Field of the invention

The invention relates to methods to prevent or treat adrenal insufficiency in critically ill patients. The invention relates to the medicinal use of corticotropin releasing hormone or a synthetic derivative thereof.

Introduction

Patients suffering from critical illnesses typically reveal high plasma (free)cortisol concentrations and low-normal plasma adrenocorticotropic hormone (ACTH) [Boonen et a/. (2013) N Engl J Med 368, 1477-1488; Van den Berghe (2016) Am J Respir Crit Care Med 194, 1337-1348]. Boonen & Van den Berghe (2014) J. Clin. Endocrinol. Metab. 99, 1569-1582 review endocrine responses to critical illness. Experts have interpreted the absence of elevated plasma ACTH, particularly in patients with severe infections, as caused by inflammation or hypoperfusion- induced damage to cells of the hypothalamus whereby synthesis of corticotropin- releasing hormone (CRH) and arginine vasopressin (AVP) is hampered. Shock or inflammation could also directly damage the anterior pituitary gland. Also, direct inhibition at the hypothalamus and/or pituitary level by various drugs have been suggested. However, an alternative explanation could be that high circulating free cortisol levels, brought about by suppressed cortisol binding proteins and by reduced cortisol breakdown, exert negative feedback-inhibition at the pituitary and/or the hypothalamic level, as such lowering ACTH, CRH, and AVP- expression/secretion. Nevertheless, during critical illness, ACTH secretion is not completely suppressed unlike what is observed with high doses of exogenous glucocorticoids or in patients with adrenal Cushing's syndrome. This could be explained by other concomitant central activation, such as via stress-induced AVP- increase which could potentiate CRH effects. Also, during the first weeks of critical illness, the frequency of the ACTH and cortisol pulses was found to be normal, whereas pulse amplitudes were lower than normal. However, a recent study has shown that a central suppression of ACTH is present during critical illness [Peeters et at. (2018) Intensive Care Med 44, 1720-1729]. Suppressed ACTH sustained over an extended period of time could predispose to adrenocortical atrophy and dysfunction. Differentiation between hypothalamic lesions, damage to the pituitary corticotropes and adrenal/ectopic causes of Cushing's syndrome can be done by analysing plasma ACTH (and cortisol) responses to an intravenous CRH bolus injection.

Despite the knowledge of the interplay between CRH, ACTH and AVP in adrenal function, there is currently no adequate prevention nor causal treatment of adrenal failure that occurs in critically ill patients.

Summary of the invention

The present invention demonstrates that CRH, known as a diagnostic, can be used as a therapeutic agent to prevent adrenal insufficiency in critically ill patients.

The present data indicate that sustained elevation of circulating free cortisol, brought about by suppressed cortisol binding proteins and by reduced cortisol breakdown, can reduce ACTH responses to a CRH injection specifically in the prolonged phase of critical illness, irrespective of the presence of sepsis/septic shock and irrespective of survival. To confirm this concept, we performed a randomized, double-blind, placebo-controlled crossover cohort study to compare the ACTH (and cortisol) responses to a synthetic human CRH-analogue, in the acute, subacute and prolonged phases of critical illness with those of healthy subjects, in relation to presence of sepsis/septic shock and survival.

Critically ill patients reveal high plasma free-cortisol and low plasma ACTH. The latter has been explained either by shock/inflammation-induced cell damage to hypothalamus and/or pituitary or by feedback-inhibition exerted by free-cortisol, possibly predisposing to central adrenal insufficiency. One can expect augmented/prolonged ACTH-responses to a CRH-injection with hypothalamic damage, immediately suppressed responses with pituitary damage, whereas delayed decreased responses only in prolonged critical illness with feedback- inhibition.

This randomized, double-blind, placebo-controlled crossover cohort study, compared ACTH-responses to 100pg IV corticorelin (CRH) and placebo in 3 cohorts of 40 matched patients in the acute (ICU-day 3-6), subacute (ICU-day 7-16) or prolonged phase (ICU-day 17-28) of critical illness, with 20 matched healthy subjects. CRH or placebo was injected in random order on two consecutive days. Blood was sampled repeatedly over 135 min and ACTH-AUC-responses to placebo were subtracted from those to CRH. The order of the CRH/placebo injections did not affect the hormone-responses, hence results could be pooled. Patients in the acute phase of illness had normal mean ± SEM ACTH -responses (5149 ± 848 pg/mL.min versus 4120 ± 688 pg /mL.min in healthy subjects; P=0.77), whereas those in the subacute (2333 ± 387 pg/mL.min, P=0.01) and the prolonged phases (2441 ± 685 pg/mL.min, P=0.001) were low, irrespective of the presence of sepsis/septic shock or survival. Suppressed ACTH -responses to CRH in the more prolonged phases, but not acute phase, of critical illness are compatible with feed-back inhibition exerted by elevated free-cortisol, rather than by cellular damage to hypothalamus and/or pituitary.

Based upon the present data, CRH administration is a plausible method to prevent or treat central adrenal insufficiency especially in long-stay ICU-patients.

Prolonged feedback-inhibition exerted by elevated free cortisol, and not inflammation/shock-induced hypothalamic or pituitary cell damage, explained suppressed ACTH-responses to CRH exclusively in the more prolonged phases of critical illness. These findings indicate that CRH infusion can prevent the development of central adrenal insufficiency in long-stay ICU patients.

The invention is further summarized in the following statements:

1. Corticotropin releasing hormone or a synthetic derivative thereof, for use in the prevention or treatment of adrenal insufficiency in a critically ill patient.

2. The corticotropin releasing hormone or a synthetic derivative thereof, for use in accordance with claim 1, wherein the synthetic derivative thereof is corticorelin.

3. The corticotropin releasing hormone or a synthetic derivative thereof, for use in accordance with claim 1 or 2, wherein the corticotropin releasing hormone or a synthetic derivative thereof is administered as a bolus at a concentration between 0.5 to 2.5 pg/kg bodyweight at a frequency from 1 to 24 injections per 24h. For example 0.5, 0.75, 1, 1.25, 1.5, 1,75, 2, 2.25 or 2.5 pg/kg bodyweight and ranges with any pair of the above values a lower and upper concentration. For example 1, 2, 4, 6, 8, or 12 injections per 24 h.

4. The corticotropin releasing hormone or a synthetic derivative thereof, for use in accordance with claim 1 or 2, wherein the corticotropin releasing hormone or a synthetic derivative thereof is administered intravenously at a concentration of 0.5 pg/kg/h to 2.5 pg/kg/h. For example 0.5, 0.75, 1, 1.25, 1.5, 1,75, 2, 2.25 or 2.5 pg/kg/h and ranges with any pair of the above values a lower and upper concentration.

5. Corticotropin releasing hormone or a synthetic derivative thereof, for use in the prevention or treatment of adrenal insufficiency in a critically ill patient in accordance with any one of claims 1 to 4, wherein the administration starts between 2 to 7 days after admission of the patient on an intensive care unit. For example 2, 3, 4, 5, 6 or 7 days after admission of the patient on an intensive care unit.

6. Corticotropin releasing hormone or a synthetic derivative thereof, for use in the prevention of adrenal insufficiency in a critically ill patient in accordance with any one of claims 1 to 5, wherein the administration starts 2 days after admission of the patient on an intensive care unit.

7. Corticotropin releasing hormone or a synthetic derivative thereof, for use in the prevention of adrenal insufficiency in a critically ill patient in accordance with any one of claims 1 to 6, wherein the administration is performed for a period between 1 to 28 days. For example during 1, 2, 5, 7, 10, 24, 21, or 28 days.

8. Corticotropin releasing hormone or a synthetic derivative thereof, for use as a medicament.

9. A method of increasing free cortisol in a critically ill patient, comprising the step of administering corticotropin releasing hormone or a synthetic derivative thereof, thereby preventing or treating adrenal insufficiency in said critically ill patient.

FIGURE LEGENDS

Fig. 1 Flowchart of the study participants and study design, a. Flowchart of the study participants b. Randomization into crossover subgroups. ICU denotes intensive care unit. *** blood sample.

Fig. 2 Plasma ACTH, total and free cortisol concentrations after CRH or placebo injection over time in ICU. Data are shown as mean ± SEM on a logarithmic scale. ICU denotes intensive care unit.

Fig. 3 Incremental (a) ACTH, (b) total cortisol and (c) free cortisol- responses to CRH and placebo in 3 patient cohorts. The AUC hormone- responses to placebo were subtracted from the AUC hormone-responses to CRH and indicate the incremental hormone-responses. Data are shown as mean ± SEM on a logarithmic scale. ICU denotes intensive care unit. The horizontal areas represent the mean ± SEM incremental hormone-responses from the 20 healthy subjects. * P<0.05, ** P<0.01, and *** P<0.0001 for the comparisons with healthy subjects. The numerical P-values are those for the comparisons between patient cohorts.

Fig. 4 Incremental ACTH-responses to CRH and placebo in 3 patient cohorts, in (a) survivors and non-survivors, (b) patients with and without sepsis, and (c) patients with and without septic shock. The AUC ACTH- responses to placebo were subtracted from the AUC ACTH-responses to CRH and indicate the incremental ACTH-responses. Data are shown as mean ± SEM on a logarithmic scale. ICU denotes intensive care unit. The horizontal areas represent the mean ± SEM incremental hormone-responses from the 20 healthy subjects. The numerical P-values are those for the comparisons between patient groups. Fig. 5 Estimated half-life of plasma (a) ACTH, (b) total cortisol and (c) free cortisol in patients compared to healthy subjects.

The plasma half-lives were estimated by dividing In2 by the estimated elimination rate constant, calculated from the slope of the regression line of the log- transformed linear decline of the concentration over time, h denotes hour. The numerical P-values are those for the comparisons between patients and healthy subjects.

A critically ill patient suffers from adrenal insufficiency when circulating cortisol levels are not sufficiently elevated to deal with the levels of stress of the illness and which can result in hypotension, vasopressor dependency, unexplained neurological dysfunction and other organ dysfunctions. A plasma cortisol in a critically ill patient that is not higher than normal (higher than values from healthy subjects) would be an indication. A central adrenal insufficiency is diagnosed when in this condition, the circulation plasma ACTH is also not elevated above normal values. Other diagnostic tests such as a short corticotropin (Synachten) test cannot be used in ICU patients as the results of this test are flawed by the increased distribution volume [Peeters et al (2018) Intensive Care Med 44, 1720- 1729].

"Prevention" in the context of the present invention refers to a medical intervention wherein the disease or its symptoms are completely or partially prevented, as reflected by the maintenance of elevated (fully or in part) above normal plasma cortisol in critically ill patients that are in proportion to the severity of illness. "Treatment" in the context of the present invention refers to a medical intervention wherein the disease or its symptoms are completely or partially reversed as indicated by a clearly detectable rise in plasma cortisol in response to the intervention.

"Corticotropin-Releasing Factor (CRF)" is an endogenous 41 amino acid peptide first identified as the major hypothalamic hormone responsible for stimulation of the pituitary-adrenal axis. (Vale et al. (1981) Science 213, 1394- 1397 ). CRF is also known in the art as corticotrop(h)in-releasing hormone (CRH), corticoliberin, corticorelin and CRF-41.

Homologous of rat, ovine, sheep, goat, porcine and fish, whether isolated from natural source extraction and purification, from recombinant cell culture systems or synthesized using peptide synthesis technology.

"Corticorelin (CRH)" is a synthetic agent which is chemically identical or similar to the endogenous form of human corticotropin-releasing hormone (hCRH), and commercially available as corticorelin, and is a potent stimulator of ACTH release from the pituitary gland. Typically human CRH is used, although ovine CRH is equally suitable.

Equally envisaged in the context of the present application are CRF-related peptides which share one or more of the biological activities of the native CRF peptides such as urocortin (Vaughan et al. (1995) Nature 378, 287-292, urotensin I (Lederis et al. (1982) Science 218, 162-164) and sauvagine (Montecucchi et al., Int. J. Pep. Prot. Res. 16, 191-199. Peptides differing in one or more amino acids in the overall amino acid sequence as well as substitutional, deletional, insertional and modified amino acid variants of CRF which substantially retain the biological activity normally associated with the intact CRF peptide, are equally envisages in the context of the present invention.

Further envisaged are other CRF-related peptides which share one or more of the biological activities of the native CRF peptides such as urocortin (Vaughan et al. (1995) Nature 378, 287-292), urotensin I (Lederis et al. (1982) Science 218, 162-164) and sauvagine (Montecucchi et al. (1980) Int. J. Pep. Prot. Res. 16,

191-199). The CRF peptides employed in the formulations of the present invention are preferably synthesized using solid- or solution-phase peptide synthesis techniques, however, other sources of the CRF peptide are readily available to the ordinarily skilled artisan. The amino acid sequences of the human, rat and ovine CRF peptides are presented in Figure 1. The terms "corticotropin releasing factor" and "CRF" likewise cover biologically active CRF equivalents; e.g., peptides differing in one or more amino acids in the overall amino acid sequence as well as substitutional, deletional, insertional and modified amino acid variants of CRF which substantially retain the biological activity normally associated with the intact CRF peptide.

US5869450 describes CRH analogs in which the fifth amino acid from the N- terminus is D-Pro or in the case of urocortin or sauvagine where the fourth amino acid from the N-terminus is D-Pro or D-Ser.

Cyclic CRH agonists are disclosed in US5844074 and US5824771. These CRH analogs, modified by cyclization of residues 30-33 of CRH via a glutamic acid- lysine bridge, are more potent than native CRH in the release of ACTH and have lower molecular weight than native CRH. The elimination of residues 1-3 or 1-11 at the N-terminus of CRH has been shown to not alter biological activities or ACTH- release potency.

Other variants of CRH, have amino acid sequences of the CRH superfamily but the amino residue at the 20 position has been replaced with a D-amino acid residue. Yet further variants are disclosed in EP1558760.

"Critically ill patient", as used herein refers to a patient who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to disease or injury, a patient who is being operated and where complications supervene, and a patient who has been operated in a vital organ within the last week or has been subject to major surgery within the last week. In a more restricted sense, the term a "critically ill patient", as used herein refers to a patient who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to disease or injury, or a patient who is being operated and where complications supervene. In an even more restricted sense, the term a "critically ill patient", as used herein refers to a patient who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to disease or injury. Similarly, these definitions apply to similar expressions such as "critical illness in a patient" and a "patient is critically ill".

"Adrenal insufficiency" is defined as, and diagnosed by circulating cortisol levels that are not sufficiently elevated to deal with the levels of stress of the illness and which can result in hypotension, vasopressor dependency, unexplained neurological dysfunction and other organ dysfunction. A plasma cortisol in a critically ill patient that is not higher than normal (which is not higher than values from healthy subjects, depending on the type of assay used, for example plasma total cortisol not higher than 20 pg/dl, and free cortisol not higher than 1.5 pg/dl), would be an indication. One absolute level of plasma cortisol cannot be identified as diagnostic, as this depends on the individual plasma binding proteins, distribution volume and severity and stage of illness. A central adrenal insufficiency is present when in this condition also plasma ACTH is not higher than normal (for example not exceeding 50 pg/ml). This would then be accompanied by a lower than normal incremental ACTH response to a CRH 100 pg IV bolus injection. Other diagnostic tests such as a short corticotropin (Synachten) test cannot be used in ICU patients as the results of this test are flawed by the increased distribution volume [Peeters et al. (2018) Intensive Care Med 44, 1720- 1729].

A critically ill patient is diagnosed as protected from adrenal insufficiency by CRH administration when the disease or its symptoms are completely or partially prevented or reversed, as reflected by the maintenance or restoration of elevated plasma cortisol concentrations that are higher than in healthy subjects and are elevated in proportion to the severity of illness.

The example of the present invention is designed to distinguish the following possibilities. If during critical illness, the hypothalamus would be acutely damaged by shock or inflammation, and the anterior pituitary gland would be intact, one would expect augmented/prolonged ACTH-responses. If the pituitary would be acutely damaged by shock or inflammation, suppressed ACTH-responses would be expected from the early phase onward. Alternatively, if ACTH is suppressed by feedback-inhibition at the level of the pituitary and hypothalamus, as in patients with adrenal/ectopic Cushing's syndrome or on high doses of glucocorticoids, the ACTH-responses to a CRH injection expectedly depend on the duration of hypercortisolism, with initially normal ACTH-responses to CRH injection followed by lowered ACTH-responses in the prolonged phase of illness.

The examples of the present invention demonstrate that, in the presence of low/normal baseline plasma ACTH and increased plasma (free)cortisol concentrations, incremental ACTH-responses to CRH in patients in the acute phase of critical illness were normal, whereas ACTH-responses became ± 55% lower than normal in the later phases, irrespective of the presence of sepsis/septic shock or survival. Interestingly, the total cortisol-responses to CRH were always lower than in healthy subjects whereas the free cortisol-responses were always normal, in line with increased cortisol distribution volume during critical illness. The time courses of the ACTH-responses to CRH were thus compatible with feed-back inhibition exerted by elevated free cortisol, rather than with hypothalamic and/or pituitary cell damage. These findings indicate that CRH can offer potential for prevention of central hypoadrenalism in ICU patients who require intensive care for several weeks, for whom it has been shown that free cortisol levels are no longer elevated [Peeters (2018) Intensive Care Med 44, 1720-1729]. The absence of hemodynamic instability in response to the CRH injections in the patients of this study is an important safety aspect for future studies.

The observation of a normal ACTH response to CRH in the first few days of critical illness argues against a damaged hypothalamus or pituitary by hypoperfusion or inflammation. The finding that presence of sepsis or septic shock did not affect ACTH-responses at any time during the course of critical illness further supports this interpretation. The 55% lowering of the ACTH-responses to CRH in the subacute and prolonged phase of critical illness corroborates sustained feedback- inhibition by elevated circulating free cortisol and is in line with the previously documented suppressed nocturnal pulsatile ACTH secretion during critical illness. Indeed, a similar degree of suppression of the ACTH-response to CRH has been reported for patients after surgical treatment for Cushing's syndrome and for patients after withdrawal of >2 weeks of therapeutic glucocorticoid treatment. The suppressed ACTH-responses to CRH observed in the subacute/prolonged phases of critical illness is compatible with low endogenous CRH and/or low vasopressin signalling, that both can be suppressed by high circulating levels of glucocorticoids. During health, hypothalamic CRH-neurons co-express CRH and AVP, which synergistically activate distinct signalling pathways within pituitary corticotropes. It is well known that AVP is only a weak direct stimulator of ACTH but a much more powerful synergiser of CRH, and thus AVP action may be required for a normal ACTH-response to exogenous CRH. Vice versa, experiments in CRH knockout mice have shown that ACTH secretion depends on CRH. Reactivation of hypothalamic CRH secretion is indeed crucial for the reactivation of ACTH secretion after withdrawal of chronic glucocorticoid treatment. Downregulation of CRH expression, via activating the glucocorticoid receptor, can be brought about by elevated free cortisol and/or by high circulating levels of bile acids that have previously shown to characterize subacute and prolonged critical illness. A postmortem study of human patients who died from septic shock after an illness of approximately one week, reported reduced ACTH mRNA levels in the pituitary gland [Polito et at. (2011) PLoS One 6; e25905]. This suppressed ACTH gene expression occurred in the absence of a compensatory rise in the expression of CRH and vasopressin in the hypothalamus and without altered expression of the CRH-receptor 1 and the vasopressin-receptor (Vlb), supporting our current findings. The results of the current study however cannot rule out a direct pituitary defect due to effects of inflammation and/or hypoxia selectively in the more prolonged phases of illness.

Remarkably, in all patients, irrespective of the duration of illness, total cortisol- responses to CRH were lower than normal whereas free cortisol-responses were always normal. This is in line with a recent study of long-stay patients who received weekly short ACTH stimulation tests for 4 weeks in the ICU, that revealed uniformly low incremental total cortisol-responses but normal incremental free cortisol-responses, explained by low plasma binding and increased cortisol distribution volume. In the current study, with increasing duration of critical illness, both total and free cortisol-responses tended to further decrease. This could be partially explained by the suppressed ACTH release in response to CRH and/or by the onset of decline of adrenocortical function. Indeed, appropriate ACTH signalling is essential to maintain integrity and function of the adrenal cortex. A postmortem study of adrenal glands harvested from patients who had been critically ill for several weeks showed loss of zonational structure, lipid droplet depletion, and suppressed ACTH -regulated gene expression [Boonen et a/. (2014) J Clin Endocrinol Metab 99, 4214-4222]. Suppressed ACTH secretion could thus negatively affect adrenal function in long-stay ICU patients. Such a negative effect of suppressed ACTH could also explain why critically ill patients beyond the fourth week in the ICU were recently shown to have circulating (free)cortisol levels that were not higher than those of healthy subjects, despite their severe illness and high risk of death [Peeters et at. (2018) Intensive Care Med 44, 1720-1729]. One week after ICU discharge on the regular ward, survivors had higher than normal plasma ACTH and (free)cortisol levels, although they were recovering. This further suggested a central adrenocortical suppression during the ICU phase, which could predispose long-stay ICU patients to central adrenal insufficiency.

In the present study no hypothalamic and pituitary tissues were available for quantification of expression of CRH, vasopressin, ACTH, and of the CRH-receptor 1 and vasopressin-receptor. This should be done in validated animal models of prolonged critical illness. It also cannot be excluded that additional suppression at the hypothalamic level from analgo-sedative drugs that are used throughout ICU stay, of which opioids are the main component. Indeed, intra-operative opioids and prolonged opioid use for chronic pain have shown to lower plasma ACTH concentrations. Furthermore, in healthy subjects, morphine blunts the ACTH- response to CRH injection at a supra-pituitary level. However, given the normal ACTH-responses to CRH, observed during the acute phase, when opioid doses are usually higher than in the later phases, an important role of opioids is unlikely. The strengths of the study were the randomized, double-blind, placebo-controlled crossover design, which allowed to compare matched patients in different phases of critical illness while minimizing confounders.

Our findings open perspectives for novel strategies to protect long-stay ICU patients against the risk of developing adrenal insufficiency. If the lack of priming of the corticotropes by CRH would be responsible for reduced ACTH expression and secretion, providing CRH could potentially allow (re)activation of ACTH synthesis and release in response to any fall in cortisol and could hereby prevent adrenal atrophy in the prolonged phase of illness. It has been shown that continuous infusion of CRH can reactivate ACTH secretion with preservation of circadian rhythmicity and pulsatility.

Studies of CRH infusion in the critically ill should initiate this intervention rather early, for example after the first 2 days in the ICU when a longer ICU dependency is foreseen, because at that time, the corticotropes are still fully responsive to CRH . If corticotropes remain sensitive to feedback-inhibition, CRH infusion may not result in too high plasma cortisol and would respect any eventual tissue- specific regulation of cortisol action, which are important safety aspects. In the current study, no side effects of a CRH bolus were noted. However, caution is warranted given that CRH has also been involved in anxiety disorders, depression, memory and learning, and is able to increase catecholamines and heart rate. If a direct pituitary defect would be present in the prolonged phases of illness, which we could not exclude, CRH will not be able to prevent this.

In conclusion, the results of the CRH tests did not support the presence of shock/inflammation-induced hypothalamic and/or pituitary damage in critically ill patients, in which case CRH would not be effective as a prevention or treatment. Instead, the consequences of prolonged feedback-inhibition exerted by elevated (free)cortisol are compatible with suppressed ACTH-responses to CRH in the prolonged phases of critical illness. These findings indicate CRH infusion can prevent or treat the development of a central adrenal insufficiency in long-stay ICU patients. Example 1: administration of CRH to critically ill patients Study participants and sample size calculation

This randomized, double-blind, placebo-controlled crossover cohort study was performed in 5 medical/surgical ICUs at the University Hospitals of Leuven, Belgium. The study aimed at comparing 3 cohorts of unique adult (age > 18y) critically ill patients, matched for demographics, comorbidities and type/severity of critical illness upon ICU admission (Table 1), assessed in the acute (ICU day 3- 6), subacute (ICU day 7-16) or prolonged phase (ICU day 17-28) of critical illness, with demographically matched healthy control subjects. All patients with a stabilized condition for at least 48h, and an expected stay in ICU for at least another 48h, were screened for eligibility.

Table 1: characteristics

Legend Table 1: Participant characteristics

aThe body-mass index (BMI) is the weight in kilograms divided by the square of the height in meters. ^b The Acute Physiology and Chronic Health Evaluation II (APACHE II) score reflects severity of illness, with higher values indicating more severe illness, and can range from 0 to 71. ^C ICU denotes intensive care unit. ^d Incidence of sepsis and septic shock was defined according to the Bone criteria and the 2016 SEPSIS definitions. ^e ci denotes critical illness. The * indicates the comparison between healthy subjects and all patients. The **indicates the comparison between patient cohorts

Exclusion criteria were administration of glucocorticoids within the last 72 hours, chronic treatment with glucocorticoids or other steroids within the last three months, use of etomidate within the last 72h, use of azoles within the last 7 days, other drugs predisposing to adrenal insufficiency (phenytoin, rifampicin, glitazones, imipramin, phenothiazine, phenobarbital), no longer requiring vital organ support, no arterial or central venous catheter in place, referral from another ICU, cerebral disease with intracranial hypertension threatening the neuroendocrine system, pituitary disorders, known adrenal disease, enrolment in another trial, or expected death within 12h (Fig. la).

The sample size of the study was determined based on an estimated effect size of a long duration of critical illness on the ACTH- responses to corticorelin, a synthetic human CRH analogue that is further referred to as CRH. Twenty unique patients per cohort would allow to detect, with an alpha error of 1% or less and a power of 80% or more, a suppression of the ACTH-response to CRH in long-stay critically ill patients of the same size ( ± 60% decrease) as previously reported for Cushing's patients on replacement hydrocortisone treatment 7-9 days after surgical removal of the tumour, in comparison with the response of 20 healthy volunteers. To further account for confounding by various illness-related aspects, the required number of patients was doubled to 40 unique patients per cohort (total of 120) (Fig. la). If a patient, who had been included in a certain time cohort was still in ICU and eligible for including in a later time cohort, this patient was tested again. The results from these repeated tests within the same patient were not included in the primary analysis but were analysed separately as a secondary, additional, longitudinal analysis of the impact of duration of illness. Screening for eligible patients started on July 1, 2016, and continued until the preset number of 40 patients in all 3 cohorts was reached (May 10, 2018), with comparable proportions of 4 diagnostic categories (Table 1 and Fig. la). The study protocol was in accordance with the 1964 Declaration of Helsinki and its later amendments, was approved by the Institutional Ethical Review Board (S58941) and made available prior to study start (ISRCTN14587520).

Clinical data, study design, and sample collection

Demographic, ICU admission and patient characteristics at study inclusion in a time cohort, and patient outcomes were documented (Table 1). After obtaining written informed consent from the healthy volunteers and from the patients or the patients' next of kin, intravenous injections of either 100pg of the synthetic human CRH analogue (CRH Ferring®) in 1ml 0.9%NaCI or of placebo (1ml 0.9%NaCI) were given on two consecutive days at 11 :00 AM, in a random order (Fig. lb). Concealment of order assignment was ensured by the use of a central computerized randomization system. The randomization was stratified in permuted blocks of 2 according to cohort number and the 4 diagnostic admission categories. The block size was unknown to the medical and research teams. Members from the clinical staff who were not involved in the study or patient care, were responsible for preparation and blinding of study medication. Patients, healthy subjects, and the research team were blinded for CRH or placebo injection.

Undiluted blood was sampled 15 minutes before injection of placebo or CRH, immediately prior to injection, as well as 5, 10, 15, 30, 45, 60, 90, and 120 minutes after injection. Samples were taken via an arterial catheter in place for clinical purposes for ICU patients and via a venous puncture for the healthy subjects. As required for accurate quantification of plasma ACTH concentrations, blood samples were collected in pre-chilled EDTA tubes and immediately placed on ice, centrifuged at 4°C and stored at -80°C until assay.

Plasma ACTH concentrations were measured with a double-monoclonal immunoradiometric assay (Brahms Diagnostics, Berlin, Germany). Total plasma cortisol concentrations (Immunotech, Prague, Czech Republic) and plasma cortisol- binding-globulin (CBG) concentrations (Riazen, Louvain-La-Neuve, Belgium) were quantified by competitive radio-immunoassay. Plasma albumin was quantified by the bromocresol green colorimetric method (Sigma-Aldrich, St. Louis, Missouri, USA). Plasma free cortisol was calculated using the Coolens' formula adapted for albumin and CBG concentrations, which has been previously validated as representative of measured free cortisol concentrations in the ICU context. Data and statistical analyses

Within the crossover design, each patient or healthy subject served as his/her own control. First, it was investigated whether the order of administration of placebo and CRH affected the hormonal responses and if this was not the case, the results for placebo and CRH could be pooled for further analysis. To determine the change in the area under the curve (AUC) of plasma ACTH and (free)cortisol in response to placebo or CRH, the plasma concentrations of sample 1 and 2 (before injection) were averaged and served as baseline, after which the AUC was calculated by the trapezoidal rule, on the placebo and the CRH test day. The AUC of the hormone- responses to placebo were than subtracted from the AUC of the hormone-responses to CRH, to determine the "delta AUC", which is further referred to as the "incremental hormone-response". In addition, plasma half-life of ACTH and of cortisol were estimated by dividing In2 by the estimated elimination rate constant, calculated from the slope of the regression line of the log -transformed linear decline of the concentration over time.

All data are presented as mean ± standard error of the mean (SEM), median and interquartile range (IQR), or numbers and percentages. Comparisons of normally distributed data were performed with use of unpaired Student's t-tests, and Wilcoxon rank-sum test was used to compare non-normally distributed data. Proportions were compared with the use of chi-square tests. To compare time-series, repeated measures ANOVA was used, where necessary after transformation to obtain a near- normal distribution. Statistical analyses were performed with use of JMP® Pro 13.0.0 (SAS Institute, Cary, NC, USA). Two-sided P-values at or below 0.05 were considered to indicate statistical significance.

Results

Patient characteristics and baseline plasma concentrations of ACTH and (free)cortisol

One hundred and twenty critically ill patients and 20 healthy subjects were studied (Table 1). The 3 time cohorts (median 4 days, 9 days or 19 days in ICU) had equal proportions of patients within the 4 admission diagnostic categories and of emergency admissions and had similar admission APACHE II scores. For each time cohort, as compared with healthy subjects, patients had similar morning plasma ACTH concentrations, higher plasma (free)cortisol concentrations, lower plasma cortisol binding proteins (CBG and albumin) concentrations (Table 1). With increasing time in ICU, plasma ACTH and CBG concentrations increased slightly, whereas plasma (free)cortisol remained high and albumin concentrations remained low. Of the 120 patients, 87 (73%) suffered from sepsis and 55 (46%) suffered from septic shock at study inclusion, 26 (22%) patients died in the ICU, and 42 (35%) died while in hospital.

Plasma incremental ACTH-responses to CRH over time in ICU

For patients as well as healthy subjects, the order of the CRH/placebo injections did not affect the ACTH-responses (P=0.15 for the acute phase, P=0.08 for the subacute phase, P= 1.00 for the prolonged phase, and P=0.16 for the healthy subjects) (Fig.2). Accordingly, results could be pooled for further analysis.

As compared with ACTH-responses of healthy subjects, the ACTH-responses of patients in the acute phase of critical illness were similar, whereas those in the subacute and the prolonged phases were lower (Fig.3a). The mean ACTH-responses to CRH decreased by 55% from the acute to the subacute phase, and remained constant from the subacute to the prolonged phase (Fig.3a).

Of the 120 unique patients, 30 patients were tested more than once. Of these 30 patients, 19 were tested in the acute and the subacute phase, and 14 were tested in the subacute and prolonged phase. Longitudinal analyses of these repetitive tests within patients confirmed the results of the unique patient cohorts, with a decrease of the mean ACTH-responses to CRH by 60% from the acute to the subacute phase (P=0.01) and no further change from the subacute to the prolonged phase (P=0.74). Plasma incremental (free)cortisol-responses to CRH over time in ICU

As compared with total cortisol-responses to CRH of healthy subjects, total cortisol- responses of patients in the acute, subacute, and prolonged phases of critical illness were always lower (Fig.3b), whereas the free cortisol-responses were always normal (Fig.3c). As compared with the acute phase of critical illness, total cortisol-responses to CRH tended to further lower (Fig.3b) and free cortisol-responses further lowered (Fig.3c) in the prolonged phase.

In healthy subjects, the ACTH-responses to CRH correlated positively with the total cortisol-responses (P=0.001, R ² =0.43) and with free cortisol-responses (P=0.004, R ² =0.37). Patients also showed positive correlations between ACTH- and total cortisol-responses (P=0.001, R ² =0.09) and between ACTH- and free cortisol- responses (P=0.0003, R ² =0.10), but these correlations were much weaker than in healthy subjects.

Estimated haif-iife of plasma ACTH and (free)cortisoi over time in ICU

The estimated plasma half-life of ACTH in patients was always similar to that in healthy subjects (P=0.57, Fig. SI). The estimated plasma half-life of total cortisol was a mean 3.25-fold longer in patients than in controls (P=0.0002), and the estimated plasma half-life of free cortisol was a mean 3.10-fold longer in patients than in controls (P=0.006).

Comparison of survivors with non-survivors , and patients with and without sepsis/septic shock

The ACTH-responses were always similar for hospital survivors and non-survivors, for patients with and without sepsis, and for patients with and without septic shock (Fig.4). This also applied to the (free)cortisol-responses (data not shown).

Side effects of CRH injection

None of the patients revealed hemodynamic instability in response to any of the test injections, whereas a sense of flushing was reported by all healthy subjects on one of the 2 study days.

Example 2 : animal and human pilot experiments

A mouse study to assess the impact of CRH treatment on the development of critical illness-induced central hypoadrenalism is performed as follows:

A mouse model of fluid-resuscitated, antibiotic-treated, sepsis-induced prolonged critical illness, is used to compare the impact of continuous peripheral or central CRH infusion (daily dose between 0.25-1 pg/day) with placebo over time. The intervention aims to prevent the development of adrenal atrophy and maintain plasma ACTH and cortisol with increasing duration of illness.

As an alternative to a continuous infusion strategy wherein fading effects may occur, a bolus strategy (per repeated 10pg/kg subcutaneous injections) is used.

A human experiment includes ICU patients still requiring intensive medical care after 48h with the perspective of a longer ICU stay. Corticorelin (CRH) is infused continuously via a central or peripheral venous catheter at a dose of 1 pg/kg/h (ranging from 0.5 pg/kg/h to 2.5 pg/kg/h) for up to 5 days (first experiment) and throughout ICU stay up to 4 weeks. Time series of plasma ACTH and (free) cortisol are documented, as well as clinical outcomes and safety. The intervention prevents the development of adrenal atrophy in the ICU.

As an alternative to continuous infusion strategy to prevent fading effects, a bolus strategy (per repeated 100 pg or ranging from 0.5 to 2.5 pg/kg bodyweight injections) is used.