Laboratory for Endocrinological Chemistry, Gemeenteziekenhuis aan de Bergweg, the Department of Internal Medicine, Zuiderziekenhuis, Rotterdam, Holland
EFFECT OF METOPIRONE IN A PATIENT WITH ADRENAL CORTICAL CARCINOMA By H. Daniels, W. J. van Amstel, W. Schopman and C. van Dommelen
ABSTRACT
1. The effect of metopirone® (SU-4885)” in a patient with metastatic adrenal cortical carcinoma was studied. The intention was to depress the symptoms of Cushing’s syndrome, due to a high glucocorticoid production by the carcinoma.
2. The excretion of 17-KS, 17-KGS, 11-oxo(y)-17-OHCS and 11-desoxy- 17-OHCS was followed for seven weeks.
3. The steroid excretion was variable. The 11-oxo(y)-17-OHCS showed a significant decrease during treatment with SU-4885 for seven weeks. The ratio 11-oxo(y)-17-OHCS: 11-desoxy-17-OHCS fell from 1.7 to 0.1 during this period.
4. The excessive glucocorticoid production of the metastatic adrenal car- cinoma was suppressed as long as treatment continued.
5. It was not possible to judge the clinical effect of SU-4885 because of the progressive nature of the disease.
* The authors gratefully acknowledge a generous gift of SU-4885 by Ciba N. V., Arn- hem, Holland.
The following abbreviations will be used:
17-Ketosteroids: 17-KS
total 17-hydroxycorticosteroids (Appleby et al.): 17-KGS 17,21-dihydroxy-20-ketosteroids: 17-OHCS 11-oxo-and 11-oxy-17,21-dihydroxy-20-ketosteroids: 11-oxo(y)-17-OHCS 11-desoxy-17,21-dihydroxy-20-ketosteroids: 11-desoxy-17-OHCS 3a,118,17a,21-tetrahydroxy-5}-pregnan-20-one: THF 3a,118,17a,21-tetrahydroxy-5a-pregnan-20-one: allo THF 3a,17a,21-trihydroxy-58-pregnan-11,20-dione: THE 3a,17a,21-trihydroxy-58-pregnan-20-one: THS 17a,21-dihydroxy-pregn-4-ene-3,20-dione: Compound S
The effect of metopirone® (SU-4885) on the steroid excretion of patients with adrenal cortical carcinoma was studied by Fukushima et al. (1960) and Jailer et al. (1960) during short term treatment with the drug. We had the opportunity of studying the effect of SU-4885 in a patient with adrenal carcinoma during fifty days.
Our intention was to lessen the symptoms of Cushing’s syndrome, which were caused by the high glucocorticoid production of the carcinoma. This seemed to be possible by means of oral treatment with the relatively non-toxic drug SU-4885.
CASE REPORT
A 49 years old married woman was admitted to hospital on January 19th, 1961. She showed the typical picture of Cushing’s syndrome with plethora (haemoglobin 16.9 80%), obesity of the trunk (85 kg, 180 cm), purple abdominal striae, hirsutism of face and abdomen and a blood pressure of 180/110. There was a diabetic glucose tolerance test and the urine was positive for sugar. The clitoris was not enlarged. Her com- plaints dated from August 1960.
The excretion of steroids in a 24-hour urine sample was 17-KS 242 mg, 17-KGS 81 mg and 17-OHCS 86 mg.
On X-ray examination of the chest the right half of the diaphragm was found to be much higher than the left, and scarcely moved. Intravenous urography showed a considerable downwards displacement of the right kidney. Operation was performed on January 24th (Prof. P. J. Kooreman, M. D.).
By means of a postero-lateral lumbar incision on the right side a yellow-grey tu- mour was removed. This tumour had a diameter of 12 cm and weighed 740 g. At some distance of this large tumour another tumour was found, with a diameter of 2.5 cm. The diagnosis of the pathologist (J. A. M. van Unnik, M. D.) was: »adrenal cortical carcinoma«. (Klinisch-Pathologische Conferentie (1961).
The postoperative course was unremarkable. Shortly before she left the hospital on February 18th, 1961, the excretion of steroids was normal (Table 2). No radio- therapy was given. Symptoms of Cushing’s syndrome partly disappeared after the operation. On July 6th, four months after the operation, the patient was admitted again to the hospital with complete relapse of Cushing’s syndrome. Tumours were felt on the right side in the abdomen. The excretion of steroids was very high (Table 2). On July 12th, oral therapy with SU-4885 (metopirone®) was started. The dosage was 1 g daily in four divided doses during the first week and afterwards 2 g daily in four divided doses. This therapy was given for 50 days. In addition a course of gamma-ray irradiation (60Co) was given on the right side of the abdomen. (K. Breur, radiologist, Rotterdamsch Radiotherapeutisch Instituut), from July 25th to August 8th inclusive. The tumour dose was 2000 r.
Irradiation was stopped because of serious diarrhoea. She left hospital and died at home in the first week of September 1961.
METHODS
The excretion of five groups of steroids was estimated before and during treatment with SU-4885 on aliquots of 24-hour urines. Creatinine values are given as a measure of the completeness of the collection.
17-KS and 17-KGS were determined according to the methods of Norymberski et al. (1953) and Appleby et al. (1955), respectively. 17-OHCS were estimated after en- zymatic hydrolysis. The procedure of Silber & Porter (1954) was followed with slight modifications. Allen’s correction was used for the calculation of the final colour in- tensity. 11-oxo(y)-17-OHCS and 11-desoxy-17-OHCS were separated paperchromato- graphically and estimated after elution from the paper as follows:
Estimation of 11-oxo(y)-17-OHCS and 11-desoxy-17-OHCS
50 ml of a mixed 24-hour urine was filtered and brought to PH 4.5 with acetic acid and sodiumacetate. Hydrolysis was effected by incubation at 37º C for 18 hours with 1200 units of ß-glucuronidase (Ketodase) per ml of urine. The hydrolyzed urine was extracted twice with 150 ml of chloroform.
To prevent formation of an emulsion, the extractions were performed in cylindrical separators by rolling them mechanically for two hours per extraction. The extracts were combined and washed twice with 0.1 N sodiumhydroxide solution (15 ml) and twice with water (15 ml), and then dried with anhydrous sodium sulfate. The solute was evaporated in vacuo.
The residue was dissolved in absolute ethanol (1 ml).
Paperchromatographic separation
0.25 ml of the alcoholic solution was put on a strip of filterpaper (Whatman no. 2; 4.5 X 45 cm) in a small zone uniformly over the whole width of the paper.
The paperstrip was equilibrated overnight in the tank for chromatography with the vapour of the aquous and organic phase of system Bush B5 and then developed for 4 hours at 28 + 1º C. After drying the strip in air in the dark, one third was cut off over the whole length and coloured with alkaline blue tetrazolium reagent (0.01 % in 2 N NaOH). Two tetrazolium positive zones develop. With small variations they are found at 2-12 and 20-30 cm from the origin.
The slow moving zone contains the 11-oxo(y)-17-OHCS, the other zone contains the 11-desoxy-17-OHCS. The corresponding zones of the remaining part of the strip were cut out and extracted three times with chloroform-methanol (1:1 ; 5 ml) for 24 hours, 5 minutes and 5 minutes resp. The combined extracts were filtered through a sintered glass funnel and brought to dryness with a stream of air.
Estimation of the Porter-Silber chromogens
The residue was suspended in water (2 ml) and the suspension extracted with chloroform (15 ml). The Porter-Silber chromogens were determined in an aliquot of the chloroform-extracts (Silber & Porter 1954) after re-extraction with reagent. Op- tical densities were read at 380, 410 and 440 mu in a Unicam SP 600 spectrophoto- meter. The 11-oxo(y)-17-OHCS and the 11-desoxy-17-OHCS concentrations were cal- culated by applying the formula of Allen (1950) in order to correct for atypical Porter-Silber chromogens. Extracts of paperblanks gave negligible extinctions with this method. A solution of 20 ug/ml of cortisol in water was used as a standard and was treated in the same way as the paperextracts. The results of both groups are ex- pressed in mg cortisol colour equivalents. They are not corrected with the recovery percentages of extraction and paperchromatography (for cortisol and compound S about 90 %).
Table 1. Running rates of 17,21-dihydroxy-20-ketosteroids in paperchromatographic system Bush B5.
Distance of steroid from origin R$ = Distance of cortisol from origin
| 6a-hydroxycortisol | 0 | 1) |
| 6-hydroxycortisol | 0 | 1) |
| 6-hydroxycortisone | 0.12 | 1) |
| 3-allo-tetrahydrocortisol | 0.33 | 2) |
| tetrahydrocortisol | 0.38 | 2) |
| 3a-allotetrahydrocortisol | 0.55 | 2) |
| allotetrahydrocortisone | 0.70 | 2) |
| tetrahydrocortisone | 0.89 | 2) |
| cortisol | 1.00 | |
| cortisone | 1.91 | 2) |
| tetrahydro S | 2.81 | 2) |
| compound S | 2.86 | 3) |
1) Frantz et al. (1961), Whatman 3.
2) Neher & Wettstein (1956), Whatman 1
3) This work, Whatman 2.
Discussion of the method
The migration rate of steroids which give a positive reaction with phenylhydrazine and sulphuric acid, in the system Bush B5 is given in Table 1. It is evident that the distance between the area of the metabolites of cortisol and cortisone and the area of tetrahydro S and compound S is considerable.
Therefore and in view of the specificity of the colour-reaction with phenylhydrazine with the modification of Silber & Porter, a single chromatographic separation in the system Bush B5 for the determination of the 11-oxo(y)-17-OHCS and the 11-desoxy- 17-OHCS in groups seems to be sufficient.
Cortisone has a position between the two groups (Table 1). The concentration of cortisone in urine is low in comparison with the tetrahydrometabolites so that the interference is within the limits of error of the method. Cortisone was not found on the chromatograms.
4 16-21-hydroxy-20-ketones give a positive reaction with the reagent of Porter & Silber (Mattox et al. 1953). However, these steroids are not detected in human urine.
68-Hydroxycortisol, found in human urine by Burstein et al. (1954), which may be increased in adrenal disorders (Touchstone & Blakemore 1961) remains at the origin in the system Bush B 5. This is also true for 6a-hydroxycortisol (Frantz et al. 1960, 1961). Tetrazolium positive areas at the origin of the chromatograms were not found. however, possibly because only small aliquots of 24-hour urines were used.
| Date | Day of SU-4885 treatment* | Creatinine g/day | mg/day | ratio 11-desoxy 11-oxo(y) | ||||
|---|---|---|---|---|---|---|---|---|
| 17- KS | 17- KGS | 17- OHCS | 11- oxo(y)- 17-OHCS | 11- desoxy- 17-OHCS | ||||
| 1-21 | 242 | 81 | 86 | |||||
| 22 | 175 | 73 | 65 | |||||
| 24 | operation: adrenal tumour and metastasis removed | |||||||
| 2- 9 | 14 | 57 | 36 | |||||
| 10 | 15 | 42 | 23 | |||||
| 11 | 13 | 25 | 13 | |||||
| 12 | 11 | 21 | 8.9 | |||||
| 13 | 7.9 | 16 | 8.8 | |||||
| 14 | 9.0 | 14 | 8.3 | |||||
| 15 | 7.8 | 15 | 9.1 | |||||
| 4-18 | 53 | 28 | 9.8 | |||||
| 19 | 53 | 24 | 10 | |||||
| 6-18 | 1.0 | 260 | 74 | 21 | ||||
| 19 | 1.0 | 248 | 69 | 25 | ||||
| 20 | 0.9 | 258 | 88 | 34 | ||||
| 7- 7 | 1.4 | 400 | 74 | 42 | 33 | 17 | 1.94 | |
| 8 | 1.5 | 500 | 160 | 62 | 36 | 27 | 1.39 | |
| 9 | 1.7 | 541 | 143 | 63 | 32 | 19 | 1.75 | |
| 12 | 1 | 1.3 | 583 | 185 | 63 | 15 | 26 | 0.57 |
| 14 | 3 | 0.9 | 343 | 74 | 31 | 6.7 | 17 | 0.39 |
| 16 | 5 | 1.5 | 671 | 200 | 59 | 6.9 | 30 | 0.23 |
| 18 | 7 | 1.4 | 416 | 195 | 49 | 9.0 | 43 | 0.20 |
| 20 | 9 | 1.4 | 330 | 122 | 40 | 6.5 | 37 | 0.18 |
| 22 | 11 | 1.2 | 403 | 156 | 50 | 6.0 | 46 | 0.13 |
| 24 | 13 | 1.0 | 262 | 140 | 38 | 3.5 | 25 | 0.15 |
| 30 | 19 | 0.8 | 360 | 190 | 33 | 5.5 | 33 | 0.17 |
| 31 | 20 | 1.1 | 386 | 146 | 34 | 3.6 | 24 | 0.15 |
| 8- 6 | 26 | 1.4 | 537 | 171 | 39 | 4.5 | 24 | 0.19 |
| 7 | 27 | 1.1 | 386 | 99 | 31 | 3.9 | 24 | 0.16 |
| 20 | 40 | 1.1 | 366 | 290 | 61 | 7.2 | 86 | 0.08 |
| 21 | 41 | 1.1 | 714 | 474 | 94 | 8.1 | 78 | 0.10 |
| 28 | 48 | 1.3 | 592 | 538 | 24 | 0.9 | 14 | 0.06 |
| 29 | 49 | 0.6 | 354 | 318 | 14 | 1.5 | 11 | 0.14 |
* day 1 to 7 incl. 1 g SU-4885 per day, from day 8: 2 g SU-4885 per day.
RESULTS AND DISCUSSION
In Table 2 the results of the determination of different groups of steroids are given with the creatinine content of the 24-hour urine. Figure 1 shows the steroid excretion during the period of treatment with SU-4885 and during a control period preceding the treatment. The results of the routinely estimated group of 17,21-dihydroxy-20-ketosteroids determined according to the method of Silber & Porter (1954) are not shown in this figure.
METOPIRONE ( g/24 h. ) IRRADIATION 2000”
2
1
40
11-oxo(y) -17 - OHCS ( mg / 24 h.)
20
80
11- desoxy - 17 - OHC S ( mg /24 h.)
60
40
20
600
400
17 - KGS ( mg / 24 h.)
200
600
400
17 - KS ( mg / 24 h.)
200
- 5 4 3 1 3 5 7 9 11 13 19 20 26
27
40 41
48 49
Table 3. Highest, mean and lowest value of 17-KS, 17-KGS, 11-oxo(y)- and 11-desoxy-17-OHCS during a period of three days before SU-4885 (3 determinations) and a period of seven weeks during SU-4885 (15 determinations) expressed in mg per g creatinine.
| 17-KS | 17-KGS | 11-oxo(y)-17-OHCS | 11-desoxy-17-OHCS | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| mg/g creatinine | highest value | x | lowest value | highest value | x | lowest value | highest value | x | lowest value | highest value | x | lowest value |
| before SU-4885 3 days, 3 determinations | 334 | 313 | 286 | 107 | 81 | 53 | 24 | 22 | 19 | 18 | 14 | 11 |
| during SU-4885 7 weeks, 15 determinations | 649 | 399 | 236 | 530 | 205 | 82 | 12 | 5.2 | 0.7 | 78 | 31 | 10 |
The deviation of these determinations and the sum of the 11-oxo(y)-17- OHCS and the 11-desoxy-17-OHCS determined after paperchromatography, was more than 10 mg in five urine samples examined. The cause of the dif- ferences is probably interference of the 17-OHCS estimation by aspecific material. The decrease in the 11-oxo(y)-17-OHCS excretion in our patient is in agreement with the known pharmacological action of SU-4885. The en- zymatic system responsible for the 118-hydroxylation of adrenal steroids is inhibited. When SU-4885 is given to normal subjects, the disturbance of the hypophyseal-adrenal axis results in a three to four fold increase in the ex- cretion of 17-OHCS (Frantz et al. 1960; Liddle et al. 1959). Frantz et al. (1960) reported that the sum of THE, THF and allo THF decreased from an average of 3.5 mg to 2.0 mg and THS increased from 0.1 mg to 14.8 mg in three subjects. None of these increases were consistently seen in the patient under study.
Table 3 gives the highest mean and lowest values of the different steroids calculated per gram of creatinine before and during SU-4885 administration. If the mean values can be taken as some indication of what happens in the growing neoplasm then there is no further increase in corticoid hormone synthesis, as the decrease in 11-oxo(y)-corticoid excretion is equal to the in- crease in 11-desoxy-corticoid excretion. However, there is an increase in the means of the 17-KS and 17-KGS excretion. These values may indicate that the growing adrenal metastatic tissues loose their ability to synthesize the 17,21-di-OH-20-O-side-chain or that there is an increased metabolism of these compounds. An effect of SU-4885 on corticoid sidechain synthesis or on the metabolism of the di-OH-20-O-steroids is not known.
Fukushima et al. (1960) compared the steroid excretion of a patient with adrenal carcinoma in two pooled urines, some days before and during SU- 4885 treatment.
The sum of metabolites of cortisol and compound S increased by 12 %. They concluded, that stimulation of the carcinoma tissues by endogenous ACTH was possible. A patient studied by Jailer et al. (1960) showed a de- crease in the sum of tetrahydrometabolites of cortisol and compound S.
It is known that patients with Cushing’s syndrome and adrenal hyperplasia often show a very fluctuating steroid excretion (Birke et al. 1956; Bassoe et al. 1958). That this can also be the case in adrenal carcinoma is shown in this study (Table 2). Calculation of the sum of 17-KS, 11-oxo(y)-17-OHCS an 11-desoxy-17-OHCS shows an increase above the control period on days 1, 5, 26, 41 and 48 of the SU-4885 period of treatment with values of resp. 450-592 mg (control) and 607-800 mg (SU-4885). The lowest excretion was found on day 13 of the SU-4885 period (291 mg). That any interpretation of these results is difficult, is also shown by the high excretion of 11-desoxy-17-OHCS on days 40 and 41 of the SU-4885 period. It is unlikely that this high excretion
after six weeks of treatment with SU-4885 can be explained by stimulation of the carcinoma by endogenous ACTH.
The course of gamma-ray irradiation (60Co)- 2000 r tumour dose in 15 days from day 13 to day 28 in the period of SU-4885 treatment - is considered to be a low palliative dose. Ramioul (1961) irradiated the adrenals of patients with metastatic mammary carcinoma with X-ray doses of 5000 r. He could not demonstrate a real inhibiting effect on the endocrine functions of the cortex. It is clear that this work has no bearing on the effect of irradiation on adrenal tumours. Still we are inclined to assume that the irradiation did not influence the steroid excretion of the patient. In any case a consistent change was not observed.
It is clear, however, that the excessive glucocorticoid-production of meta- static adrenal carcinoma can be suppressed by SU-4885 over a long period. The worsening of the condition of the patient because of the progressive nature of the disease obscured the clinical effect of the treatment with SU-4885.
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Received on March 28th, 1963.