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, pituitary adenoma cells). Osilodrostat has minor effects on adrenal androgens and suppresses the build-up of corticosterone and 11-deoxycortisol [2].

---

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.

---

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].

---

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.

---

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).

---

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].

---

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]

---

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).

---

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].

---

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: Male Ang-II- and ACTH-stimulated Sprague Dawley rats[1]
Doses: 0.1, 0.3, 1 and 3 mg/kg (Ang-II-stimulated rats) and 1, 3, 10, 30 and 100 mg/ kg (ACTH-stimulated rats)
Route of Administration: Oral administration; once
Experimental Results: Inhibited the increase in plasma aldosterone concentrations stimulated by Ang II or ACTH in a dose-dependent manner.

---

Animal/Disease Models: dTG rats[1]
Doses: 3, 10, 30 and 100 mg/kg
Route of Administration: Oral administration; daily, for 52 weeks
Experimental Results: Increased fractional LV (systolic and diastolic) shortening, normalized LV isovolumic relaxation time to RR (IVRT/RR) ratio and myocardial cell size and decreased LV weight in a dose-dependent manner.

---

\n\nLCI699/Osilodrostat formulation [1]
\nLCI699/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, 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.\n

\nExperimental protocol for rat models [1]
\nStudy 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).\n

\nExperimental protocol for monkey model [1]
\nSix 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.\n

\nBlood 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).\n\n

---

\n\nSixty 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.\n

\nOsilodrostat 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.
\n\nThe 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.\n

\nCompounds and formulation [2]
\nOsilodrostat 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.\n

---

\n\nEndpoints and assessments [2]
\nThis 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
Oselostat is rapidly absorbed orally, with a time to peak concentration (Tmax) of approximately 1 hour, and absorption is essentially complete. Within the standard dose range, the increase in drug exposure (i.e., AUC and Cmax) is slightly greater than the dose-proportional increase. Co-administration of oselostat with food has no significant effect on its pharmacokinetics. Age and sex do not affect its pharmacokinetics, but Asian patients have higher bioavailability and total exposure (although not clinically significant). Patients with moderate to severe hepatic impairment are at higher risk of exposure to oselostat—prescription information recommends a starting dose of 1 mg twice daily for patients with moderate hepatic impairment (Child-Pugh B) and a starting dose of 1 mg at bedtime for patients with severe hepatic impairment (Child-Pugh C). Following oral administration of radiolabeled oselostat, 90.6% of the radioactive material is excreted in the urine, and only 1.58% is excreted in the feces. Only 5.2% of the administered dose is excreted unchanged in the urine, indicating that urinary excretion is the primary route of elimination for osimertinib after metabolism. The median apparent volume of distribution for osimertinib is 100 L. Data on the oral clearance of osimertinib are currently unavailable. Osimertinib is extensively metabolized—approximately 80% of the oral dose is excreted as metabolites, which is the primary route of drug clearance. The most abundant metabolites in plasma are M35.4 (dioxoosilopertinib), M16.5, and M24.9, accounting for 51%, 9%, and 7% of the administered dose, respectively. The half-lives of metabolites M34.5 and M24.9 are longer than that of the parent drug; therefore, twice-daily dosing may lead to drug accumulation. Of the 13 metabolites observed in urine, the most abundant were M16.5 (osilostat glucuronide), M22 (a glucuronide conjugate of M34.5), and M24.9, accounting for 17%, 13%, and 11% of the administered dose, respectively. Metabolite M34.5 was excreted in urine at less than 1% of the administered dose, but its glucuronide conjugate (M22) accounted for approximately 13%. The biotransformation of osimertin is mediated by multiple cytochrome P450 (CYP) and UDP-glucuronyltransferase (UGT), but no single enzyme contributed more than 25% to the total clearance. Of the total clearance, approximately 26% was mediated by CYP, 19% by UGT, and 50% by other enzymes. The formation of the major osimertin metabolite M34.5 may not be mediated by CYP. The formation of its main urinary metabolite, osimertinib glucuronide (M16.5), is catalyzed by UGT1A4, UGT2B7, and UGT2B10. In vitro data indicate that these metabolites do not affect the therapeutic efficacy of osimertinib, but metabolite M34.5 has been shown to inhibit and/or induce various enzymes and transporters.

---

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

Hepatotoxicity
In the pre-registration trial, 37 out of 137 patients (27%) receiving osimerceptin for Cushing's disease experienced mild, transient elevations in serum transaminases. Only 8 patients (6%) had transaminase levels exceeding 3 times the upper limit of normal (ULN), and only 1 patient exceeded 5 times the ULN (probability score: E (unlikely to be a cause of clinically significant liver injury)). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the use of osimerceptin during lactation. Because breastfed infants may experience serious adverse reactions, such as adrenal insufficiency, breastfeeding is not recommended during osimerceptin treatment and should be avoided within one week of the last dose. ◉ Effects of Lactation on Infants As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

---

Protein binding
The protein binding rate of osilodrostat and its metabolite M34.5 in plasma is less than 40%. The degree of protein binding is independent of drug concentration. The specific plasma proteins bound by osilodrostat have not been identified.

---

Toxicokinetics[3]
On day 1, when the dose of osilodrostat was increased from 0.5 mg/kg/day to 20 mg/kg/day and used in combination with paretide (dose range 0.03–0.3 mg/kg/day), the plasma maximum concentration (Cmax) and the area under the concentration-time curve from 0 to 24 hours also increased in approximately dose-proportional manner. Table 4 shows the AUC0-24h of osilodrostat in male and female rats. For each dose group, the AUC0-24h and Cmax of osimertin after multiple dosing (day 77) were similar to those after a single dosing (day 1), and were also similar overall between males and females. On day 1, increasing the parecide dose from 0.03 mg/kg/day to 0.3 mg/kg/day in combination with osimertin (dose range: 0.5–20 mg/kg/day) resulted in a dose-proportional increase in the Cmax and AUC0-24h of parecide in male and female rats (Table 4); the Cmax and AUC0-24h of parecide were slightly increased after multiple dosing compared to a single dosing. Slight sex differences (male/female AUC0-24h ratio 1.4–1.8) were observed in parecide exposure at all tested doses. Toxicokinetic parameters were not assessed in rats administered osimertin or parecide 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.

Osilodrostat is an 11β-hydroxylase (also known as CYP11B1) inhibitor, which catalyzes the final step in the biosynthesis of endogenous cortisol. It is used to lower circulating cortisol levels to treat Cushing's disease, a condition characterized by persistently elevated, supraphysiological levels of cortisol. Cushing's disease is typically caused by excessive secretion of adrenocorticotropic hormone (ACTH) due to pituitary tumors, and surgical removal of the tumor is usually the preferred treatment. As a highly bioavailable oral medication, oselostat offers a new treatment option for patients whose causative tumor cannot be surgically removed or whose previous pituitary surgery has failed to cure the condition. Oselostat is manufactured by Novartis and marketed as Isturisa. The drug underwent Phase II clinical trials for the treatment of solid tumors, hypertension, and heart failure, but Novartis terminated development for these indications in January 2013. Oselostat was approved in the European Union in January 2020 for the treatment of endogenous Cushing's syndrome (Cushing's disease) and received orphan drug designation from the US FDA in March 2020 for the same indication. Oselostat is a cortisol synthesis inhibitor. Its mechanism of action is as an inhibitor of cytochrome P450 11B1, cytochrome P450 1A2, cytochrome P450 2C19, cytochrome P450 2D6, cytochrome P450 3A4, and cytochrome P450 3A5. Oselostat is a cortisol synthesis inhibitor used to treat Cushing's disease that is not controlled by standard therapy. No elevated serum transaminases or clinically significant liver damage were observed during oselostat treatment. Osilistopa is a highly bioavailable oral inhibitor that simultaneously inhibits steroid 11β-hydroxylase (cytochrome P450 (CYP) 11B1) and aldosterone synthase (CYP11B2; steroid 18-hydroxylase), exhibiting potential anti-adrenal activity and may be used to treat Cushing's disease (CD). After administration, osilistopa binds to and inhibits the activity of CYP11B1 (the enzyme catalyzing the final step in the synthesis of cortisol from the precursor 11-deoxycortisol) and CYP11B2 (the enzyme catalyzing the synthesis of aldosterone from corticosterone and 11-deoxycorticosterone in the adrenal gland). Inhibition of CYP11B1 prevents excessive cortisol production, thereby lowering and normalizing cortisol levels. The most common cause of Cushing's disease (CD) is a pituitary tumor that secretes adrenocorticotropic hormone (ACTH). See also: Osilistopa phosphate (active ingredient).

---

Drug Indications
Osilositol is indicated for the treatment of adult patients with Cushing's disease who are unsuitable for or cannot be cured by pituitary surgery.

FDA Label

Isturesa is indicated for the treatment of endogenous Cushing's syndrome in adults.

Treatment of Hyperadrenocortical Insufficiency
Mechanism of Action
Cushing's syndrome is an endocrine disorder caused by prolonged excessive exposure to glucocorticoids. Symptoms may include thinning of the skin and hair, weight gain, muscle weakness, osteoporosis, and a range of mental, cardiovascular, and immune deficiencies. The most common precipitating factor for Cushing's syndrome is the exogenous use of supraphysiological doses of glucocorticoids, such as those in nasal sprays, creams, and inhalers. Cushing's disease—another, less common cause of Cushing's syndrome—is usually caused by an excessive secretion of adrenocorticotropic hormone (ACTH) by a pituitary adenoma, leading to increased endogenous cortisol exposure.
Oselostat is an 11β-hydroxylase (CYP11B1) inhibitor with a weaker inhibitory effect on aldosterone synthase (CYP11B2). CYP11B1 is responsible for catalyzing the final step in cortisol synthesis—by inhibiting this enzyme, oselostat helps normalize endogenous cortisol levels and alleviate Cushing's disease symptoms. In summary, we found that at pharmacological concentrations, oselostat effectively inhibits the secretion of cortisol and aldosterone from human adrenal cortex cells in vitro. We found significant differences in the sensitivity of adrenal tissue to steroid synthesis inhibitors among different patients, which, along with differences in pharmacokinetics, may explain the clinically observed differences between patients treated with the same compound. Under the conditions of this study, oselostat and metheprone had highly similar effects on the steroid spectrum. Oselostat appears to inhibit CYP11B1 and CYP11B2, and in some cases, also mildly inhibits CYP17A1 lyase activity, and affects proximal steps of the steroid synthesis pathway. The difference between osimertin and metheprone in vivo is likely due to pharmacokinetic differences rather than differences in pharmacodynamic effects on the adrenal cortex. These data suggest that osimertin may be a treatment option for patients with Cushing's syndrome. More information from the phase 3 clinical trial will provide important data on the efficacy and safety of osimertin. [2] In rats, the combination of parretin and osimertin did not alter the toxicity profile and plasma exposure observed when either drug was used alone, while the addition of parretin to the osimertin treatment regimen may have reduced potential adrenal hypertrophy and hepatocellular hypertrophy. Therefore, this study suggests that the combination of parretin and osimertin has an acceptable safety profile. However, other safety issues, particularly the effect on the QT interval, should be considered before the combination of parretin and osimertin is used in humans. [3]

C13H10FN3

227.24

227.085

C, 68.71; H, 4.44; F, 8.36; N, 18.49

928134-65-0

Osilodrostat phosphate;1315449-72-9

44139752

White to yellow solid powder

1.3±0.1 g/cm3

433.8±45.0 °C at 760 mmHg

216.2±28.7 °C

0.0±1.0 mmHg at 25°C

1.664

1.13

0

3

1

17

337

1

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

USUZGMWDZDXMDG-CYBMUJFWSA-N

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

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

Osilodrostat;LCI699; Isturisa; LCI 699; osilodrostat; (+)-Osilodrostat; LCI699-NX; LCI-699-NX; 5YL4IQ1078; UNII-5YL4IQ1078; ...; 928134-65-0; LCI-699

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (11.00 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (11.00 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (11.00 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: ≥ 2.5 mg/mL (11.00 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 5: ≥ 2.5 mg/mL (11.00 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

Solubility in Formulation 6: ≥ 2.5 mg/mL (11.00 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

---

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