ADRENOCORTICAL CARCINOMA IN MAN. THE EFFECT OF AMPHENONE ON INDIVIDUAL KETOSTEROIDS*
T. F. GALLAGHER, PH.D Sloan-Kettering Institute for Cancer Research, New York, N. Y.
ABSTRACT
Five patients with adrenocortical carcinoma were studied with respect to the production of various individual 17-ketosteroids during control periods and during treatment with amphenone (3,3-bis(p-aminophenyl)butanone-2). In all instances the drug caused a decrease in some or all of the steroid hormone metabolites. A detailed discussion of each major component of the 17-ke- tosteroids leads to the conclusion that amphenone invariably lowers the produc- tion of hydrocortisone but exerts a variable influence upon other components of adrenal secretion. The results support the conclusion that the biosynthesis of adrenocortical hormones can be differentially altered, even in a neoplastic tissue.
T HE steroid hormones secreted by the human adrenal eventually ap- pear in the urine as 2 chemical classes of metabolites. Hormones such as hydrocortisone are converted to metabolites which retain an atom of oxygen at carbon 11 of the steroid nucleus; other adrenal hormones which initially lack the C-11 oxygen appear in urine as 11-deoxysteroids-among others, androsterone and etiocholanolone. Metastatic carcinoma of the adrenal cortex in man is invariably accompanied by a marked increase in the production and secretion of both classes of these steroid hormones, although often a great disparity in the relative proportion of either compo- nent can be demonstrated with suitable analytical methods. The purpose of this report is to describe steroid isolation studies in 5 patients with ad- renocortical carcinoma during control periods and while being treated with amphenone.1 The principal focus is on the alteration in production of the 2 classes of metabolites that may be achieved with this agent. It is hoped that the results reported will clarify the interpretation of previous data
Received March 24, 1958.
* This investigation was supported in part by a grant from the American Cancer Society and a research grant (CY-3207) from the National Cancer Institute of the National Institutes of Health, United States Public Health Service.
1 Amphenone is 3,3-bis(p-aminophenyl)butanone-2 (cf. W. L. Benczl and M. J. Allen: Structure of amphenone B and related amphenones, J. Org. Chem. 22: 352, 1957). The compound for these studies was graciously furnished by Dr. Roy Hertz of the National Cancer Institute, Bethesda, Maryland.
and will direct attention to the need for application of more searching ana- lytical methods in the evaluation of similar potential chemotherapeutic agents.
Peterson, Hertz and Lubs (1) and Thorn and associates (2) described marked decreases during amphenone treatment in the excretion of “urinary corticoids,” which are presumed to be derived from hydrocortisone. The same authors reported “unimpressive” changes or no change in the urinary 17-ketosteroids. Both groups commented that alterations in the 17-ke- tosteroids might have been detected had more exact methods of separation been employed. It was previously demonstrated (3, 4) that administration of amphenone to a patient with widely metastatic adrenocortical car- cinoma produced a highly significant fall in all the chromatographically isolated steroids which together constitute the “urinary 17-ketosteroids.” In view of these apparently conflicting findings and because of the import for a clearer knowledge of secretory function of neoplastic adrenals, de- tailed consideration is given to the effect of amphenone on the major individual components of the “urinary 17-ketosteroids.”
SUMMARY OF CASES
S-52 (study made possible through the cooperation of Drs. Olof Pearson, Leon Hell- man, C. D. West and Mortimer Lipsett of this Institute). The patient, a 52-year-old white woman, was admitted to Memorial Center for the first time in June 1956, for study and evaluation. Signs and symptoms of Cushing’s syndrome had developed in May 1954, and in April 1955 an adrencortical carcinoma had been removed surgically at another hospital. Postoperatively, there had been a striking clinical remission lasting until February 1956. On admission to Memorial Center in June, the classic clinical and chemical findings of Cushing’s syndrome were present, and there was also evidence of multiple pulmonary metastases.
While the patient was in the hospital, a continuous metabolic balance regimen was maintained and 24-hour urine collections were made for steroid analysis. Control and treatment periods were alternated. Treatment periods were as follows:
July 3- 5: Oral hydrocortisone, 150 mg. per day.
July 12-17: Intramuscular ACTH-gel, 240 units per day.
July 26-28: Oral amphenone, 3 Gm. per day. Results of steroid isolation studies dur- ing this period are shown in Figure 1.
At the conclusion of these studies, the patient was discharged. No specific medication was recommended. Her clinical status was essentially the same as on admission.
In September 1956, the patient was re-admitted to Memorial Center for the second and the last time. In the interval since discharge, the signs and symptoms of Cushing’s syndrome had increased, and signs of congestive heart failure had appeared. Complete metabolic balance studies were again started, and 24-hour urine collections were made for steroid analysis. After control studies from September 10 to 15, oral amphenone was begun in a dosage of 3 Gm. per day. The data charted in Figure 2 are the results ob- tained on the third, fourth and fifth days of this dosage, with the data of 2 prior control periods for comparison. On September 28, the daily dose was raised to 4 Gm. On October
S-52우
CONTROL
CONTROL
Mg./24Hrs.
AMPHENONE
II=OE
10
5
0
20
OH-E
15
10
5
S-52¥
0
20
CONTROL
AMPHENONE
CONTROL
15
Mg./24 Hrs.
OH-A
10
11=OE
10
F
5
5
0
0
90
15
80
OH-E
10
70
5
60
D
50
0
40
OH-A
10
30
5
20
0
10
50
0
40
7=OD
20
D
30
10
20
0
10
35
0
30
7=OD
10
C
25
0
E
20
15
30
10
E
20
5
10
A
0
L
0
10
15
C
0
A
10
DATE
7/17-19/56
7/26-28/56
7/31-8/2/56
5
DATE
0
7/31-8/2/56
9/10-15/56
9/17-20/56
2, amphenone was discontinued because of progressive lethargy. Despite stopping the drug, the patient deteriorated rapidly and died on October 12. Autopsy revealed that the cause of death was cryptococcal meningitis.
G-37 (study made possible through the cooperation of Dr. Sidney C. Werner of Presbyterian Hospital, New York). The patient was a 37-year-old white woman, first seen at Presbyterian Hospital, New York City in January 1957, complaining of amenor- rhea of six weeks’ duration and bloating of the face for two months. On physical exam- ination, she had the appearance of a woman with Cushing’s disease. Facial hair was excessive, and purpura was present over the breasts and abdomen, but there were no striae. The abdomen contained a hard mass in the region of the right kidney. The over- lying liver was readily palpable. There was a mild anemia. The serum sodium level was slightly increased (146.3 mEq./L) and the potassium level was slightly decreased (2.4 mEq./L). At operation, a typical carcinoma of the adrenal cortex was found-confirmed
by biopsy. On the third postoperative day, a series of psychotic episodes set in, ending with a lapse into coma for twelve hours. These episodes could not be explained, and subsided spontaneously. On February 20, amphenone was started in an oral dosage of 500 mg. every six hours. The results charted in Figure 3 were obtained during the fourth and fifth days of medication. Approximately two weeks after the last dose, the patient became blind suddenly, but slowly recovered within two weeks. She returned home, where she died about a month later. There was no autopsy.
L-7 (study made possible through the cooperation of Dr. George Thorn, Peter Bent Brigham Hospital, Boston). The patient (previously described by Sobel (5) ) was a 72-year-old girl who was hospitalized on the metabolic ward at the Peter Bent Brigham Hospital until her death three months later. At the age of 7 years she first manifested mild, and then rapidly progressing, signs of virilization. At the age of 7 years and 3 months surgical excision of a left adrenal tumor was performed and followed by irradiation of the tumor bed. Prior to operation, urinary 17-ketosteroid excretion was elevated. The immediate response to excision of the tumor was gratifying, both in terms of clinical signs and 17-ketosteroid excretion. However, a palpable mass developed in the left lower quadrant within two months, and pulmonary metastases became evident at the age of 7 years and 6 months. On admission to the metabolic ward, the patient was found to be markedly virilized, with only slight manifestations of Cushing’s syndrome, and mod- erate hypertension. During the period of observation, there was marked aggravation of signs and symptoms of the endocrine disorder, including progressive virilization
G-37 9
L-7 9
CONTROL
AMPHENONE
CONTROL
AMPHENONE
Mg./24 Hrs.
Mg./24 Hrs.
3
10
II=OE
2
II=OE5
0
0
4
OH-E’S
3
OH-E
2
0
20
0
15
OH-A
2
OH-A
10
-
5
O
0
D
4
”
D
20
0
10
6
0
5
7=OD
10
0
E
4
3
40
2
30
1
E
20
O
10
A
2
0
A
10
DATE
O
2/15-18
2/23-25/55
O
DATE
11/28-12/3
12/11-13
12/16-19/55
hypertension and the development of obvious Cushing’s syndrome with a moon face, striae, voracious appetite and glucosuria. Since there had been a suggestive response to adrenocorticotropin (ACTH) shortly after admission, in terms of urinary steroid excretion, the patient underwent hypophysectomy. The clinical course and the changes in urinary steroid excretion suggested a very brief (ten days) decrease in the metabolic activity of the metastatic tumor. Other attempts to control the rapidly progressing malignant lesion, including the administration of amphenone and of N, N’,N”-triethylene- thiophosphoramide (“thio-TEPA”), were unsuccessful. Amphenone was begun on December 3, 1955 in dosage of 4 Gm. per day, increased gradually to 9 Gm. per day during the period December 11 to 13. This was the first medication period during which steroid isolation studies were performed. The daily dose was increased gradually to 12 Gm. from December 16 to 19, which was the second period of the study. The results are shown in Figure 4. On the third day after the last course of amphenone therapy, the patient had a generalized convulsion and died despite efforts at resuscitation. Post- mortem examination confirmed the presence of a widely metastasizing, relatively un- differentiated, recurring adrenocortical carcinoma.
D-41 (study made possible through the cooperation of Dr. George Thorn, Peter Bent Brigham Hospital, Boston). The clinical description of the patient has been published previously (2). The length of the amphenone period (Fig. 5) was four days, during which the dosage was 3 Gm. per day orally. This was preceded by a day during which a dose of 3 Gm. of amphenone was administered; the urine of this day was not included in the study. Two weeks carlier, 10 Gm. of amphenone had been given during two days.
D-41 8
CONTROL
AMPHENONE
Mg./24 Hrs.
11=OE
10
E
O
OH-E
1ºF 10
0
OH-A
10
E
0
D
40
E
0
80
E
40
0
A
40
0 .
E
4/27-5/6/55
5/25-29/55
P-51º
CONTROL
AMPHENONE
CONTROL
Mg./24 Hrs.
11=OE
5
1
0
10
OH-E
5
0
OH-A
5
I
0
D
10
5
0
25
20
E
15
10
5
0
A
10
5
DATE
0
2/26-27/56 2/28-3/3 3/4-8/56 3/14-17/56
P-51 (study made possible through the cooperation of Drs. Olof Pearson, Leon Hell- man, C. D. West and Mortimer Lipsett of this Institute). The patient was a 51-year-old white woman who had experienced gradual onset of weakness and loss of weight be- ginning in March 1954. This was followed by the development of moon facies, truncal obesity, edema and hypertension. In February 1955, a diagnosis of Cushing’s syndrome was made and the patient was referred to Memorial Center for therapy. An adreno- cortical carcinoma was removed on April 5, 1955.
The patient improved postoperatively and was discharged. Her condition remained normal until January 23, 1956, when the weakness, moon facies and edema returned. She also complained of pain in the right side of the chest. Physical examination revealed an enlarged liver and hypertension. X-ray examination showed pulmonary metastases, but no definite bony involvement. Urinary 17-ketosteroids were moderately elevated. The glucose tolerance curve was of the diabetic type. The patient was admitted on February 4, 1956, for study during amphenone therapy.
A metabolic balance period was started on February 8, 1956 and was continued throughout the study. She received 9a-fluorohydrocortisone, 10 mg. daily for four days (February 12 to 16); ACTH-gel, 240 units daily for four days (February 19 to 23); and amphenone according to the following schedule:
| Feb. 28-29 | 1.0 Gm. |
| Feb. 29-Mar. 1 | 3.0 Gm. |
| Mar. 1-2 | 4.5 Gm. |
| Mar. 2-3 | 4.0 Gm. |
| Mar. 3-4 | 5.0 Gm. |
| Mar. 4-5 | 5.0 Gm. |
| Mar. 5-6 | 6.0 Gm. |
| Mar. 6-7 | 6.0 Gm. |
| Mar. 7-8 | 6.0 Gm. |
Combined urines for 2 periods were analyzed, and the results are shown in Figure 6.
There was no significant change in potassium, nitrogen, phosphorus or calcium bal- ances during these treatment periods. The sodium and chloride balances showed minimal changes. There was retention of both sodium and chloride during the 9a-fluorohydro- cortisone and ACTH periods, reaching a maximum positive balance of 12 to 14 mEq. daily. Sodium and chloride diuresis followed these periods of retention. During amphe- none therapy there was a negative sodium and chloride balance, reaching 24 mEq. daily. The creatinuria decreased during treatment with amphenone. The patient had a severe drug reaction with shock, fever and a rash. Amphenone had to be stopped on March 9. Upon recovery, 41-9a-fluorohydrocortisone, 4.0 mg. per day, was given from March 18 to 27, 1956.
ANALYTICAL METHODS
Urines were received in the laboratory as individual 24-hour specimens; collections were complete, as judged by constancy of the creatinine content. Individual 24-hour specimens were combined into suitable pools (as indicated in the charts) and were treated with beef liver B-glucuronidase2 at pH 5 for five days at 37° C. At the end of incubation
2 B-Glucuronidase, obtained from the Warner-Chilcott Laboratories, a division of Warner-Lambert Pharmaceutical Company, New York, N. Y. It is available under the trade name Ketodase.
the urine was adjusted to pH 1 and extracted 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. The urine plus alkali-soluble fraction after neutralization was acidified to 1 N with sulfuric acid and re-extracted continuously with other for forty-eight hours. Again the neutral ether-soluble fraction was separated. Each of these neutral extracts was separately processed into ketonic and nonketonic fractions by means of Girard’s reagent T, and the ketonic fractions were further separated into @ and ß ketosteroid subfractions by means of digitonin. These methods have been de- scribed in detail (6). The separate subfractions from cach extract were quantitatively chromatographed on paper, using minor modifications of the systems developed by Burton, Zaffaroni and Keutmann (7). The individual steroids eluted from the paper were measured quantitatively by the Zimmermann reaction, according to the method of Talbot, Butler, MacLachlan and Jones (8). The identity of each substance eluted from the chromatograms was confirmed by infrared spectrometry. A number of compounds other than those recorded were identified in all of the patients studied. These will be described elsewhere.
The extracts from Subject L-7 contained a large amount of tarry material, which made processing of the extracts difficult. With this subject, during both control and amphenone periods, it was necessary to re-treat the insoluble tar with Girard’s reagent T. The ketonie fractions so obtained were chromatographed separately and the values for the isolated compounds were added in order to obtain the daily steroid production. This difficulty was not encountered with any of the other patients; it may have been due to the large amounts of various drugs with which the patient was treated, or per- haps due to her nearly terminal condition.
RESULTS AND DISCUSSION
11-Hydroxyetiocholanolone (OH-E) and 11-ketoetiocholanolone (11 =OE)
These 2 metabolities are derived primarily from hydrocortisone and should be considered together as representative metabolities of that hor- mone. Quantitatively the greatest production was found in Subject S-52, in whom the amount exceeded 20 mg. per day. Assuming that these 2 com- pounds account for about 5 to 10 per cent of the metabolities of hydro- cortisone,3 the tumor of this patient was secreting between 200 and 400 mg. of hydrocortisone per twenty-four hours. A reasonable estimate of the daily production of this hormone by normal subjects would be less than 20 mg. per day. It is thus evident that the metastases of this patient pro- duced a great excess of hydrocortisone. The result of this high production was manifest in the extreme symptoms of Cushing’s syndrome exhibited by this patient. A comparable situation existed in Subject L-7 whose daily production of the same metabolites during control periods was 18 mg. per twenty-four hours. These 2 patients had the highest excretion of OH-E
3 This approximation is based on the percentage recovery of these metabolites after the administration of tracer doses of hydrocortisone-4-C14 to human subjects. Personal communication of D. K. Fukushima, H. Leon Bradlow and others from these labora- tories.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName} ] on 22 November 2015. at 18:33 For personal use only. No other uses without permission. . All rights reserved.
and 11 = OE. Among the other 3 patients, the least elevation in 11-oxy- genated hormone production (G-37) was a fivefold increase over normal values.
In all of the subjects studied there was a highly significant diminution in the production of these metabolites during administration of amphenone. It is emphasized that this diminution occurred even when the dosage of the drug was 2 grams per day (Subject G-37). There is an important technical factor in connection with the effect of amphenone on these 11-oxygenated metabolites. The extracts of urine obtained during the administration of large quantities of amphenone were contaminated with the drug and its metabolic transformation products. Even though the extracts had been extensively fractionated prior to chromatography, a certain portion of the drug or its metabolites was carried along with the steroids. In the paper chromatograms, a background of brownish material moved into the area where the 11-oxygenated compounds were separated. This background material was not found during control periods without amphenone. Al- though this material did not interfere with the separation or identification of the steroids studied, it did contribute nonspecific chromogenic material which resulted in a falsely high value for the 11-oxygenated metabolites during the amphenone periods. It was felt that further fractionation in an attempt to remove this background contaminant might result in an ex- cessive loss of steroid and, for this reason, no attempt was made to obtain the steroids in purified form, nor was any correction introduced to take ac- count of the added chromogen. For these reasons it is believed that the estimated values for these 11-oxygenated compounds are maximal and that the true values are probably somewhat lower than those recorded. It was clearly demonstrated that without exception amphenone caused a sig- nificant decrease in the two 11-oxygenated ketosteroids derived from hydrocorti- sone.
11-Hydroxyandrosterone (OH-A)
This compound is the major metabolite of another component of the ad- renal secretion, 118-hydroxy-44-androstene-3,17-dione (9). It is likewise a principal metabolite of adrenosterone, if this substance is produced by the adrenal gland (10). OH-A is a very minor metabolite of hydrocortisone and accounts for less than 2 per cent of the transformation products of that hormone.3 In contrast with the other two 11-oxygenated ketosteroid metab- olites (OH-E and 11 = OE), 11-hydroxyandrosterone to a major extent re- flects adrenal secretion of a C19 11-oxygenated hormone. It is not sur- prising, therefore, to find examples among these cases of carcinoma in which the production of this metabolite is disproportionate when compared with 11-hydroxyetiocholanolone and 11-ketoetiocholanolone. Thus Sub- ject L-7, who in control periods excreted 19 mg. of OH-A per twenty-four
hours, produced more of this substance than of the other 2 major 11- oxygenated ketosteroids combined. Under the influence of amphenone, Subject L-7 showed a greater diminution of this metabolite, so that in fact the greatest percentage decrease was demonstrated with OH-A. In Sub- ject G-37, on the other hand, there was no suppression of this component under the influence of a smaller dose of amphenone. In the rest of the sub- jects there was a major decrease in the production of the precursors of this metabolite during treatment with amphenone.
Dehydroisoandrosterone (D)
This compound has been present in the urine of all human subjects thus far examined, whether normal or abnormal. Under most circumstances the amount is relatively small, i.e., 1 mg. or less per day. It was once believed that excessive amounts of this compound are produced in tumors of the adrenal cortex. It is clear from the present series that this is not invariably true. Subject G-37, who had a highly malignant metastatic carcinoma, ex- creted only 0.4 mg. of D per day during the control period, and the amount was not materially lessened during the period when a small dosage of am- phenone was given. Similarly, although excretion was out of the normal range in Subjects D-41 and P-51, both produced less dehydroisoandro- sterone than might have been anticipated from the very high values for “total 17-ketosteroids.” On the other hand, there was a tremendous pro- duction of D by Subjects S-52 and L-7 during control periods. It is appar- ent that some, but not all, adrenal tumors are capable of excessive production of dehydroisoandrosterone.
All patients, with the exception of G-37, exhibited a pronounced de- crease in the production of D during treatment with amphenone. Similarly, in a patient previously reported (3, 4), D production decreased from a level of approximately 175 mg. per day during the control period to ap- proximately 2 mg. per day during administration of amphenone. In general, it appeared to be true in this series of patients that with higher production of dehydroisoandrosterone during the control period, there was a greater per- centage fall during treatment with amphenone.
7-Ketodehydroisoandrosterone (7=OD)
This compound,4 like dehydroisoandrosterone, is a normal urinary con- stituent and the amount excreted per day is usually small. The substance is
4 In the report that described the first identification of 7-ketodehydroisoandrosterone from human urine (11), it was stated that although the compound might have been an artifact formed by autoxidation of dehydroisoandrosterone during the isolation pro- cedure, in the lack of direct evidence it was preferable to accept the view that the sub- stance was a true metabolite. The results with S-52 provide excellent evidence for the correctness of this interpretation. The amount of dehydroisoandrosterone produced by this patient during the first control period was about 35 mg. per day and the 7-keto-
probably produced by the adrenal rather than as a secondary metabolite of peripheral tissues. Among the carcinoma patients of this series, only Sub- jects S-52 and L-7 exhibited elevated amounts of this metabolite. During the first course of amphenone the excretion of 7 = OD by Subject S-52 re- mained essentially at the control level, whereas the excretion of dehydro- isoandrosterone was diminished to less than 10 mg. per day; in Subject L-7, similarly, there was little change in the production of 7= OD during administration of amphenone despite a transient effect of the drug on dehy- droisoandrosterone. When Subject S-52 received a second course of am- phenone, the tumor and its metastases had materially altered secretory capacity, as shown in Figures 1 and 2. At this time amphenone caused a decrease in 7 = OD excretion to about half the control level. By contrast, however, dehydroisoandrosterone was less than one-tenth of the control value.
Androsterone (A)
The production of this metabolite was characterized by wide individual variations among the patients in this series. Subject G-37 exhibited essen- tially normal production whereas Subject D-41 showed the highest level- approximately 30 mg. per day. Since, however, Subject L-7 was a child, it is clear that in relation to her age the amount of A was the greatest in any of the patients studied. In all the patients, amphenone lowered the formation of A, although not to the same extent that was evident with the 11-oxygenated metabolites.
Etiocholanolone (E)
In all of the patients in this series there was an increase in the formation of this metabolite when compared with normal women of comparable age. However, as with A, there were wide individual differences. Thus, in Sub- ject G-37 the production of E was 5.4 mg. per day during the control period — a normal value for girls in the early twenties. Subject D-41, with ap- proximately 100 mg. per day, represented the other extreme in this series.
The effect of amphenone on the production of E was markedly different among this group of patients. Subject G-37, who received the smallest dose,
dehydroisoandrosterone amounted to almost a third of this value; during the second treatment with amphenone, dehydroisoandrosterone was about 7 mg. per day but 7-ketodehydroisandrosterone was nearly 6 mg. If the a,ß unsaturated ketone had been formed as an artifact, it would be anticipated that the relative proportions of the 2 steroids would have remained virtually similar during both periods, since the manipula- tion of the extracts was identical insofar as possible. As indicated previously, this was not the fact. In other patients in whom dehydroisoandrosterone production was at an even greater level than in Subject S-52 (e.g., Subject D-41) there was no concomitant production of significant amounts of 7-ketodehydroisoandrosterone. This is further verification of the conclusion that 7-ketodehydroisoandrosterone is a true metabolite rather than an artifact.
showed only a minor fall in the production of this metabolite. In Subject L-7 the excretion of E actually increased from 28.5 mg. per day during the control period to 42 mg. per day during the last period of amphenone ad- ministration. In Subject S-52, during the first course of amphenone treat- ment, the amount of E was intermediate between the levels in the 2 control periods (preceding and following administration of the drug). In these patients it must be concluded either that amphenone failed to sup- press the biosynthesis of the precursor of this metabolite or that there was an increase in production despite the suppression of other adrenal hor- mones. In the other 2 patients there was a marked diminution in the pro- duction of E during administration of amphenone. The results in Subject P-51 are especially interesting, in that the progressive decline in E as well as of the other metabolites with continued administration of am- phenone, is clear evidence of cumulative action. This is significant in view of the relatively rapid elimination of the drug and its metabolites. The prompt return to pre-treatment levels after cessation of treatment in 2 sub- jects (P-51 and the previously reported patient, R-42 (3, 4)) in whom E was greatly suppressed, is also clear evidence that amphenone altered steroid biosynthesis and was not lethal to the neoplastic cells.
The carcinoma of Subject S-52 exhibited 2 dissimilar responses at differ- ent times to the same agent. This is a highly significant result because of the implications that can be derived from the response of the metastases in different stages of development and function. During the first course of amphenone in Subject S-52, there was an increase in the excretion of E. At a later time when the disease had progressed materially, as judged not only from the quantitative changes in steroid production but also from the clinical condition, amphenone caused a distinct reduction in the amount of E. The decrease was from more than 30 mg. per day to less than 10 mg. per day (Fig. 2). This is the only patient in whom courses of amphenone were studied during different phases of the disease. For this reason gen- eralizations were unwarranted, but it is clearly established that the car- cinoma showed a grossly different behavior at 2 stages of its progress toward termination of the patient’s life. It seems highly probable that other similar neoplasms may show altered responsiveness to both hormones and drugs at various stages of the disease. In this respect Subject S-52 may be considered comparable to previously reported Subject R-42 (3, 4) who ex- hibited gross alteration in the pattern of steroid production when the tumor was in the “localized phase” as compared with the later pattern when the cancer was widely metastatic.
Thus it is evident that amphenone decreased the production of etio- cholanolone in the majority of patients, although in 2 subjects there was an absolute increase. In 1 of these 2 patients, at a later stage of the disease, there was a marked diminution in E during administration of amphenone.
COMMENT
By separation of the individual components of the urinary “17-keto- steroids” in a series of 5 patients with widespread metastatic adrenocorti- cal carcinoma, it has been shown that in all instances some or all of the com- ponents were decreased during treatment with amphenone. In most patients reported, the diminished production and output extended to both chemical classes of steroid metabolites, i.e., compounds oxygenated at C-11 of the steroid nucleus, as well as the 11-deoxyketosteroids. The significance of the results lies in the demonstration that amphenone invariably lowers the adrenocortical production of 11-oxygenated adrenal hormones. In addi- tion there is often, though not invariably, a pronounced diminution in the 11-deoxysteroids. The distinction between these effects of the drug can be recognized only by separation of the individual steroids which comprise the “17-ketosteroids.” This valuable information is obscured or lost com- pletely by the application of the group reaction employed in the routine clinical determination of this mixture of compounds. Together with other evidence reported elsewhere (12, 13), these results support the conclusion that the biosynthesis of adrenocortical hormones results in 2 different classes of steroids, each subject to different control mechanisms. These control mechanisms can be differentially altered, even in a neoplastic tissue.
The interest in amphenone is not primarily because of any value in the treatment of carcinoma. Its importance stems from the fact that this rela- tively toxic compound, which has a number of undesirable side-effects, can materially alter one or both pathways in the biosynthesis of adreno- cortical hormones leading to hydrocortisone and related 11-oxygenated hormones on the one hand, and 11-deoxysteroids or “adrenal androgens” on the other. Rosenfeld and Bascom (14) in studies of the isolated perfused calf adrenal concluded that amphenone exerts a relatively selective inhibi- tion of steroid biogenesis, somewhat similar to the alterations in steroid metabolism in human subjects, as described in the present report. With a better drug, it should be possible to achieve these or related effects without the disagreeable reactions which limit application of the present compound. A means for the control of either component of the adrenal steroid secre- tion through such an agent should have the most profound and far-reaching implications for biology and medicine. By application of the refined chemi- cal techniques available today, the proper evaluation of such compounds can be readily achieved. It is important that their real potential be recog- nized. Valuable information can be lost by the use of simpler but less reliable group reactions, which may obscure rather than clarify the underlying phenomena.
September, 1958
Acknowledgment
I wish to express my deep appreciation to the clinicians who by their cooperation made this study possible. At this Institute these were Drs. Leon Hellman, Olof Pearson, C. D. West and Mortimer Lipsett; at the Presbyterian Hospital, Dr. Sidney Werner; and at the Peter Bent Brigham Hospital, Dr. George Thorn with the assistance of Drs. Attallah Kappas, W. J. Reddy, Edna Sobel and Albert Renold. In the chemical work I wish to thank the technicians who carried out the analyses, especially Ruth Jandorek, Mildred Smulowitz and Voldemar Bankers.
REFERENCES
1. PETERSON, R. E .; HERTZ, R., and LUBS, H. A .: Suppression of biosynthesis of ad- renal cortical steroids in man by amphenone, Proc. Soc. Exper. Biol. & Med. 94: 421, 1957.
2. THORN, G. W .; RENOLD, A. E .; GOLDFIEN, A .; NELSON, D. H .; REDDY, W. J., and HERTZ, R .: Inhibition of corticosteroid secretion of amphenone in a patient with adrenocortical carcinoma, New England J. Med. 254: 547, 1956.
3. GALLAGHER, T. F., and KAPPAS, A .: Influence of invasiveness, hormones, and am- phenone on steroids in adrenal carcinoma, Science, 124: 487, 1956.
4. SPENCER, H .; LEWIN, I .; LASZLO, D .; HERTZ, R .; KAPPAS, A., and GALLAGHER, T. F .: Adrenocortical carcinoma with hyperadrenocorticism; a clinical, metabolic and hormonal study, A. M. A. Arch. Int. Med. 100: 658, 1957.
5. SOBEL, E. H .; RENOLD, A. E .; BETHUNE, J. E .; HOET, J. J .; REDDY, W. J., and THORN, G. W .: Effects of hypophysectomy and of amphenone administration in a child with functioning metastatic adrenal carcinoma, Am. J. Med. 24: 482, 1958.
6. KAPPAS, A., and GALLAGHER, T. F .: Studies in steroid metabolism. XXVIII. The a-ketosteroid excretion pattern in normal females and the response to ACTH, J. Clin. Invest. 34: 1566, 1955.
7. BURTON, R. B .; ZAFFARONI, A., and KEUTMANN, E. H .; Paper chromatography of steroids. II. Corticosteroids and related compounds, J. Biol. Chem. 188: 763, 1951.
8. TALBOT, N. B .; BUTLER, A. M .; MACLACHLAN, E. A., and JONES, R. N .; Definition and elimination of certain errors in the hydrolysis, extraction and spectrochemical assay of a and B-neutral urinary 17-ketosteroids, J. Biol. Chem. 136: 365, 1940.
9. BRADLOW, H. L., and GALLAGHER, T. F .: Metabolism of 113-hydroxy-44-androstene- 3,17-dione in man, J. Biol. Chem. 229: 505, 1957.
10. SAVARD, K .; BURSTEIN, S .; ROSENKRANTZ, H., and DORFMAN, R .: The metabolism of adrenosterone in vivo, J. Biol. Chem. 202: 717, 1953.
11. FUKUSHIMA, D. K .; KEMP, A. D .; SCHNEIDER, R .; STOKEM, M. B., and GALLAGHER, T. F .: Studies in steroid metabolism. XXV. Isolation and characterization of new urinary steroids, J. Biol. Chem. 210: 129, 1954.
12. GALLAGHER, T. F .: Steroid hormone metabolism and the control of adrenal secretion, Harvey Lectures 52: 1, 1957.
13. GALLAGHER, T. F .: On alteration in adrenal function, especially with adrenocortical carcinoma, Cancer Res. 17: 520, 1957.
14. ROSENFELD, G., and BASCOM, W. D .: The inhibition of steroidogenesis by am- phenone B: studies in vitro with the perfused calf adrenal, J. Biol. Chem. 222: 565, 1956.