The Effect of d-2 (p-Aminophenyl)-2-Phenylethylamine (SK&F-12185) in Cushing’s Syndrome Associated with Adrenocortical Carcinoma
By J. L. GABRILOVE, G. L. NICOLIS AND T. F. GALLAGHER
A 55-year-old woman with Cushing’s syndrome and adrenocortical carcinoma was treated with d-2(p-amino-phenyl)- 2 - phenylethylamine (SK & F-12185). Marked improvement in the clinical manifestations of Cushing’s syndrome, including the severity of the diabetes
mellitus, was observed. The urinary ex- cretion of tetrahydrocortisol was re- duced whereas that of tetrahydro S was increased. The progression of the tumor itself appeared to be unaffected. (Me- tabolism 17: No. 10, October, 936-942, 1968)
T MITHIS REPORT is concerned with the effects of d-2-(p-aminophenyl)- 2-phenylethylamine (SK&F-12185) in a patient with adrenal cortical carcinoma and Cushing’s syndrome. In previous reports we have demon- strated the effects of SK&F-12185 in primary aldosteronism1 and in Cush- ing’s syndrome due to non-tumorous adrenocortical hyperfunction.2
SK&F-12185 (d1-2(p-aminophenyl)-2-phenylethylamine) inhibits in vitro steroidogenesis by normal rat and guinea pig adrenal stimulated with ACTH. Both the chronic and acute administration of SK&F-12185 prevent the in- crease in peripheral plasma corticosterone levels of cold-stressed rats. Sim- ilarly, the rise in peripheral plasma cortisol levels induced by the administration of ACTH to guinea pigs is prevented by the oral adminis- tration of SK&F-12185. Physiologic manifestations of adrenal insufficiency induced in the rat by this compound include an inability to excrete a water load and to survive cold stress. In addition, the administration of SK&F- 12185 to the sodium-deficient rat given a water and sodium load results in natriuresis.3
MATERIALS AND METHODS
Daily 24-hour urines were obtained on the metabolic ward and the urine was assayed for its content of neutral 17-ketosteroids, 17-hydroxycorticoids and creatinine. A con-
From the Endocrine Research Laboratory and Clinic of the Department of Medicine, The Mount Sinai Hospital and the Institute for Steroid Research, Montefiore Hospital, New York, N. Y.
Aided by a grant-in-aid from Smith Kline and French, by Grants FR-71 and CA-07304 from the National Institutes of Health, and by a grant from the American Cancer Society. Received for publication March 20, 1968.
J. LESTER GABRILOVE, M.D .: Associate Clinical Professor of Medicine, Mount Sinai School of Medicine. Attending Physician, The Mount Sinai Hospital, New York, N. Y. GIORGIO L. NICOLIS, M.D .: Special Research Fellow of the N.I.H., The Mount Sinai School of Medicine, New York, N. Y. THOMAS F. GALLAGHER, PH.D .: Chief, Institute for Steroid Research, Montefiore Hospital and Medical Center; Professor of Biochemistry, Albert Einstein College of Medicine, New York, N. Y.
trol urine and 24-hour specimens obtained on several days during the course of treat- ment with SK&F-12185 were partitioned into various corticosteroids, neutral 17-ketosteroids and pregnane and pregnene metabolites.
The urinary excretion of the neutral 17-ketosteroids was measured by the method of Drekter et al.4 and the 17-hydroxycorticoids by the Peterson modification5 of the pro- cedure of Silber and Porter.6 The partition of the glucocorticoids, neutral 17-ketosteroids and pregnane and pregnene derivatives was carried out by previously published methods.7,8
The individual compounds were isolated by quantitative paper chromatography. Met- abolites containing a dihydroxyacetone side chain were isolated from the unfractionated neutral steroid extract obtained after treatment with B-glucuronidase.8 17-Ketosteroids were isolated from a separate portion of the same extract after preparation of the ketonic fraction by treatment with Girard’s reagent T; additional 17-ketosteroids were obtained from a cold acid hydrolyzed fraction after treatment with Girard’s reagent T. The values recorded for individual 17-ketosteroids are the sum of the amounts obtained in these two extracts. Reducing ketols were measured by the method of Weichselbaum, Mar- graf and Mack.9 A modified micro-Zimmerman reaction10 was employed to measure the 17-ketosteroids. Pregnanediols, pregnanetriols and 45-pregnenetriols were measured in the nonketonic steroid extract by the method of Rosenfeld et al.11
Urinary testosterone was measured by a modification of the method of Futterweit.2 The secretory rates of cortisol was measured by a previously published technique.2
CASE REPORT
This was the first Mount Sinai Hospital admission of a 55-year-old white Italian house- wife with a chief complaint of swelling for the past eight months. She had known of mild hypertension for approximately two years but had never received any therapy for this. She had not had any symptoms of congestive heart failure. Diabetes mellitus, of unknown duration, associated with polyuria was also present. She had apparently been otherwise well until eight months prior to admission when she noted the rapid onset of pedal edema. The eyes and face became puffy, the abdomen became protuberant and her legs became massively swollen. She also noted an increase in appetite and weight gain. She encountered great difficulty in climbing the steps, at first because of weakness of the legs and later because of the onset of shortness of breath. Marked facial hirsutism and redness of the face developed. The generalized weakness became increasingly severe and she was admitted to another hospital. While there, she noted episodes of crushing pain in the chest and left arm which gradually improved. She was told she had Cushing’s syndrome and admission to The Mount Sinai Hospital was recommended.
Following her discharge from the other hospital, she remained at home. She noted marked increase in weakness and edema and a punched out ulcer on her right leg. Be- cause of the progression of her symptomatology, she entered The Mount Sinai Hospital. The past medical history included pneumonia as a young woman and gall stones which she had had for many years. The family history was essentially negative.
Physical examination revealed a Cushingoid, weak, chronically ill appearing woman lying in bed. Marked pedal edema and truncal obesity were present. The temperature was 100° F. The blood pressure was 158/104 mm. Hg. Grade II hypertensive retino- pathy was present. The face and eyes were puffy. The face was rather plethoric and mark- edly hirsute. The tongue was coated with many white plaques. The thyroid was not palpable. A buffalo type of cervico-dorsal fat pad was present. Intertrigo was present under the breasts. There were a few hypostatic rales audible in the chest. There were poor excursions of the diaphragm. The heart was not enlarged to percussion. No mur- murs were audible, and rhythm was regular. The abdomen was markedly protuberant and she had an umbilical hernia. The liver was felt two to three finger breadths below the costal margin. Marked pedal and pretibial edema was present and there was a punched out ulcer on the right leg. There were furuncles over both elbows. Neurological examina- tion revealed proximal muscle weakness accompanied by atrophy. The impression was that of Cushing’s syndrome, probably due to adrenal cortical carcinoma, and diabetes mellitus.
The laboratory examination revealed the specific gravity of the urine to be 1.024; 4+ glycosuria and a heavy trace of albumin were present. On microscopic examination, two to three red blood cells and four to five white blood cells were visible per high power field. The blood hemoglobin was 12 Gm./100 ml. The white blood cell count was 11,540/mm.3 with 64 per cent segmented polymorphonuclear leukocytes, 18 per cent band forms and 18 per cent lymphocytes. The serum sodium was 133 meq./L., potassium 4.3 meq./L., carbon dioxide content 30 meq./L., chlorides 98 meq./L., creatinine 0.7 mg./100 ml., bilirubin 0.3 mg./100 ml., serum glutamic oxalacetic transaminase 26 units/ml., alkaline phosphatase 4.5 Bessey-Lowry units. The blood urea nitrogen was 30 mg./100 ml., and sugar 346 mg./100 ml. The urinary excretion of the neutral 17- ketosteroids ranged from 21.7 to 48.3 mg./24 hours and of the 17-hydroxycorticoids (Porter-Silber) from 32.0 to 89.8 mg./24 hours, Urinary testosterone was 98 ug./24 hours. The urinary excretion of estrone was 9 ug./24 hours (normal 2-25 ug.), of estradiol 1 ug./24 hours (normal 0-10 µg.), of estriol 5 µg./24 hours (normal 2-30 µg.) and of total estrogens 15 µg./24 hours (normal 4-60 µg) .* The electrocardiogram was within normal limits. Roentgenogram of the chest revealed the cardiac contour to be normal in size. Multiple large nodules were seen in both lung fields with pleural scalloping on the left. Multiple old fractures of the ribs were present. An intravenous pyelogram revealed good kidney function. The right kidney was depressed by a large mass. Retroperitoneal gas insufflation confirmed the presence of this mass which was approximately 10 cm. in dia- meter and contained flecks of calcium. Moderate demineralization of the lumbar spine was noted on roentgenography.
Following a control period of observation, she was placed on d-SK&F-12185 in a dosage of 200 mg., then 300 mg. and subsequently 400 mg. a day. During the course of ad- ministration, she developed a mild erythematous eruption which had been seen in other patients and which was presumed to be related to depression of the endogenous pro- duction of corticoids. On the eighth day of treatment, she developed a cough and fever to 102°. It could not be certain whether this represented drug fever, adrenal cortical insufficiency, or a pulmonary or urinary tract infection. Accordingly, the drug was stopped. She was given cortisol and antibiotics. She improved and after an interval period of five days, she was again started on a dosage of 300 to 400 mg. of d-SK&F- 12185.
Again, after 11 days of treatment, she developed fever to 104° associated with marked lethargy and weakness and the drug was again stopped. The temperature fell promptly and after an interval of seven days, she was again started on slowly increasing dosages of SK&F-12185, this time to a maximum of 200 mg., which she seemed to tolerate quite well. While she was treated with SK&F-12185, the requirement for orinase fell from 2 Gm. a day to as little as 0.5 Gm. a day. The glycosuria decreased markedly even on the lower dosages and the fasting blood sugars also were lower. During the administration of SK&F-12185, the floridness of the face improved markedly although she still appeared Cushingoid and severe hirsutism persisted. The urinary excretion of the neutral 17-keto- steroids and of 17-hydroxycorticoids are seen in Fig. 1.
She was discharged on the dosage of 300 mg d-SK&F-12185 per day, feeling improved and with less edema.
She was readmitted one month later because of worsening of the clinical status includ- ing progressive weakness, anorexia and increased abdominal girth.
On physical examination she appeared slightly Cushingoid but according to her fam- ily her facies were now normal in contour. The facial hirsutes had markedly receded although a fair amount of hirsutism was still present. The blood pressure was 120/80 mm. Hg. There were ecchymoses visible on the left hip. The neck veins were not dis- tended. The heart was not enlarged. No murmurs were audible. The diaphgrams were high. No rales were present in the lungs. The liver was markedly enlarged and irregular. No shifting dullness was present. There was 3 to 4+ pretibial and modest presacral edema.
* Bio-Science Laboratories.
Di B. 2 AGE : 55 CUSHING’S SYNDROME ADRENAL CARCINOMA
180
160
140
URINARY 17-OH
mg / 24 hrs
120
100
80
60
40
20
0
URINARY 17-KS mg./ 24 hrs
60
40
20
0
84
WEIGHT
82
Kg
80
78
76
S.K.F (d) 12185
DISCHARGED
400
mg.
200
0
0
10
20
30
40
50
60
70
80
DAYS
Because of her deteriorating state, she was given 5-Fluorouracil in addition to SK&F- 12185. The former was given intravenously in a dosage starting with 250 mg., followed by 500 mg. a day for four days and then 500 mg. twice a week for one and one-half weeks. A liver scan demonstrated many “cold” areas indicative of metastatic disease. She remained extremely weak and anorectic. No diarrhea or stomatitis was noted. She developed abdominal pain, weakness, increased anorexia and vomiting. The blood pres- sure fell to 75/50 mm. Hg and the pulse rate rose to 120/min. Generalized abdominal tenderness was present. She was started on cortisol to exclude the possibility of adrenal insufficiency and given parenteral fluids. She developed marked oliguria. The urea nitro- gen rose to 92 mg./100 ml. and the bilirubin to 2.9 mg./100 ml. The serum alkaline phosphatase was 42 units/ml. Two days after the acute episode she had a Jacksonian seizure, starting in the right face and right arm. She died the next morning. No post mortem examination was obtained.
RESULTS
During the first two short courses of d-SK&F-12185, no demonstrable alterations in either the urinary excretion of the neutral 17-ketosteroids or of the 17-hydroxycorticoids were noted. However, during the third course both fractions increased in titer (Fig. 1).
It can be seen from Table 1 that the administration of SK&F-12185 was followed by a marked fall in the urinary excretion of tetrahydrocortisol as well as a decrease in urinary allo-tetrahydrocortisol, tetrahydrocortisone and
| Day of Treatment | Control | Control | SK&F-12185 Day 1 (Fig. | 1) SK&F-12185 Day 31 (Fig. | 1) SK&F-12185 Day 57 (Fig. 1) |
|---|---|---|---|---|---|
| mg./Gm. Creatinine | |||||
| Tetrahydrocortisol | 56.0 | 46.2 | 12.4 | 9.6 | 20.8 |
| Allo-tetrahydrocortisol | 5.6 | 2.0 | 0.8 | 0.9 | 1.2 |
| Tetrahydrocortisone | 16.5 | 11.3 | 6.1 | 4.5 | 9.9 |
| Cortisol | 7.0 | 1.9 | 0.9 | 0.9 | 1.1 |
| 6 8-OH-cortisol | 3.6 | ||||
| 6 B-OH-cortisone | 2.3 | 1.2 | |||
| Cortisone | 3.3 | ||||
| Tetrahydro B | 3.0 | 2.2 | 2.1 | 4.0 | 2.7 |
| Tetrahydro S | 10.9 | 9.0 | 53.3 | 65.4 | 87.1 |
| Tetrahydro A | 3.2 | 2.0 | 1.3 | 2.6 | 3.6 |
| Androsterone | 4.4 | 4.9 | 4.9 | 6.7 | 7.0 |
| Etiocholanolone | 13.5 | 10.7 | 28.1 | 26.5 | 43.7 |
| Dehydroisoandrosterone | 11.5 | 15.2 | 3.9 | 6.0 | 8.5 |
| Creatinine | 0.66 Gm. | 0.85 Gm. | 0.85 Gm. | 0.78 Gm. | 0.73 Gm. |
cortisol. The urinary excretion of tetrahydro S increased markedly. Urinary androsterone was increased modestly and urinary etiocholanolone rose mark- edly. Urinary dehydroisoandrosterone decreased. Urinary tetrahydro B and tetrahydro A were unaltered.
The administration of d-SK&F-12185 resulted in a marked amelioration of the diabetes mellitus as evidenced by a decrease in the requirement for crinase as well as in the levels of the fasting blood sugar even on the re- duced dosage of orinase.
The secretory rate of cortisol was 20.7 mg./24 hours on day 67 in Fig. 1. At this time, marked clinical improvement in the symptoms of Cushing’s syndrome had been noted. Unfortunately, control studies in this regard were not done although the urinary excretion of tetrahydrocortisol on days 12, 31 and 57 was markedly reduced as compared to the control values.
No toxic effects of the drug were noted on the blood count, the electro- cardiogram, or on renal or hepatic function. Terminally the urea nitrogen rose as did the serum glutamic oxalacetic transaminase and the serum bili- rubin. These were probably due to the metastatic disease and/or the effects of 5-Fluorouracil. It is of note that terminally the urinary excretion of the 17-hydroxycorticoids decreased to 15.6 mg./24 hours from titers as high as 185.4 mg./24 hours four days previously.
DISCUSSION
It is apparent that in this patient with an adrenocortical carcinoma and Cushing’s syndrome, the administration of SK&F-12185 reduced the secretory rate of cortisol to normal levels and resulted in a marked decrease in uri- nary tetrahydrocortisol concomitant with a rise in urinary tetrahydro S. In the control specimen, tetrahydro S was present as is usually observed in the urine of patients with functioning adrenocortical carcinoma. The presump-
tion is that one of the sites of action of SK&F-12185 is on the 118-hydroxy- lation of 11-deoxycortisol to cortisol. Evidence for this was adduced in a previous report.2 Corticotropin production is ordinarily suppressed as the result of the excessive production of cortisol by adrenal tumors causing Cushing’s syndrome and this effect is of long duration following removal of such a tumor. The self regulatory cortisol-corticotropin axis is thus frequently of little importance in patients with untreated adrenal carcinoma. However, the rise in titer of the urinary 17-hydroxycorticoids during treatment with SK&F-12185 would suggest either that corticotropin secretion had been in- creased or more likely that the growth of the tumor had resulted in increased secretion of corticosteroids. Nonetheless the data would suggest that the block exerted by SK&F-12185 was maintained, at least partially, in spite of these factors since the urinary excretion of tetrahydrocortisol decreased and a normal secretory rate for cortisol was obtained during treatment.
The administration of SK&F-12185 in this patient markedly improved the clinical manifestations, including the severity of the diabetes mellitus. It would therefore be anticipated that this compound might be of value in minimizing the deleterious metabolic effects of Cushing’s syndrome in pa- tients with adrenocortical carcinoma.
The progression of the metastatic disease would indicate that the com- pound exerts no beneficial effect on the tumor itself. There is inadequate evidence in this patient to permit any statement to be made in regard to the effectiveness of 5-Fluorouracil in treating adrenocortical carcinoma.12
The following Trivial Names have been used:
| Tetrahydrocortisol | 3a, 118, 17,21-tetrahydroxy-5 ß-pregnan-20-one |
| Allo-tetrahydrocortisol | 3a, 118, 17,21-tetrahydroxy-5 a-pregnan-20-one |
| Tetrahydrocortisone | 3a, 17,21-trihydroxy-5 B-pregnane-11,20-dione |
| Androsterone | 3a-hydroxyandrostan-17-one |
| Etiocholanolone | 3a-hydroxyetiocholan-17-one |
| Dehydroisoandrosterone | 3B-hydroxy-4 5-androsten-17-one |
| Tetrahydrocorticosterone | 3a, 118, 21-trihydroxy-5 ß-pregnan-20-one |
| Tetrahydro S | 3a, 17,21-trihydroxy-5 ß-pregnan-20-one |
| Tetrahydro A | 3a, 21-dihydroxy-5 -pregnane-11,20-dione |
| 6 B-OH-cortisone | 66, 17,21-trihydroxy-44-pregnene-3,11,20-trione |
ACKNOWLEDGMENT
The authors gratefully acknowledge the cooperation of Dr. Robert Rosenfeld for the analysis of the monketonic alcohols by vapor phase chromatography. The assistance of Ruth Jandorek and Thelma Freeman is acknowledged with thanks.
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10. Wilson, H .: Chromogenic values of various ketosteroids in a micro modification of the Zimmerman reaction: comparison with the macro procedure. Arch. Biochem. 52: 217, 1954.
11. Rosenfeld, R. S., McLebeau, R. D., Jandorek, R. D., and Salumoa, T .: Analysis of urinary extracts by gas chromatography. J. Chromatogr. 8:355, 1962.
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