1β-hydroxylase (CYP11B1) (IC50 = 35 nM)

Cortisol and aldosterone are inhibited by osilodrostat (LCI699; 0.01-10 µM; HAC15 cells, 17 primary human adrenocortical cell cultures, and pituitary adenoma cells). Osilodrostat has a minor impact on adrenal androgens and inhibits the build-up of corticosterone and 11-deoxycortisol [2].

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In vitro enzyme inhibition [1]
LCI699/Osilodrostat dose-dependently inhibited the activity of recombinant human aldosterone synthase (IC50 = 0.7 nmol/L) with 3.6-fold selectivity over 11β-hydroxylase (IC50 = 2.5 nmol/L) (Table 1). Lineweaver–Burk plots (Figure 1) showed that LCI699 is a reversible competitive inhibitor of recombinant human aldosterone synthase (Ki = 1.4 ± 0.2 nmol/L, mean ± SEM) and, at higher concentrations, of 11β-hydroxylase (Ki = 2.4 ± 0.3 nmol/L).
In vitro enzymatic studies using rat recombinant enzymes showed that LCI699 was approximately 230-fold less potent at inhibiting rat aldosterone synthase than the human enzyme (Table 1). Nevertheless, LCI699 had similar weak selectivity for recombinant rat aldosterone synthase and 11β-hydroxylase (2.6-fold difference) compared with that for the recombinant human enzymes.
In monkey adrenal homogenates, the IC50 of LCI699 for aldosterone synthase was 17-fold higher than that for the human recombinant enzyme, but was 67-fold lower than the IC50 measured in rat adrenal homogenates. Selectivity for aldosterone synthase over 11β-hydroxylase was 5.2-fold in monkey adrenal homogenates.
In summary, the relative species rank order of LCI699/Osilodrostat potency for inhibiting aldosterone synthase was human > monkey > rat, whereas the 3- to 5-fold selectivity for aldosterone synthase over 11β-hydroxylase was similar across these species.

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Context: Metyrapone and ketoconazole, frequently used steroidogenesis inhibitors for treatment of Cushing syndrome, can be associated with side effects and limited efficacy. Osilodrostat is a CYP11B1 and CYP11B2 inhibitor, with unknown effects on other steroidogenic enzymes.
Objective: To compare the effects of osilodrostat, metyrapone, and ketoconazole on adrenal steroidogenesis, and pituitary adenoma cells in vitro.
Methods: HAC15 cells, 17 primary human adrenocortical cell cultures, and pituitary adenoma cells were incubated with Osilodrostat, metyrapone, or ketoconazole (0.01 to 10 µM). Cortisol and ACTH were measured using chemiluminescence immunoassays, and steroid profiles by liquid chromatography-mass spectrometry.
Results: In HAC15 cells, osilodrostat inhibited cortisol production more potently (IC50: 0.035 µM) than metyrapone (0.068 µM; P < 0.0001), and ketoconazole (0.621 µM; P < 0.0001). IC50 values of osilodrostat and metyrapone for basal cortisol production varied with a 25- and 18-fold difference, respectively, with comparable potency. Aldosterone production was inhibited more potently by osilodrostat vs metyrapone and ketoconazole. Osilodrostat and metyrapone treatment resulted in strong inhibition of corticosterone and cortisol, 11-deoxycortisol accumulation, and modest effects on adrenal androgens. No pituitary-directed effects of osilodrostat were observed.
Conclusions: Under our study conditions, osilodrostat is a potent cortisol production inhibitor in human adrenocortical cells, comparable with metyrapone. All steroidogenesis inhibitors showed large variability in sensitivity between primary adrenocortical cultures. Osilodrostat might inhibit CYP11B1 and CYP11B2, in some conditions to a lesser extent CYP17A1 activity, and a proximal step in the steroidogenesis. Osilodrostat is a promising treatment option for Cushing syndrome, and in vivo differences with metyrapone are potentially driven by pharmacokinetic differences [2].

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Effects of Osilodrostat, metyrapone, and ketoconazole on basal and ACTH-stimulated cortisol production in vitro [2]
HAC15 cell line [2]
After 3 days, osilodrostat inhibited cortisol production at significantly lower concentrations (IC50 0.0347 µM; 95% CI, 0.0294 to 0.0410) than metyrapone (0.0678 µM; 95% CI, 0.0543 to 0.0848; P < 0.0001), and ketoconazole (0.621 µM; 95% CI, 0.488 to 0.833; P < 0.0001) (Fig. 1A). Mean cortisol stimulation by ACTH was 42% (±4%) in all experiments (Fig. 1C). For osilodrostat, the IC50 value increased 1.7-fold when HAC15 cells were stimulated with ACTH (P < 0.0001 vs basal condition), whereas potency under ACTH stimulation did not significantly change for metyrapone and ketoconazole. Comparing the three compounds under ACTH stimulation, osilodrostat inhibited cortisol production as potently as metyrapone (IC50 0.0605 µM; 95% CI, 0.0514 to 0.0714 vs IC50 0.0739 µM; 95% CI, 0.0645 to 0.0847; P = 0.0669), and more potently compared with ketoconazole (IC50 0.709 µM; 95% CI, 0.523 to 0.962; P < 0.0001). Addition of the inhibitors of steroidogenesis did not affect cell amounts.

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Primary adrenocortical cultures [2]
Effects of Osilodrostat, metyrapone, and ketoconazole were also assessed in 17 primary cultures of human adrenocortical tissue: 8 cortisol-producing ACA, 3 ACTH-dependent adrenal hyperplasia, 2 ACTH-independent adrenal hyperplasias, 2 cortisol-producing ACCs, and 2 Conn syndrome–associated adrenal hyperplasias. Patient and tissue characteristics are outlined in Table 1. IC50 values of osilodrostat, metyrapone, and ketoconazole for cortisol production in primary adrenocortical cultures are listed in Table 2; dose-response curves are displayed in Fig. 2 and (26). DNA measurement was performed in 37 of the 58 adrenal culture plates in which dose responses of the compounds on either cortisol or aldosterone were assessed, and showed no effects of any of the drugs on cell number in these cultures. The 85 pM ACTH-induced cortisol increase varied from 48% to 737% in primary adrenocortical cultures (Table 2).
In unstimulated primary ACA cultures, IC50 values of Osilodrostat for cortisol production varied with a 25-fold difference (Table 2, Fig. 2A; 0.0217, 95% CI, 0.0102 to 0.0461; 0.534, 95% CI, 0.360 to 0.793), whereas there was an 18-fold difference for metyrapone, and 84-fold difference for ketoconazole. The mean IC50 of osilodrostat in ACA was higher (n = 7; 0.104 µM; 95% CI, 0.0716 to 0.151) compared with the mean IC50 in adrenal hyperplasia (n = 2; 0.0269 µM, 95% CI, 0.0210 to 0.0346; P < 0.0001 vs ACA), and not statistically significantly different from the IC50 of ACC (n = 2; 0.0644 µM; 95% CI, 0.0419 to 0.0988; P = 0.1889 vs ACA), although groups were small. The mean IC50 of osilodrostat was lower in adrenal hyperplasia compared with ACC (P = 0.0007). In eight conditions (basal or ACTH-stimulated), a direct comparison between osilodrostat and metyrapone could be made (Table 2). Metyrapone inhibited cortisol production more potently in three conditions compared with osilodrostat (P < 0.05), whereas osilodrostat inhibited cortisol more potently in ACTH-dependent adrenal hyperplasia no. 1 (P < 0.0001). Osilodrostat inhibited cortisol more potently compared with ketoconazole in 8 of the 11 cultures in which efficacy of both compounds were studied in the basal condition (P < 0.05 to P < 0.0001). Under ACTH stimulation, a lower IC50 was found for osilodrostat in two of the six primary cultures compared with ketoconazole (P < 0.01 and P < 0.001).
Efficacy of osilodrostat changed in two of the three cultures in which efficacy was compared in the basal and ACTH simulated conditions, with in one culture a higher and in the other culture a lower potency in the ACTH-stimulated condition (P < 0.01 and P < 0.05, respectively).

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Effects of Osilodrostat, metyrapone, and ketoconazole on aldosterone production in human adrenocortical cells [2]
In angiotensin II–stimulated HAC15 cells (Fig. 3D; mean increase of aldosterone, 282%; P < 0.0001), Osilodrostat inhibited aldosterone levels at more than 10 times lower concentrations compared with metyrapone (Fig. 3A; IC50, 0.0354 μM; 95% CI, 0.0269 to 0.0465 vs 0.413 μM; 95% CI, 0.306 to 0.557; P < 0.0001). The aldosterone concentration in unstimulated HAC15 cells was too low to adequately assess the inhibitory effect of the compounds. Osilodrostat also inhibited aldosterone production much more potently compared with metyrapone in an aldosterone-producing adrenal hyperplasia causing Conn syndrome (Figure 3B; IC50, 0.00281 μM; 95% CI, 0.000910 to 0.00866 vs 0.822 μM; 95% CI, 0.471 to 1.433; P < 0.0001). In a second aldosterone-producing adrenal hyperplasia, no differences were observed in suppressive effects of osilodrostat and metyrapone on basal aldosterone concentrations in two concentrations tested (0.1 and 5 μM, data not shown). In ACTH-dependent adrenal hyperplasia no. 1 in the basal condition, osilodrostat inhibited aldosterone significantly more potent compared with metyrapone (Fig. 3C; IC50, 0.00469 μM; 95% CI, 5.516E-5 to 0.398 vs 0.364 μM; 95% CI, 0.05515 to 2.397; P < 0.0001), and ketoconazole (0.315 μM; 95% CI, 0.0516 to 1.916; P < 0.0001 vs osilodrostat). In this primary culture, osilodrostat inhibited aldosterone production at significantly lower concentrations compared with those needed for cortisol inhibition (IC50 aldosterone, 0.00469 μM; 95% CI, 5.516E-5 to 0.398 vs cortisol, 0.0311 μM; 95% CI, 0.0242 to 0.0399; P = 0.0164).

In Ang-II and ACTH-stimulated Sprague Dawley rats, Osilodrostat (LCI699; 0.1-100 mg/kg; oral; once) inhibits the synthesis of aldosterone and corticosterone [1]. Osilodrostat (LCI699; 3-100 mg/kg; oral; once daily for 52 weeks) prolongs the survival of dTG rats by lowering mean arterial pressure [1].

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Background: Aldosterone synthase inhibition provides the potential to attenuate both the mineralocorticoid receptor-dependent and independent actions of aldosterone. In vitro studies with recombinant human enzymes showed LCI699 to be a potent, reversible, competitive inhibitor of aldosterone synthase (K i = 1.4 ± 0.2 nmol/L in humans) with relative selectivity over 11β-hydroxylase.
Methods: Hormonal effects of orally administered Osilodrostat/LCI699 were examined in rat and monkey in vivo models of adrenocorticotropic hormone (ACTH) and angiotensin-II-stimulated aldosterone release, and were compared with the mineralocorticoid receptor antagonist eplerenone in a randomized, placebo-controlled study conducted in 99 healthy human subjects. The effects of LCI699 and eplerenone on cardiac and renal sequelae of aldosterone excess were investigated in a double-transgenic rat (dTG rat) model overexpressing human renin and angiotensinogen.
Results: Rat and monkey in vivo models of stimulated aldosterone release predicted human dose- and exposure-response relationships, but overestimated the selectivity of Osilodrostat/LCI699 in humans. In the dTG rat model, LCI699 dose-dependently blocked increases in aldosterone, prevented development of cardiac and renal functional abnormalities independent of blood pressure changes, and prolonged survival. Eplerenone prolonged survival to a similar extent, but was less effective in preventing cardiac and renal damage. In healthy human subjects, LCI699 0.5 mg selectively reduced plasma and 24 h urinary aldosterone by 49 ± 3% and 39 ± 6% respectively (Day 1, mean ± SEM; P < 0.001 vs placebo), which was associated with natriuresis and an increase in plasma renin activity. Doses of LCI699 greater than 1 mg inhibited basal and ACTH-stimulated cortisol. Eplerenone 100 mg increased plasma and 24 h urinary aldosterone while stimulating natriuresis and increasing renin activity. In contrast to eplerenone, LCI699 increased the aldosterone precursor 11-deoxycorticosterone and urinary potassium excretion.
Conclusions: These results provide new insights into the cardiac and renal effects of inhibiting aldosterone synthase in experimental models and translation of the hormonal effects to humans. Selective inhibition of aldosterone synthase appears to be a promising approach to treat diseases associated with aldosterone excess. [1]

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The somatostatin analog pasireotide and the 11β-hydroxylase inhibitor oOsilodrostat (LCI699) reduce cortisol levels by distinct mechanisms of action. There exists a scientific rationale to investigate the clinical efficacy of these two agents in combination. This manuscript reports the results of a toxicology study in rats, evaluating different doses of osilodrostat and pasireotide alone and in combination. Sixty male and 60 female rats were randomized into single-sex groups to receive daily doses of pasireotide (0.3mg/kg/day, subcutaneously), osilodrostat (20mg/kg/day, orally), osilodrostat/pasireotide in combination (low dose, 1.5/0.03mg/kg/day; mid-dose, 5/0.1mg/kg/day; or high dose, 20/0.3mg/kg/day), or vehicle for 13weeks. Mean body-weight gains from baseline to Week 13 were significantly lower in the pasireotide-alone and combined-treatment groups compared to controls, and were significantly higher in female rats receiving osilodrostat monotherapy. Osilodrostat and pasireotide monotherapies were associated with significant changes in the histology and mean weights of the pituitary and adrenal glands, liver, and ovary/oviduct. Osilodrostat alone was associated with adrenocortical hypertrophy and hepatocellular hypertrophy. In combination, osilodrostat/pasireotide did not exacerbate any target organ changes and ameliorated the liver and adrenal gland changes observed with monotherapy. Cmax and AUC0-24h of osilodrostat and pasireotide increased in an approximately dose-proportional manner. In conclusion, the pasireotide and osilodrostat combination did not exacerbate changes in target organ weight or toxicity compared with either monotherapy, and had an acceptable safety profile; addition of pasireotide to the osilodrostat regimen may attenuate potential adrenal gland hyperactivation and hepatocellular hypertrophy, which are potential side effects of osilodrostat monotherapy [3].

In vitro enzyme inhibition [1]
Experimental design [1]
Cell lines and tissue samples [1]
Recombinant human cytochrome P450 (CYP) 11B2 and CYP11B1 enzymes were prepared from the cell lines V79-4 CYP11B2-adrenodoxin-adrenodoxin reductase (AAR) #317 and V79-4 CYP11B1-AAR #618, respectively. Recombinant rat CYP11B2 and CYP11B1 enzymes were prepared similarly. All cell lines were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 0.5× antibiotic, 800 μg/mL geneticin and 250 μg/mL hygromycin.
Rat adrenal homogenates were prepared from the adrenal glands of male Sprague-Dawley (S-D) rats as described previously. Monkey CYB11B2 and CYB11B1 homogenates were prepared from the adrenal glands of female cynomolgous monkeys. Monkey adrenal gland tissue was minced and homogenized on ice in a glass tissue grinder in 1 mL of ice-cold homogenization buffer per 100 mg tissue (adding 2.7 mmol/L CaCl2 and one ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablet per 50 mL buffer). The homogenized material was centrifuged at 450 g for 5 min at 4°C, and the supernatant brought to a final glycerol concentration of 5%, flash-frozen in liquid nitrogen, and stored at −80°C until analysis. Aldosterone, cortisol and corticosterone concentrations were quantified using 96-well plate assays (see Additional file 1).

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CYP11B2 and CYP11B1 enzyme assays [1]
Human CYP11B2 and CYP11B1 assays were performed as described previously. The rat and monkey assays were conducted similarly, using 11-DOC as substrate.

Stock solution of Osilodrostat, metyrapon, and ketoconazole were dissolved in 0.01N hydrochloric acid, distilled water, and absolute ethanol, respectively, according to manufacturer’s instructions, and stored at −20°C at a stock concentration of 10−2 M. At the start of each experiment, osilodrostat, metyrapone, and ketoconazole were diluted to the correct concentration in the same solution as it was dissolved in. Synacten stock concentration was stored at 4°C and diluted in culture medium at the day of use. Angiotensin II stock concentration was stored at −20°C and diluted in distilled water at the day of use. The concentrations of ACTH and angiotensin II used were based on a dose-response curve performed in HAC15 cells on cortisol and aldosterone production, respectively, and according to previously reported studies. One day after seeding the cells, incubations were started. Control cells were vehicle treated. For HAC15, cells were plated at a density of 100,000 cells per well in 0.5 mL medium. Osilodrostat, metyrapone, or ketoconazole (0.01 to 5 µM) were added for 3 days, with or without 10 nM ACTH or 100 nM angiotensin II, for evaluation of the effects on the steroid profile, and aldosterone production, respectively. To assess the effect of osilodrostat on mouse corticotroph pituitary cells, AtT20 cells were incubated with osilodrostat for 1, 3, and 7 days (0.01 to 10 µM), to evaluate potential effects of the drug in multiple conditions (different incubation times and higher concentrations). For 7-day experiments, medium and compounds were refreshed after 3 days [2].

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Primary human adrenal and pituitary adenoma culture experiments were similar to experiments in HAC15 and AtT20 cells, with small adjustments: ACTH was used at a concentration of 85 pM, angiotensin II was used at a concentration of 10 nM, treatment was started 3 to 4 days after plating the cells and preceded by medium refreshment, and in primary corticotroph pituitary adenoma cultures, Osilodrostat was only tested at a concentration of 1 µM. The concentrations of ACTH and angiotensin II used in the primary cultures were lower compared with those in HAC15 cells because of the generally higher sensitivity to these compounds in primary cultures. Owing to a limited number of cells obtained from some specimens, not all experiments could be carried out in every primary culture [2].

Animal/Disease Models: dTG rat[1]
Doses: 3, 10, 30 and 100 mg/kg
Route of Administration: Oral; one time/day for 52 weeks
Experimental Results: Shortened LV fractional shortening (systolic and diastolic), LV Isovolumic relaxation time to RR (IVRT/RR) ratio and cardiomyocyte size were normalized and diminished LV mass in a dose-dependent manner. LCI699/Osilodrostat formulation [1]
LCI699/Osilodrostat solution was freshly prepared (from powder) before each experiment. In the rat models, Osilodrostat/LCI699 (free base) was first dissolved in 1.5 molar equivalents of 1 N HCl plus 10 parts of water and then diluted in 3% cornstarch (1 mL/kg volume). In the monkey model, Osilodrostat/LCI699 (phosphate salt) was dissolved in water (1 mL/kg volume). LCI699 was administered by oral (rat and monkey) or nasogastric gavage (monkey). Compound doses in the monkey model are quoted as free base equivalents.

Experimental protocol for rat models [1]
Study protocols for the rat models of Ang-II- and ACTH-stimulated aldosterone synthesis followed a published protocol. For the Ang-II-infusion model, an initial loading dose of 300 ng/kg angiotensin II (Ang II) was followed by 100 ng/kg/min intravenous (i.v.) infusion for 9 h. For the ACTH-infusion model, the loading and infusion doses of ACTH were 100 ng/kg and 30 ng/kg/min, respectively. After 1 h of Ang II or ACTH infusion, a blood sample was collected for determining the post-Ang II or ACTH ‘baseline’ (i.e., secretagogue-elevated) plasma aldosterone and corticosterone concentrations. Osilodrostat/LCI699 was administered at doses of 0.1, 0.3, 1 and 3 mg/kg in the Ang-II-infusion model, and 1, 3, 10, 30 and 100 mg/kg in the ACTH-infusion model. In both models, infusion continued for a further 8 h. Blood samples were withdrawn in heparin (final concentration 15 U/mL) from the arterial cannula at 15 and 30 min, and 1, 2, 3, 4, 5, 6, 7, 8, and 24 h post-dosing. Plasma aldosterone and cortisol were determined by radioimmunoassay and LCI699 by liquid chromatography separation coupled with tandem mass spectrometric detection (LC-MS/MS) (see Additional file 1).

Experimental protocol for monkey model [1]
Six monkeys (4.9–8.8 kg) were selected and were divided into two groups of three animals. Experiments were not initiated until after at least 2 weeks of recovery from the catheter/vascular access port (VAP) surgeries. Thirty minutes before the start of the experiment, a Huber needle was inserted transdermally into the VAP for the collection of blood samples and injection of ACTH. Between samplings, catheters/VAPs were flushed with saline and kept patent with 10 U/mL heparin. In all cases, the total blood withdrawn did not exceed 1% of body weight per week, and at least 1 week of recovery was allowed between sequential experiments.

Blood samples (0.3 mL in 15 U/mL heparin) for baseline pharmacokinetic and pharmacodynamic assessments were collected at 0.5 h, 0.25 h and immediately before dosing. Osilodrostat/LCI699 (5, 15, 50 or 150 μg/kg) or vehicle (water) was administered followed 3 h later by ACTH(1–24) 3000 ng/kg i.v. in 0.1 mL/kg (over ~2 min). The 3000 ng/kg dose of ACTH was determined from a pilot dose–response experiment, which showed a consistent and maximal stimulation of plasma aldosterone and cortisol. Blood samples were collected at 0.125, 0.25, 0.5, 0.75 and 1 h after ACTH injection to assess the time course of plasma aldosterone and cortisol stimulation. Further blood samples were collected up to 8 h and at 23.5 and 24 h after LCI699/vehicle administration. Between the 8 h and 23.5 h collections, the Huber needles were removed and the monkeys were returned to their home cages. All instrumentation was removed after the last sample at 24 h. Plasma aldosterone and cortisol were determined by radioimmunoassay and LCI699 by LC-MS/MS (see Additional file 1).

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Sixty male and 60 female rats were randomized into single-sex groups to receive daily doses of vehicle, low-dose Osilodrostat/pasireotide, mid-dose osilodrostat/pasireotide, high-dose osilodrostat/pasireotide, high-dose osilodrostat monotherapy, or high-dose pasireotide monotherapy (Table 1). Prior to initiation of dosing, all animals were weighed, and randomization was stratified by body weight. Rats received their specified treatment regimen daily for 13 weeks; the 13-week treatment period was chosen according to current European Medicines Agency recommendations (ICH M3 [R2]). Three female sentinel rats were used for health screening procedures.

Osilodrostat was administered orally using a plastic gavage tube, followed by, where applicable, sc injection of pasireotide into the interscapular area within 5 min of osilodrostat administration. Animals were dosed at approximately the same time each day, except during designated procedures. Oral and sc routes of administration were selected for osilodrostat and pasireotide, respectively, as they represent the intended routes of administration in humans.
The low, mid, and high doses of Osilodrostat (1.5, 5, and 20 mg/kg/day) and pasireotide (0.03, 0.1, and 0.3 mg/kg/day) were considered appropriate based on the results of previous monotherapy studies in rats (osilodrostat dose range: 0.2–50 mg/kg/day, orally; pasireotide dose range: 0.08–0.24 mg/kg/day, sc; Novartis Pharma AG, unpublished data); these doses were expected to provide sufficient exposure multiples against human systemic exposure at therapeutic doses. Osilodrostat and pasireotide doses of up to 20 and 0.24 mg/kg/day, respectively, were tolerated during two 6-month monotherapy studies; the no-observed-adverse-effect levels (NOAELs) for osilodrostat and pasireotide were 2 and 0.024 mg/kg/day, respectively.

Compounds and formulation [2]
Osilodrostat was formulated in ultrapure water for administration by oral gavage. Pasireotide was formulated with acetate-buffered solution (pH 4.5), acetic acid, and d-mannitol in sterile water for sc injection. Vehicle control consisted of ultrapure water for oral gavage and acetate-buffered solution (pH 4.5), acetic acid, and d-mannitol in sterile water for sc injection. Dosing volumes were 5 mL/kg for oral gavage (osilodrostat and vehicle) and 1 mL/kg for sc injection (pasireotide and vehicle). Agents were stored at 4 °C and protected from light. For administration of osilodrostat, dosing solutions were removed from the refrigerator and stirred for at least 10 min at room temperature prior to initiation of dosing. Dosing solutions of pasireotide were removed from the refrigerator and allowed to warm to room temperature for at least 30 min prior to initiation of dosing.

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Endpoints and assessments [2]
This study sought to determine the effects of daily Osilodrostat and pasireotide, alone and in combination, on the pituitary–adrenal axis and to report any other treatment-related toxicities. The toxicokinetic characteristics of osilodrostat and pasireotide were also determined.

Absorption, Distribution and Excretion
The oral absorption of osilodrostat is rapid, with a Tmax of approximately 1 hour, and assumed to be essentially complete. Exposure (i.e. AUC and Cmax) increases slightly more than dose-proportionately over the standard dosing range. Coadministration of osilodrostat with food does not affect its pharmacokinetics to a clinically significant extent. Age and gender do not affect pharmacokinetics, but bioavailability and total exposure is higher (though not clinically significant) in patients of Asian descent. Exposure to osilodrostat is greater in patients with moderate-severe hepatic impairment - prescribing information recommends a starting dose of 1mg twice daily in patients with moderate hepatic impairment (Child-Pugh B) and a starting dose of 1mg each evening in patients with severe hepatic impairment (Child-Pugh C).
Following oral administration of radiolabeled osilodrostat, 90.6% of the radioactivity was eliminated in the urine with only 1.58% in the feces. Only 5.2% of the administered dose was eliminated in the urine as unchanged parent drug, suggesting that metabolism followed by urinary elimination is osildrostat's primary means of clearance.
The median apparent volume of distribution of osilodrostat is 100 L.
Data regarding the oral clearance of osilodrostat are not currently available.

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Metabolism / Metabolites
Osilodrostat is extensively metabolized - approximately 80% of an orally administered dose is excreted as metabolites, and this is the predominant means of drug clearance. The most abundant metabolites in plasma are M35.4 (di-oxygenated osilodrostat), M16.5, and M24.9 at 51%, 9%, and 7% of the administered dose, respectively. The M34.5 and M24.9 metabolites have longer half-lives than the parent drug which may lead to accumulation with twice-daily dosing. Of the thirteen metabolites observed in the urine, the most abundant are M16.5 (osilodrostat glucuronide), M22 (a glucuronide conjugate of M34.5), and M24.9 at 17%, 13%, and 11% of the administered dose, respectively. The M34.5 metabolite accounts for less than 1% of the dose excreted in urine, but its glucuronide conjugate (M22) accounts for approximately 13%. The biotransformation of osilodrostat is mediated by multiple cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes, though no single enzyme appears to contribute >25% to the total clearance. Of the total clearance, approximately 26% is CYP-mediated, 19% is UGT-mediated, and 50% is mediated by other enzymes. The formation of M34.5, the major metabolite of osilodrostat, is likely non-CYP-mediated. The formation of osilodrostat glucuronide (M16.5), its major urinary metabolite, is catalyzed by UGT1A4, UGT2B7, and UGT2B10. _In vitro_ data suggest that none of the metabolites contribute to the therapeutic efficacy of osilodrostat, but the M34.5 metabolite has been implicated in the inhibition and/or induction of multiple enzymes and transporters.

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Biological Half-Life
The elimination half-life of osilodrostat is approximately 4 hours.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of osilodrostat during breastfeeding. Because of the potential for serious adverse reactions, such as adrenal insufficiency, in the breastfed infant, breastfeeding is not recommended during treatment with osilodrostat and should be avoided until 1 week after the final dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Hepatotoxicity
In preregistration trials, mild, transient serum aminotransferase elevations occurred in 37 of 137 (27%) patients receiving Osilodrostat for Cushing disease, but in only 8 (6%) patients were values above 3 times the upper limit of normal (ULN), and only 1 was above 5 times ULN (
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Protein Binding
Both osilodrostat and its M34.5 metabolite are minimally protein-bound in plasma at less than 40%. The extent of protein-binding is independent of drug concentration. The specific plasma proteins to which osilodrostat binds have not been elucidated.

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Toxicokinetics [3]
On Day 1, an increase in Osilodrostat dose from 0.5 to 20 mg/kg/day in combination with pasireotide (dose range, 0.03–0.3 mg/kg/day) was accompanied by approximately dose-proportional increases in maximum plasma concentration (Cmax) and area under the concentration–time curve from 0 to 24 h (AUC0–24h) for osilodrostat in male and female rats (Table 4). For each dose group, the osilodrostat AUC0–24h and Cmax were similar after multiple doses (Day 77) compared with those after a single dose (Day 1) and were, in general, similar between sexes.
An increase in pasireotide dose from 0.03 to 0.3 mg/kg/day in combination with Osilodrostat (dose range, 0.5–20 mg/kg/day) on Day 1 led to dose-proportional increases in Cmax and AUC0–24h for pasireotide in male and female rats (Table 4); the Cmax and AUC0–24h of pasireotide increased slightly after multiple doses compared with those after a single dose. A slight gender difference was observed in pasireotide exposure at all doses tested (male/female AUC0–24h ratio, 1.4–1.8). Toxicokinetic parameters were not assessed for rats administered osilodrostat or pasireotide alone.

[1]. Aldosterone synthase inhibition: cardiorenal protection in animal disease models and translation of hormonal effects to human subjects. J Transl Med. 2014 Dec 10;12:340.

[2]. Osilodrostat Is a Potential Novel Steroidogenesis Inhibitor for the Treatment of Cushing Syndrome: An In Vitro Study. J Clin Endocrinol Metab. 2019 Aug 1;104(8):3437-3449.

[3]. Osilodrostat (LCI699), a potent 11β-hydroxylase inhibitor, administered in combination with the multireceptor-targeted somatostatin analog pasireotide: A 13-week study in rats. Toxicol Appl Pharmacol. 2015 Aug 1;286(3):224-33.

See also: Osilodrostat (has active moiety).

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Drug Indication
Isturisa is indicated for the treatment of endogenous Cushing's syndrome in adults.

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Osilodrostat is an inhibitor of 11β-hydroxylase (also referred to as CYP11B1), the enzyme that catalyzes the final step in the biosynthesis of endogenous cortisol. It is used to lower circulating cortisol levels in the treatment of Cushing's disease, a disorder in which cortisol levels are chronically and supraphysiologically elevated. Cushing's disease is often the result of ACTH hypersecretion secondary to a pituitary tumor, and surgical resection of the tumour is generally the treatment of choice. As an orally bioavailable drug therapy, osilodrostat provides a novel treatment option for patients in whom removal of the causative tumor is not an option or for whom previous pituitary surgery has not been curative. Osilodrostat is manufactured by Novartis under the brand name Isturisa. It has undergone phase II clinical trials for the treatment of solid tumours, hypertension, and heart failure, but development for these indications was discontinued by Novartis in January 2013. Osilodrostat was approved for use in the EU in January 2020 for the treatment of endogenous Cushing's syndrome (i.e. Cushing's disease), and was granted FDA approval and Orphan Drug designation in the US in March 2020 for the same indication.
Osilodrostat is a Cortisol Synthesis Inhibitor. The mechanism of action of osilodrostat is as a Cytochrome P450 11B1 Inhibitor, and Cytochrome P450 1A2 Inhibitor, and Cytochrome P450 2C19 Inhibitor, and Cytochrome P450 2D6 Inhibitor, and Cytochrome P450 3A4 Inhibitor, and Cytochrome P450 3A5 Inhibitor.
Osilodrostat is an inhibitor of cortisol synthesis that is used in the treatment of Cushing disease not controlled by standard therapy. Osilodrostat therapy has not been linked to serum aminotransferase elevations during therapy or with instances of clinically apparent liver injury.
Osilodrostat is an orally bioavailable inhibitor of both steroid 11beta-hydroxylase (cytochrome P450 (CYP) 11B1) and aldosterone synthase (CYP11B2; steroid 18-hydroxylase), with potential anti-adrenal activity and ability to treat Cushing disease (CD). Upon administration, osilodrostat binds to and inhibits the activity of CYP11B1, the enzyme that catalyzes the final step of cortisol synthesis from the precursor 11-deoxycortisol, and CYP11B2, the enzyme that catalyzes aldosterone synthesis from corticosterone and 11-deoxycorticosterone in the adrenal gland. The inhibition of CYP11B1 prevents the production of excess cortisol, thereby decreasing and normalizing the levels of cortisol. CD is most often caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor.
See also: Osilodrostat Phosphate (active moiety of).

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Drug Indication
Osilodrostat is indicated for the treatment of adult patients with Cushing's disease for whom pituitary surgery is not an option or has not been curative.
FDA Label
Isturisa is indicated for the treatment of endogenous Cushing's syndrome in adults.
Treatment of adrenal cortical hyperfunction

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Mechanism of Action
Cushing’s syndrome is an endocrine disorder resulting from chronic and excessive exposure to glucocorticoids, the symptoms of which may include thinning of the skin and hair, weight gain, muscle weakness, and osteoporosis, as well a constellation of psychiatric, cardiovascular, and immunological deficiencies. Cushing’s syndrome is most commonly precipitated by exogenous treatment with supraphysiological doses of glucocorticoids such as those found in nasal sprays, skin creams, and inhalers. Cushing’s disease - another less common cause of Cushing’s syndrome - is generally the result of increased endogenous cortisol exposure due to excessive secretion of adrenocroticotrophic hormone (ACTH) from a pituitary adenoma. Osilodrostat is an inhibitor of 11β-hydroxylase (CYP11B1) and, to a lesser extent, aldosterone synthase (CYP11B2). The CYP11B1 enzyme is responsible for catalyzing the final step of cortisol synthesis - by inhibiting this enzyme, osilodrostat helps to normalize endogenous cortisol levels and alleviate symptoms of Cushing’s disease.

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In conclusion, we show that osilodrostat in pharmacological concentrations is a potent inhibitor of in vitro cortisol and aldosterone secretion in human adrenocortical cells. We demonstrate highly variable sensitivity to steroidogenesis inhibitors with respect to cortisol production between adrenal tissues of patients, which together with differences in pharmacokinetics, potentially explain clinically observed differences between patients treated with the same compound. Under the conditions of our study, effects of osilodrostat and metyrapone on the steroid profile are highly comparable, where osilodrostat seems to block CYP11B1 and CYP11B2, in some conditions to a lesser extent CYP17A1 lyase activity, and a proximal step in the steroidogenesis pathway. Differences between osilodrostat and metyrapone in vivo are potentially the result of pharmacokinetic differences rather than the pharmacodynamic effects on the adrenal cortex. These data indicate that osilodrostat is a promising treatment option for patients with CS. Additional information from phase 3 trials will provide important further data on efficacy and safety of osilodrostat.[2]

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The combination of pasireotide and osilodrostat in rats did not change the toxicity profile and plasma exposure seen during administration of either agent alone, whereas the addition of pasireotide to the osilodrostat regimen may attenuate potential adrenal gland hypertrophy and hepatocellular hypertrophy. As such, this study suggests that the combination of pasireotide and osilodrostat has an acceptable safety profile. However, other safety concerns, in particular effects on QT interval, should also be considered prior to using the pasireotide and osilodrostat drug combination in humans.[3]

C13H13FN3O4P

325.2321870327

325.062

C, 48.01; H, 4.03; F, 5.84; N, 12.92; O, 19.68; P, 9.52

1315449-72-9

Osilodrostat;928134-65-0; 928134-31-0; 1315449-72-9 (phosphate)

71009737

White to off-white solid powder

3

7

1

22

387

1

C1CC2=CN=CN2[C@H]1C3=C(C=C(C=C3)C#N)F.OP(=O)(O)O

FMCPYRDGUZBOJZ-BTQNPOSSSA-N

InChI=1S/C13H10FN3.H3O4P/c14-12-5-9(6-15)1-3-11(12)13-4-2-10-7-16-8-17(10)13;1-5(2,3)4/h1,3,5,7-8,13H,2,4H2;(H3,1,2,3,4)/t13-;/m1./s1

4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluorobenzonitrile;phosphoric acid

Osilodrostat phosphate; 1315449-72-9; LCI699-AZA; UNII-Y6581YAW9V; Y6581YAW9V; Isturisa; 4-((5R)-6,7-Dihydro-5H-pyrrolo(1,2-c)imidazol-5-yl)-3-fluorobenzonitrile dihydrogen phosphate; BENZONITRILE, 4-((5R)-6,7-DIHYDRO-5H-PYRROLO(1,2-C)IMIDAZOL-5-YL)-3-FLUORO-, PHOSPHATE (1:1);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ≥ 200 mg/mL (~614.95 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)