CONCENTRATION OF DOPAMINE ANALOGS

IN THE ADRENAL. MEDULLA

Rodney D. Ice, Donald M. Wieland, William H. Beierwaltes, Richard G. Lawton, and Michael J. Redmond

College of Pharmacy, The University of Michigan Medical Center, Ann Arbor, Michigan

Seven labeled sulfonanilide analogs of dopa-mine were synthesized and their tissue distribution in rats were determined as a function oftime. The methanesulfonanilide derivative, \f-27, showed significant uptake and retentionin the rat adrenal, with 0.66% dose/gm at 5min and 0.27% dose/gm at 24 hr. The 24-hrtarget-to-nontarget concentration ratios for theadrenal versus liver, blood, kidney, and heartwere 13, 27, 30, and 60, respectively. These results compared reasonably with the corresponding target-to-nontarget ratios of 23, 45, 8, and15 obtained for "'C-dopamiree. The six other

analogs showed considerably less uptake andretention. Further evaluation of NP-27 in dogsindicated selective uptake in the adrenal medulla.

pharmacologically similar to their unmodified counterparts (6). We report here that the methanesulfonanilide analog of dopamine (NP-27, where R =35SO2CH:,) shows specific uptake and retention in

the rat adrenal and the dog adrenal medulla, the firstknown case in which a radiolabeled noncatecholiccompound has shown specific uptake in the adrenalmedulla.

MATERIALS AND METHODS

Labeled compounds. The labeled compounds weresynthesized in the Nuclear Pharmacy Laboratory of

Received Feb. 15, 1975; revision accepted June 27, 1975.For reprints contact: Rodney D. Ice, Nuclear Medicine

Section, University of Michigan Medical Center, 1405 E.Ann St., Ann Arbor, Mich. 48104.

Previous work from this laboratory has demonstrated that I4C-labeled dopamine shows a striking

concentration in the dog adrenal medulla, reachingconcentrations greater than any other 14C-labeled precursor of epinephrine (7). A similar uptake of 14C-

dopamine has been demonstrated in the humanadrenal medulla and neuroblastoma (2) and inpheochromocytoma (3). The recent syntheses ofnC-dopamine and lsF-dopamine have given further

impetus to the search for a scanning agent for thehuman adrenal medulla (4,5). However, no gamma-emitting dopamine analogs utilizing long-lived isotopes (T,/2 > 6 hr) have yet been synthesized.

In a continuing effort aimed at understandingstructure-distribution relationships, which wouldthen lead to agents suitable for visualization of theadrenal medulla, we synthesized seven labeled sul-fonanilide analogs of dopamine and determined theirtissue distribution in rats as a function of time (Fig.1). The use of the NHSO2R moiety as a bioisostericsubstitute for the 3-OH group of dopamine wasprompted by the observation that the sulfonanilideanalogs of phenylethanolamines are in some cases

NHR

CH2CH2NH2

FIG. 1. Structural formula of dopamine analogs.

TABLECompoundNP-27NP-11NP-19NP-44NP-42NP-46NP-52'

See Fig.1.

SPECIFIC ACTIVITIES OFSEVENDOPAMINEANALOGSR'^'SO^CH,•^SOïPh-l-pSOsPh-'H-pSOïCHïPh-^H-pSCMCHd-Ph-'H-pSCMCHj-iPh-'H-pS02CHjml1

for structural formula.Specific

activity5.1

/¿Ci/mg370¿iCi/mg1.67mCi/mg8.50mCi/mg9.55mCi/mg14.0mCi/mg52.0iuCi/mg

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ICE, WIELAND, BEIERWALTES, LAWTON, AND REDMOND

5 min afterdosage"C-DopamineNP-27NP-11NP-19NP-44NP-42NP-46at

30min"C-DopamineNP-27NP-11NP-19NP-44NP-42NP-46at

1hr"C-DopamineNP-27NP-11*

Other tissuesAdrenal0.97±0.050.66±0.100.42±0.020.46±0.051.12±0.020.91±0.741.63±0.090.83±0.050.32±0.170.29±0.020.31±0.030.53±0.040.33±0.011.11±0.321.33±0.220.530.32±0.02studiedand notTABLELiver1.14±0.320.47±0.133.93±0.331.12±0.402.26±0.203.48±0.511.52±0.440.50±0.021.04±0.900.52±0.051.15±0.201.59±0.081.70±0.090.92±0.090.58±0.011.770.33±0.02listedwere2.

RELATIVE TISSUE DISTRIBUTION OF RADIOACTIVITY(%Spleen0.43±0.050.05±0.010.36±0.040.45±0.030.99±0.103.82±0.071.16±0.150.23±0.040.08±0.060.10<±0.010.20±0.010.83±0.041.55±0.210.59±0.030.30±0.020.170.06<±0.01Pancreas0.21±0.010.21±0.110.34±0.030.37±0.111.20±0.140.63±0.101.23±0.060.15±0.050.15±0.130.18±0.000.25±0.021.45±0.070.40±0.010.97±0.030.11±0.010.140.11±0.01renal

medulla, intestine,Renal

cortex1.39±0.120.40±0.123.39±0.401.13±0.184.07±0.043.23±0.573.03±0.350.11±0.171.57±1.400.66±0.100.10±0.074.41±0.660.91±0.092.41±0.260.59±0.262.880.61±0.15testes,

fa».lung0.55±0.042.14±0.550.57±0.050.59±0.131.54±0.163.42±1.501.44±0.120.22±0.090.75±0.700.33±0.060.33±0.041.32±0.111.04±0.081.23±0.070.20±0.011.170.20±0.04thyroid.Heart1.01±0.080.93±0.260.33±0.020.33±0.101.15±0.110.66±0.141.37±0.060.43±0.110.45±0.420.15<±0.010.23±0.020.83±0.090.25±0.031.37±0.130.26±0.020.500.12±0.01andurine. ABlood0.46±0.090.64±0.240.30±0.030.54±0.140.40±0.051.45±0.390.34±0.030.17±0.020.22±0.140.15±0.010.37±0.090.39±0.070.26±0.070.30±0.130.16±0.030.320.19±0.034-hrintervalDOSE/GM)Muscle0.14±0.010.09±0.020.12±0.010.19±0.160.21±0.040.21±0.030.45±0.190.06±0.010.09±0.080.07±0.030.17±0.010.31±0.030.20±0.030.28±0.020.09±0.020.120.07±0.01wasalso de-

the Phoenix Memorial Building, University of Michigan. Details of the synthesis will be published elsewhere. The 14C-labeled dopamine (specific activity,

67 fiCi/mg) used as a standard in this study waspurchased from New England Nuclear Corp. Table 1shows the resultant specific activities of the compounds evaluated. All compounds were formulatedas the hydrobromide salts. The NP-27 and 14C-

dopamine were administered in 0.9% saline solution; all other compounds were administered with20% ethanol in 0.9% saline solutions.

All compounds were characterized by infraredand nuclear magnetic spectroscopy and correct carbon, hydrogen, and nitrogen analysis. Radiochemicalpurity was determined by thin-layer chromatographyon silica gel-G, with all compounds giving single ra-diopeaks coincident with their respective cold spots.

Animals and tissue specimen. Sprague-Dawleymale rats weighing 150 ±20 gm were anesthetized

with sodium pentobarbital (5 mg/100 gm body wt)and injected via the femoral vein with 20 fid of thecompound in 0.2-0.5 ml solution, allowing 3 minto complete the injection. Three or more rats werekilled by decapitation at each time interval shownin Table 2.

The organs and tissues were removed and cleanedof fat and connective tissue, and representative 20—40 mg samples were placed in counting vials containing 0.3 ml 10% NaOH. These were left to digestovernight, then dissolved by warming to 70 °C,agi

tated for a few seconds, and left to cool. Next, twodrops of glacial acetic acid and three drops of 30%H2O- were added and mixed thoroughly. Then 10 mlPCS solubilizer (Amersham/Searle) and 2.5 ml H-..Owere added and vortexed until the solution becameclear. The vials were then wiped clean with a softcloth and placed in the liquid scintillation counter(Searle Radiographies, Unilux HA) for cooling and

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RADIOCHEMISTRY AND RADIOPHARMACEUTICALS

FROM ' »C-DOPAMINE AND DOPAMINE ANALOGS INTHE(at

1hr)NP-19NP-44NP-42NP-46at

6hr"C-DopamineNP-27NP-11NP-19NP-44NP-42NP-46at

24hr"C-DopamineNP-27NP-11NP-19NP-44NP-42NP-46fermined

but notAdrenal0.36±0.090.37±0.030.25±0.03—0.54±0.120.310.03±0.010.22±0.070.13±0.010.22±0.010.16±0.020.91±0.180.27±0.020.02<±0.010.06±0.010.10±0.010.15±0.030.06±0.01includedin thisLiver0.52±0.141.15±0.221.57±0.69—0.03±0.010.060.02<±0.010.30±0.060.36±0.131.02±0.640.32±0.010.04<±0.010.02<±0.010.02<±0.010.22±0.040.35±0.050.68±0.210.19±0.02table.ASpleen0.40±0.230.41±0.181.01±0.05—0.11±0.030.040.01<±0.010.14±0.080.32±0.091.52±0.170.40±0.100.08±0.010.02<±0.010.02<±0.010.08±0.020.15±0.020.92±0.220.25±0.09minimumofPancreas0.25±0.040.94±0.040.18±0.03—0.05<±0.010.010.01<±0.010.10±0.010.22±0.040.28±0.180.27±0.020.03<±0.01<0.01<±0.01<0.01<±0.010.02<±0.010.08±0.020.24±0.070.04±0.01RAT*Renal

cortex1.60±0.351.61±0.260.91±0.07—0.13±0.010.040.04<±0.010.43±0.200.41±0.111-66±1.600.89±0.290.11±0.030.01<±0.010.05±0.010.12±0.040.34±0.070.26±0.020.50±0.14Lung0.53±0.241.00±0.150.81±0.26—0.03±0.010.040.02<±o.oi0.20±0.040.25±0.052.69±2.550.36±0.050.03±0.010.02<±0.010.01<±0.010.13±0.060.09±0.010.38±0.090.10±0.04Heart0.18±0.040.65±0.120.15±0.02—0.13±0.030.010.01<±0.010.06±0.000.12±0.010.40±0.300.17±0.030.06±0.010.00<±0.010.01<±0.010.03<±0.010.06±0.010.20±0.080.06±0.02three

rats were killed each time interval. ResultsBlood0.17±0.050.30±0.050.20±0.09—0.01<±0.010.030.05±0.010.10±0.070.45±0.251.07±1.040.32±0.110.02±0.020.01<±0.010.03<±0.010.11±0.020.14±0.010.71±0.060.07±0.01inmean ±1Muscle0.13±0.040.30±0.050.22±0.08—0.02<±0.010.010.01<±0.01——0.06<±0.010.35±0.280.12±0.03

;0.01<±0.01<0.01<±0.010.01<±0.010.04±0.010.05±0.010.54±0.170.03±0.01s.d.

dark adaptation. Samples were counted for 10 mineach and quenching was corrected using the two-channel-ratio method. Data are expressed as percentage of given dose per gram of fresh tissue.

The procedure used to determine the tissue distribution in dogs has been outlined in a previouspublication (7).

RESULTS

Table 2 presents the relative tissue concentrationof the synthesized compounds in % dose/gm in nineselected rat tissues. The other tissues analyzed included the renal medulla, intestine, testes, fat, andthyroid, and all had concentrations less than 0.1%dose/gm. The distribution data on 14C-dopamine are

included for comparison. The highest radioactivityconcentration in the adrenal was evident with 14C-dopamine. However, NP-27 showed a marked uptake and retention in the adrenal, with 0.66% dose/

gm at 5 min and 0.27% dose/gm at 24 hr. The24-hr distribution pattern for NP-27 is presentedin Fig. 2. Figure 3 shows the relative concentrationratios of dopamine against its methanesulfonanilideanalog, NP-27. Carbon-14-dopamine showed an adrenal uptake one and a half to three times greaterthan NP-27 at all time intervals. The 24-hr target-to-nontarget concentration ratios of NP-27 for the adrenal versus liver, blood, kidney, and heart were 13,27, 30, and 60, respectively. These results comparereasonably with the corresponding ratios of 23, 45,8, and 15 obtained for 14C-dopamine.

The arylsulfonanilide analogs NP-11 and NP-19gave moderate uptake in the adrenal at short intervals but the radioactivity was rapidly released. At notime interval were the adrenal-to-liver ratios greaterthan 2.

Concentration of the tritiated chain-extended analogs NP-44, NP-46, and NP-42 in the rat adrenal,

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ICE, WIELAND, BEIERWALTES, LAWTON, AND REDMOND

028

0.26

0.24

0.22

0.20

I 0.18

LU 0.16

O 0.14OSÌ0.12

0.10

0080.06004

002

0.00ADRENAL LIVER BLOOD KIDNEY SPLEEN MUSCLE THYROID

FIG. 2. Tissue :"S concentration of

methanesulfonanilide NP 27 at 24 hr afterinjection.

although again high for short time intervals, diminished quickly after 1 hr. The adrenal-to-liver ratiosnever rose above 1. These more lipophilic compounds showed greater retention in the major organsat longer intervals than the other analogs tested.

The 125Iderivative NP-52 showed low adrenal up

take (0.06% dose/gm at 30 min). At 4 hr, 96%of the radioactivity counted appeared in the thyroid,suggesting in vivo deiodination.

The results of the NP-27 tissue distribution indogs are presented in Table 3. Long-term retentionof NP-27 was evident in the adrenal medulla. AHother organs, except the spleen, indicated rapidelimination of NP-27. The spleen concentration decreased by a factor of 6 whereas the adrenal medullaonly decreased by a factor of 2. At 48 hr the concentration ratio of adrenal medulla to adrenal cortexwas 8.5.

DISCUSSION

In light of the in vivo instability of radioiodinatedtyramine derivatives (7), it is apparent that if dopa-mine is to be successfully labeled with radioiodine,the iodine label cannot be substituted directly onthe aromatic ring. Larsen, et al (6,8,9) found thatcertain alkylsulfonamide moieties can be substitutedfor the 3-hydroxy group of phenylethanolamineswithout loss of adrenergic potency. Thus, the possi-

60

50

40

coo

tr

20

10

| "C-DOPAMINE

| | 3SS-METHANESULFONANILIDE

ADRENAL ADRENAL ADRENAL ADRENALLIVER BLOOD KIDNEY HEART

FIG. 3. Tissue radioactivity ratios for "C-dopamine and meth

anesulfonanilide NP-27 at 24 hr.

bility arises that radioiodine might show greater invivo stability if it was substituted on the 3-arylsul-fonamide group of a dopamine analog as in NP-11.

To verify the feasibility of the overall approach,the 35S-methanesulfonanilide analog NP-27 was syn

thesized and its tissue distribution determined. Thespecific localization of NP-27 in the rat adrenal,

TABLE 3. TISSUE DISTRIBUTION OF RADIOACTIVITY (% DOSE/GM) FROMNP-27 IN THEDOGTime(hr)62448Adrenolmedulla0.00480.00170.0021Adrenalcortex0.00440.00020.0003Liver0.02000.00110.0005Spleen0.00530.00240.0009Pancreas0.00740.00060.0003Renalcortex0.00430.00070.0004Lung0.00440.00030.0002Heart0.00660.00020.0002Blood0.00220.00100.0004Muscle0.00040.00020.0002

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RADIOCHEMISTRY AND RADIOPHARMACEUTICALS

presented in Fig. 2, was indeed encouraging. Thehigh uptake in the rat adrenal is even more notablein view of the low specific activity of the compound,i.e., the 20 /¿Cior 4 mg dose per rat represents approximately 15% of the total weight (25 mg) ofthe average rat adrenal. Wolf, et al found a significant dependence of adrenal uptake on the specificactivity of nC-dopamine (4). Thus the adrenal uptake and target-to-nontarget ratios obtained forNP-27 may be greater with higher specific activities.

The logical extension of these initial findings wasevaluation of the p-iodophenyl analog NP-11. Sinceselective adrenal localization did not occur withNP-11, the phenyl analog NP-19 was studied to determine if the bulky iodine atom itself caused thelack of retention of NP-11 in the adrenal. However,essentially the same adrenal uptake and distributionpattern was observed for the two compounds. Theadded failure of the chain-extended analogs NP-44,NP-46, and NP-42 to show retention in the adrenalemphasized the strict structural constraints placed onpotential adrenal-scanning agents.

Phenylethanolamine N-methyl transferase (PNMT)which transfers a methyl group from S-adenosyl-methionine to a phenylethanolamine or a phenyl-ethyldiamine, is known to exist in high concentrations in the adrenal medulla (10,11). PNMT maybe inhibited by phenylethylamines or indolaminesand this specificity is due in part to a lack of a/3-hydroxy group (72). Thus dopamine and its sul-fonanilide analog NP-27 may possibly localize because of their specific inhibition of PNMT, as wellas by virtue of their intermediacy in the norepineph-rine pathway.

An important consideration is the possibly greaterin vivo stability of NP-27 over dopamine itself dueto the failure of catechol-O-methyl transferase to initiate metabolic breakdown of NP-27. In any case,NP-27 with a gamma emitter like 75Semust be con

sidered a potentially useful agent for visualizationof the adrenals.

Although visualization of the human adrenal me

dulla is not yet a reality, we hope the present workwill widen the possible approaches to solving theproblem.

ACKNOWLEDGMENTS

This paper was presented in part at the Annual Meetingof the Society of Nuclear Medicine, San Diego, Calif., June1974.

This work was supported in part by the Nuclear MedicineResearch Fund, by NCI Grants CA-05134-13, and by AECContract AT(11-1)-2031.

REFERENCES

/. MORALESJO, BEIERWALTESWH, COUNSELLRE, et al:The concentration of radioactivity from labeled epinephrineand its precursors in the dog adrenal medulla. J NucíMed8: 800-809, 1967

2. LlEBERMAN LM, BEIERWALTES WH, VARMA VM, et al:

Labeled dopamine concentration in human adrenal medullaand in neuroblastoma. / NucíMed 10: 93-97, 1969

3. ANDERSONBG, BEIERWALTESWH, HARRISONTS, et al:Labeled dopamine concentration in pheochromocytomas.J NucíMed 14: 781-784, 1973

4. FOWLER JS, ANSARI AN, WOLF AP, et al: Synthesisand preliminary evaluation in animals of carrier-free "C-L-dopamine hydrochloride. J NucíMed 14: 867-869, 1973

5. WOLF AP, et al: Personal communication6. LARSEN AA, GOULD WA, ROTH HR, et al: Sulfonani-

lides. II. Analogs of catecholamines. J Med Chem 10: 462-

472, 19677. COUNSELL RE, SMITH TD, RANADEVV, et al: Personal

communication, 19698. ULOTH RH, KIRK JR, GOULD WA, et al: Sulfonani-

lides. I. Monalkyl- and arylsulfonamidophenethanolamines.J Med Chem 9: 88-97, 1966

9. LARSEN AA, LISH PM: A new bio-isostere: Alkylsul-phonamidophenethanolamines. Nature 203: 1283-1284,1964

10. AXELRODJ: Purification and properties of phenylethanolamine N-methyl transferase. J Biol Chem 237: 1657-1660, 1962

11. KRAKOFF LR, AXELROD J: Inhibition of phenylethanolamine N-methyl transferase. Biochem Pharmacol 16:1384-1386, 1967

12. FULLER RW, MILLS J, MARSH MM: Inhibition ofphenylethanolamine N-methyl transferase by ring substituteda methylphenethylamines (amphetamines). J Med Chem 14:322-325, 1971

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1975;16:1147-1151.J Nucl Med.   Rodney D. Ice, Donald M. Wieland, William H. Beierwaltes, Richard G. Lawton and Michael J. Redmond  Concentration of Dopamine Analogs in the Adrenal Medulla

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