Adrenal imaging agents: Rationale, synthesis, formulation and, metabolism

Adrenal Imaging Agents: Rationale, Synthesis, Formulation and, Metabolism William H. Beierwaltes, Donald M. Wieland, Terry Yu, Dennis P. Swanson, and Stephen T. Mosley In this introductory paper on radionuclide adrenal imaging, the rationale, synthesis, formulation, and metabolism of t w o clinically well-established and one promising adrenocortical imaging agents are reviewed. Their present clinical utility is reviewed in a separate presentation in this issue. Progress to date

in developing a positive imaging agent of the adrenal medulla and tumors of chromaffin tissue will be given brief consideration because there is, as yet, no clinically successful radiolabeled adrenal medulla imaging agent.

H O L E S T E R O L is the principal precursor of adrenocortical steroids 1,2 (see Figs. 1 5). Although cholesterol is found in rather high concentration in the plasma and is present in almost every tissue of the body, it is stored normally in only three locations and always in the ester form: the adrenal cortex, the corpora lutea cells of the ovary, and the Sertoli cells of the testis. Two to six per cent of the wet weight of canine adrenal glands consist of cholesterol, of which 80%-85% is in esterified form. 2 The most common pathological site of cholesterol storage is in atheromatous plaques. Beierwaltes and co-workers were encouraged to do their first experiments in dogs with '4C cholesterol because of the uniquely high concentration of stored cholesterol ester in the adrenal cortex 3,4 and to a lesser extent in the adrenal medulla? ,6 They found that the uptake of '4C cholesterol was relatively uniform in all 16 tissues studied, but the retention of ~4C cholesterol in the adrenal gland after 1 or 2 days was relatively unique2 Eighty-five per cent of the radioactivity in the adrenal of the dog at 5 days after the tracer was in the "storage" esterified cholesterol lipid fraction. 4 A p p e l g r e n dosed mice with 14C-labeled pregnenolone, progesterone, and cholesterol. 7 A u t o r a d i o g r a p h y and tissue radioassays established that 14C-labeled cholesterol concentration in the adrenal cortex exceeded that of any other tissue and concentrated to a greater extent than other steroid analogues. Nagai et al. 8 determined the biodistribution of various radio labeled steroids in the mouse, rabbit, and man. The highest adrenal-to-liver ratio was observed at 48 hr, with 3H stigmasterol reaching a value of 27 in mice; lower values were noted with '3'I-labeled cholesterol and stigmasterol. A scan of one patient with Cushing's disease, 5 hr after injection of '31I-stigmasterol, provided suggestive but not definitive evi-

dence that a left adrenal adenoma was visualized. Figure 6 presents the chemical configuration of our first t h r e e s u c c e s s f u l r a d i o l a b e l e d adrenal imaging agents.

C

Seminars in Nuclear Medicine, Vol. VIII, No. 1 (January), 1978

Iodine-131-19-Iodocholest erol ( NM-145 ) (19-iodocholest-5-en-3 ~-ol-131I ) I-131-19-iodocholesterol was first synthesized by our c o l l e a g u e , C o u n s e l l , in 1969. 9 Preliminary experiments in dogs 4 resulted in adrenal concentrations of 12'H-19-iodocholesterol up to 3.75 #Ci/g in the ACTH-stimulated animal at 8 days after injection. Adrenal-toliver ratios of 12'~I-19-iodocholesterol were as

William H. Beierwaltes, M.D.: Professor of Medicine, Department of Internal Medicine, Physician-in-Charge, Division of Nuclear Medicine, University of Michigan Medical Center," Donald M. Wieland, Ph.D.: Director of Radiopharmaceutical Research and Development, Division of Nuclear Medicine, Department of Internal Medicine, University of Michigan Medical Center; Terry Yu, Ph.D.: Organic Chemist, Division of Nuclear Medicine, Department of Internal Medicine, University of Michigan Medical Center," Dennis P. Swanson, M.S.: Acting Director of the Clinical Nuclear Pharmacy, Division of Nuclear Medicine, Department of lnternal Medicine, University of Michigan Medical Center; Stephen T. Mosley, M.S.: Research Associate, Laboratory Supervisor, Radiopharmaceutical Research Laboratory, University of Michigan Medical Center, Ann Arbor, Mich. From the Department of Internal Medicine, Division of Nuclear Medicine, University of Michigan Medical Center, Ann Arbor, Mich. Supported by Grant CA-09015-02, Cancer Research Training in Nuclear Medicine, from the National Cancer Institute, DHEW, by ERDA Contract EY-76-S-02-2031 and by the Nuclear Medicine Research Fund. Reprint requests should be addressed to William H. Beierwaltes, M.D., Division of Nuclear Medicine, University of Michigan Medical Center, Ann Arbor, Mich. 48109. | 1978 by Grune & Stratton, Inc. 0001-2998/78/0801-000/$2.00/0 5

6

BEIERWALTES ET AL.

Cholesterol

CtH~ j ~ - N I ~ Or H

20= - Hydroxyioos

20r

[Man, ordmall~] (Ellpt~n)

Hydrox~/cholesterol

2 2 - Hydtox~oll4 I

....

2 0 ~ , 22 - DihydroxyCholesterol 20, 22- Desmolose Complex cHs

AS - Pre~ner~one

i

~OH I) 3D-ol-Oeh~lrogenaN 2) ~ - 3 - Oxosteroid JsomoroIo

[

0

(BOcterio, rots, bovine]

(C~n#xetone) A

Fig. 1. Biochemical steps in the synthesis of progesterone from cholesterol (enzymes on left and k n o w n enzyme inhibitors on right),

ProQesterone

Pr'ogester~

0

17, 0 ~"~r ~ 9 - Hydroxylo~ C

cH3 I ~ (SU- 8OO0) 0

[Dog~

(Mort,dog) 1SU-90551 17= - Hydroxyproge

OH

21- Hydroxylose

[

II- Deoxyc

OH

o~",~

I ,B-Hyarox~o=e J

c~ o

[ICon.dog. rot,

k~'~c.~ ky,~

gu~o p~)

(Metyra~n4) rot, g~n*a p~I)

[Men.

(SKF -

Fig. 2. Biochemical progesterone.

steps

in s y n t h e s i s

of c o r t i s o l

from Corlisol

12185)

ADRENAL IMAGING AGENTS

7

Progesterone

[:Bovine3 21- Hydroxylose

~

N

(Bo - 40028) I CHzCH t NH - C - NH 2

HO

NH ~

CH30

~.,~T, ci_ c . T ~ ~.'~.) CH3 ~ ' I '

lIB- Hydroxylose

NHz~

9

[:Man,dog, rot, guineopig:] (Metyropone)

CHCHzNHz

:::72:;

HO

Corhcoster~Oneo (Metyropone) ~ N N

(:Rot]

18 - Hydroxylose

(SU-9055)

T HO

Ho HOCH ~,,~0

I8-Hydroxycorhcosterone 0 ~.

18 - Dehydrogenose

Fig. 3. Biochemical steps in synthesis of aldosterone from progesterone.

high as 168:1 at 6 days postinjection, and adrenal:kidney radioactivity ratios were 300:1. The u p t a k e in dog adrenals with ~2'~I-19iodocholesterol was similar to 4-14C cholesterol both with and without ACTH. Successful imaging of the canine adrenals in vivo, utilizing the rectilinear scanner and the scintillation camera, led to application of '3'I19-iodocholesterol for the investigation of human adrenal disease.l~ The 19-iodocholesterol had the same kinetics as an irreversible enzyme inhibitor in that the adrenals have been imaged successfully in the human for as long as

cH3 ~.t- ~

['C"~T'- ~-~/('-')~

I ct"~'~"

~-'-'N

[Rat] (SU- 8000)

Aldosterone

23 days after a tracer dose of 1311-19-iodocholesterol.

Iodine-131-6-beta-Iodomethyl-19-norcholesterol (NP-59) (~31I-6fl-iodomethyl-19-norcholest5( lO)-en-3fl-ol) Figure 6 also presents the chemical configuration of our second successful adrenal imaging agent, 131I-6fl-iodomethyl-19-norcholesterol. This agent was initially identified as an impurity in l~q-19-iodocholesterol and was synthesized s i m u l t a n e o u s l y by both K o j i m a and co-

8

BEIERWALTESETAL.

Progesterone

o~",,~ ~,... ~ o CH3

(SU-8000)

17a- Hydroxylose C I ~ ~ N 0

(SU-10'603)

0 17. -

~OoH

Hydroxyprogesetron___o___~e 17- Desmolase I

(SU- 9055)

(SU-8000)

I

OH ~4-Androstenedione o ~ I

I

\ O

\

~

OH t ~ CH3

:3-,8-oI-Dehydrogenase N~~(C CH3 CH3

H O ' ~ Androsterone

Test~176

yonoketone)

0 HO' J H ~ Etiocholanolone

workers '~ and our group. ~3 Experiments in rats 14,]5 and dogs 1~ demonstrated a fivefold higher uptake of radioactivity in the adrenal cortex from this compound than from the same dose of ~3q-19-iodocholesterol, without a corresponding increase in background radioactivity c o n c e n t r a t i o n . T h e c o n s i d e r a b l y less radioactivity in the unblocked thyroid gland also suggested that there was less in vivo deiodination of this agent. The fact that the human adrenals are imaged sooner, more intensely, and with less background activity has now been established. 18 T a b l e 1 presents the radiation dose in R a d s / m C i in the h u m a n from ~ I - 6 iodomethyl- 19-norcholesterol (NP-59). Table 2 shows the absolute and relative uptake of radioactivity in normal thyroid and in

Fig. 4. Biochemical steps in synthesis of androgens from progesterone. thyroid cancer tissue from N a - ~ l I and in normal adrenal and in adrenocortical carcinoma tissue from NM-145 and NP-59 as determined by radioactivity assay of the excised tissue. Whereas, the uptake of radioactivity in normal thyroid tissue due to 131I is comparable to the uptake of radioactivity in normal adrenal tissue from radioiodinated cholesterol, u p t a k e of radioiodinated cholesterol activity in adrenal carcinoma tissue is at least an order of magnitude less than the uptake of 131I from Na-131I in carcinoma of the thyroid gland. Initially, we d e m o n s t r a t e d that although normal adrenal cortical tissue c o n c e n t r a t e d 131I-19-iodocholesterol in a range of 0.1%-0.2% dose/g, adrenocortical carcinomas of two patients producing cortisol excess concentrated only 0.0001-0.0004, and 0.001% dose/g, respec-

ADRENAL IMAGING AGENTS

9

o

\

/

%0

(Metyropone) E s t ~

OH

HO~ - ~ Cl~

17-R-hydroxy-

(Su-to'6o3) o

OH

Fig. 5.

SYNTHESIS OF RADIOIODINATED CHOLESTEROLS

dehydrogenose

IN 16a-Hydroxylose

~ O H

possible beneficial effects of therapy doses. Several authors have demonstrated diagnostic localization of iodocholesterol in adrenocortical carcinoma metastatic to liver. 19-23 Seabold et al. 21 have reported a patient with an adrenocortical carcinoma per cent dose/g uptake of 0.007 in metastases to liver and to vertebra as compared to 0.01 in the primary. The patient, treated with o,p'DDD, died of adrenocortical carcinoma.

HO ~ - - ~ V

Biochemical steps in synthesis of estrogens from

androgens.

tively23 It is of interest in this regard that welldifferentiated thyroid cancer, for all practical purposes, always concentrates ~31I less avidly than normal tissue. 24,25The thyroid cancer with the highest functional activity reported 26 showed 40% of the concentrating ability of the normal thyroid gland (about 0.3%-1.5% dose/g in normal thyroid gland). It seems reasonable, therefore, that adrenocortical carcinoma metastases in humans, which concentrate 0.01%-0.2% of the dose of 131I-6/3-iodomethyl19-norcholesterol/g, may be candidates for the

I

The serendipitous discovery 12,13 that 131I-6fliodom ethyl- 19-norcholest-5-(10)-en-3/3-ol (NP59), the homoallylic isomer of ~3q-19-iodocholesterol, is a superior adrenal imaging agent demonstrates that the cholesterol structure is not necessary for adrenocortical localization. This point was further confirmed by Kojima, who observed that the parent steroid of NP-59, 3H-6/3-methyl- 19-norcholest-5-(10)-en-3/3-ol, shows greater adrenal uptake than 3H-cholesterol itself. = A thorough and systematic structure-distribution study of radioiodinated steroids has never been done. The reason is in large part due to the difficulty of synthesis of these analogues and to their in vitro instability.12 The possibility of obviating these two problems was suggested by the report of Szinai and Owoyale, who found the adrenal uptake of 12~I-3/3-iodocholestene, an easily synthesized and stable compound, to

II

Ill

NH2

NH 2

HO

'3'I-19 - I O D O C H O L E S T E R O L ( 13tI-19-1ODOCHOLEST- 5 (6) - EN-3/3 - OL )

i~1Hz

C H z '311

'3'I-N P - 5 9

~231-5 - I O D O - S K F 12185

(6/3J3'I-IOOOMETHYL- 19- NOR CHOLEST 5(IO)-EN-3B-OL)

Fig. 6. The chemical configuration of our first three successful radiolabeled adrenal (1969), ~3Zl-6-beta-iodomethyl-19-norcholesterol (1975), z2~I-SKF-12185(1976).

imaging agents.

~311-19-iodocholesterol

10

BEIERWALTES ET AL. Table 1. Rads/mCi of Iodine-131-6-Beta-lodomethyl19-Norcholesterol (N P-59) Total body Adrenals Ovaries Testes Liver

1.2 150.0* 8.0 2.3 2.4

* 150 Rads based on percent uptake data from experimental animals. Using percent uptake data observed in actual clinical studies, the dose is significantly lower, For example, the absorbed dose in the adrenal calculated for the average percent uptake in our normal subjects of 0.16% administered activity per gland is 27.5 Rads.

be 2 ~ times greater than 1311-19-iodocholesterol. 28 However, the concentration of 125I-3-fliodocholestene in the lung, liver, and spleen was many times higher than observed with 1311-19iodocholesterol. The possibility existed that the high uptake in these latter organs was due to the precipitation of the compound in particulate or colloidal form on i.v. injection. When Yu and coworkers 29 used high specific activities and a formulation that would favor molecularity in vivo, the tissue distribution data of 1251-3/3iodocholestene in dogs showed adrenal uptakes and target-to-nontarget concentration ratios similar to ~3'I-19-iodocholesterol and NP-59. Scintiphotos of the normal dog adrenals were obtained using ~3q-3fl-iodocholestene, though the images were inferior to those obtained by NP-59. Nonetheless, the work is significant on three counts: (1) The 3r group of cholesterol is not necessary for adrenal localization. (2) The l~5I-3fl-iodocholestene is exceptionally stable for an alkyl iodide--less than 2% deiodination after 12 days at room temperature. (3) The 125I-3/3-iodocholestene is rapidly synthesized ( < 1 hr) in one step from a readily available tosylate.

The implications for an effective struct u r e - d i s t r i b u t i o n study of r a d i o i o d i n a t e d steroids are substantial, for the radioiodine atom can be placed in the 3fl-position and the r e m a i n d e r of the cholesterol s t r u c t u r e systematically modified. Hopefully, such a study would help answer such questions as: (I) Which structural features will decrease liver localization without lowering adrenal uptake? (2) Which modifications will accelerate adrenal localization (i.e., <24 hr) without a concurrent increase in concentration in nontarget organs? (3) What structural changes will increase the rate of release from the adrenal following maximum uptake? Our initial structure-distribution studies 29 with 12 '25I-3/3-iodosteroids are summarized in Fig. 7. As can be seen in this summary, the uptake of 3fl-iodosteroids is extremely sensitive to minor modifications of the 17fl side chain, as evidenced by the five- to tenfold lower adrenal uptake of compounds 9 and 10. Replacement of the 17/3 side chain with a hydrogen atom (compound 8) or a keto group (compound 9) abolishes adrenal localization; an observation consistent with the low adrenal uptakes previously observed in this laboratory with 3H-labeled androgens. 3~ Reduction of the 5,6-double bond (compound 5) has no effect on adrenal concentration, but does raise liver uptake. Since the rate determining step in bile acid formation is 7 a-hydroxylation, 31 it was felt that hepatic c l e a r a n c e would be a c c e l e r a t e d by functionalization of the 7-position with oxygencontaining moieties. However, these derivatives (compounds 1-4) rapidly deiodinate in vitro. Though at present '~'I-6fl-iodomethyl-19-norcholest-5-(10)-en-ol (NP-59) remains the steroid of choice for human adrenocortical imaging, fu-

Table 2. Comparative Uptake of Iodine-131 in Normal and Cancerous Thyroid From Sodium-Iodine-131 and Adrenal From Iodine-131-19-1odocholesterol and Iodine-131-6-Beta-19-Norcholesterol (Percent Dose per Gram) Iodine- 131 (J Natl Cancer Institute 13:815, 1953) I- 131

Organ Thryoid

Normal Gland

Carcinoma of

0.3-1.5

O. 15

O. 1-0.2

0,001-0.004 0.01 (primary) 0.007 (mets.) 0,0001

Adrenal

Cholesterol (J.C.E.M., 34:36, 1972) (J.C.E.M., 1976) (J.C.E.M., 1977)

ADRENAL

IMAGING AGENTS

11

Compound

I

R

Concentration

I

H

5 . 0 9 +- 0.33

2

=0

*

3

OH

4

OCOCH 3

* -'X-

5

H

6

=0

4.70 -+ O.II I.I2 +0.15

7

OH

0.78 + 0 . 5 7

I R

R

8

H

0.28 +- 0 . 0 2 #

9

=0

0.48 -+ 0.01/f" C2H5

Z

I0

~

II

~

12

0.45 • 0 . 0 4

1.03t-0,07 --

L26 +_0.12

--

0.94 + 0 . 0 7

CHs 19 - Iodocholesterol

6/~- Iodomethyl - 19 norcholesterol [ NP- 59) 4.88 _+0 . 5 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - -

9W-Deiodinoted rapidly in vitro, /PMoximum concentration at IOmin,~ no uptake was found at 5 days.

Fig. 7. C o n c e n t r a t i o n o f 12~l-3-beta-iodosteroids in female dog adrenal cortex at 5 days.

ture structure distribution studies will bring a better understanding of the structural requirements necessary for adrenal localization and hopefully lead to the development of a radiolabeled steroid with more optimum scanning characteristics. Form ulation of lodocholes terols Since cholesterol and the majority of its derivatives are insoluble in water or normal saline and only slightly soluble in alcohol, a major problem existed in developing a suitable formulation for i.v. injection in humans. This was finally solved by utilizing a reconstituting solution consisting of 1.6% polysorbate (Tween) .80, 6.6% absolute ethanol diluted to final volume with normal saline. Following evaporation of the organic exchange medium used in the synthesis of the radioactive cholesterol derivatives, the material is redissolved in Tween 80-absolute ethanol using a minimum of heat (60~ for 30 sec). After

cooling for 5 min, normal saline is added slowly to the desired volume. Care must be taken to assure that the specific activity of the radioactive cholesterol is high enough (1.0-1.5 mCi/mg) to prevent precipitation from the limited amount of ethanol. Considerable success has been achieved using this formulation for not only the cholesterol derivatives studied, but also for the lipid soluble enzyme inhibitors discussed later in this article. It appears to be well tolerated in both humans and laboratory animals. In the above formulation, '3'I-labeled 6fliodomethyl-19-norcholesterol (NP-59)is stable at 0-5~ throughout the recommended shelf life of 2 weeks. At room temperature, however, up to 10% deiodination may occur within 4 days (Fig. 8). Recently, stability studies indicate that formulated NP-59 is no less stable than the dry u n r e c o n s t i t u t e d material. Radiochemical purity, ascertained using TLC-silica gel (Eastman) in 100% chloroform, indicates an Rf of approximately 0.4 for NP-59. The major radiochemical impurity appearing in the preparation is free iodide (Rf = 0.0) with a small amount of impurity corresponding to the R f of 19-iodocholesterol ( R f = 0.3). Apparently, an equilibrium reaction exists resulting in a shift from the 6fl-iodomethyl derivative to the 19-iodo derivative, since HPLC analysis of "cold" 6fl-iodomethyl-19-norcholesterol does not reveal the presence of any "cold" 19iodocholesterol. The established lower limit of radiochemical purity of 80% for NP-59, along with expected in vivo deiodination, necessitates the adjunct administration of Lugol's solution to I00 I

~"

9o' so

.B 7

~."6 70

""m

e---e Storage at 0-5~ ~'"'~. Storage at 22~ 11--4 Storage at 580C.

o

~

6o

~o

~

4I

~

&

,~

I ,2

i~

D0ys Post Synthesis Fig. 8. Graph of deiodination of 6-beta-iodomethyl-19norcholesterol with times and temperature,

12

BEIERWALTES ET AL.

the patient commencing 2 days prior to injection and continuing for 2 weeks. RADIOLABELED ENZYME INHIBITORS

Goldman studied the uptake and release of a radiolabeled enzyme inhibitor, '4C-isoxazole, in the adrenal cortex? 2 When he administered this compound to rats, he showed that this competitive inhibitor would bind specifically, tightly, and for a long period of time of 3-hydroxysteroid dehydrogenase and 2~5-4,3-ketosteroid isomerase. Our studies on this irreversible e n z y m e inhibitor ( ' 4 C - i s o x a z o l e ) in r a t s , resulted in an uptake in the adrenal gland of 2% of the tracer dose/g of tissue in 2 days and 1% in the ovaries? 3 This concentration in the adrenal was 30 times that in liver. The radioactivity in adrenal and ovary remained at one-half to onethird of this level for at least 35 days. 33 Following pretreatment of rats with estradiol, 34 the concentration of radioactivity from this radiolabeled "irreversible" enzyme inhibitor reached 8% dose/g in the adrenal. Unfortunately, it did not concentrate in the human adrenal gland? 3 Also, isoxazole is not easily labeled with a g a m m a emitter. As another consideration, the long persistence of a radiolabeled irreversible enzyme inhibitor in target tissue might show promise for therapy, but would not be ideal for diagnosis. In view of these problems, further studies on isoxazole were discontinued. However, it did occur to us that perhaps the best studied enzymes and their reversible enzyme inhibitors in common use in pharmacologic doses were the enzymes of the adrenal cortex (as shown in Figs. 1-5). A d r e n o c o r t i c a l e n z y m e inhibitors have achieved extensive medical application in the last two decades. 3~-36 However, they have never been systematically investigated as possible radiodiagnostic agents. As shown in Figs. 1 and 3, nearly every step in the biochemical conversion of cholesterol to aldosterone has at least one known enzyme inhibitor. With few exceptions, these inhibitors are nonsteroidal organic b a s e s - - e i t h e r aniline or p y r i d i n e derivat i v e s - w i t h short biologic half-lives. Metyrapone, a pyridine derivative, perhaps the best known of the steroid hydroxylase inhibitors, has been widely used to determine pituitary A C T H reserve. Its mechanism of action has been extensively studied and has been

shown to involve binding to ! 1/~-hydroxylase in the inner membrane of the mitochondria of all zones of the adrenal cortex. 36 The 1 lr lase enzyme system consists of three fractions: (l) a flavoprotein, (2) adrenodoxin, and (3) cytochrome P-450 as well as the cofactors N A D P H , 02, and Mg ~+. Cytochrome P-450, a heine-containing protein, is the substrate-binding and oxygen-activating component of the system, and it is to this protein that metyrapone binds. Unfortunately, metyrapone also binds to cytochrome P-450 in the liver, though various studies indicate that the concentration of cytochrome P-450 is greater in the adrenal cortex than in the liver. 37 In Fig. 9 are shown the structures of eight radiolabeled enzyme inhibitors that have been synthesized and evaluated for adrenocortical localization in the dog. 38 The ~H-compounds were labeled by catalytic e x c h a n g e m e t h o d s , purified by silica gel c h r o m a t o g r a p h y , and f o r m u l a t e d as the hydrochloride salts in physiological saline. Among the tritiated compounds evaluated, 3 H - S K F - 1 2 1 8 5 , 3 H - m e t y r a p o n e , and 3Hmetyrapol showed the highest concentration in the dog adrenal cortex (Table 3). With 3H-SKF12185 and aH-metyrapone, the adrenal cortexto-liver concentration ratios ranged from 4-8, suggesting competitive binding to hepatic P-450. However, 3H-metyrapol, the respective alcohol A

C2H~/--A~

0~'~ N./'~, 0 H [aH(G)]-4AMINOGLUTETHIMIDE

4;

CHCHzNHz

9

[~H(G)]- SKF-12185 0 CH3 II I

A

C2H~/~X

0,~'~ N/'~,0 ~a~I H 'z~I-3-IODO-4AMINOGLUTETHIMIDE

CHCHzNHa

9

I- 3-IODO-SKF-12185 H

CH3

CH~~..'~.J [3H(G)]-METYRAPONE

[3H (G)]- METYRAPOL

Fig. 9. Chemical configuration of our first 8 radiolabeled enzyme inhibitors of enzymes in the adrenal cortex. ~8

13

ADRENAL IMAGING AGENTS

Table 3. Concentration of 3H(G)--Enzyme Inhibitors in Selected Organs at Peak Adrenal Uptake in Dogs* Interval After Dose

Compound

Adrenal Cortex

Liver

Kidney

Blood

Amphenone B Aminoglutethimide SU-10603

30 min 1 hr 1 hr

0.40 • 0.01 0.68 • 0.08 0.65 • 0.06

0.35 • 0.01 0.41 • 0.08 0.86 • 0.04

0.23 • 0.05 0.20 • 0.05 0.47 • 0.40

0.73 • 0.34 0.45 • 0.08 0.27 • 0.10

SKF-12185 Metyrapone

30 min 45 min

1.86 • 0.03 2.75 • 0.17

0.64 • 0.01 0.44 • 0.04

0.23 • 0.01 0.28 • 0.04

0.06 • 0.00 0.17 • 0.04

1 hr

8.91 • 0.89

0.52 • 0.06

0.19 • 0.03

0.10 • 0.00

Metyrapol

* Peak adrenal uptake occurred at different time intervals with different compounds.

of metyrapone, was unique in its distribution, showing an adrenocortial uptake of 8.9% Kg dose/g at 1 hr and an adrenal/liver ratio of 25; this ratio increased to 57 at 2 hr. In vitro studies 39-4~ have shown that metyrapol binds more avidly to ll/3-hydroxylase P-450 than does metyrapone. The 8.9% Kg dose/g uptake is greater than that obtained with the iodocholesterols at 3 5 days. Since metyrapol showed the best adrenal uptake and target-to-nontarget concentration ratios, the radioiodination of this compound was pursued. Though numerous structure-activity studies of m e t y r a p o n e - l i k e compounds have been published, the pyridine rings of metyrapone have never been functionalized. The 5or 5'- positions of the metyrapol molecule were chosen for incorporation of radioiodine because (1) 3(5)-halopyridines act as typical aromatic halides, whereas 2- and 4-halopyridines are often unstable; (2) an iodine atom in the 3(5) position would lower the basicity of the pyridine nitrogen less than in the 2- or 4-position; 41 a possibly important consideration if the pyridine nitrogen(s) are involved in binding to 11/3h y d r o x y l a s e . U n f o r t u n a t e l y , pyridines are highly resistant to electrophilic addition, especially iodination. 42 However, pyridine-N-oxides o

are readily functionalized by a diverse number of substitution reactions, and this fact was utilized as shown in Fig. 10 to synthesize 125I-5'iodometyrapol. 43 Table 4 compares the biodist r i b u t i o n of 3 H - m e t y r a p o l and t25I-5'iodometyrapol. The latter compound shows a higher concentration in the adrenal cortex than in all other organs, but the uptake is fourfold lower than that observed with 3H-metyrapol. Efforts are in progress to radioiodinate the Aring of metyrapol in the 5-position. The 125I-p-iodobenzoate of metyrapol has also been synthesized, but failed to concentrate in the adrenal cortex. Metabolic studies indicate that the ester is rapidly hydrolyzed in vivo, suggesting the evaluation of more stable ester and amide derivatives. Further efforts are under way to determine which structural modifications of metyrapol will enhance adrenal localization and still permit ready incorporation of a ~,-label. Table 5 lists a selected number of 3H-labeled bipyridyls that have been synthesized and evaluated. As indicated, minor structural modification of the metyrapol side-chain adversely affects adrenal localization and tends to support the supposition that the best position for a foreign 3,-label is on one of the pyridine rings.

i01CIH3

CH3

I

'

O

AgNO3 _p-NOzPhCOCI

H0 I

CHz I

125

I) Pd/H z 2) HNOz/NoT 3) (l- BuO)3 LiAH

Fig. 10. S y n t h e s i s iodomethyrapol.

of

1251-6'-

Propylane Glycol 5) Fs/HAc

I01 CIH3 r

~

c - ic ~ - - ~

I O

NOz

BEIERWALTES ET AL.

14

Table 4. Concentration of Radiolabeled Metyrapols in Selected Organs at Peak Adrenal Uptake in Dogs* Adrenal Cortex

Liver

Kidney

Blood

3H-Metyrapol

8.91 • 0.89

0.78 • 0.03

0.30 • 0.07

0.14 • 0.00

1251-5'-Iodometyrapol

0.94 • 0.08

0.24 • 0.00

0.12 • 0.01

0.06 • 0.00

Compound

* Peak uptake occurred at different time intervals with the different compounds,

In contrast to metyrapol, the phenethylamine derivative, SKF-12185, surprisingly showed higher u p t a k e in the adrenal cortex after radioiodination (Table 6). The iodine label is extremely stable both in vitro and in vivo (i.e., low t h y r o i d u p t a k e ) as o p p o s e d to or thoiodinated phenols. 44 Recently, we have studied the adrenal uptake of two of these radiolabeled enzyme inhibitors in humans. Eight patients, scheduled for adrenalectomy, received 100 t~Ci each of 3H-metyrapol or 1=5I-SKF-12185 i.v. 1-2 hr before excision of the adrenals. Samples of adjacent tissues and blood were taken for comparison. Data were expressed as per cent Kg-dose/g • 1 SEM. A mean adrenal uptake of 3H-metyrapol of 5.8 4- 1.4% (four patients) occurred at 1 hr. Blood, muscle, and fat were 0.6% or less. At 2 hr, adrenal blood and liver were 3.2 4- 0.4%

(three patients), 0.1 • 0.1% (one patient), and 1 . 5 4- 0.1% (one patient), respectively. 125ISKF-12185 in adrenals was 2.3 + 0.4% (one patient) at 2 hr and 0.8 4- 0.1% (one patient) at 1 hr. Muscle and fat were 0.1% or less. The per cent uptakes in the adrenals at 1-2 hr are equivalent to that of 13~I-19-iodocholesterol at 2 days. We also produced our first successful adrenal gland imaging in the dog using '3'ISKF-1218575 Although adrenal:liver ratios with '25I-SKF12185 were low, Anger camera imaging continuously from 10 min to 3 hr on two dogs, using 6 mCi of '31I-SKF-12185 each, resulted in images of the adrenals 2 hr after injection (see Fig. 11). This dose of '3q-SKF-12185 was used to simulate the crystal photon flux expected with 2 mCi of '2~I-SKF. We are currently evaluating the efficacy of 2 hr adrenal imaging

Table 5, Concentration of 3H(G)--Bipyridyls in Dog at 1 Hour (Concentration in Percent Kilogram D o s e / G r a m ) Adrenal Cortex

Liver

Kidney

Blood

8.91 • 0.89

0.78 =~ 0.03

0.30 • 0.07

0.14 • 0.00

0.93 • 0.02

0.23 • 0.05

0.06 • 0.03

0.08 • 0.00

0.93 • 0.02

0.23 :~ 0.05

0.06 • 0.03

0.08 • 0.00

0.39 • 0.02

0.23 • 0.10

0.08 9 0.00

OH

I

py3--CH--C(CH3)2--Py 3 (Metyrapol)

OH

I I

py3--C--C(CH3)2--Py 3

121"-13

OH

I

py3--i--C(CH3)2--py3 Ph

OCOCH3

[

py3--CH--C(CH3)2--Py 3 pya = Pyridine ring.

5.58 + 0.11

ADRENAL IMAGING AGENTS

15

Table 6. Concentration of Radiolabeled SKF-12185 in Selected Organs at Peak Adrenal Uptake in Dogs* Compound

Interval After Dose

Adrenal Cortex

Liver

Kidney

Blood

3H-SKF-12185

30 rain

1,86 ~_ O.03

0.64 4- 0,O1

0.23 4- O.O1

O.06 4- O.00

'~sI-SKF-12185 '311-SKF-12185

1 hr 3 hr

2,48 • 0.54 2.90 • 0.16

0.70 • 0.14 0,65 4- 0.07

0.194- 0.02 0,32 • 0.07

0.04 4- 0.01 0.03 • 0.DO

Peak adrenal uptake occurred at different time intervals with the different compounds.

with ~23I-SKF-12185. The calculated radiation dose from 2 mCi of 123I-SKF-12185, in humans, e x p r e s s e d in Rads is: a d r e n a l = 0.76, ovaries = 0.18, liver = 0.68. ADRENAL MEDULLA

Precursors of Epinephrine Early in our work of developing radiolabeled compounds for uptake in malignant melanomas, we e x p l o r e d the u p t a k e of r a d i o l a b e l e d precursors of epinephrine. We were interested to find that, although they showed insufficient uptake in animal melanomas to make a trial in humans worthwhile, there was a striking uptake in the adrenal glands. We, therefore, explored the uptake and excretion of epinephrine and 14C-labeled precursors of epinephrine in dogs and d e m o n s t r a t e d that the u p t a k e of 14Cdopamine in adrenal medulla exceeded that of all other precursors studied 46 (Fig. 12) and

showed a t89 in 10 min in blood (Fig. 13). Dr. Counsell and his graduate students tried unsuccessfully to make a radioiodinated analogue of dopamine that would concentrate in the adrenal medulla similar to the concentration of 14Cdopamine. 47 He did succeed, however, in making an 0-, m-, and p-iodophenylalanine that conc e n t r a t e d with species specificity in the pancreas of the mouse; each compound showed a reproducibly different uptake depending upon position of the iodine label. 48 This work was later confirmed by Ulberg? 9 At the same time, our biomedical evaluation group demonstrated a concentration of 14Cdopamine in the human neuroblastoma (the second most common cause of death from cancer in children) considerably more than in the normal human adrenal medulla '~~ (Fig. 14). We also demonstrated that it concentrated similarly in three pheochromocytomas removed from two adolescent brothers (Fig. 15). The

Fig. 11. (A) First successful image of dog adrenal gland at 3 hr after 6 mCi of '~1-SKF-12185 (to simulate the crystal photon flux of 2 mCi of =~'LI-SKF-12185). (B) Image of adrenals of same dog at 5 days after 1 mCi of 6-beta-iodomethyl-19-norcholesterol.

16

BEIERWALTES ET AL.

48oF

~-,,054~

=I 4401-

739.6

6HR

24HR

TYROSINE DOPA

I

DOPAMINE I NOREPINEPHRINE I EPINEPHRINE I

320 F

I I+++++++++++++++] I

v////////////J

I I

[:

l

+f 240

Medulla/ Plasma

Medull0/ Kidney

Medulla/ Liver

Medulla/ Muscle

peak uptake was observed at 2 hr, with considerable persistence of uptake at 8 hr after the tracer dose?' Fowler and co-workers +2-+3 synthesized I'Cdopamine and observed localization in the dog adrenal medulla at short time intervals ( < 2 hr), but attempts to image the adrenals were not reported. With carrier-free 'lC-dopamine, it was observed that the concentration in the adrenal medulla increased approximately fourfold (form 0.028 to 0.117 average). Dopamine labeled in the 6-position with radioiodine ('23I and '3'I) surprisingly showed adrenal medul l ar y c o n c e n t r a t i o n s nearly identical to that obtained with 'lC-dopamine? 4 50

t -'~ 50 -~ \ -~\k

TYROSINE DOPA DOPAMINE NOREPINEPHRINE EPINEPHRINE

= = = --o----o . . . . == c-----o

I0 --.7

..

....

\% ~" ....... iI

o (5)

I

TIME (hours) Fig. 13. Plasma 14C radioactivity disappearance in dogs after t4-C labeled epinephrine and its precursors. ~9

Fig. 12. Uptake of five 14C precursors of epinephrine in dog adrenal at 6 hours. 69

Also significant was that only minor in viva deiodination occurred; other workers have observed extensive in viva deiodination of radioiodinated t yram i nes 5~ and phenylethylamine. 46 Fowler and associates concluded that the u p t a k e of radioiodinated 6iododopamine in the adrenal medulla was probably not high enough to permit imaging, though they conceded that carrier-free radioiodinelabeled dopamine, if it could be synthesized, would show enhanced uptake and might be useful as a scanning agent. Dop am in e A n alogu es

A possible alternative to labeling dopamine directly on the catechol ring was prompted by the finding that the NHSO2R moiety can be used as a bioisosteric substitute for the 3-OH group of catecholamines? 6 Ice, Wieland, and co-workers+7 synthesized the seven radiolabeled sulfonanilide analogues of dopamine shown below. Compound I (R = 3%O2CH3) localized in the adrenal of both the rat and dog, with uptake and T I N T values similar to dopamine itself. However, the remaining analogues containing iodo-, aryl-, and alkylaryl groups failed to localize in the adrenals attesting to low bulk tolerance in this region of the molecule. Other bioisoteric substitutes for the 3-OH group are known, 58 some of which may be more amenable to "r-label incorporation than are the sulfonanilide analogues. These c a t e c h o l a m i n e analogues do not act as substrates for catecholO - m e t h y l t r a n s f e r a s e (COMT), and their

A D R E N A L I M A G I N G AGENTS

17

CPM/MG • 120 II0 100 90 8O "to 6O 5O 40 30 2O 10

Fig. 14. Uptake of t4C from x4C-dopamine in neuroblastomas in children.

enhanced metabolic stability 5~ suggests that peak adrenal uptake may occur at longer time intervals than observed with labeled catecholamines. COMPO UND

R

I 1I III IV V VI VII

~SO2CH3 a%O2Ph-I-p SO~Ph-3H-p SO2CHzPh-aH-p SO2(CH2)2Ph-3R-p SO2(CH2)3Ph-aH-p SO~CH2~I

S el enium- 75-19-S el enoc hol es t erol

We have published that Z%e-cholesterols (labeled in the 19 position) concentrated better in % DOSE/GM (xlO-3: 40 --

55

30

I--I 65 MIN P'A 117 MIN

25

9

8 HRS

20

15

I0

5

TUMOR

LIVER

FAT

MUSCLE

Fig. 15. Uptake of 14C from 14C-dopamine in three pheochromocytomas at 1, 2, and 8 hr in two brothers who had surgical resections of three pheochromocytomas.

D

Hepotomo

a

Neurolemmoma Neuroblastoma Adrenal Medulla TISSUES ASSAYED

the adrenal medulla than in the adrenal cortex of dogs at two different time intervals, s~ We apparently imaged one pheochromocytoma with this agent. Further work with this agent has been tabled, however, because the radiation dose is unnecessarily high. RADIOLABELED ENZYME INHIBITORS OF ENZYMES IN ADRENAL MEDULLA

Since diagnostic concentrations of radiolabeled enzyme inhibitors in the adrenal cortex have been achieved, a similar approach might be applied to the development of a scanning agent for the adrenal medulla. The enzymes responsible for the sequential conversion of tyrosine to epinephrine have been characterize& 1-63 and are represented in Fig. 16. Their distribution in the mammalian body is of paramount consideration for such an approach. Phenylethanolamine - N - methyltransferase (PNMT) is unique to the adrenal medulla (a very minute amount is found in the brain), and, thus, radiolabeled inhibitors of this enzyme represent perhaps the most feasible approach to developing a scanning agent for the adrenal medulla. An added advantage to this enzyme is that it is ACTH responsive. Activity, therefore, may be stimulated with ACTH or suppressed with dexamethasone. Dopa decarboxylase (DD) is an ubiquitous enzyme widely distributed in mammalian tissues such as brain, liver, kidney, lung, adrenals, various neoplasms, and sympathetic nerve endings. Tyrosine hydroxylase (TH) and dopamine-flhydroxylase (DBH) are found in sympathetic tissue, adrenal medulla, and the brain. DBH is also found in the heart. Possible competitive

18

BEIERWALTES ET A L

Tyros• ~CH2-CHHO~...~J

NH2 COOH

rCH3

[ * * T ~ C H 2 - Ci- NH2 He.*L~__~ COOH

TyrOs• hydroxylose

(3-Iodo -0(-methyltyrosine) Dope HO~CH2-CIHHOJ~__~

NH2

COOH

1

Dope 1 decerboxylose Dopomlne ~

cHz- cHz-NH2

Dopm'nine-,8,-

n_- Bu

CH2-CH-CH2Brl Brl -CHz " ~

l

Noreplnephrine

COOH Dibr0mofusaric Acid

He ~"~--~Cl H"CHz- NH2 H O ~ OH

PhenylethonolomineN-methyltronsferose

{~ ~ N T ' H SKF - 7698

Epinephrine

CH3

Cl. ~ N

H

CI SKF- E,4139

HO,~CH-CH2 -NHCH3 He ~

Fig. 16. Chemical configuration of some enzyme inhibitors of catecholamine biosynthesis.

OH

uptake of radioactivity by the brain and heart would not interfere with scanning of the adrenal medulla. Since PNMT is the enzyme unique to the adrenal medulla, initial efforts have focused on exploring radiolabeled inhibitors of this enzyme. Many synthetic phenyl amines as well as natural endogenous phenylethylamines and phenylethanolamines show varying degrees of

PNMT inhibition. 64 However, most of the compounds either do not show sufficient in vivo inhibition, or in the case of the natural catecholamines, present a difficult radiolaheling task. An attractive alternative was suggested by Pendleton and co-workers, who found that two tetrahydroisoquinoline derivatives, SKF-7698 and SKF-64139, show strong in vivo inhibition of PNMT. 6~ The latter compound also con-

Table 7. 3H-SKF-7698 (NP-97) P N M T Inhibitor Tissue Distribution in Dogs* Organ Adrenal medulla Adrenal cortex Adrenal Liver Spleen Kidney Ovary Blood Urine

30 Min (2 Dogs) 0.28 • O.16 • 0.12 • O.81 • 0.12 • 0.15 • 0.12 • 0,06 • -,14 •

0.00 0.04 0.O3 0.02 0.01 0.02 0.03 0.02 0,01

* Dose: 100/~Ci i.v.; sp. act. = 1 .O mCi/mg, percent kilogram dose/gram.

1 Hr (3 Dogs)

4 Hr (2 Dogs)

1.91 0.38 0.52 O.51 0.28 0.33 O.19 0,11 1.94

O11 0.08 0.07 0.14 0.06 0.10 0.40 0,22 3.42

• • • • • • • • •

0.79 0.03 0.17 0.03 0.01 0.04 0.01 0.04 0,43

• • :~ • • • • • •

0.01 0,02 0.02 0.02 0.04 0.02 0.00 0.13 0,08

19

A D R E N A L I M A G I N G AGENTS

Table 8. 3H-SKF-64139 (NP-t 19) PNMT Inhibitor Tissue Distribution in Dogs* Organ

15 Min (1 Dog)

30 Min (3 Dogs)

1 Hr (1 Dog)

4 Hr (1 Dog)

Adrenal medulla

1.03 • 0.06

0 . 8 8 • 0.17

0.53 • 0.33

0 . 2 0 • 0.12

Adrenal cortex

0.54 • 0.10

O.31 • 0.07

0.21 • 0 . 0 6

O.10 • 0.01

Adrenal

0.53 • 0.02

0,33 • 0 . 0 5

0.81 • 0.37

0.13 • 0.01

Liver

0.32 • 0 . 1 4

0.44 • 0.06

0.41 • 0,01

0.20 • 0.00

Spleen

0.27 • 0 . 0 8

0.17 • 0.02

0.12 • 0,01

0 . 0 4 • 0.01

Kidney

0.38 • 0 . 0 9

0 . 3 0 ~: 0.07

0.28 • 0 . 0 0

0 . 1 6 • 0.01

Ovary

1,59 • 0,87

0.17 • 0.02

0.08 • 0,01

0 . 0 5 • 0.03

Blood

0 . 7 3 • 0.26

0 . 2 4 • 0.12

0.89 • 0 . 8 0

0.09 ~: 0.07

Urine

0.22 • 0,11

1.57 • 0 . 7 0

1.33 •

1.78 • 0.02

1.O3

* Dose: 1 0 0 #Ci i.v.; sp. act.: 1 mCi/mg, percent kilogram d o s e / g r a m

centrates in the rat adrenal medulla 5 min after i.v. injection as shown by 14C- autoradiography. Axelrod and co-workers have utilized SKF-7698 to reduce the blood pressure of spontaneously hypertensive rats via the inhibition of brain stem PNMT. 66 We have determined the biodistribution of 3H-SKF-7698 and 3H-SKF-64139 in dogs at various time intervals as shown in Tables 7 and 8. The high uptake of 3H-SKF-64139 in the adrenal medulla at short time intervals has prompted the synthesis and evaluation of radioiodinated analogs; work which is still in progress.

Adrenal Medulla Korn and associates, 67 in a recent investigation of radiolabeled antiarrhythmic drugs as potential myocardial scanning agents, found that the p-iodobretylium analogue (p-RIBA), in contrast to the myocardial selective ortho isomers (o-RIBA), shows selective uptake in the dog adrenal medulla. Human studies with the 13'I-p-RIBA have not been reported. Delineation of the mechanism of uptake ofp-RIBA may lead to the design of a new class of compounds that bind preferentially to adrenal medullary tissue.

REFERENCES

1. Goodman DS: Cholesterol ester metabolism. Physiol Rev 45:747, 1965 2. Samuels LT, Uchikaua T: Biosynthesis of adrenal steroids, in Eisenstein AB (ed): The Adrenal Cortex. Boston, Little, Brown, 1967, pp 61 102 3. Beierwaltes WH, Varma VM, Lieberman LM, et al: Percent uptake of labeled cholesterol in adrenal cortex. J Nucl Med 10:387, 1969 4. Blair RS, Beierwaltes WH, Lieberman LM, et al: Radiolabeled cholesterol as an adrenal scanning agent. J Nucl Med 12:176, 1971 5. Blaschko H, Firemark H, Smith AD, et al: Lipids of the adrenal medulla lysolecithin, a characteristic of chromaffin granules. Biochem J 104:545, 1967 6. Da Prada M, Pletscher A, Tranzer JP: Lipid composi, tion of membranes of amine-storage organelles. Biochem J 127:681, 1972 7. Applegren LE: Sites of steroid hormone formation. Autoradiographic studies using labeled precursors. Acta Physiol Scand Suppl 301:1, 1967 8. Nagai T, Sotis BA, Koh CS: An approach to developing adrenal-gland scanning. J Nucl Med 9:576, 1968 9. Counsell RE, Ranade J J, Blair RJ, et al: IX. Radioiodinated cholesterol. Steroids 16:317, 1970. 10. Beierwaltes WH, Lieberman LM, Ansari AN, et al: Visualization of human adrenal glands in vivo by scintillation scanning. JAMA 216:275, 1971

11. Lieberman LM, Beierwaltes WH, Conn JW, et al: Diagnosis of adrenal disease by visualization of human adrenal glands with 1-131-19-iodocholesterol, N Engl J Med 285:1387, 1971 12. Kojima M, Minoru M: Homoally!ic rearrangement of 19-iodocholesterol. JCS Chem Comm January 15, 1975 13. Basmadjian GP, Hetzel KR, Ice RD, et al: Synthesis of a new adrenal cortex scanning agent, 613-1-131iodomethyl-19-norcholest-5(10)-en-313-ol (NP-59). J Label Comp 11:427, 1975 14. Kojima M, Maeda M, Ogawa H, et al: New adrenal scanning agent. J Nucl Med 16:666, 1975 15. Basmadjian GP, Hetzel KR, Ice RD, et al: Synthesis of a new adrenal imaging agent: 6-/3-I-131-iodomethyl-19norcholest-5(10)-en-313-ol (NP-59). J Nucl Med 16:514, 1975 16. Sarkar SD, Ice RD, Beierwaltes WH, et al: 6/3-1-131iodomethyl- 19-norcholest-5(10)-en-3/3-ol (NP-59) concentrates in the adrenal gland better than 1-131-19iodocholesterol. J Nucl Med 16:565, 1975 17. Sarkar SD, Beierwaltes WH, Ice RD, et al: A new and superior adrenal scanning agent: NP 59. J Nucl Med 16:1038, 1975 18. Sarkar SD, Cohen EL, Beierwaltes WH, et al: A new and superior adrenal imaging agent, NP 59: Evaluation in humans. J Clin Endocrinol Metab 45:163, 1977 19. Forman BH, Antar MA, Touloukian RJ, et al: Local-

20

ization of a metastatic adrenal carcinoma using 1-131-19iodocholesterol. J Nucl Med 15:332, 1973 20. Watanabe M, Kamoi I, Nakayama C, et al: Scintigraphic detection of hepatic metastases with 1-131 labeled steroid in recurrent adrenal carcinoma: Case report. J Nucl Med 17:904, 1976 21. Seabold JE, Haynie TP, DeAsis DN, et al: Detection of m e t a s t a t i c adrenal carcinoma using 1-131-6fliodomethyl-19-norcholesterol total body scans. J Clin Endocrinol Metab 45:788, 1977 22. Chatal JF, Carbonnel B, Le Mevel BP, et al: Uptake of 1-131-19-iodocholesterol by an adrenal cortical carcinoma and its metastases. J Clin Endocrinol Metab 43:248, 1976 23. Morita R, Lieberman LM, Beierwaltes WH, et al: Percent uptake of 1-131 radioactivity in the adrenal from radioiodinated cholesterol. J Clin Endocrinol Metab 34:36, 1972 24. Fitzgerald PV, Foote FW: The function of various types of thyroid carcinoma as revealed by the radioautographic demonstration of radioactive iodine (I131). J Clin Endocrinol 9:1153, 1949 25. Wollman SH: Analysis of radioiodine therapy of metastatic tumors of the thyroid gland in man. J Nat Cancer Inst 13:815, 1953 26. Dobyns BM, Maloof F: The study and treatment of 119 cases of carcinoma of the thyroid with radioactive iodine. J Clin Endocrinol 11 : 1323, 1951 27. Kojima M, Maeda M, Ogawa H, et al: Comparison of uptake of 6fl-H-3-methyl-19-norcholest-5(10)-en-3fl-ol and H-3 cholesterol by rat adrenal. Radioisotopes 25:222, 1976 28. Szinai SS, Owoyale JA: Presented before the Medicinal Chemistry Division of the American Chemical Society, 169th National Meeting, Philadelphia, April 1975 29. Yu T, Wieland DM, Ice RD, et al: Synthesis of labeled 3fl-iodocholesterols for adrenal imaging. J Label Comp Radiopharm 13:274 1977 30. Sturman MF, Beierwaltes WH, Prakash S, et al: Uptake of radiolabeled testosterone, 5-a-dihydrotestosterone, estradiol and pregnenolone by canine prostate. J Nucl Med 15:94, 1974 31. Balasubraminiam S, Mitropoulos KA, Myant NB: Evidence for the compartmentation of cholesterol in ratliver microsomes. Eur J Biochem 34:77, 1973 32. Goldman AS: Specific retention of an inhibitor of 3flhydroxysteroid dehydrogenase in enzyme-containing tissues of the rat. Endocrinology 86:678, 1970 33. Ryo UY, Beierwaltes WH: Distribution of 14C-isoxazole in adrenals, ovaries and breast carcinoma. J Nucl Med 14:321, 1973 34. Ryo UY, Beierwaltes WH, Ice RD: Enhancement of uptake with estradiol treatment of radiolabeled irreversible competitive enzyme inhibitor in the adrenal cortices and ovaries of rats with endocrine "autonomous" breast carcinomas. J Nucl Med 15:187, 1974 35. Temple TF, Liddle GW: Inhibitors of adrenal steroid biosynthesis. Ann Rev Pharmacol 10:199, 1970 36. Gower DB: Modifiers of steroid-hormone metabolism: A review of their chemistry, biochemistry and clinical application. J Steroid Biochem 5:501, 1974 37. Wickramasinghe RH: Biological aspects of

BEIERWALTES ET AL.

cytochrome P450 and associated hydroxylation reactions. Enzymes 19:348, 1975 38. Beierwaltes WH, Wieland DM, Ice RD, et al: Localization of radiolabeled enzyme inhibitors in the adrenal glands. J Nucl Med 17:998, 1976 39. Williamson DG, O'Donnell VJ: The interaction of metopirone with adrenal mitochondrial cytochrome P-450. A mechanism for the inhibition of adrenal steroid 11 flhydroxylation. Biochemistry 8:1306, 1969 40. Satre M, Vignais PV: Steroid 11 fl-hydroxylation in beef adrenal cortex mitochondria. Binding affinity and capacity for specific [14C] steroids and for [~H] metyrapol, an inhibitor of the 11 fl-hydroxylation reaction. Biochemistry 13:2201, 1974 41. Abramovitch RA, Singer GM: Properties and reactions of pyridines, in Abramovitch RA (ed): The Chemistry of Heterocylic Compounds, Vol 14, Part I, New York, Wiley, 1974, pp 3 5 42. Acheson RM: An Introduction to the Chemistry of Heterocyclic Compounds, ed. 2. New York, Interscience, 1967, p 168 43. Wieland DM, Kennedy WP, Ice RD, et al: Radioiodinated pyridines Potential adrenocorticalimaging agents. J Label Comp Radiopharm 13:229, 1977 44. Counsell RE, Ice RD: The design of organ imaging radiopharmaceuticals, in Ariens EJ (ed): Drug Design, Vol. VI. New York, Academic 1975, pp 184-187 45. Mosley ST, Wieland DM, Beierwaltes WH, et al: Radiolabeled enzyme inhibitors as adrenal scanning agents: Imaging of dog adrenals and tissue distribution in humans. J Nucl Med, June 1977 (abstr) 46. Morales JO, Beierwaltes WH, Counsell RE, et al: The concentration of radioactivity from labeled epinephrine and its precursors in the dog adrenal medulla. J Nucl Med 8:800, 1967 47. Counsell RE, Smith TD, DiGiulio W, et al: Tumor localizing agents. VIII. Radioiodinated phenylalanine analogs. J Pharm Sci 57:1958, 1968 48. Varma VM, Beierwaltes WH, Lieberman LM, et al: Pancreatic concentration of 1-125-1abeled phenylalanine in mice. J Nucl Med 10: 219, 1969 49. Ulberg S, Blomquist L: Selective localization to pancreas of radioiodinated phenylalanine analogs. Acta Pharm Seuciea 5:45, 1968 50. Lieberman LM, Beierwaltes WH, Varma VM, et al: Labeled dopamine concentration in human adrenal medulla and in neuroblastoma. J Nucl Med 10:93, 1969 51. Anderson BG, Beierwaltes WH, Harrison TS, et al: Labeled dopamine concentration in pheochromocytomas. J Nucl Med 14:781, 1973 52. Fowler JS, Ansari AN, Wolf AP, et al: Synthesis and preliminary evaluation in animals of carrier-free HC-labeled dopamine hydrochloride, J Nucl Med 14:867, 1973 53. Fowler KS, Wolf AP, Christman DR, et al: in Subramanian, Rhodes BA, Cooper, JT, et al (eds): Radiopharmaceuticals. New York, Society of Nuclear Medicine, 1975, pp 196-204 54. Fowler JS, MacGregor RR, Wolf AP: Radiopharmaceuticals. Halogenated dopamine analogs: Synthesis and radiolabeling of 6-iododopamine and tissue distribution studies in animals. J Med Chem 19: 356, 1976

ADRENAL IMAGING AGENTS

55. Counsell RE, Smith TD, Ranade VV, et al: Potential organ or tumor imaging agents. XI. Radioiodinated tyramines. J Med Chem 16:684, 1973 56. Larsen AA, Gould WA, Roth HR, et al: Sulfonanilides. II. Analogs of catecholamines. J Med Chem 10:462, 1967 57. Ice RD, Wieland DM, Beierwaltes WH, et al: Concentration of dopamine analogs in the adrenal medulla. J Nucl Med 16:1147, 1975 58. Kaiser C, Schwartz MS, Colla DF, et al: Adrenergic agents. III. Synthesis and adrenergic activity of some catecholamine analogs bearing a substituted sulfonyl or sulfonylakyl group in the meta position. J Med Chem 18:674, 1975 59. Uloth RH, Kirk JR, Gould WA, et al: Sulfonanilides. I. Monoalkyl- and Arylsulfonamidophenethanolamines. J Med Chem 9:88, 1966 60. Sarkar SD, Ice RD, Beierwaltes WH, et al: Selenium-75-19-selenocholesterol--A new adrenal scanning agent with high concentration in the adrenal medulla. J Nucl Med 17:212, 1976 61. Melmon KL: Catecholamines and the adrenal

21

medulla, in William RH (ed): Textbook of Endocrinology, ed. 5. Philadelphia, Saunders, 1974, pp 283 316. 62. Sandier M, Rushsen CRJ: The biosynthesis and metabolism of catecholamines, in Ellis CP, West GS, (eds): Progress in Medicinal Chemistry, Vol. 6, London, Butterworth, 1964, pp 200 265 63. Axelrod J: Purification of phenylethanolamine-Nmethyl-transferase. J Biol Chem 237:1657, 1962 64. Krakoff LR, Axelrod J: Inhibition of phenylethanolamine-N-methyl transferase. Biochem Pharmacol 16:1384, 1967 65. Pendleton RG, Kaiser C, Gessner G: Studies of adrenal phenylethanolamine N-methyltransferase (PNMT) with SKR-64139, a selective inhibitor. J Pharmacol Exp Ther 197:623, 1976 66. Saavedra JM, Grobecker H, Axelrod J: Adrenaline--Forming enzyme in brainstem: Elevation in genetic and experimental hypertension. Science 191:483, 1976 67. Korn N, Buswink A, Yu T, et al: A radioiodinated bretylium analog as a potential agent for scanning the adrenal medulla. J Nucl Med 18:87, 1977