging using 11C--CoQj0 with positron emission tomography. Nucl. Med. Biol. 14, 1. Nelson F., Murase T. and Kraus K. A. (1964) Ion exchange procedures. I. Cation exchange in concentrated HC1 and HC104 solutions. J. Chromatog. 13, 503. Subramanian G., McAfee J. G., Mehter A., Blair R. J. and Thomas F. D. (1973) 99mTc-stannouspytate: a new in vivo colloid for imaging the reticuloendothelial system. J. Nucl. Med. 14, 459.
0883-2889/91 $3.00+0.00 Copyright © 1991 PergamonPress pie
Printed in Great Britain.All rights reserved
Potential Radiopharmaceuticals Labeled with Titanium-45* K I I C H I I S H I W A T A J t , T A T S U O I D O l, M I N O R U M O N M A l, M A T S U T A R O M U R A K A M P , H I R O S H I F U K U D A 2, M O T O N O B U K A M E Y A M A 3, K E N J I Y A M A D A 2, S A T O S H I E N D O 2, S E I R O Y O S H I O K A 2, T A C H I O S A T O 2 a n d T A I J U M A T S U Z A W A z ~Division of Radiopharmaceutical Chemistry, Cyclotron and Radioisotope Center, 2Department of Radiology and Nuclear Medicine, The Research Institute for Tuberculosis and Cancer and 3Division of Neurosurgery, Institute of Brain Diseases, School of Medicine, Tohoku University, Sendai 980, Japan (Received 15 October 1990; in revised form 17January 1991)
The potential of some compounds labeled with cyclotron-produced titanium-45 (45Ti) as radiopharmaceuticals was studied. Properties of colloid formation of 45TIOC12 or 45TiO-phytate in vivo resulted in the highest radioactivity uptake in the rat liver, followed by the spleen, suggesting potential for imaging the reticuloendothelial system. Three 45TiO-complexeswith diethylenetriaminepentaacetic acid, citric acid and human serum albumin showed the highest radioactivity levels in the blood over 6 h. The binding of the 45Ti with plasma transferrin in vitro and in vivo suggested that these compounds can be used for estimating the blood volume. Also, potential as an indicator representing the breakdown of the blood-brain barrier in the rat was demonstrated by autoradiography.
Introduction Titanium-45 (45Ti) has a half-life of 3.08 and a high positron branching ratio (85%), and can be produced using even small cyclotrons in positron emission tomography (PET) centers. It is uncertain whether Ti is an essential trace element in human body; however, the formation of complexes with chelating agents or organometallic compounds (Clark, 1968, 1975) could suggest the usefulness of radiolabeled Ti in nuclear medicine as potential radiopharmaceuticals, as well as in biological and environmental studies (Merrill et al., 1978). Preliminary works aimed at the application of 45Ti-labeled compounds to PET and biological studies were carried out (Ishiwata et al., 1982; Kameyama et al., 1983; Kawamura et al., 1986). We now report our results in the preparation of some 45Ti-labeled compounds and their animal studies using a tissue sampling method and in vivo imaging.
Materials and Methods Production a n d separation o f 4STi
Preparation of 45Ti was carried out according to the method of Merrill et al. (1978) with some modifica*Presented in part at the Fourth International Symposium on Radiopharmaceutical Chemistry, Jiilich, Germany, August 1982. 1"Author for correspondence.
tions. The 45Ti was produced by the proton irradiation of 65-130 mg of scandium foil (0.127 or 0.254 mm thickness, 99.9% purity, Alfa Division, Ventron Corporation, Danvers, U.S.A.) at 11.5 MeV via the 45Sc(p, n)45Ti reaction using the AVF cyclotron at Tohoku University. Analysis of radionuclidic purity by a gamma-ray spectrometer using a Ge(pure) detector showed only 45Ti as the radioactive nuclide. The 45Ti was obtained with yields of 25 _ 2 and 48 +_ 0.5 mCi/pA saturation using 0.127 and 0.254 mm thick foils, respectively. The 45Ti was separated by the method of Nelson et al. (1964). Following the irradiation, the scandium foil was dissolved in 2 mL of 6 M HCI to give 45TIC13, and a drop of conc. HNO3 was added to oxidize the 45Ti(III) to the + 4 state. The solution was evaporated to dryness and dissolved in 6 m HC1 again. This step was repeated three times to remove HNO3 completely. The 45Ti dissolved in 2 mL of 6 M HCI was loaded on an A G 50W x 8 column (100-200mesh, Bio-Rad, 1.9cm i.d. x 13cm) prewashed with 6 M HCI. The 45Ti was eluted with 6 M HC1. The scandium was eluted with 4 M HC1 containing 0.1 M HF. After drying of the 45Ti fraction, the 45Ti was dissolved in 1-2 mL of 1 M HCI. This 45Ti(IV) preparation is probably an oxotitanium species, 45TIOC12. Separation was done within 2 h of the end of irradiation with decay-corrected radiochemical yields of 75-90%.
707
708
KIICHI ISHIWATA et al.
Table 1. Relative migrating distances of the 4STi-labeled complexes on the paper electrophoresis 4~i preparation
Ligand
pH 6.5*
pH 3.6*
*~Ti(IV) 45Ti(III) 4~Ti(1V) 4~i(IV)
DTPA DTPA CA Fluoride
1.0 0.87 0 (leading) 0 (leading)
1.0 0.63 0.48 (tailing) 0.48 (tailing)
*Migrating distances of the DTPA complex to the cathode were 5.2 cm at pH 6.5 (at 5 mA and 500 V for 30 min) and 4.8 cm at pH 3.6 (at 20 mA and 700 V for 20 rain).
Preparation of 45Ti compounds A small amount ( < 50 # L) of 45TIOC12solution was added to 0.2 mL of the chelating reagent solution in saline: 0.6 mg/mL sodium phytate (Thechne Phytate Kit, Daichi Radioisotope Lab., Tokyo), 5 mg/mL human serum albumin (HSA), 10 mg/mL diethylenetriaminepentaacetic acid (DPTA) and 21 mg/mL citric acid (CA). The solutions were adjusted to pH 4-6 with 0.1 M NaOH and passed a through 0.45 #m filter. HSA and DPPA were kindly supplied by Daichi Radioisotope Lab., Tokyo. Because the 45TIOC12solution produced the precipitate, 45TIO2 (Merrill et al. 1978) or 45Ti(OH)4, in neutral pH conditions, was used for animal studies, along with the 45TIOC12 solution adjusted approximately to pH 4-5 with NaOH. A 45Ti(III)-DPTA complex was also prepared by mixing a 45Ti(III) solution with DPTA, and a fluoride complex was prepared from a 45TIOC12 solution and KF. 45Ti-complexes were analyzed by paper electrophoresis (PE) which was performed using two solutions of pyridine, acetic acid and water in different amounts: at pH 6.5 (100:4:900, v/v) or at pH 3.6 (1:10:89, v/v). Table 1 shows the results of the analyses. By thin-layer chromatography (TLC) on silica gel with a solvent of acetone and water (1 : 1, v/v), both 45TiO-DTPA and 45TiO-CA showed a spot (Rf value = 0.60) with tailing. 45TiO-phytate and 45TIOC12preparations did not migrate in either analysis. By gel-filtration chromatography as described below, about 30% each of 45TiO-DTPA and 45TiO-HSA was recovered in the low molecular region and in the protein fraction, respectively. Tissue distribution studies Male Donryu rats weighing 140-160 g were injected i.v. with 0.37MBq 45Ti-labeled compounds. Two groups of rats implanted with AHI09A hepatoma on their back were injected with 45TiO-DTPA or 45TiOCA. The rats were killed by dislocation at various time intervals. Tissues were dissected, counted for 45Ti and weighed. The tissue uptake was expressed as the differential absorption ratio (DAR). The DAR stands for (counts of tissue/total injected counts) x (g body wt/g tissue). In vitro plasma binding assay 45TiO-DTPA (0.37 MBq/20 #L) was incubated with 80 #L of rat plasma at 37°C for 10 min. The solution was applied to the HPLC analyses. A gel filtration column, TSKgel G3000SW (7.5mm i.d. x 60cm,
Tosoh, Tokyo), was used with 20 mM sodium acetate buffer, pH 5.9, containing 0.15 M NaCI at a flow rate of 1.0 mL/min. An anion-exchange TSKgel D E A E 5PW column (7.5 mm i.d. x 7.5 cm, Tosoh) was used with Tris buffer with a gradient modifier with a flow rate of 0.5 mL/min. For the first 2 min, 20 mM TrisHC1, pH 8.0, was used for elution, after which a solvent with a linear gradient of 0-0.3 M CH3 COONa for 35 min, was eluted. For the last 30 min, Tris buffer containing 0.3 M CH3COONa was eluted. In vivo imaging Whole-body imaging of rats given 19 MBq of 45TiO-phytate or 45TiO-DTPA was performed using a gamma-camera (Pho/Gamma HP, Ohio Nuclear) equipped with a pin-hole collimator for 60 min after injection. Rats in which a small part of the brain was frozen by liquid nitrogen were injected i.v. with 80-150MBq of 45TiO-DTPA and 0.2mL of 2% Evan's Blue, and decapitated 15 min later. The frozen brain was cut into sections of 20 #m thickness in a cryostat. The sections were exposed to Kodak NMC-I film, and the image of 45Ti was obtained.
Results and Discussion Chemical and solution state of the 4STi compounds 45Ti(IV) easily takes a colloidal form under neutral conditions. When 45TIOC12 in the acidic condition 10
T
T - -
A,T
CA1
o
~o (B) 15
A 10
~
5
: .,,.. ..:'"
0 0
.... [ " . . . . . . . . 10
"""'" 20
/ 30
..., - '~ '
~ 40
50
60
Retention Time (rain)
Fig. I. HPLC profiles of 45TiO-DTPA incubated in vitro with rat plasma on a gel filtration column (A) and an anion-exchange column (B). Percentages of radioactivity in each fraction for the total radioactivity applied on the columns were calculated. Dotted lines represent the elution profiles of plasma proteins measured using a u.v. detector at 280 nm. Arrows show elution times of 45TiO-DTPA, transferrin (T) and albumin (A).
Fig. 2. Single-photon scintigrams of 45TiO-phytate (A) and 45TiO-DTPA (B). Whole-body images were obtained 10 min after injection of the tracers. Head, upper side; tail, lower side.
Fig. 3. Autoradiogram of coronal brain sections of the rat given 45TiO-DTPA and Evan's Blue. The region of the brain, indicated by the arrow, was damaged by freezing with liquid nitrogen.
709
Ti-45 labeled compounds
711
Table 2. Tissue distribution of radioactivity after i.v. injection of 45TiOC12 or 4~l'iO-phytate in rats 45TiOC12 30 min Blood Brain Heart Lung Liver Spleen Kidney Muscle
0.89 + 0.08 0.040 + 0.007 0.23 ± 0.03 1.36 __.0.48 15.49 + 1.69 3.60 _.+1.53 0.31 _+0.04 0.059 + 0.021
45TiO-phytate
60 min 0.68 + 0.026 + 0.24 + 1.26 + 14.83 + 4.87 + 0.24 + 0.072 +
0.12 0.002 0.06 0.06 1.25 2.02 0.02 0.009
Uptake, DAR* 180 min
30 rain
0.52 + 0.07 0.028 + 0.005 0.19 ± 0.01 0.81 + 0.47 17.70 + 4.02 4.53 + 1.49 0.30 5- 0.05 0.057 + 0.021
0.12 + 0.05 0.012 __.0.003 0.041 __.0.011 0.45 -[- 0.13 19.93 + 2.29 2.90 + 0.50 0.083 + 0.015 0.026 _+0.007
*Mean + SD (n = 3--4).
w a s neutralized w i t h N a O H , the colloidal precipitate, p r o b a b l y 45TiO2 o r 45Ti(OH)4 (Merrill et al. 1978), a p p e a r e d . Also, n e i t h e r the 45TiOC12 a d j u s t e d to p H 4 - 5 n o r 45TiO-phytate m i g r a t e d o n P E a n d
Tissue distribution I n rats given 45TiOC12, the h i g h e s t radioactivity
T L C analyses, s u g g e s t i n g the colloid f o r m a t i o n o f the 45Ti.
level w a s f o u n d in the liver, followed by t h a t in the spleen a n d l u n g ( T a b l e 2). U p t a k e in the b r a i n a n d m u s c l e w a s very low. 45TiO-phytate s h o w e d distribu-
45Ti-complexes m i g r a t e d to the c a t h o d e by PE in the
tion similar to 45TiOC12 30 m i n postinjection, w h i c h is
order of Ti(IV)-DTPA, Ti(III)-DTPA and Ti(IV)C A = T i ( I V ) - f l u o r i d e (Table 1). T h e 4 5 T i ( I V ) - D T P A m i g r a t e d faster t h a n the 4 5 T i ( I I I ) - D T P A u n d e r b o t h
a characteristic o f the colloid f o r m a t i o n . 45TiO--DTPA, 45TiO-CA a n d 45TiO-HSA s h o w e d
p H c o n d i t i o n s . T h e g r e a t e r negative c h a r g e o f the T i ( I V ) - D T P A c o m p l e x explains the fact t h a t D T P A
radioactivity decreased very slowly. B l o o d - t o - t i s s u e r a t i o s o f all tissues were nearly c o n s t a n t f o r the
with five c a r b o x y l g r o u p s c o o r d i n a t e s w i t h a n o x o t i t a n i u m species. T h e c o m p l e x is p r o b a b l y e x p r e s s e d as 4 5 T i O - D T P A , w i t h an electric c h a r g e o f - 3 . T h e 4~Ti(III) p r o b a b l y f o r m s an o c t a h e d r a l c o m p l e x o f 4 5 T i - D T P A w i t h - 2 electric charge. C o m p l e x e s with
first 6 0 m i n , after w h i c h the ratios for the liver, spleen, lung, kidney a n d m u s c l e increased slightly. I n A H l 0 9 A - b e a r i n g r a t s given 4 5 T i O - D T P A o r 45TiO-CA, the t u m o r u p t a k e levels 3 h p o s t i n j e c t i o n
C A a n d fluoride s e e m e d to have l o w e r c h a r g e s a n d to be less stable t h a n the D T P A c o m p l e x e s . T h e C A c o m p l e x is p r o b a b l y expressed as T i O ( O H ) ~ C A o r T i O 4 S A , a n d the fluoride c o m p l e x as TiOF42- .
similar tissue distributions (Table 3). T h e highest b l o o d
were t w o times h i g h e r t h a n t h o s e m e a s u r e d at 10 m i n b u t were l o w e r t h a n the levels in the blood. T h e results suggest t h a t ligands o f the 45TiO-complexes were displaced by p l a s m a p r o t e i n s o r t h a t the c o m p l e x e s b o u n d t h e m s e l v e s w i t h the p l a s m a proteins.
Table 3. Tissue distribution of radioactivity in rats after i.v. injection of 45TiO-DTPA, 45TiO-CA or 'STiO-HSA Uptake, DAR* Blood Brain Heart Lung Liver Spleen Kidney Muscle AH109A~"
45TiO-DTPA 45TiO-CA 45TiO-HSA 45TiO-DTPA 45TiO-CA 45TiO-HSA 45TiO-DTPA 45TiO-CA 45TiO-HSA 45TiO-DTPA 4~TiO42A 45TiO-HSA 45TiO-DTPA 45TiO-CA 45TiO-HSA 45TiO-DTPA 45TiO4~A 45TiO-HSA 45TiO-DTPA 45TiO-CA 45TiO-HSA 45TiO-DTPA 45TiO-CA 45TiO--HSA 4~I'iO--DTPA 45TiO-CA
10 min
30 min
60 min
3h
6h
12.63 + 0.78 10.97 + 0.98 14.91 4- 2.06 0.50 + 0.17 0.46 _+0.13 0.47 + 0.22 3.02 __.0.28 2.33 + 0.30 2.39 + 0.57 5.29 + 0.17 3.17+0.18 5.50 __. 1.61 2.80 + 0.21 1.87 + 0.20 2.62 + 0.53 2.05 + 0.92 1.94 __.0.28 2.61 __.0.84 3.94 + 0.19 2.25 + 0.14 3.47 __.0.80 0.71 _+0.10 0.23 + 0.02 0.31 + 0.07 0.72 + 0.14 0.51 + 0.14
10.73 + 0.53 10.08 + 0.60
9.11 __.0.83 8.82 ___0.44 10.12 _+0.85 0.26 + 0.02 0.30 __.0.13 0.36 + 0.05 2.45 __.0.38 2.56 _+0.30 2.58 + 0.63 4.26 _+0.95 3.42+0.93 3.32 __.0.67 2.50 + 0.90 1.58 ___0.18 1.89 + 0.15 1.85 + 0.15 1.82 + 0.22 1.92 + 0.28 2.55 + 0.42 1.82 + 0.22 2.50 ___0.42 0.48 __.0.04 0.30 +_0.05 0.25 ± 0.01
6.44 __. 1.39 5.06 + 1.24 5.71 _+ 1.52 0.21 + 0.08 0.23 __.0.06 0.14 + 0.02 1.84 __.0.45 1.51 + 0.44 1.39 + 0.26 2.59 + 0.48 2.00+0.51 2.72 + 0.73 2.62 ___0.84 2.24 ___0.76 1.35 _+0.33 1.71 + 0.38 1.47 ___0.29 1.36 + 0.26 1.98 + 0.42 1.50 + 0.37 1.43 ___0.33 0.43 __.0.11 0.26 + 0.08 0.26 __.0.06 1.81 + 0.48 1.31 ___0.30
3.76 + 0.88 3.00 -t- 0.31 3.44 __0.52 0.14 + 0.03 0.17 ___0.03 0.11 + 0.04 1.32 + 0.29 0.84 + 0.07 1.06 -t- 0.29 1.83 + 0.34 1.37+0.26 1.86 + 0.64 2.49 + 0.31 1.96 + 0.39 1.37 __.0.42 2.07 __.0.50 1.28 + 0.24 1.50 + 0.52 1.72 + 0.09 1.16 _.+0.53 1.62 + 0.44 0.43 + 0.03 0.31 + 0.06 0.27 __.0.12 1.61 + 0.22 1.23 __.0.21
*Mean _+SD (n = 3~,). tDifferent groups'of rats were used.
0.52 + 0.02 0.38 + 0.05 3.65 _.+0.18 2.67 + 0.53 6.20 + 1.12 3.77+0.89 2.88 + 0.28 1.68 __.0.21 2.15 _+0.17 1.69 + 0.10 3.85 + 0.34 2.08 + 0.36 0.88 + 0.04 0.22 + 0.03
712
KIICHI ISHIWATAet al.
Plasma protein binding o f ~STiO-DTPA
The 45TiO-DTPA preparation was not eluted from a DEAE-5PW column, and 23% of the total radioactivity was eluted from a G3000SW column. The in vitro sample showed very similar elution profiles of radioactivity and plasma proteins on the G3000SW column [Fig. I(A)]. Main radioactivity was eluted in an albumin fraction that included transferrin, and 2.4% of the total was recovered in the retention volume of the 45TiO-DTPA; however, the in vivo plasma sample gave a radioactive peak corresponding to the albumin fraction. On the DEAE-5PW column, both the in vitro and in vivo samples showed a radioactive peak in the same retention time as that of the transferrin [Fig. I(B)]. The recovery yields of in vivo and in vitro samples using two columns were 22-26% for the G3000SW and 29-33% for the DEAE-5PW. The results indicate that transferrin is a primary plasma binding protein with 45Ti, and the affinity of the 45Ti to other proteins seems to be very weak in vivo. It is suggested that the weak affinity may be mediated via DTPA and that the DTPA is displaced with transferrin to give a 45TiO-transferrin complex. Although DTPA is a strong chelating reagent, the analyses of 45TiO-DTPA on TLC and on a G3000SW column suggest partial dissociation of the complex. Low recovery yields of the in vivo and in vitro samples on both columns may be explained by the affinity of 45TiO-DTPA and 45Ti-plasma protein complex to the columns or partial dissociation of the complex in the analytical conditions. The dissociated 45Ti was absorbed on the columns. In vivo imaging
In a gamma scintigraphy using rats, radioactivity of 45TiO-phytate was highly concentrated in the liver [Fig. 2(A)], and diffused slightly 60 min later. The image probably represents the reticuloendothelial system (RES) as 99mTc-phytate (Subramanian et al., 1973). Some 45TiO-phytate preparation formed large colloids, most of which did not pass through a 0.45 #m membrane filter. Such preparations showed the highest uptake by the lung (Ishiwata et al., 1982). The colloid formation with different sizes may be due to small amounts of contaminants in the 45Ti preparation or to the preparation conditions such as the acidity. The size of colloid particles should be controlled to perform the optimal imaging of the RES, because the uptake by the RES is mainly dependent on the properties and sizes of the colloid particles (Kuperus, 1979). The 45TiO-DTPA was uniformly distributed in a body without any changes for 60 min [Fig. 2(B)]. The binding of the 45Ti with plasma transferrin indicates that the tracer has potential for measuring the circulating plasma volume or blood pools. Of the three complexes, a relatively stable 45TiO-DTPA may be a better tracer. Miura et al. (1987) used the 45Ti-DTPA
for evaluating the blood pool in the left ventricular cavity of the dog heart. In another application, the 45TiO-DTPA is a possible tracer as a blood-brain barrier (BBB) indicator. An autoradiographic study using rats given 45TiO-DTPA showed an image of the breakage of the BBB (Fig. 3). The image of the 45Ti was coincident with that of the Evan's Blue. The radioactivity level of the damaged region was 8.6 times higher than that of the normal brain region. The tracer will be useful for animal studies, especially those using the double radionuclide autoradiographic technique (Kameyama et al., 1983). In conclusion, 45Ti was produced by the proton irradiation to develop a new type of positron-emitting metallic radiopharmaceutical. 45TiO-phytate has potential for the imaging of RES, and 45TiO-DTPA will be useful for measuring the blood volume or as a BBB indicator. The significance of the titanium in the biological processes has not been ascertained. On the other hand, because the chemistry has been well investigated (Clark, 1968, 1975), it is hoped that several types of 45Ti-labeled compounds will be developed for biological studies. Acknowledgement--The authors thank the staff at the
Cyclotron and Radioisotope Center for their cooperation. References
Clark R. J. H. (1968) The Chemistry of Titanium and Vanadium. Elsevier, Amsterdam. Clark R. J. H. (1975) Titanium. In Comprehensive Inorganic Chemistry 3, p. 355. Pergamon Press, New York. Ishiwata K., Ido T., Monma M., Murakami M., Kameyama M., Fukuda H. and Matsuzawa T. (1982) Preparation of 45Ti-labeled compounds and their medical application. J. Label. Compds Radiopharm. 19, 1539 (abstract). Kameyama M., Ogawa A., Shirane R., Tsurumi Y., Suzuki J., Takahashi T., Ishiwata K. and Ido T. (1983) New approach to autoradiography using positron emitting radionuclide. J. Cereb. Blood Flow Metab. 3(suppl.l), S109 (abstract). Kawamura M., Ido T., Ishiwata K., Inoue K., Kimura S., Matsuda K., Kawashima K. and Kameyama M. (1986) Metabolism of 45Ti-labeled compounds: effect of ascorbic acid. J. Label. Compds Radiopharm. 23, 1360 (abstract). Kuperus J. H. (1979) The role of phagocytosis and pinocytosis in the localization of radiotracers. In Principles of Radiopharmacology, Vol. III, (Colombetti L. G., ed), p. 267. CRC Press, Boca Raton, FL. Merrill J. C., Lambrecht R. M. and Wolf A. P. (1978) Cyclotron isotopes and radiopharmaceuticals XXIV. Titanium-45. Int. J. Appl. Radiat. Isot. 29, 115. Miura Y., Kagaya Y., Nozaki E., Ishide N., Maruyama Y., Takishima T., Takahashi T., Ishiwata K., Watanuki S. and Ido T. (1987) Myocardial imaging using 11C--CoQj0 with positron emission tomography. Nucl. Med. Biol. 14, 1. Nelson F., Murase T. and Kraus K. A. (1964) Ion exchange procedures. I. Cation exchange in concentrated HC1 and HC104 solutions. J. Chromatog. 13, 503. Subramanian G., McAfee J. G., Mehter A., Blair R. J. and Thomas F. D. (1973) 99mTc-stannouspytate: a new in vivo colloid for imaging the reticuloendothelial system. J. Nucl. Med. 14, 459.
