Radiolabeled Compounds of Biomedical Interest Containing

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Radiolabeled Compounds of Biomedical Interest Containing Radioisotopes of Gallium and Indium M. J. WELCH and S. MOERLEIN The Edward Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 South Kingshighway, St. Louis, MO 63110

There has been considerable interest in the application in medicine of the four radioisotopes, Ga, Ga, In, and In, listed in Table 1. Each of the two elements has one isotope with a half-life of 2-4 days which has been used for such applications as tumor scanning [ Ga-citrate (1) and In-bleomycin (2)], abscess localization [ Ga-citrate (3) and In-labeled white cells (4)], and thrombus detection [ In-labeled platelets (5)]. As shown in Table 1, both indium-111 and gallium-67 are cyclotron-produced, and the most common method of production utilizes proton reactions. It is interesting to note that at the present time (August, 1979) there are at least eight commercial cyclotrons in the United States dedicated to isotope production, and these accelerators produce mainly four radioisotopes for radiopharmaceutical use ( In, Ga, I , and T1). TABLE I RADIONUCLIDES OF INDIUM AND GALLIUM OF INTEREST IN NUCLEAR MEDICINE 67

68

111

113m

67

111

67

111

111

111

67

123

201

113m

ISOTOPE

mm

HALF-LIFE

2.8 days 1.7 hr

METHOD OF PRODUCTION

111Cd (P,n)

In

Decay of 3 n (t = 115 days) 1 1

S

67 a

6 8

3.3 days

68 min

G

67

Ga

68

Zn(p,n) Decay of G e (t /2=275 days) 1

1 / 2

The short-lived gallium and indium isotopes are produced for medical purposes at the site of use from a radionuclide generator (6). The sn- In generator has been displaced in the United States by the Mo- Tc generator, although it continues to be utilized in countries where there are delivery problems with the much shorter-lived Mo- Tc system (7). The germanium-68/gallium generator is one of a very limited number of 113

113m

99

99m

99

99m

0-8412-05 8 8-4/ 80/47-140-121 $05.00/0 © 1980 American Chemical Society

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generator systems that produces a s h o r t - l i v e d p o s i t r o n - e m i t t i n g r a d i o n u c l i d e (£). There i s c u r r e n t l y great i n t e r e s t i n p o s i t r o n - e m i t t i n g radiopharmaceuticals because of the f a c t that t h e i r d i s t r i b u t i o n can be quantitated i n v i v o u t i l i z i n g p o s i t r o n emission t r a n s a x i a l tomography. U t i l i z i n g t h i s technique, r e c o n s t r u c t i o n of a r a d i o n u c l i d e d i s t r i b u t i o n i s p o s s i b l e by s e v e r a l techniques to give the true d i s t r i b u t i o n of a c t i v i t y i n the source ( 8 9 1 0 ) .

Inorganic Chemistry in Biology and Medicine Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SAN DIEGO on 04/09/16. For personal use only.

r

r

The design of radiopharmaceuticals l a b e l e d with indium and g a l l i u m r a d i o n u c l i d e s i s compounded by the f a c t that both indium and g a l l i u m form very strong chelates with the plasma p r o t e i n t r a n s f e r r i n (11,12). Due to t h i s l a r g e s t a b i l i t y constant and the l a r g e r amount of t r a n s f e r r i n i n human plasma (0.25 mg/100 ml), one would a n t i c i p a t e indium and g a l l i u m compounds being thermodynamically unstable i n v i v o . I t appears, however, that f o r indium and g a l l i u m complexes with s t a b i l i t y constants >1 the r a t e at which the e q u i l i b r i u m i s reached i s slow compared to the r a t e o f many b i o l o g i c a l events (11) such as the 1.6 hour glomerular f i l t r a t i o n r a t e of I n D T P A (12). I t should be noted, however, that the i n j e c t i o n o f weak c h e l a t e s o f indium and g a l l i u m leads to very s i m i l a r b i o d i s t r i b u t i o n s . Because of t h i s e f f e c t of exchange with t r a n s f e r r i n , one o f the major goals o f research i n t h i s area i s the developement of s t r o n g l y - b i n d i n g b i f u n c t i o n a l c h e l a t e s . The f i r s t o f these was 1-(p-benzenediazonium)-ethylenediamine-N,N,N•,N -tetra-acetic a c i d (azo-0-EDTA) shown i n Figure 1, which was developed by Sundberg, et a l (14). This compound forms a l i n k between the metal ETDA complex and a p r o t e i n by means o f the diazo group. Human serum albumin l a b e l e d with I n i n such a manner was found to have a b i o l o g i c a l h a l f - l i f e of 7 days and to l o s e l e s s than 5% of i t s a c t i v i t y to t r a n s f e r r i n when incubated with serum f o r 2 weeks (15). T h i s and other approaches (16) have extended the number of a v a i l a b l e indium and g a l l i u m radiopharmaceuticals. The f o l l o w i n g i s a d i s c u s s i o n o f the major uses o f each o f the four isotopes. f

111

1

1 1 1

Indium-mm The t i n - i n d i u m generator was introduced i n 1966 by Stern et a l (12). The tin-113 which i s produced by the Sn(n, Y) 3sn r e a c t i o n i n a nuclear r e a c t o r i s r e t a i n e d i n a hydrated zirconium oxide column e l u t e d with 0.05M h y d r o c h l o r i c a c i d . The generator eluate has been used d i r e c t l y as a blood pool scanning agent ( l 8 19 20). The generator e l u a t e , when i n j e c t e d d i r e c t l y , leads to the formation of 3 m i _ t r a n s f e r r i n , which remains i n the blood pool f o r s e v e r a l 3 m half-lives. I n c r e a s i n g the pH of the generator eluate leads to c o l l o i d a l formations which have been used f o r the v i s u a l i z a t i o n of the l i v e r , spleen, and bone marrow (21 22 23). Larger p a r t i c l e s where the r a d i o a c t i v e indium i s a s s o c i a t e d with i r o n hydroxide (22 23.) or with macroaggregates 1 1 2

1 1

f

f

1 1

n

1 1

I n

f

r

r


7.

Ga

W E L C H AND MOERLEIN

H00C-CH HOOC-CH

Figure 1.

2

& In

123

Radioisotopes

CH -C00H 2

-COOH

Metal binding molecule that forms a link between the In-EDTA the protein by means of a diazo bond

and

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of albumin, have been u t i l i z e d f o r lung scanning (24 25). As discussed p r e v i o u s l y , only c h e l a t e s with a slow exchange rate remain s t a b l e i n v i v o . Indium-113m c h e l a t e s with EDTA and DTPA have been u t i l i z e d f o r the d e t e c t i o n o f b r a i n tumors and f o r the study of r e n a l funcions ( 2 6 2 7 ) . Indium-113m c h e l a t e s with ethylenediamine tetra(methylene phosphonic a c i d ) (EDTMP) and d i e t h y l e n e t r i a m i n e penta(methylene phosphonic a c i d ) (DTPMP) have been u t i l i z e d to study bone tumors (28 29). These agents a l s o have promise f o r the d e t e c t i o n of myocardial i n f a r c t s (30.) • I t can be seen from the above d i s c u s s i o n that the simple compounds of indium-113m that have been prepared to date can be used to study many organs o f the human i n a non-invasive manner. Although the 393 keV decay energy and 1.7 hour h a l f - l i f e o f 3mi k i t a l e s s i d e a l n u c l i d e than 9 9 m the i g h a l f - l i f e (118 days) o f i t s parent 3 s n make i t very u s e f u l i n developing c o u n t r i e s or i s o l a t e d r e g i o n s where d e l i v e r y o f r a d i o i s o t o p e generators i s d i f f i c u l t . The tin-113/indium-113m generator may be e l u t e d s e v e r a l times a day (1.7 hours generates 50% of the e q u i l i b r i u m a c t i v i t y ) and need be replaced only twice a year. f

f


f

1 1

n

m a

e

Tc>

o n

1 1

Indium-111 The major uses of indium-111 i n medicine are l i s t e d i n Table 2. Indium-111 l a b e l e d DTPA i s the p r e f e r r e d agent f o r the study of c e r e b r a l s p i n a l f l u i d k i n e t i c s ( c i s t e r n o g r a p h y ) ( 3 1 ) . Indiuml a b e l e d bleomycin has been used f o r tumor scanning (£), although 6?Qa c i t r a t e has achieved greater c l i n i c a l use. I t appears that indium bleomycin i s i n f a c t a weak c h e l a t e and the i n v i v o d i s t r i b u t i o n i s very s i m i l a r to that of indium t r a n s f e r r i n . TABLE I I INDIUM-111 RADIOPHARMACEUTICALS Radiopharmaceutical Application Reference 1 1 1

InCl3 I n Citrate In-DTPA In-EDTA In-EDTMP In-HMDTP In-DTPMP In-Fe(0H) Colloid

1 l 1

111

111

111

111

111

1 1 1

3

111

In-Bleomycin In-HSA min-Transferrin 1ll

1 1 1

1 1 1

In-Fibrinogen In-RBC s f

Tumor and Bone Marrow Imaging Bone Marrow Imaging Cisternography Cisternography Bone Imaging Bone Imaging Bone Imaging Lymph Node Scanning Lung S c i n t i g r a p h y Tumor Scanning Cisternography Cisternography and Bone Marrow Imaging Thrombus Imaging Cardiac and Spleen Imaging

32,134 135 31,32,136 31,32,136 137 137 137 138 139 2,32 15,32,136 15,32,136 32 32

7.


Ga

& In

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Radioisotopes


Table I I continued 1 1 1

In-Platelets

Thrombus Imaging

111

In-Leukocytes

Abscess and

34,37,38, 137 39

111

In-Lymphocytes

Inflammatory S i t e Imaging Lymph Node Imaging Lymphocyte K i n e t i c s

40,140 141

The a p p l i c a t i o n s o f indium-111 that are c u r r e n t l y being i n v e s t i g a t e d i n c l u d e s t u d i e s with b i f u n c t i o n a l c h e l a t e s and the l a b e l i n g o f blood c e l l s . The b i f u n c t i o n a l c h e l a t i n g group (Figure 1) has been u t i l i z e d t o a t t a c h I n t o albumin, f i b r i n o g e n , and bleomycin (32.). Using t h i s b i f u n c t i o n a l technique i t i s p o s s i b l e t o prepare a s t a b l e indium bleomycin chelate which has great p o t e n t i a l f o r tumor l o c a l i z a t i o n . In recent years the most e x c i t i n g a p p l i c a t i o n has been the u t i l i z a t i o n o f indium-111 l a b e l e d 8-hydroxyquinoline t o l a b e l blood c e l l s . I t has been shown t h a t when the 8-hydroxyquinoline complex i s mixed with c e l l s separated from plasma, the indium becomes f i r m l y bound i n s i d e the c e l l (33*31) • Studies t o evaluate the mechanism o f uptake suggest that the l i p o p h i l i c c h e l a t e d i f f u s e s i n s i d e the c e l l and t h a t there a r e i n t r a c e l l u l a r binding s i t e s to which the indium exchanges (35u. 3&)• Studies u t i l i z i n g t r i t i a t e d 8-hydroxy-quinoline have shown that the 8-hydroxyquinoline i s not r e t a i n e d i n the c e l l but i s p a r t i t i o n e d between the l i p o p h i l i c c e l l and the aqueous suspension media. Other s t u d i e s u t i l i z i n g both l a b e l e d white c e l l s (33) and p l a t e l e t s (36) have shown t h a t when the c e l l s a r e l y s e d the a c t i v i t y i s attached to p r o t e i n s . As the indium i s attached i n s i d e the blood c e l l a s t a b l e l a b e l r e s u l t s f o r r e i n j e c t i o n i n t o a p a t i e n t because the c e l l membrane p r o h i b i t s plasma t r a n s f e r r i n access t o the l a b e l e d p r o t e i n . Labeled p l a t e l e t s (34, 37 3 8 ) l a b e l e d white c e l l s (32.), and l a b e l e d lymphocytes (4Q.) have a l l been s t u d i e d e x t e n s i v e l y . P l a t e l e t s have been shown i n a s e r i e s of normal volunteers to behave i n the same manner as unlabeled p l a t e l e t s (38), and i n p a t i e n t s with thrombosis o r a t h e r o s c l e r o s i s (31) t o l o c a l i z e a t or v i s u a l i z e the s i t e o f the l e s i o n s . Labeled white c e l l s accumulate i n abscesses (33) and have been used f o r abscess d e t e c t i o n i n humans (32.) • This a b i l i t y o f ^ I n - S - h y d r o x y q u i n o l i n e t o l a b e l blood c e l l s combined with the good imaging c h a r a c t e r i s t i c s o f indium-111 allows t h i s v a l u a b l e a p p l i c a t i o n o f t h i s n u c l i d e . I t should be noted that the k i n e t i c s o f uptake o f the l a b e l e d c e l l s are such that the h a l f - l i f e o f 3 m i i s too s h o r t f o r many a p p l i c a t i o n s as a c e l l label. 1 1 1

r

11

1 1

n

Gallium-68 As discussed p r e v i o u s l y , the germanium-68/gallium-68

f

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generator i s of p a r t i c u l a r i n t e r e s t because i t i s a convenient generator to produce a p o s i t r o n - e m i t t i n g r a d i o n u c l i d e . A commercially a v a i l a b l e generator i s based on the system i n i t i a l l y described by Greene and Tucker (41). In t h i s system the germanium68 i s loaded onto an a c t i v a t e d alumina column and the gallium-68 i s extracted with 0.005M EDTA. The gallium-EDTA s o l u t i o n a t the time of e l u t i o n contains l e s s than 10~ ? o f 6 8 as a contaminant. Owing to the l a r g e d i f f e r e n c e s i n the h a l f - l i v e s of the daughter and parent, the breakthrough o f the parent must be very low, as the r a d i a t i o n dose t o a p a t i e n t from ^ Ge i s many orders of magnitude g r e a t e r than that from ^ G a . Although ^ G a EDTA can be used d i r e c t l y f o r b r a i n or r e n a l scanning (42), the production of any other compound r e q u i r e s one to i n i t i a l l y decompose the EDTA complex. Although s e v e r a l methods have been used to accomplish t h i s (43.), they are a l l time consuming and l o s e a s i g n i f i c a n t f r a c t i o n o f the 68-minute h a l f - l i v e d gallium-68. Because of t h i s problem, there has r e c e n t l y been considerable e f f o r t to develop a generator producing the gallium-68 i n e i t h e r an i o n i c form or as a weak c h e l a t e . Both solvent e x t r a c t i o n and column systems have been developed t o accomplish t h i s . In the solvent e x t r a c t i o n technique ( M ) , gallium-68 i s extracted from an aqueous s o l u t i o n i n t o chloroform or methylene c h l o r i d e as the g a l l i u m 8-hydroxyquinoline complex. A f t e r evaporation o f the solvent the 68Q _8-hydroxyquinoline can e i t h e r be used d i r e c t l y f o r c e l l l a b e l i n g (45) or exchanged with stronger l i g a n d s to form other g a l l i u m - l a b e l e d radiopharmaceuticals. This type of generator has r e c e n t l y been automated (46) to produce the g a l l i u m 8-hydroxyquinoline without operator manipulation. In a new chromatographic generator system (41), the c a r r i e r - f r e e germanium-68 i s adsorbed on polyantimonic a c i d i n sodium oxalate s o l u t i o n at pH 5-10. Gallium-68 can be eluted as the g a l l i u m o x a l a t e over a pH range of 7 t o 11, and the germanium-68 breakthrough i s l e s s than 0.06$. Other approaches to the production o f a generator f o r i o n i c gallium-68 have been described by N e i r i n c k x and Davis (48), who have described two systems. In one o f these, gallium-68 i s e l u t e d with d i l u t e h y d r o f l u o r i c a c i d from a s t r o n g l y basic anion exchange r e s i n , Bio-Rad AG1-X8, onto which s t r o n g l y adsorbed. The d i s t r i b u t i o n c o e f f i c i e n t s f o r germanium and g a l l i u m were determined and at the optimum c o n d i t i o n s y i e l d s o f gallium-68 of >95% with germanium breakthrough of l e s s than 10~3j were obtained. When the c o n c e n t r a t i o n o f h y d r o f l u o r i c a c i d was l i m i t e d to 0.01N HF to decrease the p r o b a b i l i t y of f l u o r i d e t o x i c i t y , gallium-68 y i e l d s of 90J with germanium-68 breakthrough of these changes allow more o^Ga t o be d e l i v e r e d to the tumor s i t e . This simple mechanism o f f e r s the advantage o f e x p l a i n i n g why g a l l i u m accumulates only i n v i a b l e (not n e c r o t i c ) neoplasms (£3.) and that i t may operate f

f

f

2

f

P

r

P

f

G a

f

G a

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c o n c u r r e n t l y with the a l t e r n a t i v e mechanisms which w i l l now be discussed. The s i m i l a r i t y between g a l l i u m and the a l k a l i n e earth elements has been developed i n a d i f f e r e n t explanation f o r g a l l i u m tumor uptake (87,88,89), Because the l a c k o f tumor s p e c i f i c i t y i n °?Ga uptake suggests that a simple mechanism i s i n v o l v e d , perhaps a simple competition between g a l l i u m and magnesium or calcium f o r macromolecular l i g a n d s occurs. Since the i o n i c r a d i i o f Ga3+ (0.62) and M g (0.65) are s i m i l a r , the exchange should occur and favor g a l l i u m complexation because o f i t s higher valence and hence greater complex s t a b i l i t y . Exchange with C a ( i o n i c radius 0.99) i s a l s o p o s s i b l e because the macromolecules involved are able t o generate s t e r i c f i t t i n g t o accommodate c a t i o n s i z e i n accordance with the r a d i u s - r a t i o p r i n c i p l e . These magnesium and calcium l i g a n d s include RNA, DNA, p r o t e i n s , a c i d aminoglycans, and phospholipids, a l l o f which show a c t i v e metabolic synthesis during c a l c i f i c a t i o n ( 9 0 9 1 ) . I t i s not suggested that g a l l i u m s u b s t i t u t e s f o r the a l k a l i n e e a r t h metals throughout t h e i r metabolic pathways, but rather than ^Ga remains i n the s o l u b l e f r a c t i o n o f tumor c e l l s , bound t o the r e s p e c t i v e macromolecules. The higher tumor content o f calcium and magnesium (92,93) i m p l i e s g r e a t e r concentration o f the metal-binding l i g a n d s , and t h e r e f o r e greater c e l l u l a r uptake o f gallium-67 v i a competitive i o n i c exchange.


2 +

2 +

f

In a follow-up study o f the s i m i l a r i t i e s o f calcium and a l l i u m b i o k i n e t i c s , a comparative i n v e s t i g a t i o n o f the uptake o f ^Ca and 6?Ga l a c t a t i n g dogs showed that s i m i l a r s u b c e l l u l a r d i s t r i b u t i o n s i n mammary gland c e l l s (94). However, the uptake of the two r a d i o n u c l i d e s d i d not c o r r e l a t e when t r a n s m i s s i b l e venereal tumor was used. I t was concluded t h a t although l a c t a t i n g mammary gland uptake o f calcium and g a l l i u m shows s i m i l a r c h a r a c t e r i s t i c s , there i s no s i m i l a r i t y i n the mechanism of uptake o f these two elements by tumor t i s s u e . Using c u l t u r e d mammalian sarcoma c e l l s , i t has been found that t r a n s f e r r i n i s necessary i n the growth medium f o r gallium-67 uptake t o occur (Q5.Q6.Q7). A " t r a n s f e r r i n r e c e p t o r " on EMT-6 sarcoma c e l l s f o r 5 i _ i e l e d t r a n s f e r r i n was c h a r a c t e r i z e d by Scatchard a n a l y s i s t o have an average a s s o c i a t i o n constant K = 4.54 x 10 1/mole and approximately (with v a r i a t i o n ) 500,000 receptors per c e l l (95). I t was proposed that tumor accumulation of gallium-67 can occur only i f the metal i s complexed with t r a n s f e r r i n so that i t can i n t e r a c t with the r e c e p t o r s o f tumor, as w e l l as non-malignant c e l l s (.23.) • The complex then enters the c e l l v i a an "adsorptive endocytosis" process (Q5 Q6.Q7.Q8.QQ) s i m i l a r to the manner i n which i r o n i s taken up by r e t i c u l o c y t e s and bone marrow c e l l s (100,101). These t r a n s f e r r i n receptors are saturable (that i s , a p l o t o f 5 i _ t r a n s f e r r i n uptake versus e x t r a c e l l u l a r t r a n s f e r r i n c o n c e n t r a t i o n reaches a peak ( a t about 200 yg/ml) as more c a r r i e r t r a n s f e r r i n i s added t o the medium) (.25.). Since uptake i s a l s o p r o p o r t i o n a l t o the f r a c t i o n o f i

n

1 2

a D

6

r

1 2


7.

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g a l l i u m which i s bound to t r a n s f e r r i n , t h i s mechanism p r e d i c t s that t o t a l c e l l u l a r uptake i s p r o p o r t i o n a l to the " c a l c u l a t e d c e l l u l a r uptake" (the f r a c t i o n of 6?Ga as 6?Ga t r a n s f e r r i n times the f r a c t i o n of t o t a l t r a n s f e r r i n which i s c e l l - b o u n d ) ( 9 6 ) . Once the g a l l i u m - t r a n s f e r r i n i s i n s i d e the c e l l , i t i s deposited i n the lysosomes i n i t i a l l y and then d i s t r i b u t e d to other regions of the c e l l s (some g a l l i u m i s stored i n f e r r i t i n , but most of the element i s deposited i n the m i c r o v e s i c l e s and rough endoplasmic reticulum). The i n t r a c e l l u l a r g a l l i u m must be i r r e v e r s i b l y bound to macromolecules to prevent i t from d i f f u s i n g i n t o the e x t r a c e l l u l a r space (102)• Only c e l l s with a " t r a n s f e r r i n r e c e p t o r " as w e l l as " i n t r a c e l l u l a r r e c e p t o r " w i l l accumulate and retain ?Ga. The above hypothesis o f f e r s a simple d e s c r i p t i o n of gallium-67 uptake s i n c e g a l l i u m complexes r a p i d l y exchange with plasma p r o t e i n s which r e s u l t s i n p r i m a r i l y g a l l i u m - t r a n s f e r r i n ( 5 2 5 3 5 4 ) . I t a l s o e x p l a i n s how p r e l a b e l e d t r a n s f e r r i n w i l l give a higher tumor uptake of g a l l i u m (102), how a d m i n i s t r a t i o n of scandium w i l l increase the tumor/blood r a t i o by c o m p e t i t i v e l y d i s p l a c i n g the g a l l i u m from serum t r a n s f e r r i n (58), and the c o r r e l a t i o n between unsaturated i r o n b i n d i n g c a p a c i t y (UIBC) and tumor uptake of g a l l i u m (103). However, i t does not o f f e r reasons f o r the discrepancy between 6?Ga and 59pe d i s t r i b u t i o n c h a r a c t e r i s t i c s (104), the reported i n h i b i t o r y e f f e c t s of t r a n s f e r r i n on tumor uptake (105), or why g a l l i u m accumulation i n tumors of i r o n - d e f i c i e n t animals i s not greater than that of animals fed a normal i r o n d i e t (106). Doubting that endocytosis o f f o r e i g n m a t e r i a l was the primary uptake mechanism f o r tumors (59), other workers chose to examine the intramolecular d i s t r i b u t i o n of g a l l i u m i n an attempt to e l u c i d a t e the i n t r a c e l l u l a r r e c e p t o r s i n v o l v e d i n the process. E a r l y r e p o r t s from autoradiographic (107), zonal u l t r a c e n t r i f u g a l and enzymatic (108), and conventional (109) techniques show that gallium-67 l o c a l i z e s i n the lysosomes of both l i v e r and tumor t i s s u e . Large amounts o f gallium-67 i n the s o l u b l e p o r t i o n of t i s s u e homogenates (84 110) are a t t r i b u t e d to the d i s r u p t i o n of these o r g a n e l l e s during homogenation (111) because o f the l a r g e amount of 6 7 - a c i d phosphatase (a lysosomal enzyme) i n the preparations (59). More r e f i n e d methods have shown that gallium-67 binds to a microsomal f r a c t i o n which probably represents rough endoplasmic r e t i c u l u m (111). In normal r a t l i v e r , most of the g a l l i u m l o c a l i z e s i n lysosomal p a r t i c l e s ( M ) > but the hepatomas sequestered the m a j o r i t y o f t h e i r °?Ga i n the smaller o r g a n e l l e s (111). I t has been f u r t h e r shown that a majority (approximately 60?) o f the e x t r a c t a b l e ? G a (about 70% o f the c e l l u l a r gallium) from tumor and l i v e r c e l l s of the r a t i s a s s o c i a t e d with two macromolecular f r a c t i o n s o f molar weight 1-1.2 x 10^ Daltons and 4-5 x 10 * Daltons (64). The 1-1.2 x 10^ D band i s found i n both l i v e r and tumor c e l l s , whereas the 4-5 x 10^ D band i s found 6

r

r

r

G a

6

1


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p r i m a r i l y i n tumor c e l l s , although at minute concentrations only (25 yg o f c a r r i e r g a l l i u m w i l l s a t u r a t e the b i n d i n g o f t h i s component). Most of the l i v e r uptake i s a s s o c i a t e d with the heavy macromolecule, but 50% o f the gallium-67 e x t r a c t e d from tumor c e l l s a s s o c i a t e s with the low molecular weight f r a c t i o n (64). T h i s d i f f e r e n c e i n complexation may represent an a l t e r e d physiology between tumor and normal t i s s u e . Both molecules are g l y c o p r o t e i n s , and unstable to heat and a l k a l i n i t y . Because plasma-bound °7oa i s s t a b l e at pH 8.0 f o r s e v e r a l hours ( M ) , the pH l a b i l i t y o f these complexes i s evidence that there are i n t r a c e l l u l a r receptors f o r g a l l i u m which d i f f e r from the plasma p r o t e i n s that bind to t h i s metal (64). T h i s pH-dependent process may represent the d i s s o c i a t i o n of a complex or the change o f an i o n i c s p e c i e s [Ga(0H)3 Ga(0H)]j~] w i t h i n the i n t r a c e l l u l a r space. L a c t o f e r r i n , with a molecular weight of 8.5-9.0 x 10 * and a s t r u c t u r e s i m i l a r to t r a n s f e r r i n (112) has been suggested as an a l t e r n a t i v e i n t r a c e l l u l a r g a l l i u m - b i n d i n g agent (113). L a c t o f e r r i n binds i r o n with a g r e a t e r a f f i n i t y than t r a n s f e r r i n (114) and i s found i n t i s s u e s and s e c r e t i o n s ( e s p e c i a l l y milk) which l o c a l i z e gallium-67 (115 1I6 117.118.119). I t was proposed that 6?Ga l a b e l e d to t r a n s f e r r i n and other plasma p r o t e i n s i s t r a n s f e r r e d to c e l l u l a r l a c t o f e r r i n due to the l a t t e r s greater c h e l a t i n g a b i l i t y (113-120). Such a t r a n s f e r of gallium-67 has been demonstrated i n v i t r o (121), and increased c o n c e n t r a t i o n s of the p r o t e i n has been found i n tumors (122 123 124). T h i s mechanism has been c r i t i c i z e d s i n c e l a c t a t i o n not only produces l a c t o f e r r i n (hence breast uptake of gallium-67) but a l s o increased lysosomal a c t i v i t y which may account f o r increased r a d i o g a l l i u m uptake i n b r e a s t , milk, and tumor (125). However, i t o f f e r s s e v e r a l advantages as w e l l , one o f which i s e l i m i n a t i o n of the " d i m e r i z a t i o n " of the 4-5 x 10^ MW molecule to e x p l a i n the 83,000 MW r e s u l t s when tumor c e l l homogenates were analyzed using SDSrpolyacrylamide g e l e l e c t r o p h o r e s i s (126). The l a c t o f e r r i n hypothesis agrees with r e p o r t s that (only) about o n e - t h i r d o f the gallium-67 i n tumors i s a s s o c i a t e d with f e r r i t i n (52.) and that gallium-67 was a s s o c i a t e d with 85,000-90,000 MW "fragments of degraded i n t r a c e l l u l a r t r a n s f e r r i n " (127). The author o f t h i s mechanism emphasized however, that l a c t o f e r r i n l e v e l s are not elevated i n a l l tumors, and f e r r i t i n may act as an a l t e r n a t i v e pathway f o r b i n d i n g (128). Perhaps the most e x c i t i n g aspect of t h i s proposed mechanism i s the manner i n which i t c o r r e l a t e s with r e c e n t l y - i s o l a t e d ion-binding molecules c a l l e d siderophores (129). The primary f u n c t i o n o f l a c t o f e r r i n i s to d i m i n i s h the amount o f e x t r a c e l l u l a r f r e e i r o n and thereby i n h i b i t b a c t e r i a l growth (130). L a c t o f e r r i n deposited by polymorphonuclear leukocytes i s attached to the surface of monocytes and macrophages i n inflammatory responses (130). Siderophores are synthesized by b a c t e r i a l c e l l s to sequester i r o n needed f o r growth, and 1

f

f

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1

P

P


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t h e r e f o r e compete with l a c t o f e r r i n ( 1 3 1 ) . Upon the recent r e p o r t of a s i d e r o p h o r e - l i k e substance i s o l a t e d from the i r o n - d e f i c i e n t growth medium of a mammalian tumor t i s s u e ( 1 3 2 ) , i t i s l i k e l y that l a c t o f e r r i n i s deposited as a r e a c t i v e response during competition f o r a v a i l a b l e e x t r a c e l l u l a r i r o n . Since both l a c t o f e r r i n and siderophores bind g a l l i u m r e a d i l y ( 1 3 3 ) , the g a l l i u m - 6 7 uptake i s expected to be higher i n the r e g i o n of a neoplasm. In c o n c l u s i o n , one may see that the t i s s u e d i s t r i b u t i o n o f g a l l i u m i s w e l l known and c l i n i c a l l y u s e f u l , but the problem of s u b c e l l u l a r l o c a l i z a t i o n and uptake mechanism remains to be s o l v e d . Studies to date seem to be i n agreement on some p o i n t s and i n o p p o s i t i o n on others. This s t a t e of a f f a i r s may merely r e f l e c t the v a r i a t i o n i n the pathology o f the d i f f e r e n t neoplasms, or i t may be showing us the complexity of the g a l l i u m uptake mechanism. The pharmacology o f the g a l l i u m i o n may be so ubiquitous that there i s no s i n g l e uptake mechanism f o r any given tumor type. T h i s s i t u a t i o n may a l s o hold f o r other radiometals (such as I n or the r a d i o l a n t h a n i d e s ) which f o l l o w a l a r g e number o f biochemical pathways. In any case more work i s needed to f i n d the various i n t r a c e l l u l a r d i s t r i b u t i o n s of g a l l i u m and the p o s s i b l e uptake mechanism(s) i n the hope that a mechanism can be i s o l a t e d which w i l l be used to optimize radiopharmaceuticals to s e l e c t i v e l y and r a p i d l y p a r t i t i o n the r a d i o n u c l i d e from the plasma i n t o the tumor c e l l . 1 1 1

Acknowledgements This authors thank t h e i r many colleagues i n various phases o f t h i s work, p a r t i c u l a r l y Ter-Pogossian, B.A. S i e g e l , B. Kumar, and G. T h i s work was supported by DOE Contract Literature

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Radiolabeled Compounds of Biomedical Interest Containing

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