Steroidogenic responses to varying parenteral routes of administered α1–18 ACTH

Steroidogenic Responses to Varying Parenteral Routes of Administered (~1-18 ACTH J. Howard

Pratt, Sidney

Pave1 1. Dale,

Komanicky, and

In healthy human subjects, the steroidogenie responses to a single injection of al-1 8 ACTH by the subcutaneous, intramuscular, and intravenous (IV) routes were assessed by measurement of plasma cortisol levels, 17-hydroxycorticosteroid excretion, and cortisol secretion rates. Results showed that regardless of the parenteral route used, (Yl-l 8 ACTH at doses of 1.0 and 0.5 mg was an effective adrenocorticotropic agent; however, with the IV route, the effect was considerably prolonged. The patterns of disappearance

James

Thomas

E. Wilson,

C. Melby

within the plasma of al-18 ACTH and al-24 ACTH were compared following a single IV injection of these compounds labeled with “‘I. al-l 8 ACTH produced only a slight initial rise and a slower decline in plasma radioactivity compared to crl-24 ACTH, suggesting slower degmdation of al-18 ACTH. Administerod as a single IV injection, al-18 ACTH has a prolonged effect on steroidogenesis and has potential for being a useful diagnostic and therapeutic ACTH preparation.

YNTHETIC POLYPEPTIDE DERIVATIVES of ACTH can have equal adrenocorticotropic activity and less allergenic potential than the natural hormone. The steroidogenic capacity of the synthetic ACTH, aACTH’-‘* [D-Ser,’ Lys, ” LysNH,‘*]* (al-18 ACTH) has been shown in animals and man to be greater than ACTH with 24 amino acids.lm5 In the present study, the steroidogenic responses to three different parenteral routes of administered (Y1-18 ACTH were compared. Unexpectedly, it was found that al-18 ACTH given as a single intravenous (IV) injection had a more prolonged stimulatory effect on adrenocorticoid function than an equal quantity given by either the intramuscular (IM) or subcutaneous (SC) route.

S

MATERIALS

AND

METHODS

All subjects were healthy male volunteers between the ages of 21 and 55 yr; each group consisted of five subjects. Beginning at 800 a.m., al-18 ACTH, I.0 mg, was given by either the IM, SC, or IV route. A group that received no injection served as controls. Plasma was drawn from all subjects prior to al-18 ACTH administration and at time intervals over the next 48 hr. Twenty-four-hr urine collections were obtained during the 2 days post-ACTH administration.

*In this paper, all synthetic ACTH derivatives are referred_& as a in compliance with previous recommendations for ACTH nomenclature (Li CH, Science 129:969, 1959). It should be noted that in some instances references are made to ACTH derivatives here called (Y and in previous studies referred to asp. From the Section of Endocrinology and Metabolism, Robert Dawson Evans Department of Clinical Research, Department of Medicine, Boston University School of Medicine, Boston, Mass. Receivedforpublication May 21, 1975. Supported in part by Grants-in-aid AM-12027-07. TOI-AM-07002-01. P02-AM08657-1 I, and ROI-HL15732-03 from the National Institutes of Health. Reprint requests should be addressed to J. Howard Pratt, M.D., Section of Endocrinology and Metabolism, Boston University Medical Center, University Hospital, 75 East Newton Street, Boston, Mass. 02118. Q 1976 b.v Grune & Stratton, Inc.

Metabolism,

Vol.

25,

No.

2 (February),

1976

221

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ET Al.

50 r

TIMElh)

2

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I6

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1

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11 30

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24-40

h

I I

q

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g&

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: 1:

I o-24h Q I-18

ACTH

1.0 mg

24-48

h

Fig. 1. A comparison of responses to a single injection of al-18 ACTH, 1.O mg, by the subcutaneous (SC), intramuscular (IM), and intravenous (IV) routes as measured by plasma cortisol concentrations, cortisol secretion rates (CSR), and 17OHCS excretions ( mean f SE).

A similar protocol was followed with a lower dose of (Y l-18 ACTH, 0.5 mg, except that plasma and urine samples collected on the day preceeding ACTH administration served as control values. Plasma cortisol levels were measured by the competitive protein-binding method as described by Murphy.6 The 17-hydroxycorticosteroid (III-OHCS) excretion was determined by quantitation of At the beginning of each urine collection, 1,2,3H-cortisol urinary Porter-Silber chromogens.7 was administered intravenously for the purpose of measuring the cortisol secretion rate (CSR).* A comparison of the disappearance of al-18 ACTH and al-24 ACTH (Cortrosyn@) was made using the following protocol: 50 pg of each ACTH compound was iodinated with 0.4 pCi of

STEROIDOGENIC

RESPONSES

223

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-0

CONTROL

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SC

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IM I”

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TIME(h)

20

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6

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42

48

24.48h

Fig. 2. A comparison of responses to a single injection of al-18 ACTH, 0.5 mg, by the subcutaneous (SC), intmmuscular (IM), and intmvenous (IV) routes as measured by plasma cortisol concentmtions, cortisol secretion rates (CSR), and 17OHCS excretions (mean f values were determined

SE). *Control in the 24-hr

period preceding the study and are a summation of the mean f SE of all the groups.

24-48

h

12sI.9 The iodinated ACTH was found to be more than 90% pure when subjected to paper chromatoelectrophoresis.’ Four normal human subjects (two males and two females) received either iodinated al-18 ACTH or al-24 ACTH injected as a rapid intravenous bolus. Plasma samples were subsequently obtained at timed intervals over the next 2 hr. and the plasma radioactivity measured for each sample. Results were expressed as percentage of injected dose, and the patterns of disappearance of plasma radioactivity were compared. The paired t test was used for statistical analysis.

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ET AL.

RESULTS

al-18

ACTH,

1.0 mg (Fig. 1)

A rise in plasma cortisol concentration followed administration of 1.0 mg of al-18 ACTH regardless of the parenteral route; however, with the IV route, a more prolonged steroidogenic response was found. In all three groups, cortisol levels had increased significantly at 30 min (p < 0.05) following administration of al-18 ACTH and had reached maximal values at 6 hr. Whereas in the IM and SC groups, cortisol levels had returned to below control values at 24 hr, in the IV group, plasma cortisol levels were still nearly maximal and did not return to control values until 42 hr after administration of al-18 ACTH. Seventeen-OHCS excretion in the first 24-hr period following administration of al-18 ACTH was substantially increased in all treatment groups (p < 0.01). In the second 24-hr period, 17-OHCS excretion in the IV group remained elevated in comparison to controls (p < O.Ol), whereas in the IM and SC groups, the excretion rate had returned to control values. Similarly, the CSR increased over control values in all groups in the initial 24 hr following treatment (p < O.Ol), but only in the IV group during the second 24-hr period did the CSR remain high (p < 0.01). al-18 ACTH,

0.5 mg (Fig. 2)

The steroidogenic response to 0.5 mg of al-18 ACTH given intravenously was still of longer duration than that seen by the other parenteral routes; how36

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30

-

b

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5

IO

IS

I

I

20

25 TIME

30

&l-l8

ACTH

ACTH

C_a

35

’

40

lHI--LI 60

90

(MINUTES)

Fig. 3. Disappeamnce within the plasma of “‘1 derivatives of al-18 lowing single intmvenous injections of these compounds.

and al-24

ACTH fol-

STEROIDOGENIC

225

RESPONSES

ever, the effect was less sustained than with 1.0 mg. Plasma cortisol levels were significantly higher in the IV group at 18 hr compared to the IM and SC groups (p < 0.05), but were not significantly different by 24 hr. The 17-OHCS excretion in all groups was substantially increased in the first 24-hr period post-ACTH administration compared with the control period (p < 0.02); and in the IV group, the 17-OHCS excretion was significantly greater than the IM and SC groups (p < 0.05 in both instances). During the second 24-hr period, the 17OHCS excretion was significantly increased over control values only in the IV group (p < 0.05); however, the excretion was only 47% of that observed in the first 24-hr period. Measurements of the CSR showed similar results. In the initial 24-hr period, the CSR in the IV group was greater than the IM and SC groups (p < 0.05 in both instances). In the second 24-hr period, the CSR increased over controls in the IV group only (p < 0.05), and the value was again 47% of that observed in the first 24 hr. Plasma Disappearance Rate of’25i-al-18

ACTH versus al-24

ACTH (Fig. 3)

Subsequent to the intravenous administration of 12’I-al-24 ACTH, there was an early and intense increase followed by a rapid decline in plasma radioactivity. In contrast to this, intravenously administered 125I-~1-18 ACTH resulted in only a modest initial rise with a subsequent slower decline in plasma radioactivity. Clearly, different patterns of disappearance within the plasma were observed. DISCUSSION

Synthetic analogues of the pituitary hormone ACTH with shorter chain lengths retain biologic potency” and have reduced allergenic potential. The more recently synthesized al-18 ACTH” has been shown in human subjects to be more potent than (Yl-24 ACTH,’ and when administered intramuscularly or subcutaneously, produces a more prolonged adrenocorticotropic effect than similar doses of a!l-24 ACTH .’ The present study points to still another feature of the steroidogenic potential of al-18 ACTH. Administered by the IV route, ar1-18 ACTH produced a greater and a more sustained steroidogenic response when compared to other parenteral routes. This effect was seen with doses of both 1.0 and 0.5 mg of al-18 ACTH, but with the higher dose, the sustained response was greater. When a comparison was made of the pattern of disappearance in plasma of a!1-18 ACTH versus (Yl-24 ACTH after the IV injection of lz51 derivatives of these compounds, levels of al-18 ACTH never reached the initial high levels found with al-24 ACTH, and subsequently, plasma radioactivity levels of (Y1- 18 ACTH showed a slower decline suggesting slower metabolism of cy1- 18 ACTH. The actual half-life cannot be accurately estimated by this method since metabolic products of the iodinated ACTH compounds probably contributed to the level of plasma radioactivity; however, a qualitative difference in the metabolic fate of al-18 ACTH versus (~I-24 ACTH can be demonstrated. Daly et al.,12 using a cytochemical method for measurement of ACTH, demonstrated a half-life for al-18 ACTH of 53 min, compared with a half-life of 9 min for al-24 ACTH. The slower metabolism of (Y1-18 AC‘TH might be based on intravascular and/or protein binding that could retard metabolism and provide

226

PRATT

for a slower and more sustained release of the ACTH than would exposure of the material to tissues in subcutaneous or intramuscular It is concluded that with a single IV injection, (~1-18 ACTH potent and prolonged adrenocorticotropic effect that could serve diagnostic and therapeutic agent.

ET AL.

occur with sites. produces a as a useful

ACKNOWLEDGMENT The authors would like to express appreciation to Dr. Rosina B. Dixon of CIBA-Geigy, Summit, N.J. for providing al-18 ACTH (Ba-41795) and for her generous assistance during the execution of this study. REFERENCES I. Desaulles PA, Riniker B, Rittel W: High corticotrophic activity of a short-chain synthetic corticotrophin analogue, in Margoulies M (ed): Proceedings of the International Symposium, Liege, 1968. Amsterdam, Excerpta Medica, 1968, p 489 2. Walser A, Muller T: The adrenocorticotropic effect in humans of several synthetic peptides related to fi l-24 corticotrophin, in Margoulies M (ed): Proceedings of the International Symposium, Liege, 1968. Amsterdam, Excerpta Medica, 1968, p 487 3. Maier R, Barthe PL, Schenkel-Hulliger L, Desaulles PA: The biological activity of [ I-D-serine, 17-18-dilysinel-@corticotrophin-( 1- I8)-octadecapeptide-amide. Acta Endocrinol 68458, 1971 4. Steinetz BG, Sherwood OD, Birkhimer ML, Butler MC, Sawyer WK: Prolonged elevation of plasma cortisol levels in dogs treated with an 18 amino acid synthetic corticotropin. Experientia 29: 1367, 1973 5. Keenan J, Thompson JB, Chamberlain MA. Besser GM: Prolonged corticotrophic action of a synthetic substituted l-18 ACTH. Br Med J 41142. 1971 6. Murphy BEP: Some studies of the proteinbinding of steroids and their application to the routine micro and ultramicro measurement of

various steroids in body fluids by competitive protein-binding radioassay. J Clin Endocrinol Metab 271973, 1967 7. Silber RH, Porter CC: The determination of 17,21-dihydroxy-20-ketosteroids in urine and plasma. J Biol Chem 210~923, 1954 8. Melby JC, Dale SL, Grekin RJ, Gaunt R, Wilson TE: 18-hydroxy-1 I deoxycorticosterone (18-OH-DOC) secretion in experimental and human hypertension. Recent Prog Horm Res 28:287, 1972 9. Berson SA, Yalow RS: Methods in Investigative and Diagnostic Endocrinology, vol 2A. New York, American Elsevier, 1973, p 84 10. Danowski TS, Hofmann K, Weigand FA, Sunder JH: Steroid responses to ACTHlike polypeptides. J Clin Endocrinol Metab 28: 1120,1968 11. Riniker B, Rittel W: (The synthesis of [ 1-D-serine 17,18-dilysinel-P corticotrophin(I-18)-octadecapeptidamide, a potent corticotrophic substance). Helv Chim Acta 53:513, 1970 12. Daly JR, Fleisher MR, Chambers DJ, Bitensky L, Chayen J: Application of the cytochemical bioassay for corticotrophin to clinical and physiological studies in man. Clin Endocrinol 3:335, 1974