as 6601-o1s1$02.ou/0
`DU
`T’2ll:32;—{(/jlixiical Endocrinology and Metabolism
`GDP“-ight © 1988 by The Endocrine Society‘
`
`Vol. 86,
`Printed in L
`
`Dose-Response Relationships Between Plasma
`1 Adrenocortic'otro1Sin (ACTH), Cortisol, Aldosterone,
`and 18-Hydroxycorticosterone After Injection of
`ACTH—(1—39) or Human Corticotropin-Releasing
`Hormone in Man*
`
`W. OELKERS. T. BOELKE, AND V. BAHR, WITH THE TECHNICAL ASSISTANCE or
`12. EXNER, B. FAUST, AND H. HARENDT
`Division of Endocrinology, Department of Internal Medicine, Klinikum Steglitz, Freie Uniuersitdt Berlin,
`Berlin, West Germany
`
`very steep. Plasma ACTH levels between 50 and 60 ng/L (11-
`ABSTRACT. The effects of sc injections (at 1500 h) of increas-
`13 pmol/L) stimulated cortisol to almost 80% of the maximal
`ing amounts of synthetic human ACTH-(1-39) (1.25—30 pg) on
`increment obtained with plasma ACTH levels above 300 ng/L
`plasma ACTH, cortisol, aldosterone, and 18~hydroxycorticoster—
`one were compared with those of iv injections of 30 and 100 pg
`(>66 pmol/L). This dose—response relationship is similar to that
`synthetic human CRH in nine normal men. Five micrograms of
`found in clinical tests of the pituitary—adrenal axis (insulin test,
`metyrapone test). The effects of plasma ACTH released by CRH
`ACTH, sc, was the lowest dose that significantly increased
`on cortisol secretion were not significantly different from those
`plasma levels of the three steroids. CRH (30 pg, iv) increased
`of injected ACTH. Our results argue against the hypothesis that
`plasma cortisol and 18—hydroxycorticosterone, but not aldoster-
`the effect of CRH on steroid secretion is mediated or modulated
`one, while 100 pg CRH also raised aldosterone secretion. The
`by POMC-derived peptides other than ACTH. (J Clin. Eridocri—
`dose—response curve (peak plasma ACTH level us. maximum
`nolMe£ab 66: 181, 1988)
`increment of plasma cortisol within the first hour) was initially
`
`
`EY et al. (1) were the first to investigate the rela-
`tionship between plasma ACTH and plasma 17-
`hydroxycorticosteroid concentration during the infusion
`; Pf ACTH-(1-39) (porcine) for 24 11. In their study, max-
`imum 17—OHCS levels were reached with plasma ACTH
`concentrations between 2 and 5 mU/mL (~200—500 ng/
`L or 44-no pmol/L). Fehm et al. (2) concluded recently
`that Dlasma ACTH levels much higher than those in the
`earl)’ morning hours in man are necessary for maximal
`flcute stimulation of cortisol secretion, as occurs during
`Insulin—induced hypoglycemia. The relationship between
`9.931‘ Plasma ACTH and cortisol levels after the iv injec-
`tmn Of Incremental doses of ovine (3) or human (4) CRH
`rllagtgegsts that cortisol secretion is nearly maximally stim-
`pmoei/Lat ACTH levels around 40 or 50 ng/L (8.8—11
`). Since plasma ACTH cannot be increased above
`30
`r.
`.
`.
`.
`.
`‘Hi/[:‘(17.o pmol/L) by injecting even 30 pg/kg ovine
`Recel
`(1
`1.
`Addxevs May1s,19s7.
`“mm 1
`s reouests for reprints to: W. Oelkers, M.D.. Department of
`denbura dM9d1cme, Klinikum Steglitz, Freie Universitat Berlin, Hin»
`2: Th‘: “mm 30. 1000 Berlin 45, West Germany.
`ge
`ppo e
`y a gran 0
`e
`eu sc e orsc ungs
`_ Work was su
`rt d b
`t
`f th D t h F
`h
`-
`mfllflschaft (O9 47/8%)‘
`
`d«
`(0) CRH (3), we investigated ACTH—cortisol
`response relationships by injecting incremental sc d:
`of human (h) ACTH—(1—39). The results are comp:
`with those obtained after iv injection of two stanc
`doses of hCRH. Since the effectiveness of CRH 2
`stimulator of aldosterone is also debated (5-7), we III!
`ured,
`in addition, plasma aldosterone and one of
`potential precursors, 18-hydroxycorticosterone(18-(
`B), after ACTH and CRH injections.
`
`Subjects and Methods
`
`Nine normal men were studied. Their age varied betwee
`and 32 yr, and their body weight between 65 and 78 kg.
`were nonsmokers and were eating an unrestricted diet.
`protocol of the study was approved by the Ethical Commi
`of the Klinikum Steglitz, and each man gave written con.-
`to participate in the study.
`ACTH or hCRH injections were always given at 1500
`the afternoon after the men had eaten a light fat—free mez
`1200 h and had been quietly seated in an armchair for 1 h
`1400 h a plastic cannula for blood sampling was placed
`large forearm vein. Blood was collected in tubes containing
`EDTA for measurements of plasma ACTH, cortisol, aldos
`
`181
`
`Amerigen Exhibit 1183
`Amerigen Exhibit 1183
`Amerigen v. Janssen IPR2016-00286
`Amerigen v. Janssen IPR20l6-00286
`
`

`
`i2
`
`OELKERS, ROELKE, AND BAHR
`
`the blood samples drawn before and at 1530, 1545, and 160%
`after ACTH injection. After hCRH injection,
`these stergii.
`were measured at 1515, 1530, and 1545 h. The BIAS we?
`prece),-(led by paper chromatographic purification of the steroid:
`(8, 9 .
`‘
`All ACTH and cortisol samples were analyzed in duplicate
`and all samples from an individual man were analyzed at m;
`same time. Statistical analyses of the results were performed
`using a STATS 2 program of Statsoft, Inc. (Tulsa, OK) and at
`IBM computer. Since all studies were performed in the Sam;
`subjects, the Wilcoxon test for correlated samples was used
`exclusively.
`
`Results
`
`Figure 1 shows the mean plasma ACTH and Cortjsoi
`concentrations after the injection of 2.5430 pg ACTH’
`sc, and 100 pg CRH, iv. After the administration of 1,2,5’
`pg ACTH (not shown), neither plasma ACTH nor cor.
`tisol increased significantly. Plasma ACTH did rise am,
`the injection of 2.5 pg ACTH, sc, from 11.3 i 2.3 (ism,
`to 18.4 :t 1.2 ng/L (P < 0.05), but plasma cortisol did?
`not increase significantly. After 5 pg ACTH, plasma 1
`ACTH rose to 27.8 :L- 1.4 ng/L (6.2 pmol/L), and cortisol 2::
`rose significantly to 399 nmol/ L (Fig. 1 and Table 1). In g;
`the first half hour after the administration of 100 jig L
`ACTH, cortisol rose almost as high as after the larger
`ACTH doses, although the mean peak ACTH level after 1:
`10 pg ACTH, sc, was much lower than that after 20 or
`30 pg ACTH. After the higher ACTH doses, the riseill “
`plasma cortisol
`lasted longer than after the smaller :._
`ACTH doses. The plasma ACTH peak after iv injection“ :
`of 30 or 100 pg hCRH was earlier than the plasma ACTH ‘V’
`‘
`peak after so ACTH injection. The peak plasma ACTH
`and cortisol levels after 100 pg CRH, iv, were similarto
`those after 10 pg ACTH, sc, while the responses to 30 pg, V;
`CRH, iv, resembled those to 5 pg ACTH, sc (Figs. 1 and V V
`3). The areas under the plasma ACTH and cortisol curves w =
`in the first 60 min after injection also were similar for 5
`pg ACTH and 30 pg hCRH and for 10 pg ACTH and 100
`pg hCRH, as shown in Table 1.
`Figure 2 is a plot of the peak plasma ACTH levels
`after the different dosages of ACTH sc or CRH iv as
`.
`function of the highest plasma cortisol increment within 7
`the first 60 min after injection. The dose—response curve » ‘
`initially was steep, but above peak ACTH levels of bll)0l3l'
`60 ng/L (13 pmol/L) it became Very flat. However, l3h"U=,
`cortisol response to 20 pg ACTH was significantly higher Fj
`.
`(P < 0.05) than that to 10 pg ACTH, but the cortis0l'_‘
`responses to 20 and 30 pg ACTH were not significantlll 3'
`different. The dose—response relationships after injecti?“ T
`of ACTH so or CRH iv fell almost on the same line (F19
`2), although the timing of the peaks was different (FlE5'
`1 and 3).
`Figure 3 shows the effects of the incremental ACTH j 1
`
`I
`
`V
`
`
`
`l
`
`ie, and 18—OH—B at 1445, 1455, 1515 (after CRH only), 1530,
`45, 1600, 1630, and 1700 h. The tubes were put into crushed
`2 and centrifuged immediately at 3 C, and the plasma was
`)red for up to 6 weeks at -70 C before RIA.
`Synthetic hACTH—(1~39) was purchased from Peninsula
`boratories. Inc. (Belmont, CA; lot 009309). Before the study
`s begun, 500 pg ACTH were dissolved in 50 mL sterile 150
`nol/L saline and passed through Millipore filters (0.22 pm;
`.llex—GS, Millipore Corp., Bedford, MA) into 20 sterile plastic
`)es which<—were immgediately frozen at -70 C. Each aliquot
`s thawed 1 h before injection. Dilutions of the thawed ACTH
`utions (theoretic concentration, 10 pg/mL) were compared
`Ieatedly with the standard ACTH used as constituent of the
`3TH RIA kit (see below). In six assays, the ACTH injection
`ution was found to contain 10.8 t 1.1 (iSD) pg/mL ACTH.
`e ACTH solution was drawn into sterile plastic syringes of
`lropriate size and injected sc, using very fine needles, into
`upper arm contralateral to that which had been cannulated.
`lore than 1 rnL ACTH solution had to be given, two or three
`s were injected. The injections caused almost no pain, Each
`[1 received 1.25, 2.5, 5, 10, 20, and 30 pg ACTH in a randorn~
`I order at intervals of at least 3 days. On other occasions,
`men received 30 and 100 pg hCRH (CRF Bissendorfa
`itide G1nbH, Wedemark, West Germany),
`iv, instead of
`PH. No adverse effects of ACTH injection were noted.
`ee men reported a mild warm flush after CRH injection,
`ch persisted for less than 5 min.
`.CTH was measured in unextracted plasma using commer«
`RIA kits obtained from Compagnie Oris Industrie S.A. (Gif
`Yvette, France). These kits contained a rabbit- a1-iti—ACTH
`body directed against the 1~24 sequence of ACTH. hACTH—
`9), calibrated against the ACTH MRC standard 74/555,
`used as standard. The antigen-antibody complex was pre-
`ated by adding a reagent containing an insoluble complex
`ieep antirabbit 'y—globulin. The minimum ACTH Concen-
`on detectable varied between 5 and 7 ng/mL (1.1—1.5 pmol/
`Fhe intraassay variability at a mean ACTH concentration
`).1 ng/ . (4.4 pmol/L) was 11% (n = 9). The interassay
`ibility of a plasma pool with a mean ACTH concentration
`ng/L (11.2 pmol/L; n = 18) was 8.6%. In 40 normal men
`40 yr of age), plasma ACTH levels (0800-0900 h) ranged
`less than 5 (n = 4) to 46.3 ng/L (<1.1—10.2 pmol/L), with
`an of 19.7 i 10.3 (ISD) ng/L (4.3 i 2.3 pmol/L). The
`rla ACTH levels at 1500 h in the study subjects were 9.5 :
`g/L (2.1 i 0.35 pmol/L).
`asma cortisol was measured using CEA Sorin kits With
`ody—coated tubes. The sensitivity ofthe assay was between
`1d 20 nmol/L. The intraassay Variabilities at different
`iol levels (100, 350, and 1000 nmol/L; n = 15 each) were
`3.9%, and 4.5%, respectively. The interassay variabilities
`3 same concentration levels were 9.6%, 5.7%, and 5.5%,
`ctively. The areas under the curve for ACTH and cortisol
`;ion were calculated by multiplying the mean of the hor-
`concentrations at the adjacent times of measurement up
`min by the time (in minutes), followed by summing the
`s of the individual time segments. Basal secretion (prein—
`a hormone level X 60) was then subtracted.
`sma aldosterone and 18-OH-B were measured by RIA in
`
`

`
`

`
`

`
`
`
`ll” They were able to mimic spontaneously occurring
`19 plasma ACTH and cortisol peaks by injecting
`9*“
`fhCRH iv
`M511 doses 0
`-
`_
`_
`‘
`3 The fact that an acute increase in plasma AC’IH to
`‘~
`,3 50 mg/L (13.2 pmol/L) stimulates cortisol secretion
`Ebou
`‘
`ll
`‘
`‘in 0 tant for cl’
`‘cal tests of the
`flmost maxima y 19:1 P T
`1,91
`_
`Situitaryadreiial axis. In most normal subjects, plasma
`TCTH rises to levels above 150 or 200 ng/L (33 or 44
`limo}/L) after insulin-induced hypoglycemia or metyra-
`lone testing (13, 14). In patients with moderately im-
`paired ACTH secretion, these tests will not ‘reveal the
`figmrbance if only the adrenal (plasma cortisol or 11-
`lwxycortisol) response is measured, since an increase in
`lasma ACTH to 50 or 60 ng/L (11 or 13 pmol/L) will
`pad to almost normal stimulation of the adrenal cortex.
`_fter comparison of steroid responses to insulin and
`jetyrapone tests in a large number of patients with
`ltuitary disease (15), we performed the short metyra-
`me test according to the method of Jubiz et al.
`(16),
`ith plasma ACTH and 11-deoxycortisol measurements
`.0800 h in 7 normal subjects and 35 patients with
`tuitary disease. In the normal subjects, plasma ACTH
`creased to 170-360 ng/L (37~'79 pmol/L) and 11-deox-
`ortisol to 210-500 nmol/L. In the patients with pitui-
`ry disease, in whom plasma ACTH increased to be-
`een 50 and 210 ng/L (11 and 46 pmol/L), 11-deoxy-
`rtisol increased into the normal range after metyra—
`ne injection. Only in patients with ACTH increases to
`5 than 55 ng/L (12 pmol/L) was the rise of 11-deoxy-
`tisol subnormal. This finding indicates that the re-
`ts presented in this paper are applicable to the inter-
`ztation of clinical tests.
`teports on the effect of CRH on plasma aldosterone
`controversial. Muller et al. (5) found no significant
`rease in plasma aldosterone after injection of 100 pg
`RH into normal subjects. We also found no significant
`in plasma aldosterone in nine normal recumbent
`1 given 100 pg OCRH at 1700 h (17), while cortisol
`18~0H-B increased markedly. Hcrmus el al. (6) and
`laglen et al. (7), however, found plasma aldosterone
`ise slightly but significantly after the injection of 200
`‘CRH. iv, in normal subjects.
`1 this study, both plasma aldosterone and 18-OH-B
`3 Significantly stimulated by the same threshold dose
`CTH‘(1—39) (5 tag) that significantly stimulated cor-
`- However, with all ACTH and hCRH doses studied,
`Iariability of the aldosterone and 18-OH—B responses
`greater than that of the cortisol responses. While 30
`CRH significantly stimulated plasma cortisol and
`iH‘B (P < 0.01), plasma aldosterone rose minimally.
`H (100 Mg) significantly stimulated aldosterone 45
`after injection, but the response was smaller than
`30f Cortisol and 18—OH-B. Since 30 pg hCRH elicited
`‘*3 ACTH responses similar to those occurring after
`
`ACTH AND CORTICOSTEROIDS
`
`1.
`
`the injection of 5 rig ACTH, it appears that the injectt
`ACTH was more effective in stimulating aldosteror
`than was the ACTH released by hCRH. This sugges’
`the existence of POMC peptides or other CRH-induce
`factors that inhibit the aldosterone response to ACTI
`released by CRH. Altogether, the smaller aldosteron
`responses to ACTH and CRH, compared with those (
`cortisol and 18-OH—B, may be due to the relatively hig
`sodium intake of our subjects, which can blunt aldostei
`one responses nonspecifically (18, 19). Differences i.
`dietary sodium also may explain the contradictory find
`ings of different researchers with regard to aldosteron
`stimulation by CRH (5-7).
`CRH injection stimulates not only the release 0
`ACTH but also that of other POMC—derived peptide.
`(20, 21). 13-Endorphin, 6-lipotropin, and proxy-melano
`tropin have been reported to stimulate aldosterone se
`cretion or enhance the effects of ACTH on aldosterone
`secretion (22-24). We found that hCRH—released ACTI-f
`tended to stimulate aldosterone secretion less than in-
`jected ACTH. This finding argues against the physiolog-
`ical significance of potential aldosterone-stimulating fac-
`tors, other than ACTH, that may be released after hCRH
`injection.
`
`,
`
`.
`
`. TN
`I.
`
`References
`‘By RII, Shirriizii N, Nicholson VVE, Island DP, Iiiddlo GW 1963
`orrelation of plasma ACTH concentration with adrenocortical
`‘response in normal human subjects, surgical patients, and patients
`Vliliatli Cushing’s disease. J Clin Invest 42: I669
`2. Felhm HL, Klein E, Holl R, Voigt KII 1984 Evidence of extrapi-
`t viitary mechanisms mediating the morning peak of plasma cortisol
`n man. J Clin Endocrinol Metab 58:4l0
`3. Orth DN, Jackson RV, de Cherney GS, de Bold CR, Alexander
`AN, Island DP, Rivier J 1983 Effect of synthetic ovine corticotropiri
`releasing factor. Dose response of plasma adreiiocnrticotropin and
`cortisol. J Clin Invest 71:587
`4. Schurmeyer TH, Avgerinos PC, Gold PW, Gallucci WT, Tomai
`TP, Cutler GB, Loriaux DL, Chrousns GP 1984 Human corticotro-
`pin-releasing factor in man: pharmacokinetic properties and dose-
`responso of plasma adrcnocorticotropin and cortisol secretion. J
`Clin Endocrinol Metab 59:l103
`5. Muller 0A, Dorr HG, Hagen B, Stalla GK. von Werder K 1982
`Corticotropin releasing factor stimulation test in normal controls
`and patients with disturbances of the hypothalaino—pituitary—ad-
`renal axis. Klin Wochenschr 60: 1485
`6. Hcrmus ARMM, Pieters GFFM, Smals AGH, Benrziad ThJ, Klop-
`penborg PVVC 1984 Plasma adrenocorticotropin, cortisol, and al-
`dosterone responses to corticotropin-releasing factor: modulatory
`effect of basal cortisol levels. J Clin Endocrinol Metab 58:18-7
`. Conaglen JV, Donald RA, Espiiier EA, Livesey JH, Nicholle MG
`1984 The effect of ovine corticotropin-releasing factor on cate-
`uholamine, vasopressin, and aldosterone secretion in normal man.
`J Clin Endocrinol Metab 587163
`8. Schoneshofer M 1977 Simultaneous determination of eight adrenal
`steroids in human serum by radioimmunoassay. J Steroid Biochem
`8:995
`9. Belkien L, Schoneshofer M, Oelkers VV 1980 Development and
`characterization of antisera to 18~hydroxy-corticosterone and 18-
`hy droxydeoxycorticosterone. Steroids 351427
`10. Oelkers W, Kohler A, Belkien L, Fuchs-Hammoser R, Maiga M,
`Scherer B, Weber PC 1982 Studies on the niochaiiism by which
`ACTH stimulates renin activity and angiotensin II formation in
`
`-2
`
`

`
`186
`
`OELKERS, BOELKE.
`
`AND BAHR
`
`JCE
`
`penh) 112:150
`Gordon R, Nicholls MG, Tree M, Fraser R, Rohertsnn ,,
`Influence of sodium balance on ACTH/adrenal cortioster
`‘V
`response curves in the dog. Am J Physiol 238:E543
`Tuck ML, Sowers JR, Asp ND, Viosca SP, Berg (‘,1 Ma
`1981 Mineralocorticoid response to low dose adrenocgl-ti
`infusion. J Clin Endocrinol Metab 52:440
`
`
`
`Jackson RV, de Cherney GS, de Bold CR, Sheldon wg, Al
`AN, Rivier J, Vale W, Orth DN 1984 Synthetic ovine com
`releasing hormone: simultaneous release of proopiomela
`peptides in man. J Clin Endocrinol Metab 58:740
`. Motomatsu T, Takahashi H, Ibayashi H, Nohunaga M1
`man plasma prnnpiomelanocortin, N-terminal peptide an
`ocorticotropin: circadian rhythm dexamethasone S\lpp1‘e§m'
`corticotropin releasing hormone stimulation. J Clin En
`Metab 591495
`. Matsuoka H, Mulrow PJ, Li CH 1980 B—Lipotropin: a new.
`terone stimulating factor. Science 209:307
`. Al Dujaili RA, Hope J, Estivariz FE, Lowry PJ, Edwards V
`1981 Circulating human pituitary progamma—me1anon-op’
`hances the adrenal response to ACTH. Nature 291:156
`. Giillner HG, Gill JR 1983 Beta endorphin selectively an
`aldosterone secretion in hypophysectomized, nephrectomized
`J Clin Invest 71:124
`
`18.
`
`19.
`
`20.
`
`11.
`
`14.
`
`15.
`
`16.
`
`17.
`
`man. Acta Endocrinol (Copenh) 100:573
`Pedersen RC, Brownie AC 1980 Adrenocortical response to corti—
`cotropin is potentiated by part of the amino-terminal region of
`pro-corticotropin/endorphin. Proc Natl Acad Sci USA 77:22.39
`. Schiirmeyer TH, Avgerinos PC, Booth JD, Gold PW, Ton-iai TI’,
`Lnriaux T)I., Chrofisos GP 1985 Injections of human CRF——a
`rapidly metabolised peptide—cause elevations of plasma ACTH
`and cortisol that mimic endogenous secretory episodes in man.
`Acta Endocrinoi: [Suppl 2571 (Copenh) lO8:177
`. Staub JJ, Noelpp B, Girard J, Baumann JB, Graf S, Ratcliffe JG
`1979 The short metyrapone test: comparison of the plasma ACTH
`response to metyrapone and insulin-induced hypoglycemia. Clin
`Endocrinol (Oxf) 10:595
`Lindholm J, Kehlet H, Blichert—Toff M, Riishede J, Hummer L,
`Dinesen B 1978 Discrepancy between ACTH and cortisol responses
`to insulin induced hypoglycemia. Clin Endocrinol (Oxf) 9:371
`Achenbach K, Oelkers W 1985 Comparison of insulin hypoglycemia
`and short metyrapone tests in patients with pituitary disease. Klin
`Wochenschr 63:769
`Jubiz W, Meikle AW, West DC, Tyler FH 1970 Single dose metyr-
`apone test. Arch Intern Med 1252472
`Haller H, Hensen J, Biihr V, Oelkers W 1986 Effects of angiotensin
`II infusion on the early morning surge of ACTH and on o-CRH-
`provoked ACTH secretion in normal man. Acta Endocrinol (Co—