STUDIES WITH AN ADRENAL INHIBITOR IN ADRENAL CARCINOMA*
DAVID K. FUKUSHIMA, PH.D., T. F. GALLAGHER, PH.D., W. GREENBERG, M.D.t AND O. H. PEARSON, M.D .; Sloan-Kettering Institute for Cancer Research, New York, N. Y.
ABSTRACT
The production of steroids in a case of widely disseminated adrenal carci- noma was studied with SU-4885. There was a striking decrease of both C19- and C21-11-oxygenated steroids in the urine with a concomitant increase in the corresponding 11-deoxy derivatives. The results support the view that the tumor and its metastases were responsive to ACTH and that there was in- creased steroid production during treatment with SU-4885. The isolation and characterization by partial synthesis of pregnane-3a,17a,20a,21-tetrol and its 200 epimer (reduced derivatives of Reichstein’s substance S) are reported. The isolation of pregnane-3x, 116,17a,20a-tetrol but not of 11-ketopregnane-3a, 17a, 20a-triol from the urine of a patient with adrenal carcinoma is noted.
T HERE are now available several materials which can influence the nature and amount of the steroids produced by the adrenal glands. One of the more recent of these is 2-methyl-1,2-bis-(3-pyridyl)-1-propa- none, or SU-4885, which inhibits the enzymatic system responsible for 118- hydroxylation of steroids (1-4). We have recently had the opportunity to study the steroid metabolism of a patient with a widely disseminated adrenocortical carcinoma, before and during treatment with this com- pound. Because of the amount and variety of steroid metabolites present in the urine of this patient, there was an excellent opportunity for detailed ex- amination of the effects of the drug.
SUBJECT AND METHODS
Subject
G.G., a 30-year-old white woman with previously diagnosed Cushing’s syndrome was admitted to Memorial Center on July 31, 1958 complaining of profound weakness. Her illness dated from June 1957, when hypertension, proteinuria and pedal edema were discovered during the patient’s fifth pregnancy. These findings disappeared with the delivery of a normal full-term infant in August 1957. In November 1957, the patient
Received March 5, 1960.
* This investigation was supported in part by a grant from the American Cancer Society and research grants (CY-3207 and CY-3809) from the National Cancer Institute of the National Institutes of Health, United States Public Health Service.
t Present address: Flower and Fifth Avenue Hospital, New York, N. Y.
# Present address: Western Reserve University, Cleveland, Ohio.
September, 1960 ADRENAL INHIBITOR IN ADRENAL CARCINOMA 1235
complained of a 20-pound weight gain, a “rounded” face and pedal edema. Examination at that time demonstrated recurrence of the proteinuria and hypertension. A diagnosis of “kidney trouble” was made, and therapy with prednisone was instituted. This medi- cation was continued until January 1958, at which time she was hospitalized with lobar pneumonia.
Upon recovery from the pneumonitis, the prednisone therapy was discontinued. The presence of emotional lability and moon facies was related to the preceding steroid therapy, but failure of these symptoms to regress after prednisone withdrawal led to a diagnosis of adrenocortical hyperfunction. An excretory urogram revealed a mass adja- cent to the superior pole of the left kidney. In April 1958, surgical exploration of the left adrenal gland revealed an encapsulated yellow tumor weighing 450 Gm., which was removed. Microscopic examination of sections led to the diagnosis of adrenocortical carcinoma. Following extirpation of the tumor, there was prompt regression of the Cushing’s syndrome. In July 1958, signs and symptoms of adrenocortical hyperfunction returned.
Physical examination. The patient’s appearance was classically “Cushingoid” with a rotund, plethoric face, centripetal obesity, and a cervicodorsal fat pad. Blood pressure was 158/108 mm. Hg. Acneform lesions were present over the face, anterior chest and breasts. Cutaneous veins were strikingly prominent and blue striae were present on the buttocks and thighs. There was dullness and an absence of breath sounds at the right pulmonary base. The liver was enlarged to 11 cm. below the right costal margin in the midclavicular line, and was hard and nodular. No significant muscle weakness was evi- dent, despite the obvious wasting and flabbiness of the thigh and calf muscles.
Laboratory findings. The serum potassium level was 2.4 mEq. per liter, sodium 144.0 mEq. per liter, chloride 83.0 mEq. per liter, and plasma carbon dioxide 49 mEq. per liter; serum pH, 7.57; concentration of fasting blood sugar 129 mg. per 100 ml., serum protein- bound iodine 4.3 ug. per 100 ml., hemoglobin 12.9 Gm. per 100 ml., hematocrit 40 per cent, leukocyte count 4,300 per cu. mm. with 4 per cent lymphocytes, and eosinophil count 18 per cu. mm. Urine specific gravity was 1.008 and the urine was alkaline to litmus. The titer of urinary gonadotropins was negative for 6.5 m.u. The 24-hour excretion of 17- hydroxycorticoids was 114 mg., and of 17-ketosteroids, 74 mg. The concentration of serum 17-hydroxycorticoids was 49.1 ug. per 100 ml. The electrocardiogram showed low T waves and prominent U waves.
Treatment and course. The hypokaliemia and alkalosis were corrected with potassium chloride. On the ninth through the fourteenth hospital day the patient received SU-4885, 2 Gm. daily by mouth for the first three-day period, and 4 Gm. daily for the second three- day period. During administration of this compound there was a clear-cut lowering of the fasting blood sugar level. No alteration in hepatic size or function was detected and blood pressure, electrolyte pattern and the patient’s general status remained unchanged.
The patient died of bronchopneumonia on the fortieth hospital day. Postmortem examination revealed a massively enlarged liver filled with tumor nodules. Apart from some retroperitoneal nodes, no other sites of metastatic spread were found. It is of in- terest that the right adrenal gland weighed 6 Gm. and was unremarkable histologically.
Methods
The control urine collection was obtained from August 4 to 8, 1958, and was complete as judged from the constancy of the daily creatinine content. The collection during the SU-4885 period was obtained from August 11 to 13, after the patient had been treated with 2 Gm. per day of the drug from August 9, 1958. The individual 24-hour specimens
were combined for each interval and were treated with beef liver (-glucuronidase1 at pH 5 for five days at 37° C. At the end of incubation the material was adjusted to pH 1 and subjected to extraction continuously with ether for forty-eight hours. The ether- soluble neutral fraction was separated and the alkali-soluble material was combined with the extracted urine. This latter mixture, after neutralization, was acidified to 1 N with sulfuric acid and again subjected to extraction continuously with ether for forty- eight hours. The neutral ether-soluble fraction from this extract was separated. A por- tion of each of the neutral ether-soluble extracts was separately processed into ketonic and nonketonic fractions by means of Girard’s reagent T. The ketonic fraction obtained from the neutral steroid extract after the urine had been acidified to 1 N with sulfuric acid was further separated into a and B ketosteroid subfractions by means of digitonin. These methods have been described in detail (5). The separate subfractions were chro- matographed on paper in systems described subsequently. The individual steroids after elution from the paper were measured quantitatively by appropriate color reactions or by enzymatic analysis, i.e., 17-ketosteroids by the modified micro-Zimmermann reac- tion (6); reducing ketols by the method of Weichselbaum, Margraf and Mack (7); 3a- hydroxysteroids such as the cortols, cortolones and pregnanetriol, by the method of Hurlock and Talalay (8) using a-hydroxysteroid dehydrogenase prepared from Pseu- domonas testosteroni and measuring the formation of diphosphopyridine nucleotide (DPNH) at 340 mu; ß-hydroxysteroid dehydrogenase was used for 45-pregnene-36,17a, 20a-triol. When necessary, the identity of the steroids eluted from the chromatograms was established by infrared spectrometry, usually by examination of the acetate of the compound in question, in solution in carbon disulfide or chloroform, and comparison of the spectrum with a reference standard (9, 10). The values obtained by the color re- actions were corrected to milligram equivalents of the compound isolated and analyzed.
The values reported for compounds other than 17-ketosteroids were obtained from the unfractionated crude neutral steroid extract after enzymatic hydrolysis; because of numerous artifacts present and attendant difficulties of separation, identification and interpretation, the extract obtained from the urine acidified to 1 N was examined only for 17-ketosteroids, particularly dehydroisoandrosterone.
Chromatography of these neutral steroid extracts was performed on Whatman No. 1 paper, with sheets measuring 7X48 inches and a cylindrical tank with a height of ap- proximately 4 feet. The following systems were used for the separation of indicated compounds :
System A: Benzene: methanol: water : ethyl acetate (1:1:1:0.1), for separation of pentahydroxy-, tetrahydroxymonoketo-, tetrahydroxy- and trihydroxydiketo-C21 ster- oids by chromatography for thirty-two hours at 25° C.
System B: Toluene : isooctane : methanol : water (3:1:4:1), for twenty-four hours for separation of trihydroxymonoketo-, trihydroxy- and dihydroxydiketo-C21 steroids; and for four hours for dihydroxy-C21 and C19 steroids.
System C: Toluene :isooctane : methanol : water (1:3:3:1), for ninety-two hours for separation of dihydroxymonoketo- and hydroxydiketo-C19 steroids.
System D: Isooctane: methanol : water (1:1:0.1), for twenty hours on paper measuring 7 ×22 inches for monohydroxymonoketo-C19 steroids.
1 B-Glucuronidase, known as Ketodase, was obtained from the Warner-Chilcott Laboratories, a division of Warner-Lambert Pharmaceutical Company, New York, N. Y.
ISOLATION AND CHARACTERIZATION OF CERTAIN STEROID DERIVATIVES Pregnane-3a,17a,200,21-tetrol
This compound was prepared by the reduction of 3a,21-diacetoxy-17a- hydroxypregnane-20-one with lithium aluminum hydride. A small portion of the crude pregnanetetrol was chromatographed on Whatman No. 1 paper in System A for twenty-four hours. Two spots were obtained on staining with phosphomolybdic acid. The less polar material (28-29 inches from the origin) was the principal reaction product and the more polar ma- terial (26-27 inches from origin) was present in only trace amount. The former was assumed to be the 208-hydroxy epimer and the latter the 20x- hydroxy epimer from knowledge of analogous reduction with lithium alumi- num hydride of dihydroxyacetone side-chain steroids. The relative mobili- ties of other C-20 epimeric glycerol side-chain steroids supported this con- clusion. Recrystallizations of the crude reduction product from methanol- ethyl acetate afforded pregnane-3a, 17a,208,21-tetrol, m.p. 217.5-218.5°; [a][26+37.7º (ethanol).
Analysis: Calculated, for C21H3604: C, 71.55; H, 10.29. Found: C, 71.09; H, 10.24.
Paper chromatography showed only one spot at 28-29 inches from the ori- gin in System A for twenty-four hours. Oxidation with periodic acid af- forded etiocholanolone, as evidenced by mobility on paper. Acetylation with acetic anhydride and pyridine at room temperature afforded preg- nane-3a,17a,208,21-tetrol 3,20,21-triacetate, m.p. 147-150°; [a][26+73.9º (chl.).
Pregnane-3a,17a,20a,21-tetrol
This compound was prepared from 3a,21-diacetoxy-17a-hydroxypreg- nane-20-one by the method previously described for the synthesis of corto- lone from 3a,21-diacetoxy-17a-hydroxypregnane-11,20-dione (11). The catalytic hydrogenation of 3@,21-diacetoxy-17a-hydroxypregnane-20-one in acetic acid with Adam’s catalyst was assumed to yield principally the 200- hydroxy epimer, pregnane-3a,17a,200,21-tetrol 3,21-diacetate, m.p. 170- 172°; [@]23+3.5° (chl.).
Analysis: Calculated, for C25H4006: C, 68.78; H, 9.28. Found: C, 68.76; H, 9.18.
The p-toluenesulfonate ester of the C-20 hydroxy group was then prepared with p-toluenesulfonyl chloride in pyridine solution. The product had an m.p. 120-121° (dec.); [a]p23+64.8º (chl.).
Analysis: Calculated, for C32H46OBS: C, 65.06; H, 7.67. Found: C, 65.04; H, 7.84.
The 17a-hydroxy group was acetylated with acetic anhydride, acetic acid and p-toluenesulfonic acid and the product without isolation was sub- jected to acetolysis in acetic acid and potassium acetate. The reaction product was chromatographed on alumina. Elution with benzene afforded pregnane-3a,17a,20a,21-tetrol 3,20,21-triacetate. Recrystallizations from ethyl acetate and ethanol yielded the analytical sample, m.p. 169.5-171°; [a]D23-7.7º (chl.).
Analysis: Calculated, for C27H4207: C, 67.75; H, 8.85. Found: C, 67.64; H, 8.65.
Hydrolysis with alkali yielded pregnane-3a,17a,20a,21-tetrol which had a double m.p., 251-253.5° and 257-258°; [a]D24+9.1º (ethanol).
Analysis: Calculated, for C21 H3604: C, 71.55; H, 10.29. Found: C, 71.79; H, 10.21.
The tetrol moved 26-27 inches from the origin as a single spot upon chromatography on paper in System A for twenty-four hours.
The catalytic reduction of 3a,21-diacetoxy-17a-hydroxypregnane-20- one afforded 2 isomeric side-products in addition to the desired pregnane- 3a,17a,200,21-tetrol 3,21-diacetate. One of the compounds was a product of acetyl migration, pregnane-3a,17a,200,21-tetrol 3,20-diacetate, m.p. 145-148.5°; [a]D23+11.0° (chl.).
Analysis: Calculated, for C25H4006 : C, 68.78; H, 9.24. Found: C, 68.44; H, 9.17.
Acetylation of this isomer afforded the 3,20,21-triacetate identical in all respects with pregnane-3a,17a,200,21-tetrol 3,20,21-triacetate. The other side-product obtained in about 10 per cent yield was a tetrol diacetate epimeric at C-20, pregnane-3a,17a,20a,21-tetrol 3,21-diacetate, m.p. 185-187°;[a]D28+8.4 (chl.).
Analysis: Calculated, for C25H4006 : C, 68.78; H, 9.24. Found: C, 68.77; H, 9.24.
Acetylation yielded pregnane-3a,17a,20a,21-tetrol-3,20,21-triacetate.
DISCUSSION
Steroid production before treatment (control period)
The number of hormones and the amount of each produced by this metastatic carcinoma was truly remarkable. The metabolites recorded in Tables 1, 2, and 3 represent a minimum of 10 hormones, of which hydro- cortisone was quantitatively the most significant. The metabolites of this substance alone (including unchanged hormone) totaled 70 mg. per day.
| Metabolites | Urinary excretion (mg./24 hrs.) | |
|---|---|---|
| Control period | SU-4885 period | |
| Of hydrocortisone | ||
| Hydrocortisone | 3.3 | - |
| Cortisone | 1.3 | - |
| 3a, 118,17a,21-Tetrahydroxypregnane-20-one (THF) | 25.7 | 4.2 |
| 3a, 17a,21-Trihydroxypregnane-11,20-dione (THE) | 12.3 | 2.0 |
| 3a, 118,17a,21-Tetrahydroxyallopregnane-20-one | 2.7 | - |
| Cortolones (a+B) | 6.6 | 4.3 |
| Cortol | 2.4 | 1.1 |
| ฿ Cortol | 2.7 | 0.9 |
| 3a, 11B-Dihydroxyetiocholane-17-one | 8.9 | 2.7 |
| 3a-Hydroxyetiocholane-11,17-dione | 4.6 | 1.8 |
| Total | 70.5 | 17.0 |
| Net decrease | 53.5 | |
| Of Substance S | ||
| 3a, 17a,21-Trihydroxypregnane-20-one (tetrahydro S) | 9.8 | 64.0 |
| Pregnane-3a, 17a, 20a,21-tetrol | 2.3* | 9.4* |
| Pregnane-3a, 17a, 208,21-tetrol | 1.4* | 3.7* |
| Total | 13.5 | 77.1 |
| Net increase | 63.6 | |
* Semiquantitative estimates because of contamination with other metabolites. The 2 isomers of pregnane-3a, 17a20,21-tetrol were obtained in crystalline form and directly com- pared with synthetic products.
In tracer studies with normal human subjects after the administration of hydrocortisone-4-C14 the recovery of these metabolites has been about 70 to 90 per cent of the amount of radioactivity found in the neutral steroid extract of urine (12); the latter corresponds generally to between 50 and 60 per cent of the dose. Therefore, the minimal production of hydro- cortisone by this neoplasm must have been 130 mg. per day-at least six times the estimated maximal production of a normal person. The adrenal
| 17-Ketosteroid metabolites | Urinary excretion (mg./24 hrs.) | |
|---|---|---|
| Control period | SU-4885 period | |
| 11-Oxysteroid | ||
| 118-Hydroxyandrosterone | 12.9 | 4.2 |
| Net decrease | 8.7 | |
| 11-Deoxysteroids | ||
| 3a-Hydroxyandrostane-17-one (androsterone) | 4.6 | 9.9 |
| 3a-Hydroxyetiocholane-17-one (etiocholanolone) | 10.8 | 17.4 |
| Total | 15.4 | 27.3 |
| Net increase | 11.9 | |
| Metabolites | Urinary excretion (mg./24 hrs.) | |
|---|---|---|
| Control period | SU-4885 period | |
| Dehydroisoandrosterone | 14.9 | 21.6 |
| 7-Ketodehydroisoandrosterone | 3.1 | 1.6 |
| 45-Pregnene-33,17a, 20a-triol | 3.3* | 7.8* |
| 45-Pregnene-36,17a,208-triol | 2.0* | 2.8* |
| Pregnane-3a, 17a, 20a-triol | 4.1* | 4.9* |
| Aldosterone | 20-25+ | 35-50+ |
| 3a,118,21-Trihydroxypregnane-20-one (tetrahydro B) | 3.6 | |
| 3a,21-Dihydroxypregnane-11,20-dione (tetrahydro A) | 2.1 | - |
| Corticosterone | +# | - |
| 3,20-Diketo-44-pregnene-17a,21-diol | + | + |
| Pregnane-3a, 116,17a, 20a-tetrol | + | - |
| Pregnane-3a, 118,17a,200-tetrol | + | - |
| 3a, 17a-Dihydroxypregnane-20-one | + | + |
| 38,118-Dihydroxyandrostane-17-one | + | + |
| 38,118-Dihydroxyetiocholane-17-one | + | + |
| 3a-Hydroxyandrostane-11,17-dione | + | - |
| 36-Hydroxyetiocholane-17-one | + | + |
* Semiquantitative estimates because of contamination with other metabolites.
t Micrograms per 24 hours.
# The compounds for which no analytical values are given were identified qualitatively beyond reasonable doubt by the respective mobilities, by rechromatography of the acetates and by infrared spectrometric analysis.
androgen, as characterized by androsterone and etiocholanolone, yielded more than 15 mg. of metabolites per day. In terms of testosterone or 44-androstene-3,17-dione, this quantity is estimated to represent about 25 mg. of the total daily androgen production. Average normal values for this component of the adrenal secretion cannot be given with certainty be- cause of the diminution with age. However, the amount of hormone es- timated is at least five times that in a woman of comparable age. Dehydro- isoandrosterone production was eight to fifteen times that of a normal subject but considerably less than in many other adrenocortical car- cinomas that have been reported. Aldosterone secretion was clearly ele- vated but was not at the level frequently seen in cases of primary aldo- steronism due to a tumor.
The urinary end-product, 116-hydroxyandrosterone, is the chief metab- olite of 110-hydroxy-44-androstene-3,17-dione (13). The amount of this metabolite present was almost 13 mg. This corresponds to a minimum production of 20 mg. of hormone per day, or approximately ten times the normal level. From the relatively small amount of “tetrahydro A” and “tetrahydro B” present, it would appear that corticosterone was not a major secretory product of the tumor, but this must be a tentative con- clusion because of the gaps in our knowledge of the metabolism of that hormone.
It is clear, nevertheless, that all components of the adrenal secretion were greatly elevated as result of this carcinoma. Particularly noteworthy
is the relatively large amount of the metabolite “tetrahydro S” and its further reduction products, the isomeric pregnanetetrols, the synthesis of which is described in this report. “Tetrahydro S” is found in large amounts in the urine of patients with adrenal carcinoma (14, 15) and in amounts of less than 1 mg. per day in normal subjects (14), although after three days of intravenous infusion of ACTH the amount can be increased to about 10 mg. per day. The excretion of this metabolite during the control period without treatment is pertinent because of the pronounced increase that was apparent when the drug was administered.
The metabolites, pregnane-3a, 116,17a,20a-tetrol and its epimer at C-20, have special significance. These undoubtedly represent end-products of the metabolism of 21-deoxyhydrocortisone, since it has been shown that the 21-hydroxyl group is not eliminated from hydrocortisone in the course of its metabolic transformation (16). These metabolites, and in particular the 11-keto analog, are prominent in the urine of patients with one type of congenital adrenal hyperplasia (17, 18). It has been reported that 11- ketopregnane-3a,17a,20a-triol was absent in the few examples of adreno- cortical carcinoma studied (19, 20). In view of the unusually large steroid production by this tumor, and the fact that normal biosynthetic pathways may be partly deranged in the metastatic cells, it would not be unreason- able to find small quantities of these metabolites of an “incomplete” hydro- cortisone molecule. The estimates of the amount present are unreliable because the separation from other steroids of similar polarity required re- peated chromatography with sensible material losses. The fact that 11- ketopregnane-3a,17a,20a-triol could not be detected is of some interest. This may have been in part due to technical difficulties, but the fact that the 118-hydroxy metabolites were identified during both periods would indicate that the 11-keto analog would have been found, if present, in comparable amount. There was clearly less precursor hormone produced by the carcinoma and its metastases than in the one example of congenital adrenal hyperplasia reported from these laboratories (17).
The metabolite 45-pregnene-36,17a,20a-triol is of interest as an illustra- tion of the fact that in a carcinoma (21) and, at least to a limited extent, in normal people (20, 22) hydroxylation at C-17 can be achieved in vivo with a compound that lacks the 44 unsaturated 3-ketone system charac- teristic of the usual steroid hormones. Whether this product is biosyn- thetically related to dehydroisoandrosterone is unclear at present.
Steroid production during administration of SU-4885
As evident in Table 1, during administration of the drug there was a striking decrease of all the 11-oxygenated steroids examined. On a per- centage basis, the response appeared proportionate for at least 2 hormones,
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that is the metabolites of both hydrocortisone and 118-hydroxy-44- androstene-3,17-dione, which decreased to somewhat less than 30 per cent of the control values. Corticosterone metabolites were not detected during this time.
Concomitantly there was a significant increase in all of the 11-deoxy- steroids except 7-ketodehydroisoandrosterone. The striking increase in the metabolites of Reichstein’s substance S was slightly greater than the decrease in hydrocortisone secretion. Androsterone and etiocholanolone increased appreciably under the influence of the drug and the increased excretion of these compounds was roughly equivalent to what might have been anticipated from the decreased production of 116-hydroxy-44- androstene-3,17-dione. Dehydroisoandrosterone increased significantly during administration of SU-4885, as. did the related metabolite 45- pregnene-38,17a,20a-triol. The change in pregnane-3a,17a,20a-triol was probably not significant, especially in consideration of the difficulty en- countered in the chromatographic separation of isomeric metabolites of similar polarity.
Two alternative hypotheses may be entertained as explanations for these facts. The first of these is that the tumor was autonomous and that inhibition of one reaction in the biosynthetic sequence resulted in diversion of 11-oxygenated hormone to 11-deoxysteroids without any net alteration in total hormone production. On the other hand, Liddle and his collab- orators (4) have supplied convincing evidence that inhibition of 11- hydroxylation by SU-4885 results in adrenal production of steroids unable to suppress pituitary adrenocorticotropin (ACTH) secretion. As a con- sequence there may be increased steroid secretion compared with the basal period without treatment. Liddle and his collaborators (4) failed to obtain an increase in urinary 17-hydroxycorticoids in 3 cases of Cush- ing’s syndrome associated with adrenocortical tumor. The approach em- ployed by these investigators differed from that of the present study in that group reactions were employed rather than isolation and measure- ment of individual steroid metabolites.
It is amply clear from the steroid isolation studies that SU-4885 sub- stantially depressed 116-hydroxylation in Patient G.G., with both the C21 and C19 steroids. An examination of the material balance between the hydrocortisone metabolites and the substance S metabolites during the control period and while under the influence of the drug reveals that the increase in 11-deoxysteroids was greater than the net increase in the 11- oxygenated analogs. Although the difference is not great and might possi- bly result from the accumulation of errors in the determination of so many different compounds, it is in the direction suggested by Liddle’s hypoth- esis. Moreover, the results are consistent for the two classes of C21
metabolites, namely, those with the dihydroxyacetone side-chain (tetra- hydrocortisone and tetrahydrocortisol for the 11-oxygenated metabolites, and tetrahydro-S for the 11-deoxy series) and the metabolites with the glycerol side-chain (cortols and cortolones which represent the 11-oxy- genated metabolites, and pregnane-3a,17a,20a+6,21-tetrols which repre- sent the 11-deoxy metabolites). The net increase of 11-deoxy metabolites for each class was greater under the influence of SU-4885 than can be accounted for by the decrease in the corresponding class of 11-oxygenated compounds.
This finding is verified by the comparison of the material balance of the 17-ketosteroid metabolites shown in Table 2. The increase in androsterone and etiocholanolone under the influence of the drug more than equaled the decrease in 118-hydroxyandrosterone. Although this is a somewhat less trustworthy material balance, in that substance S probably yielded small amounts of androsterone and etiocholanolone, it is again in the di- rection suggested by Liddle’s hypothesis. The results with dehydroiso- androsterone and 45-pregnene-36,17a,20a-triol, shown in Table 3, simi- larly support the view that the tumor and its metastases were stimulated by increased production of endogenous ACTH during administration of SU-4885. This is significant because the increased production of these 2 compounds was substantial on a percentage basis and because no corre- sponding 11-oxygenated analogs of these 2 metabolites are known.
Thus, if a strictly literal interpretation be given to the observed changes in steroid production, it must be concluded that this tumor and its metas- tases were still capable of response to an increase of endogenously pro- duced ACTH. This interpretation, however, should be accepted with a measure of caution, not only because of the facts already noted but be- cause our present knowledge of the metabolism of Reichstein’s substance S is not nearly as complete as that of hydrocortisone. In addition, there is always the possibility that the peripheral metabolism of hormones may have been influenced by the drug, although there is no evidence available upon this point. Since it is known that some adrenal carcinomas can be stimulated by exogenous ACTH (4, 23) the possibility that this carcinoma was likewise responsive is credible, although direct evidence was not ob- tained. Alternatively the presumably normal contralateral adrenal gland may have been the source of the increase in steroid production under the influence of SU-4885. If this explanation were correct, it would imply that steroid production by the tumor and metastases had been altered in kind by the drug, without a change in amount. However, ACTH secretion was increased as a consequence of the fall in circulating hydrocortisone, and the normal adrenal while under the influence of SU-4885 responded to this stimulation with a greater amount of 11-deoxysteroids. If either provi-
sional interpretation be accepted, the interesting corollary must be drawn that the daily secretion of an amount of hydrocortisone which yielded 17 mg. of identifiable urinary metabolites was insufficient to suppress the pituitary of this patient. This amount of hormone is considerably greater than that effective for suppression of a normal hypophysis, and it can therefore be suggested that the pituitary of this patient had reached a new and different homeostatic level while still capable of increased hormone production in response to a drop in the circulating level of hydrocortisone.
The exception to these tentative conclusions is aldosterone. There was an increased production of this 11-oxygenated hormone under the in- fluence of SU-4885, in contrast to the suppressed production of the other 11-oxygenated compounds. Jenkins and associates (24) reported a similar finding but thought that the metabolites of the drug made evaluation difficult. Gold, DiRaimondo and Forsham (25) likewise reported an in- crease in aldosterone production during administration of SU-4885. These findings emphasize the different factors controlling the production of aldosterone in distinction to the other adrenal hormones, even in adreno- cortical carcinoma. Similar reservations, however, must be expressed about this finding, since our knowledge of the metabolism of aldosterone is fragmentary. In fact, as with all attempts at generalizations about the metabolic properties of adrenocortical carcinoma, definitive judgment should be reserved until even more extended and searching studies are possible.
Acknowledgment
The authors gratefully acknowledge the invaluable technical assistance of Mildred Smulowitz and Ruth Jandorek and the determination and interpretation of infrared spectra by Beatrice S. Gallagher.
REFERENCES
1. CHART, J. J .; SHEPPARD, H .; ALLEN, M. J .; BENCZE, W. L., and GAUNT, R .: New amphenone analogs as adrenocortical inhibitors, Experientia 14: 151, 1958.
2. JENKINS, J. S .; MEAKIN, J. W .; NELSON, D. H., and THORN, G. W .: Inhibition of adrenal steroid 11-oxygenation in the dog, Science 128: 478, 1958.
3. LIDDLE, G. W .; ISLAND, D .; LANCE, E. M., and HARRIS, A. P .: Alterations of adrenal steroid patterns in man resulting from treatment with a chemical inhibition of 118-hydroxylation, J. Clin. Endocrinol. & Metab. 18: 906, 1958.
4. LIDDLE, G. W .; ESTEP, H. L .; KENDALL, J. W., JR .; WILLIAMS, W. C., JR., and TOWNES, A. W .: Clinical application of a new test of pituitary reserve, J. Clin. Endocrinol & Metab. 19: 875, 1959.
5. KAPPAS, A., and GALLAGHER, T. F .: Studies in steroid metabolism. XXVIII. The a-ketosteroids excretion pattern in normal females and response to ACTH, J. Clin. Invest. 34: 1566, 1955.
6. WILSON, H .: Chromogenic values of various ketosteroids in a micro modification of the Zimmermann reactions: comparison with the micro procedure, Arch. Biochem. & Biophys. 52: 217, 1954.
September, 1960 ADRENAL INHIBITOR IN ADRENAL CARCINOMA 1245
7. WEICHSELBAUM, T. E .; MARGRAF, H. W., and MACK, R .: Determination in plasma of free 17-hydroxy and 17-desoxy corticosteroids and their glucuronic-acid conju- gates, J. Clin. Endocrinol. & Metab. 15: 970, 1955.
8. HURLOCK, B., and TALALAY, P .: Principles of the enzymatic measurement of steroids, J. Biol. Chem. 227: 37, 1957.
9. DOBRINER, K .; KATZENELLENBOGEN, E. R., and JONES, R. N .: Infrared Absorption Spectra of Steroids: An Atlas. New York, Interscience Publishers, Inc., 1953, vol. 1.
10. ROBERTS, G .; GALLAGHER, B. S., and JONES, R. N .: Infrared Absorption Spectra of Steroids: An Atlas. New York, Interscience Publishers, Inc., 1958, vol. 2.
11. FUKUSHIMA, D. K .; LEEDS, N. S .; BRADLOW, H. L .; KRITCHEVSKY, T. H .; STOKEM, M. B., and GALLAGHER, T. F .: The characterization of four new metabolites of adrenocortical hormones, J. Biol. Chem. 212: 449, 1955.
12. FUKUSHIMA, D. K .; BRADLOW, H. L .; HELLMAN, L .; ZUMOFF, B., and GALLAGHER, T. F .: Metabolic transformation products of hydrocortisone-4-C14 in normal men, J. Biol. Chem. (in press).
13. BRADLOW, H. L., and GALLAGHER, T. F .: Metabolism of 116-hydroxy-44-androstenc- 3,17-dione in man, J. Biol. Chem. 229: 505, 1957.
14. TOUCHSTONE, J. C .; BULASCHENKO, H .; RICHARDSON, E. M., and DOHAN, F. C .: The excretion of pregnane-3a-17a,21-triol-20-one (tetrahydro S) in normal and pathologic urine, J. Clin. Endocrinol. & Metab. 17: 250, 1957.
15. ROSSELET, J. P .; OVERLAND, L .; JAILER, J. W., and LIEBERMAN, S .: The isolation of 3a,17a,21-trihydroxypregnane-20-one from human urine, Helvet. chim. acta 37: 1933, 1954.
16. FUKUSHIMA, D. K., and GALLAGHER, T. F .: Absence of 21-dehydroxylation in con- genital adrenal hyperplasia, J. Clin. Endocrinol. & Metab. 18: 694, 1958.
17. FUKUSHIMA, D. K., and GALLAGHER, T. F .: Steroid isolation studies in congenital adrenal hyperplasia, J. Biol. Chem. 229: 85, 1957.
18. Cox, R. I., and FINKELSTEIN, M .: Pregnane-3a-17a, 20a-triol and pregnane-3x, 17a,20a-triol-11-one excretion by patients with adrenocortical dysfunction, J. Clin. Invest. 36: 1726, 1957.
19. FINKELSTEIN, M .: Adrenocortical heterofunction, Acta endocrinol. 30: 489, 1959.
20. Cox, R. I .: The separation and quantitative estimation of pregnane-3x, 17a,20a- triol, pregnane-3a, 17a,20a-triol-11-one and other urinary acetaldehydogenic steroids, J. Biol. Chem. 234: 1693, 1959.
21. HIRSCHMANN, H., and HIRSCHMANN, F. B .: Steroid excretion in case of adreno- cortical carcinoma. V. 45-Pregnenetriol-33,17a,20a, J. Biol. Chem. 187: 137, 1950.
22. FOTHERBY, K .: The isolation of 30-hydroxy-45-steroids from the urine of normal men, Biochem. J. 69: 596, 1958.
23. GALLAGHER, T. F .: On alterations in adrenal function, especially with adrenocortical carcinoma, Cancer Res. 17: 520, 1957.
24. JENKINS, J. S .; POTHIER, L .; REDDY, W. J .; NELSON, D. H., and THORN, G. W .: Clinical experience with selective inhibition of adrenal function, Brit. M. J. 1: 398, 1959.
25. GOLD, E. M .; DIRAIMONDO, V. C., and FORSHAM, P. H .: Quantitation of pituitary corticotropin reserve in man by use of an adrenocortical 11-beta hydroxylase in- hibitor (SU-4885), Metabolism 9: 3, 1960.