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Abstract (1) Background: Cushing’s disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that stimulates the adrenal glands to overproduce cortisol. Chronic exposure to excess cortisol has detrimental effects on health, including increased stroke rates, diabetes, obesity, cognitive impairment, anxiety, depression, and death. The first-line treatment for CD is pituitary surgery. Current surgical remission rates reported in only 56% of patients depending on several criteria. The lack of specificity, poor tolerability, and low efficacy of the subsequent second-line medical therapies make CD a medical therapeutic challenge. One major limitation that hinders the development of specific medical therapies is the lack of relevant human model systems that recapitulate the cellular composition of PitNET microenvironment. (2) Methods: human pituitary tumor tissue was harvested during transsphenoidal surgery from CD patients to generate organoids (hPITOs). (3) Results: hPITOs generated from corticotroph, lactotroph, gonadotroph, and somatotroph tumors exhibited morphological diversity among the organoid lines between individual patients and amongst subtypes. The similarity in cell lineages between the organoid line and the patient’s tumor was validated by comparing the neuropathology report to the expression pattern of PitNET specific markers, using spectral flow cytometry and exome sequencing. A high-throughput drug screen demonstrated patient-specific drug responses of hPITOs amongst each tumor subtype. Generation of induced pluripotent stem cells (iPSCs) from a CD patient carrying germline mutation CDH23 exhibited dysregulated cell lineage commitment. (4) Conclusions: The human pituitary neuroendocrine tumor organoids represent a novel approach in how we model complex pathologies in CD patients, which will enable effective personalized medicine for these patients. Keywords: organoids; neuroendocrine tumors; induced pluripotent stem cells; CDH23 1. Introduction Cushing’s disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that stimulates the adrenal glands to overproduce cortisol [1,2,3,4]. The WHO renamed pituitary adenomas as PitNETs [5]. While PitNETs have been defined as benign, implying that these tumors cause a disease that is not life threatening or harmful to health, in fact chronic exposure to excess cortisol has wide-ranging and detrimental effects on health. Hypercortisolism causes increased stroke rates, diabetes, obesity, depression, anxiety, and a three-fold increase in the risk of death from cardiovascular disease and cancer [4,6,7,8]. The first-line treatment for CD is pituitary surgery, which is followed by disease recurrence in 50% of patients during the 10-year follow-up period after surgery in the hands of an experienced surgeon [9,10,11]. Studies have demonstrated that surgical failures and recurrences of CD are common, and despite multiple treatments, biochemical control is not achieved in approximately 30% of patients. This suggests that in routine clinical practice, initial and long-term disease remission is not achieved in a substantial number of CD patients [7,12]. Hence, medical therapy is often considered in the following situations: when surgery is contraindicated or fails to achieve remission, or when recurrence occurs after apparent surgical remission. While stereotactic radiosurgery treats incompletely resected or recurrent PitNETs, the main drawbacks include the longer time to remission (12–60 months) and the risk of hypopituitarism [3,13,14]. There is an inverse relationship between disease duration and reversibility of complications associated with the disease, thus emphasizing the importance of identifying an effective medical strategy to rapidly normalize cortisol production by targeting the pituitary adenoma [4,7,12]. Unfortunately, the lack of current standard of care treatments with low efficacy and tolerability makes CD a medical therapeutic challenge. The overall goal of medical therapy for CD is to target the signaling mechanisms to lower cortisol levels in the body [15,16]. The drugs offered for treatment of CD vary in the mechanism of action, safety, tolerability, route of administration, and drug–drug interactions [15,16]. In the era of precision medicine [17], where it is imperative to identify effective therapies early, there is an urgent need to accelerate the identification of therapies targeted to the ACTH-secreting pituitary tumor which are tailored for each individual patient. The absence of preclinical models that replicate the complexity of the PitNET microenvironment has prevented us from acquiring the knowledge to advance clinical care by implementing therapies specifically targeting the tumor, which would have a higher efficacy and tolerability for CD patients. In this instance, organoids can replicate much of the complexity of an tumor. An “organoid” is defined as a three-dimensional cell structure, grown from primary cells of dissociated pituitary tumors in Matrigel matrix, which proliferate, and differentiate in three dimensions, eventually replicating key biological properties of the tissue [18]. While pituitary cell lines predominantly represent hormonal lineages, these cultures do not reproduce the primary pituitary tissue because of the tumor transformation and non-physiological 2D culture conditions [19,20,21]. Pituitary tissue-derived organoids have been generated from mouse models [22,23]. While several human and rat pituitary spheroid/aggregate/tumoroid models have been reported, these cultures consist of poorly differentiated cells with high replicative potential which can affect drug response and produce data that poorly translate to the clinic [24,25]. In this study, we developed an organoid model derived from human PitNETs that replicated much of the cellular complexity and function of the patient’s tumor. Organoids derived from corticotroph PitNETs retained the genetic alterations of the patient’s primary tissue. 2. Materials and Methods 2.1. Generation and Culture of Human Pituitary Neuroendocrine Tumor (PitNET) Organoids Patients with planned transsphenoidal surgery for pituitary tumors were identified in the outpatient neurosurgery clinics. Tissues were collected under the St. Joseph’s Hospital and Barrow Neurological Institute Biobank collection protocol PHXA-05TS038 and collection of outcomes data protocol PHXA-0004-72-29, with the approval of the Institutional Review Board (IRB) and patient consent. Samples were de-identified and shipped to the Zavros laboratory (University of Arizona) for processing. Pituitary tumor tissue was collected in Serum-Free Defined Medium (SFDM) supplemented with ROCK inhibitor (Y27632, 10 µM), L-glutamine (2 mM), A83-01 (activin receptor-like kinase (Alk) 4/5/7 inhibitor, 0.5 mM), penicillin/streptavidin (1%), kanamycin (1%), amphotericin/gentamycin (0.2%), CHIR-98014 (4 mM), and thiazovivin (TZV, 2.5 mM). Tissues that contained red blood cells were incubated with Red Blood Cell (RBC) Lysis Buffer according to the manufacturer’s protocol (Thermo Fisher Scientific, San Fransisco, CA, USA). Tissues were dissected into small pieces, transferred to digestion buffer (DMEM/F12 supplemented with 0.4% collagenase 2, 0.1% hyaluronic acid, 0.03% trypsin-EDTA) and incubated for 5–10 min at 37 °C with gentle shaking. Tissue was further incubated with Accutase™ (Thermo Fisher Scientific) for 5 min at 37 °C. Enzymatically dissociated cells were pelleted and washed in DPBS supplemented with antibiotics at a 400 relative centrifugal force (RCF) for 5 min. Dissociated adenoma cells were resuspended in Matrigel™, and Matrigel™ domes containing the cells were then plated in culture dishes and overlaid with pituitary growth media (Supplemental Table S1). The culture was maintained at 37 °C at a relative humidity of 95% and 5% CO2. Organoid growth medium was replenished every 3–4 days and passaged after 15 days in culture. 2.2. Generation of Induced Pluripotent Stem Cells (iPSCs) Induced pluripotent stem cell lines (iPSC lines) were generated from control individuals (no reported disease) or CD patients according to published protocols by the University of Arizona iPSC Core [26]. All human iPSC lines were tested and found to be negative for mycoplasma contamination using the Mycoalert Mycoplasma testing kits (LT07-318, Lonza), and no karyotype abnormalities were found (KaryoStat+, Thermo). 2.3. Pituitary Organoids Generated from iPSCs Six well culture plates were coated with 2 mL/well 0.67% Matrigel (diluted in E8 media, UA iPSC core, 151169-01) and incubated at 37 °C at a relative humidity of 95% and 5% CO2 overnight. The iPSC lines were reprogrammed from the blood of either a healthy donor (JCAZ001) or a CD patient (iPSC7 and iPSC1063) at the University of Arizona iPSC Core. Passage 12 iPSCs were plated onto the coated plates and incubated at 37 °C at a relative humidity of 95% and 5% CO2. At 70% confluency, cells were passaged to freshly coated 24 well plates at a ratio of 1:8 and grown to 85–90% confluency before beginning the directed differentiation schedule. From days 0 to 3, cells were cultured in E6 media supplemented with 1% penicillin/streptomycin, 10 μM SB431542, and 5 ng/mL BMP4. BMP4 was withdrawn from the culture at day 3. Starting on day 4, the cells were cultured in E6 media, supplemented with 10 μM SB431542, 30 ng/mL human recombinant SHH, 100 ng/mL FGF8b, 10 ng/mL FGF18, and 50 ng/mL FGF10. Fifteen days after culture, the cells were harvested in cold E6 media by pipetting and resuspended in Matrigel™ (20,000 cells/50 mL Matrigel™). Matrigel™ domes containing the cells were plated in culture dishes and overlaid with differentiation media containing E6 media which was supplemented with 10 μM Y-27632, 30 ng/mL human recombinant SHH, 100 ng/mL FGF8b, 10 ng/mL FGF18, and 50 ng/mL FGF10 (Supplemental Table S2). Organoids were cultured for a further 15 days at 37 °C at a relative humidity of 95% and 5% CO2. 2.4. Spectral Flow Cytometry (Cytek™ Aurora) The multicolor flow cytometry panel was designed using the Cytek® Full Spectrum Viewer online tool to calculate the similarity index (Supplemental Figure S1). The organoids were harvested in cold SFDM media and centrifuged at 400× g for 5 min. Supernatant was discarded and organoids were dissociated to single cells using Accutase® (Thermo Fisher Scientific 00-4555-56). The enzymatic reaction was stopped using prewarmed DPBS, and cells were then centrifuged at 400× g for 5 min and incubated with fluorochrome-conjugated/unconjugated primary surface or cytoplasmic antibodies (Supplemental Figure S1) at 4 °C for 30 min. Cells were then washed with Cell Staining Buffer (BioLegend # 420-201) and incubated with secondary antibodies (Supplemental Figure S1) at 4 °C for 30 min. Cells were fixed using Cytofix/Cytoperm™ Fixation/Permeabilization Solution (BD Biosciences # 554714) at 4 °C for 20 min, followed by washing with Fixation/Permeabilization wash buffer. Cells were labeled with fluorochrome-conjugated/unconjugated intracellular primary antibodies (Supplemental Figure S1) at 4 °C for 30 min, then washed and incubated with secondary antibodies at 4 °C for 30 min. Cells were resuspended in cell staining buffer and fluorescence and measured using the Cytek Aurora 5 Laser Spectral Flow Cytometer. An unstained cell sample was fixed and used as a reference control. UltraComp eBeads™, Compensation Beads (Thermo Fisher Scientific # 01-2222-42) were stained with the individual antibodies and used as single stain controls for compensation and gating. Data were acquired using the Cytek™ Aurora and analyzed using Cytobank software (Beckman Coulter, Indianapolis, IN, USA). 2.5. Whole Mount Immunofluorescence Organoids were immunostained using published protocols by our laboratory [27,28,29]. Proliferation was measured by using 5-ethynyl-2′-deoxyuridine (EdU) incorporation according to the Manufacturer’s protocol (Click-IT EdU Alexa Fluor 555 Imaging Kit, Thermo Fisher Scientific C10338). Co-staining was performed by blocking fixed organoids with 2% donkey serum (Jackson Immuno Research, # 017-000-121) diluted in 0.01% PBST for 1hr at room temperature. Organoids were then incubated overnight at 4 °C with primary antibodies, followed by secondary antibodies and Hoechst (Thermo Fisher Scientific H1399, 1:1000 in 0.01% PBST) for 1 h at room temperature. Human specific primary antibodies used included: rabbit anti-ACTH (Thermo Fisher Scientific 701293, 1:250), rabbit anti-Synaptophysin (Thermo Fisher Scientific PA5-27286, 1:100), species PIT1 (Thermo Fisher Scientific PA5-98650, 1:50), rabbit anti-LH (Thermo Fisher Scientific PA5-102674, 1:100), mouse anti-FSH (Thermo Fisher Scientific MIF2709, 1:100), mouse anti-PRL (Thermo Fisher Scientific CF500720, 1:100), Alexa Flour conjugated GH (NB500-364AF647, 1:100), and mouse anti-CAM5.2 (SIGMA 452M-95, 1:250). The secondary antibodies used included Alexa Fluor 488 Donkey Anti Rabbit IgG (H+L) (Thermo Fisher Scientific A21206, 1:100) or Alexa Fluor 647 Donkey Anti Mouse IgG (H+L) (Thermo Fisher Scientific A31571, 1:100). Organoids were visualized and images were acquired by confocal microscopy using the Nikon CrestV2 Spinning Disk (Nikon, Melville, NY, USA). Fluorescence intensity and percentage of EdU positive cells of total cells, were calculated using Nikon Elements Software (Version 5.21.05, Nikon, Melville, NY, USA). 2.6. Nuclear Morphometric Analysis (NMA) Nuclear Morphometric Analysis (NMA) using treated organoids was performed based on a published protocol that measures cell viability based on the changes in nuclear morphology of the cells, using nuclear stain Hoechst or DAPI [30]. Images of organoid nuclei were analyzed using the ImageJ Nuclear Irregularity Index (NII) plugin for key parameters, which included cell area, radius ratio, area box, aspect, and roundness. Using the published spreadsheet template [30], the NII of each cell was calculated with the following formula: NII = Aspect − Area Box + Radius Ratio + Roundness. The area vs. NII of vehicle-treated cells were plotted as a scatter plot using the template, and was considered as the normal cell nuclei. The same plots were generated for each condition, and the NII and area of treated cells were compared to the normal nuclei, and classified as one of the following NMA populations: Normal (N; similar area and NII), Mitotic (S; similar area, slightly higher NII), Irregular (I; similar area, high NII), Small Regular (SR; apoptotic, low area and NII), Senescent (LR; high area, low NII), Small Irregular (SI; low area, high NII), or Large Irregular (LI; high area, high NII). Cells classified as SR exhibited early stages of apoptosis, and cells classified as either I, SI, or LI exhibited significant nuclear damage. The percentage of cells in each NII classification category were calculated and plotted as a histogram using GraphPad Prism. 2.7. ELISA Concentration of secreted ACTH in conditioned media that was collected from organoid cultures was measured using the Human ACTH ELISA Kit (Novus Biologicals, NBP2-66401), according to the manufacturer’s protocol. The enzyme–substrate reaction was measured spectrophotometrically (BioTek Gen5 Micro Plate Reader Version 3.11, Santa Clara, CA, USA) at a wavelength of 450 nm, and the ACTH concentration (pg/mL) was interpolated by a standard curve with a 4-parameter logistic regression analysis, using GraphPad Prism (Version 9.2.0, San Diego, CA, USA). 2.8. Drug Assay Patient adenoma-derived pituitary organoids were grown in 96-well plates and treated with 147 small molecules taken from the NCI AOD9 compound library for 72 h. (https://dtp.cancer.gov/organization/dscb/obtaining/available_plates.html (accessed on 22 August 2021)). Drugs were diluted from 10 mM DMSO stock plates into 100 M DMSO working stocks with a final concentration of 1μM. All vehicle controls were treated with 0.1% DMSO. Organoid proliferation was measured using a CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (MTS, Promega, G3582, Madison, WI, USA) according to the manufacturer’s instruction. Organoid death was calculated based on the absorbance readings at 490 nm, collected from the MTS assay relative to the vehicle controls. Drug screens were performed with biological replicates in the same screen. Drugs were selected based on their ability to target key signaling pathways as well as clinical relevance to the treatment. Drug sensitivity is represented by cell viability, and is significant at <0.5 suppressive effect of the drugs. The percent of cell viability relative to the vehicle control was calculated. Correlation coefficients across each organoid were calculated using the Pearson method to assess confidence in replication. The variance component was detected for each drug across all organoids. A random effect model was run with a single random factor for each drug, and estimated variance was calculated by rejecting the null hypothesis that variation was not present among samples. The drug responses were grouped by variance factor, into large (vc > 100), median (100 > vc > 50), and small (vc < 50). A heatmap was used to display the differential responses in cell viability for the drugs. Drugs that clustered together and showed response within corticotrophs were investigated further based on their mode of action. Pathways (Kegg and Reactome) and gene ontology mapping were conducted for the genes that were being targeted by the drugs, in order to evaluate the key responses in cellular processes. A network was constructed in Cytoscape v 3.8.2 (San Diego, CA, USA) for the purpose of association between the drugs and genes. 2.9. Drug Dose Responses Organoids were grown in Matrigel™ domes within 96-well round-bottom culture plates. Recombinant human SHH was removed from the pituitary organoid growth media, 24 h prior to drug treatment. Organoids were treated with either vehicle (DMSO), cabergoline (Selleckchem S5842), ketoconazole (Selleckchem S1353), roscovitine (Selleckchem S1153), GANT61 (Stemcell Technologies 73692), pasireotide (TargetMol TP2207), mifeprostone (Selleckchem S2606), etomidate (Selleckchem S1329), mitotane (Selleckchem S1732), metyropane (Selleckchem S5416), or osilodrostat (Selleckchem S7456) at concentrations of 0, 1, 10, 100, 1000, and 10,000 nM, for 72 h. The percentage of cell viability was measured using an MTS assay (Promega G3580). Absorbance was measured at 490 nm and normalized to the vehicle. Concentrations were plotted in a logarithmic scale, and a nonlinear dose response curve regression was calculated using GraphPad Prism. An IC50 value for each drug treatment was determined based on the dose response curve, using GraphPad Prism analysis software. 2.10. Calculation of Area under the Curve (AUC) AUC (area under the curve) was determined by plotting the normalized % cell viability versus transformed concentration of the drugs, using a trapezoidal approximation for the area [31]. The formula was based on splitting the curve into trapezoids with bases equal to the % viability (V) and height equal to the interval length (difference in concentrations (C), and then summing the areas of each trapezoid: ∑n0(Vn+Vn−1)2∗(Cn−Cn−1) 2.11. Quantitative RT PCR (qRT-PCR) RNA was collected from patient-derived organoid cultures using the RNeasy Mini Kit (Qiagen). cDNA was generated from the extracted RNA, and then pre-amplified using TaqMan PreAmp Master Mix (Thermo Fisher Scientific 391128). The primers used were human-specific GAPDH (Thermo Fisher Scientific, Applied Biosystems Hs02786624_g1), NR5A1 (SF1) (Thermo Fisher Scientific, Hs00610436_m1), PIT1 (Thermo Fisher Scientific, Hs00230821_m1), TPit (Thermo Fisher Scientific, Hs00193027), and POMC (Thermo Fisher Scientific, Hs01596743_m1). Each PCR reaction was performed using a final volume of 20 µL, composed of 20X TaqMan Expression Assay primers, 2X TaqMan Universal Master Mix (Applied Biosystems, TaqMan® Gene Expression Systems), and a cDNA template. Amplification of each PCR reaction was conducted in a StepOne™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), using the following PCR conditions: 2 min at 50 °C, 10 min at 95 °C, denaturing for 15 s at 95 °C, and annealing/extending for 1 min at 60 °C, for a total of 40 cycles. Relative fold change was calculated using the 2 − ∆∆Ct method [32], where CT = threshold cycle. Results were analyzed as the average fold change in gene expression compared to the control, and GAPDH served as an internal control. 2.12. Whole Exome Sequencing WES was performed by the University of Arizona Center for Applied Genetics and Genomic Medicine. Isolated DNA from patient adenoma tissue will be quantified using the Qubit quantitation system with standard curve, as per the supplier protocol (Thermo Fisher Scientific). All samples were further tested for quality using the Fragment Analyzer (Advanced Analytical), following the manufacturer-recommended protocols. Whole exome sequencing (WES) was performed by array capture and approximately 60 Mb of exome target sequence, using the SureSelectXT Human All Exon V6 enrichment (Agilent) or equivalent (which one was used). All exome library builds were quantified via qPCR and subsequently sequenced to a minimum 20X coverage, using paired-end chemistry on the Illumina NovaSeq platform. Whole exome sequencing (WES) was performed by hybridization capture of approx. 35 Mb of the exome target sequence, using the Swift Exome Hyb Panel (Swift Biosciences 83216). All exome library builds were quantified via qPCR and subsequently sequenced to a minimum 20X coverage, using paired-end chemistry on the Illumina NextSeq500 or NovaSeq platform (Illumina). DNA reads were trimmed, filtered by quality scores and aligned to the human genome (hg38) with Burrows–Wheeler Aligner with default parameters. Picard (http://broadinstitute.github.io/picard (accessed on 22 December 2021)) was used to mark duplicates. Germline single nucleotide variants (SNV) were called using the Genome Analysis Tool Kit (GATK), using the given guidelines. Mutations were annotated using ANNOVAR for coding sequences. Variants that passed the quality filter were further investigated for similarity. Concordance between tissue and organoids was calculated using Jaccard similarity index (Jij = Mij/(Mi + Mj − Mij) where Mi is the number of variants in tissues, Mj is the number of variants in organoids, and Mij is the number of identical variants in both tissue and organoid. 2.13. Single Cell RNA Sequencing (scRNA-Seq) Cultures were collected on day 15 of the pituitary directed differentiation schedule, and cells were dissociated into a single-cell suspension using Cell Dissociation Buffer (Thermo Fisher Scientific 13151014). Cells (15,000 cells/sample) were resuspended in the sample buffer (BD Biosciences 65000062), filtered using cell strainer (40 microns), and loaded into a BD Rhapsody cartridge (BD Biosciences 400000847) for single-cell transcriptome isolation. Based on the BD Rhapsody system whole-transcriptome analysis for single-cell whole-transcriptome analysis, microbead-captured single-cell transcriptomes were used to prepare a cDNA library. Briefly, double-stranded cDNA was first generated from the microbead-captured single-cell transcriptome in several steps, including reverse transcription, second-strand synthesis, end preparation, adapter ligation, and whole-transcriptome amplification (WTA). Then, the final cDNA library was generated from double-stranded full-length cDNA by random priming amplification using a BD Rhapsody cDNA Kit (BD Biosciences, 633773), as well as the BD Rhapsody Targeted mRNA and WTA Amplification Kit (BD Biosciences, 633801). The library was sequenced in PE150 mode (paired-end with 150-bp reads) on NovaSeq6000 System (Illumina). A total of 80,000 reads were demultiplexed, trimmed, mapped to the GRCh38 annotation, and quantified using the whole transcriptome analysis pipeline (BD Rhapsody™ WTA Analysis Pipeline v1.10 rev6, San Jose, CA, USA) on the Seven Bridges Genomics platform (https://igor.sbgenomics.com (accessed on 4 April 2022)), prior to clustering analysis in Seurat. For QC and filtration, read counting and unique molecular identifier (UMI) counting were the principal gene expression quantification schemes used in this single-cell RNA-sequencing (scRNA-seq) analysis. The low-quality cells, empty droplets, cell doublets, or multiplets were excluded based on unique feature count (less than 200 or larger than 2500), as they may often exhibit either an aberrantly high gene count or very few genes. Additionally, the mitochondrial QC metrics were calculated, and the cells with >5% mitochondrial counts were filtered out, as the percentage of counts originating from a set of low-quality or dying cells often exhibit extensive mitochondrial contamination. After the removal of unwanted cells from the single cell dataset, the global-scaling normalization method LogNormalize was employed. This method normalizes the feature expression measurements for each cell by the total expression, multiplies this by a scale factor (10,000), and log-transforms the result. The molecules per gene per cell, based on RSEC error correction (RSEC_MolsPerCell file) matrix files from iPSCctrl and iPSCCDH23 samples, were imported into Seurat v4, merged, and processed (as stated above) for UMAP reduction, cluster identification, and differential marker assessment using the FindAllMarkers function within Seurat. 2.14. Statistical Analyses Sample size was based on assessment of power analysis using SigmaStat software. Data collected from each study from at least 4 in vitro technical replicates were analyzed by obtaining the mean ± standard error of the mean (SEM), unless otherwise stated. The significance of the results was then tested using commercially available software (GraphPad Prism, GraphPad software, San Diego, CA, USA). 3. Results 3.1. Generation and Validation of Human PitNET Tissue Derived Organoids Human PitNET tissue was harvested during endoscopic transsphenoidal pituitary surgery from 35 patients in order to generate organoids. These cultures are referred to as human PitNET tissue derived organoids (hPITOs). Supplementary Table S3 summarizes the neuropathology reports and clinical diagnosis from these cases. In summary, 12 corticotroph (functional, CD), and 3 silent corticotroph tumors (nonfunctional tumors), 9 gonadotroph tumors, 8 lactotroph tumors, and 3 somatotroph tumors (acromegaly) were used to generate hPITOs (Supplementary Table S3). Bright-field microscopy images of hPITOs that were generated from corticotroph adenomas from patients diagnosed with CD (Figure 1a–e). Silent/nonfunctioning tumors (Figure 1f,g) revealed morphological diversity among the organoid lines between individual patients and amongst subtypes. Confocal microscopy was used to capture a z-stack through the hPITO38, immunofluorescently stained for CAM5.2 (red), ACTH (green), and Hoechst (nuclear staining, blue) and emphasizes the 3D cellular structure of the hPITOs (Supplemental Video S1). Lactotroph, gonadotroph, and somatotroph adenomas were used to generate hPITOs, and showed the same morphological divergence amongst subtypes and between each patient line (Supplemental Figure S2). Proliferation was measured within the cultures using 5-ethynyl-2′-deoxyuridine (EdU) uptake and showed that the percentage of EdU+ve cells/total Hoechst+ve nuclei directly correlated with the pathology MIB-1 (Ki67) score (red, R2 = 0.9256) (Figure 1a–g, Supplemental Figure S2). ACTH concentration, which was measured by ELISA using organoid conditioned culture media collected from each hPITO line, showed the highest expression in the corticotroph adenoma organoids generated from CD patients (Figure 1h). Figure 1. Morphology and function of corticotroph hPITOs. (a–g) Brightfield images, immunofluorescence staining using antibodies specific for CAM5.2 (red), ACTH (green), and EdU (magenta, inset) of organoid cultures generated from patients with Cushing’s disease (hPITOs 1, 7, 10, 33, 35) or nonfunctional corticotroph adenomas (hPITO8, 12). Quantification of %EdU positive cells/total cell number is shown and compared to the Ki67 score given in the pathology report (Supplemental Table S3). An ELISA was performed using conditioned media collected from (h) corticotroph hPITO cultures and (i) lactotroph, somatotroph, and gonadotroph hPITO cultures for the measurement of ACTH secretion (pg/mL). 3.2. Characterization of Cell Lineages in Pituitary Adenoma-Derived Organoids by Spectral Cytek™ Aurora Analysis In order to validate the similarity in cell lineages identified between the organoid line and the patient’s tumor, we compared the immunohistochemistry from the neuropathology report (Supplemental Table S3) to the expression pattern of pituitary adenoma-specific markers, which were measured using Cytek™ Aurora spectral flow cytometry (Figure 2). The location of cells that are found in each cluster based on the highly expressed antigens are shown in the representative tSNE (viSNE) maps (Figure 2a). Compared to nonfunctional adenoma-derived hPITOs, organoids derived from corticotroph adenomas of CD patients highly expressed proliferating (Ki67+) T-Pit+ ACTH cells (Figure 2a). Interestingly, there was an increase in SOX2+ cells within the total cell population, associated with Crooke’s cell adenoma hPITOs (Figure 2a). Within the total cell population, cell clusters expressing CD45 and vimentin were also measured (Figure 2a). Data for the analysis of corticotroph hPITOs, derived from CD patients and individuals with nonfunctional adenomas, were summarized in a heatmap for each subtype organoid line based on quantified cell abundance (percent of total cells) using spectral flow cytometry (Figure 2b). Figure 2. Cell heterogeneity of corticotroph hPITOs. (a) viSNE maps define spatially distinct cell populations using pituitary specific cell lineage, stem cell, and transcription factor markers. Cell populations were quantified in organoids generated from CD patients with corticotroph adenomas (sparsely granulated and Crooke’s cell adenoma) or patients with nonfunctional corticotroph adenomas. (b) Quantification of the abundance of cells expressing pituitary specific markers as a percent total. viSNE maps define spatially distinct cell populations in organoid cultures generated from CD patient with (c) corticotroph adenoma (hPITO37, Crooke’s cell adenoma) and adjacent normal tissue (hPITO37N), or (d) sparsely granulated corticotroph adenomas (hPITO38) and adjacent normal tissue (hPITO38N). Organoid cultures derived from pituitary adenomas (hPITO37 and hPITO38) were compared to organoids derived from adjacent normal pituitary tissue (hPITO37N and hPITO38N) (Figure 2c,d). While Pit1 lineages including cells expressing GH and PRL, as well as SF1 lineages expressing FSH and LH, were detected in the hPITO37N and hPITO38N organoid cultures, these cell populations were significantly reduced within the patient’s matched adenoma tissue (Figure 2c,d). Overall, hPITOs derived from CD patients expressed increased stem and progenitor cell markers, including CXCR4, SOX2, and CD133 (Figure 2). Collectively, our findings of the characterization of the hPITO cultures support our prediction that this in vitro model recapitulates much of the patient’s adenoma pathophysiology. 3.3. Inherent Patient Differences to Drug Response Is Reflected in the Organoid Culture Tumor recurrence can occur in as many as 30–50% of CD patients after successful surgical treatment [10,33,34]. Unfortunately, bilateral adrenalectomy is the chosen surgical treatment for patients with persistent CD [35]. Bilateral adrenalectomy leads to the increased risk for development of Nelson’s syndrome (progressive hyperpigmentation due to ACTH secretion and expansion of the residual pituitary tumor). Although the risk of developing Nelson’s syndrome following adrenalectomy can be reduced by 50% with stereotactic radiotherapy [35], there is a need to develop medical therapies that directly target the pituitary adenoma. Thus, we established a high-throughput drug screening assay using patient-derived PitNET organoids. After 72 h of treatment, cell viability was measured using an MTS assay, and data were represented as a heatmap whereby blue indicated higher cell death, and red suggested higher cell viability. The replicates behaved consistently with the drug response, with correlation scores of >0.8 for these samples (Figure 3a). We estimated the variance component for each drug across all organoids. Variation among samples was found to be significant (p ≤ 0.05) for each of the 83 drugs. The drug responses were grouped by variance factor into large, median, and small. The larger the variance, the more variable the drug response was across the organoids. We noted a set of drugs that showed a significant differential response across the functional corticotroph organoids. Unsupervised clustering of drug responses across organoids shows a pattern that relates to our statistically calculated results (Figure 3a,c), and the replicates for each independent organoid cluster together. The drugs with higher variance components across all the functional corticotrophs cluster together as a group (Figure 3a). These drugs show cell viability of 10% to 60% across different organoids. Analyzing the pattern more closely, we observe that, within a pathologically defined group, there was a differential organoid response to drugs as well as inherent patient differences to drugs within this group. Figure 3 demonstrates a variation in drug responsiveness amongst the organoid lines generated from individual patients. Importantly, there was further divergence in drug responsiveness amongst the individual organoid lines within each pathologically defined corticotroph subtype. These data clearly demonstrate that the inherent patient difference to drug response which is often observed among CD patients is reflected in the organoid culture. Figure 3. Drug screen using hPITOs generated from CD patients. (a) High-throughput drug screening of hPITOs reveals sensitivities to a range of therapeutic agents. Cell viability with high values (indicating resistance) are depicted in red, and low values (indicating sensitivity) are in blue in the clustered heatmap. (b,c) Clusters showing response to therapeutic agents with the most variance across the organoids. (d) Network of drugs from the clusters b and c and their gene targets, showing their participation in signaling pathways and cellular processes. Drugs that clustered together and showed correlated responses were investigated further for their mode of action based on target genes (Figure 3d). The genes were analyzed for their associations in cellular pathways and gene ontology functional processes. Identified drug–gene pairs were interconnected by cellular pathways that are known to regulate cell cycle, WNT signaling, hedgehog signaling, and neuroactive ligand-receptor interaction signaling pathways (Figure 3d). These identified genes are also known to be influenced by multiple cellular functions, such as cytokine–cytokine receptor interactions and Notch signaling. Proteosome 20S subunit genes PSMAs/PSMBs and the HDAC gene family are involved in many cellular functions. The ephrin receptors (EPHs), adrenoceptor alpha receptors (ADRs), dopamine receptors (DRDs), and the 5-hydroxytryptamine serotonin receptors (HTRs) gene families influence neuronal functions and are targeted by multiple drugs in our focused cluster. These data reveal potential therapeutic pathways for CD patients. Divergent half maximal inhibitory concentration (IC50) values, as documented by an MTS cell viability assay, were observed in response to drug treatment among hPITOs lines 28, 33, 34, 35, and 37. Note that a shift of the curve to the right indicates a higher IC50 (i.e., more resistant to that drug). Cell viability assays were normalized to vehicle-treated controls in order to ensure that toxicity was specific to the drug effects (Figure 4). Dose response curves for organoid 33 and organoid 34 showed better responses at lower doses for cabergoline compared to Metyrapone and osilodrostat, but different for organoid 35, where Metyrapone and osilodrostat gave better responses than Cabergoline (Figure 4a–h). For the drugs mifepristone and GANT61, 33 and 34 had the same level of response to both the drugs. However, when the two organoid responses were compared, 34 had a better response than 33 (Figure 4a–h). Similar divergent drug responses were observed in hPITO lines 37 and 38 (Figure 4i,k). However, organoids generated from adjacent normal pituitary tissue from patients 37 and 38 were nonresponsive to the same standard of care of investigational drugs for CD (Figure 4j,l). These data were consistent with observation made in the drug screen (Figure 3a–c), and demonstrate that there was an inherent difference to drug response within the organoid cultures of the same corticotroph subtype. Figure 4. Drug dose responses by hPITOs generated from CD patients. Dose responses to mifepristone, GANT61, cabergoline, and osilodrostat. (a,e) hPITO28, (b,f) hPITO33, (c,g) hPITO34, and (d,h) hPITO35. Dose responses to cabergoline, ketoconazole, roscovitine, GANT61, pasireotide, mifepristone, etomidate, mitotane, metyrapone, and osilodrostat in (i) hPITO37, (j) organoids generated from adjacent normal pituitary tissue (hPITO37N), (k) hPITO38, (l) hPITO38N, and (m) hPITO39. (n) IC50 and integrated area under the curve in response to mifepristone, ketoconazole, and pasireotide using hPITO39 cultures. Nuclear morphometric analysis of hPITO39 cultures in response to (o,p) vehicle, (q,r) mifepristone, (s,t) pasireotide, and (u,v) ketoconazole. Morphometric classification of NII was based on the normal (N), small (S), small regular (SR), short irregular (SI), large regular (LR), large irregular (LI), and irregular (I) nuclear morphology. Representative Hoechst staining of organoids in response to drug treatments for the calculation of the nuclear irregularity index (NII) are shown in the insets in (p,r,t,v). In addition to cell viability, Nuclear Morphometric Analysis (NMA) using treated organoids was performed based on a published protocol that measures cell viability according to the changes in nuclear morphology of the cells, using nuclear stain Hoechst or DAPI [30]. Nuclear Irregularity Index (NII) was measured based on the quantification of the morphometric changes in the nuclei in response to the standard-of-care drugs mifepristone, pasireotide, and ketoconazole in hPITO39 (Figure 4o–v). The area vs. NII of vehicle-treated cells were plotted as a scatter plot using the template, and considered as the normal cell nuclei (Figure 4o). The same plots were generated for mifepristone (Figure 4q), pasireotide (Figure 4s), and ketoconazole (Figure 4u). The NII and area of treated cells were compared to those of the normal nuclei, and classified as one of the following NMA populations: Normal (N; similar area and NII), Mitotic (S; similar area, slightly higher NII), Irregular (I; similar area, high NII), Small Regular (SR; apoptotic, low area and NII), Senescent (LR; high area, low NII), Small Irregular (SI; low area, high NII), or Large Irregular (LI; high area, high NII) (Figure 4p,r,t,v). Cells classified as SR exhibited early stages of apoptosis, and cells classified as either I, SI, or LI exhibited significant nuclear damage. Data showed that mifepristone induced significant apoptosis in hPITO39 cultures (Figure 4r), compared to responses to pasireotide (Figure 4t) and ketoconazole (Figure 4v). These responses were consistent with the IC50 and the total area under the curve in response to drugs (Figure 4m,n). Measurement of NII is an approach which may be used to confirm potential drug targets identified from the drug screen. 3.4. Organoid Responsiveness to Pasireotide Correlates with SSTR2 and SSTR5 Expression Organoid lines hPITO28, 31, 33, 34, and 35 exhibited divergent IC50 values in response to SSTR agonist pasireotide (Figure 5a). hPITO34 was the most responsive to pasireotide, with a low IC50 value of 6.1 nM (Figure 5a). Organoid lines hPITO33 and hPITO35 were the least responsive, with IC50 values of 1.2 µM and 1 µM, respectively, in response to pasireotide (Figure 5a). The expression of SSTR subtypes 1–5 among the different organoid lines were measured by qRT-PCR and IHC (Figure 5b). One of the least responsive organoid lines, hPITO28, exhibited lower differential expression in SSTR2 and SSTR5 compared to the highly responsive hPITO34 line (Figure 5a,b). Gene expression levels of SSTR2 and SSTR5 within hPITO28 and 34 correlated with protein levels within the patient’s tumor tissue (Figure 5c–f). Given the greater binding affinity for SSTR5 compared to SSTR2 by pasireotide, these data were consistent with greater responsiveness to the drug by hPITO34 in comparison to hPITO28 (Figure 5a,c–f). The expression of SSTR subtypes 2 and 5 within the organoid cultures correlated with the expression patterns of the patient’s tumor tissues (Figure 5a,c–f). Figure 5. SSTR1-5 expression in hPITOs and patient’s PitNET tissue. (a) Dose response of hPITO28, 31, 33, 34, and 35 lines to pasireotide. (b) Differential expression of SSTR subtypes 1–5 (SSTR1, SSTR2, SSTR3, SSTR4, SSTR5) in hPITO28, hPITO31, hPITO33, hPITO34, and hPITO35. Immunohistochemistry of (c,e) SSTR2 and (d,f) SSTR5 expression in patient PitNET tissue (Pt28 and Pt34), from which hPITO28 and 34 were generated. 3.5. Organoids Derived from Pituitary Corticotroph Adenomas Retain the Genetic Alterations of the Patient’s Primary Tumor In order to identify the genetic features of the organoids derived from pituitary adenomas of CD patients, we performed whole-exome sequencing (WES) of hPITOs and the corresponding primary adenoma tissues. We performed WES analysis of each hPITO line, and compared the results with those for the corresponding primary adenoma tissues. We showed the concordance rate of exonic variants between the primary tumor tissues obtained from CD patients and the corresponding organoid line. We identified, on average, approximately 5000 mutations across each of the 14 paired samples of organoids and tissues. For the variants detected, all seven pairs showed a Jaccard index ranging from 0.5 to 0.8. Out of seven pairs, five (hPITO24, 25, 28 and 35) pairs had a Jaccard score of 0.8, while hPITO33 and 34 pairs had 0.7, and hPITO1 had 0.5. In order to investigate the similarity across the SNV (single nucleotide variation) sites, we calculated the Jaccard index of exon sites for synonymous and non-synonymous events, and found scores for all pairs ranging from 0.8 to 0.9. Furthermore, for only non-synonymous events, Jaccard scores also ranged from 0.8 to 0.9, except for hPITO1, which showed overall lower concordance, and had a score of 0.4 to 0.5. Figure 6 shows non-synonymous mutations found in organoid and tissue pairs for some of the key genes that are known to be involved in pituitary adenoma disease. Concordance indices between organoids and the matched patient’s adenoma tissues is reported in Figure 6. Therefore, WES data demonstrated that organoids derived from pituitary corticotroph adenomas retained the genetic alterations of the patient’s primary tumor tissue. Figure 6. Genomic landscape of hPITOs recapitulates genetic alterations commonly found PitNETs. Overview of single nucleotide variation events detected in hPITOs in genes commonly altered in PitNETs. The mutation frequency across the organoid population is depicted on the right. Color coding of the figure shows that organoid lines are derived from the same patient tumor tissue. ORG: organoid line, TIS: matched patient’s PitNET tissue. 3.6. IPSC Pituitary Organoids Generated from a CD Patients Expressing Familial Mutations Reveal Corticotroph Adenoma Pathology In Vitro Extensive research has revealed the role of somatic and germline mutations in the development of CD adenomas [36,37]. Pituitary organoids were developed from iPSCs generated from the PBMCs of CD patients and carrying germline mutations that were identified by WES (Supplemental Figure S4). Chromosomal aberrations were not found when comparing against the reference dataset in the iPSCs generated from the CD patients (Supplemental Figure S3a,b). PBMCs isolated from patients diagnosed with CD were analyzed by WES in order to determine the expression of germline mutations. WES revealed the expression of a more recently identified gene predisposing patients to CD, namely cadherin-related 23 [38] (Supplemental Figure S5). Pituitary organoids were then developed from iPSCs which were generated from the PBMCs of patients with CD (iPSCCDH23 and iPSCMEN1) and a healthy individual (iPSCctrl). Expression of PIT1 (pituitary-specific positive transcription factor 1), ACTH (adrenocorticotropic hormone), GH (growth hormone), FSH (follicle-stimulating hormone), LH (luteinizing hormone), PRL (prolactin), and synaptophysin (synaptophysin) with co-stain Hoechst (nuclei, blue) was measured by immunofluorescence, using chamber slides collected at 15 of the differentiation schedules (Supplemental Figure S6). While pituitary tissue that was differentiated from iPSCctrl expressed all major hormone-producing cell lineages (Supplemental Figure S6a), there was a significant increase in the expression of ACTH and synaptophysin, with a concomitant loss of PIT1, GH, FSH, LH, and PRL in iPSCsMEN1 (Supplemental Figure S6b,c). Interestingly, iPSCCDH23 cultures exhibited a significant increase in the expression of ACTH, GH, LH, and synaptophysin, with a concomitant loss of PIT1, FSH, and PRL (Supplemental Figure S6b,c). Immunofluorescence of iPSCs collected on the fourth day of the differentiation schedule revealed no expression of PIT1, ACTH, GH, FSH, LH, or PRL in (data not shown). Compared to control lines, iPSC lines expressing mutated CDH23 secreted significantly greater concentrations of ACTH earlier in the differentiation schedule (Supplemental Figure S7a). The upregulated expression of pituitary corticotroph adenoma-specific markers in iPSCCDH23 and iPSCMEN1 demonstrates that the iPSC-derived organoids represented the pathology of corticotroph adenomas in vitro. 3.7. ScRNA-seq Reveals the Existence of Unique Proliferative Cell Populations in iPSCCDH23 Cultures When Compared to iPSCsctrl Using Seurat to identify cell clusters, as well as Uniform Manifold Approximation and Projection 9UMAP, clustering analysis identified 16 distinct cell populations/clusters consisting of known marker genes. Clusters 1, 5, and 7 of the iPSCsCDH23 were distinct from the iPSCctrl cultures (Figure 7a,b). Pituitary stem cells were characterized in iPSCctrl and iPSCCDH23 cultures (Figure 7b). Clusters 1 and 5 expressed markers consistent with the corticotroph subtype cell lineage (Figure 5c). Markers of dysregulated cell cycles and increased proliferation were identified in cell cluster 7 (Figure 7c). Expression of the E2 factor (E2F) family of transcription factors, which are downstream effectors of the retinoblastoma (RB) protein pathway and play a crucial role in cell division control, were identified in distinct cell cluster 7, which was identified within the iPSCCDH23 cultures (Figure 7c). Stem cell markers were also upregulated in cell cluster 7, and identified within the iPSCCDH23 cultures (Figure 7c). Using Cytobank software to analyze organoids collected 30 days post-differentiation, cells were gated on live CK20 positive singlets, and 9000 events per sample were analyzed by the viSNE algorithm. ViSNE plots are shown in two dimensions with axes identified by tSNE- 1 and tSNE-2, and each dot representing a single cell positioned in the multidimensional space (Figure 7d). Individual flow cytometry standard files were concatenated into single flow cytometry standard files, from which 12 spatially distinct populations were identified (Figure 7e). Overlaying cell populations identified by traditional gating strategies onto viSNE plots identified unique cell populations within the iPSCCDH23 cultures (Figure 7e). There were distinct cell populations between the iPSCctrl and iPSCCDH23 organoids, in addition to expression of hormone and cell lineage markers such as ACTH, TPit, PRL, and PIT1 (Figure 7e). The cell populations that exhibited high expression of Ki67 within the iPSCctrl organoid cultures included SOX2+ and PIT1+ populations (Figure 7f). The highly proliferating cell populations within the iPSCCDH23 organoid cultures included those that expressed CD90+/VIM+/CXCR4+ (mesenchymal stem cells), CXCR4+/SOX2+ (stem cells), TPit+ (corticotroph cell lineage), CD133+/CD31+ (endothelial progenitor cells), and CK20+/VIM+/CXCR4+ (hybrid epithelial-mesenchymal stem cells) (Figure 7f). Overall, the iPSCCDH23 organoids were significantly more proliferative compared to the iPSCctrl cultures (Figure 7f). Immunofluorescence staining of iPSCCDH23 organoids revealed increased mRNA expression of TPit and POMC, which correlated with increased ACTH protein compared to iPSCsctrl (Supplemental Figure S6). As shown in Supplemental Figure S6b,c, iPSCCDH23 cultures also exhibited a significant increase in the expression of GH and LH (Supplemental Figure S6b,c). Figure 7. Single cell analysis of iPSCctrl and iPSCCDH23 cultures 15 and 30 days post-directed differentiation. (a) UMAP plots showing identified cell clusters 0–16 in iPSCctrl and iPSCCDH23 cultures 15 days post-directed differentiation. (b) Violin plots of representative identified markers of the corticotroph cell lineage, where 2 subpopulations were observed among iPSCctrl and iPSCCDH23 cultures. Arrows highlight clusters 1, 5, and 7. (c) Violin plots showing expression of genes representative of stem cells, Wnt, NOTCH, Hh and SST signaling, anterior pituitary (corticotroph) cell lineage, and cell cycle in clusters 1, 5, and 7 of iPSCCDH23 cultures. Plot width: cell number, plot height: gene expression. (d) viSNE maps showing concatenated flow cytometry standard files for both samples and iPSCctrl and iPSCCDH23 organoids 30 days post-directed differentiation. (e) Overlay of manually gated cell populations onto viSNE plots. (f) Fluorescent intensity of Ki67 of viSNE maps for both samples and iPSCctrl and iPSCCDH23 organoids. iPSCctrl = 22518 events; iPSCCDH23 = 17542 events. Collectively, Figure 7 demonstrates that the development of pituitary organoids generated from iPSCs of CD patients may reveal the existence of cell populations which, potentially, contribute to the support of adenoma growth and progression, as well as an expansion of stem and progenitor cells that may be the targets for tumor recurrence. 4. Discussion Our studies demonstrate the development of organoids generated from human PitNETs (hPITOs) can potentially be used to screen for the sensitivity and efficacy of responses to targeted therapies for CD patients that either fail to achieve remission or exhibit recurrence of disease after surgery. In addition, we have documented that induced pluripotent stem cells (iPSCs) generated from a CD patient expressing germline mutation CDH23 (iPSCCDH23) reveals the disease pathogenesis under directed differentiation. Many early in vitro experiments have used pituitary cell lines, spheroids, aggregates, and/or tumoroids that do not replicate the primary PitNET microenvironment [19,20,21], and lack a multicellular identity [39,40]. The development of PitNET tissue-generated organoids is limited to the use of transgenic mouse models as the source [22,23,41]. The recent organoid cultures reported by Nys et al. [42] have been generated from single stem cells isolated from PitNET tissue, and are claimed to be true organoids due to their clonality. However, multicellular complexity was not validated by the protein expression or hormone secretion from pituitary cell lineages in these cultures [42]. According to the National Cancer Institute (NCI, NIH), an ‘organoid’ is defined as “a tiny, 3-dimensional mass of tissue that is made by growing stem cells (cells from which other types of cells develop) in the laboratory” [43]. The hPITOs reported here begin from single and/or 3–4 cell clusters dissociated from the PitNET tissue that harbors the stem cells. Supplemental Video S2 demonstrates a process of ‘budding,’ as well as lumen formation as organoids grow and differentiate. We document differentiation and function by comprehensive spectral flow cytometry, ELISA, and response to standard of care drugs. The growth of PitNET organoids reported in the current study is consistent with that of gastrointestinal tissue derived cultures that begin from cell clusters, crypts, or glands [27,44,45]. Our studies report a PitNET tissue organoid culture with a multicellular identity consisting of differentiated cell lineages, stem/progenitor cells, and immune and stromal cell compartments, which replicates much of the patient’s own adenoma pathology, functionality, and complexity. We have also demonstrated that iPSCs, derived from the blood of a CD patient, can be directly differentiated into pituitary organoids that resemble similar characteristics to the tumor tissue. Many investigators have proposed the use of organoids in personalized medicine, but have focused these efforts on targeted treatment of cancers [27,46,47,48]. The findings reported in these studies are the first to implement this approach for the potential treatment of PitNETs. Collectively, we have developed a relevant human in vitro approach to potentially advance our knowledge as well as our approach to studies in the field of pituitary tumor research. Both the hPITOs and the iPSCCDH23 may be implemented in studies that strive to (1) define the molecular and cellular events that are crucial for the development of PitNETs leading to CD, and (2) accelerate the identification of effective targeted therapies for patients with CD. While published studies have advanced our understanding of the molecular mechanisms of the pathogenesis of corticotroph adenomas and elucidated candidate therapeutic targets for CD, these reports fall short of directly informing clinical decisions for patient treatment. Using organoids to screen potential drugs and compounds can potentially improve therapeutic accuracy. Figure 3 demonstrated a variation in drug responsiveness amongst the organoid lines generated from individual patients. Importantly, there was further divergence in drug responsiveness amongst the individual organoid lines within each pathologically defined corticotroph subtype. For example, hPITOs generated from patients with sparsely granulated corticotroph adenomas (hPIT0s 10, 25, 34, 35) and Crooke’s cell adenomas (hPITOs 7, 33) showed variable responses regardless of similar pathologically defined subtypes. In addition, the response of the tumor cells within the organoids to the standard of care drugs that directly target the pituitary in the body, including mifepristone and cabergoline, was only 50% in hPITO34 and hPITO35, and almost 0% in the other lines, including hPITO7, 10, and 25. These data clearly demonstrate that the inherent patient difference to drug response that is often observed among CD patients is reflected in the organoid culture. This culture system may be an approach that will provide functional data revealing actionable treatment options for each patient. Patient-derived organoids from several tumors have served as a platform for testing the efficacy of anticancer drugs and predicting responses to targeted therapies in individual patients [27,46,48,49,50]. An example of the use of organoids in identifying drug responsiveness within an endocrine gland is that of papillary thyroid cancer [51]. Organoids developed from PTC patients were used as a preclinical model for studying responsiveness to anticancer drugs in a personalized approach [51]. However, our study is the first report of the use of hPITOs for drug screening. Connecting genetic and drug sensitivity data will further categorize corticotroph subtypes associated with CD. WES analysis of each hPITO line was compared to the results for the corresponding primary adenoma tissues. We showed the concordance rate of exonic variants between the primary tumor tissues obtained from CD patients and the corresponding organoid line. On average, approximately 80% of the variants observed in the CD patients’ adenoma tissues were retained in the corresponding hPITOs. Pituitary organoids were also developed from iPSCs generated from PBMCs of a CD patient expressing a germline genetic alteration in cadherin-related 23 CDH23 (iPSCCDH23), a CD patient expressing an MEN1 mutation (iPSCMEN1), and a healthy individual (iPSCctrl). Foundational studies performed by investigators at the genome level have revealed significant knowledge regarding the pathophysiology of CD [36,37,52,53]. In some instances, CD is a manifestation of genetic mutation syndromes that include multiple endocrine neoplasia type 1 (MEN1), familial isolated pituitary adenoma (FIPA), and Carney complex [54,55]. CDH23 syndrome is clinically associated with the development of Usher syndrome, deafness, and vestibular dysfunction [56]. Several mutations in CDH23 are associated with inherited hearing loss and blindness [57]. However, none of the variants found in this study were linked to any symptoms of deafness or blindness. A possible explanation is that deafness-related CDH23 mutations are caused by either homozygous or compound heterozygous mutations [57]. In a study that linked mutations in CDH23 with familial and sporadic pituitary adenomas, it was suggested that these genetic alterations could play important roles in the pathogenesis of CD [38]. Genomic screening in a total of 12 families with familial PitNETs, 125 individuals with sporadic pituitary tumors, and 260 control individuals showed that 33% of the families with familial pituitary tumors and 12% of individuals with sporadic pituitary tumors expressed functional or pathogenic CDH23 variants [38]. Consistent with the expected pathology and function of a PitNET from a patient with CD, iPSCCDH23 organoids exhibited hypersecretion of ACTH, and expression of transcription factors and cell markers were reported in the pathology report for corticotroph PitNETs. Collectively, these findings warrant further investigation to determine whether carriers of CDH23 mutations are at a high risk of developing CD and/or hearing loss. Specifically, clinical investigation is required to determine whether pituitary MRI scans should be adopted in the screening of CDH23-related diseases, including Usher syndrome and age-related hearing loss. Pituitary organoids generated from iPSCs of a CD patient revealed the existence of cell populations that potentially contribute to the support of PitNET growth and disease progression, as well as an expansion of stem and progenitor cells that may be the targets for tumor recurrence. Organoids derived from both pituitary adenomas and iPSCs exhibited increased expression of stem cell and progenitor markers at both the protein and transcriptomic levels. Unique clusters that were proliferative in the iPSCCDH23 organoids expressed a hybrid pituitary cell population which was in an epithelial/mesenchymal state (CK20+/VIM+/CXCR4+/Ki67+). In support of our findings, a similar report of a hybrid epithelial/mesenchymal pituitary cell has been made as part of the normal developmental stages of the human fetal pituitary [58]. Previous studies have suggested that pituitary stem cells undergo an EMT-like process during cell migration and differentiation [59,60,61]. Consistent with our findings are extensive studies using single cells isolated from human pituitary adenomas to show increased expression of stem cell markers SOX2 and CXCR4 [22,23,41,62,63]. Within the clusters identified in the iPSCCDH23 culture were cell populations expressing stem cell markers, including SOX2, NESTIN, CXCR4, KLF4, and CD34. The same iPSCCDH23 cell clusters, 4, 8, 9, and 11, co-expressed upregulated genes of NOTCH, Hedgehog, WNT, and TGFβ signaling, which are pivotal not only in pituitary tumorigenesis and pituitary embryonic development, but also in ‘tumor stemness’ [22,23,41,62,63,64]. We also noted that clusters of cell populations 5 and 14 unique within the iPSCCDH23 cultures expressed upregulated genes which were indicative of high proliferation. We observed upregulated expression of the E2F family of transcription factors (E2Fs) E2F1 and E2F7. These findings are of significance, given that there is evidence to show that upregulation of E2Fs is fundamental for tumorigenesis, metastasis, drug resistance, and recurrence [65]. Within the pituitary adenoma microenvironment, whether these stem cells directly differentiate into pituitary tumors or support the growth of the adenoma is largely unknown. In addition, whether pituitary stem cell populations become activated in response to injury is also understudied. Although the role of stem cells has been identified using a mouse model through implantation of the cells within the right forebrain [66], the identification of pituitary tumor-initiating stem cells using in vivo orthotopic transplantation models is impossible in mice. Pituitary tumors harboring the stem cells may require engraftment within the environment from which the cells are derived in order to enable growth and differentiation of the tumor. However, it is technically impossible to implant cells orthotopically in the murine pituitary. The pituitary tumor organoid cultures presented in these studies may offer an approach by which isolation, identification, and characterization of this stem cell population is possible. Therefore, we would gain knowledge on the mechanisms of pituitary tumor pathogenesis and reveal potential novel targets for therapeutic interventions by using the iPSC generated pituitary organoid culture. PitNETs associated with the development of CD cause serious morbidity due to chronic cortisol exposure that dysregulates almost every organ system in the body. Overall, existing medical therapies remain suboptimal, with negative impact on health and quality of life, including considerable risk of therapy resistance and tumor recurrence. To date, little is known about the pathogenesis of PitNETs. Here, we present a human organoid-based approach that will allow us to acquire knowledge of the mechanisms underlying pituitary tumorigenesis. Such an approach is essential to identify targeted treatments and improve clinical management of patients with CD. 5. Conclusions Cushing’s disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET), which stimulates the adrenal glands to overproduce cortisol. The absence of preclinical models that replicate the PitNET microenvironment has prevented us from acquiring the knowledge to identify therapies that can be targeted to the tumor with a higher efficacy and tolerability for patients. Our studies demonstrate the development of organoids generated from human PitNETs or induced pluripotent stem cells as an essential approach to identifying targeted therapy methods for CD patients. Supplementary Materials The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells11213344/s1, Figure S1: Antibodies used and Cytek® Full Spectrum Viewer showing calculated similarity indices; Figure S2: Morphology and proliferation of lactotroph, somatotroph, and gonadotroph hPITOs; Table S1: Pituitary Growth Media; Table S2: Components used for pituitary organoids generated from iPSCs; Table S3: clinical characteristics of pituitary adenoma samples used for the generation of organoids; Table S4: Average correlation of replicates reported in Figure 3; Table S5: pituitary cell lineage or stem cell markers used in the scRNA-seq analysis; Video S1: hPITO38 EdU ACTH 3. Author Contributions Conceptualization, Y.Z.; methodology, J.C., Y.Z., J.M.C., B.N.S., S.M. and K.W.P.; software, J.C., Y.Z., J.M.C., S.M., Y.C., P.M. and R.P.; validation, Y.Z., J.C., J.M.C., A.S.L., K.C.J.Y. and R.P.; formal analysis, J.C., Y.Z., J.M.C., R.P., Y.C., S.M. and P.M.; investigation, Y.Z.; resources, Y.Z., J.C., J.E., C.A.T., B.H. and A.S.L.; data curation, J.C., Y.Z., J.M.C., R.P. and S.M.; writing—original draft preparation, Y.Z., J.C, S.M., J.M.C., Y.C., B.H. and R.P.; writing—review and editing, Y.Z., J.C., J.M.C., A.S.L., K.C.J.Y., S.M., J.E., C.A.T., K.W.P., B.H., Y.C., P.M., B.N.S. and R.P.; visualization, Y.Z., J.C., J.M.C., A.S.L., K.C.J.Y. and R.P.; supervision, Y.Z.; project administration, Y.Z.; funding acquisition, Y.Z. All authors have read and agreed to the published version of the manuscript. Funding This research was supported by the Department of Cellular and Molecular Medicine (University of Arizona College of Medicine) startup funds (Zavros). This research study was also partly supported by the National Cancer Institute of the National Institutes of Health under award number P30 CA023074 (Sweasy). Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of St. Joseph’s Hospital and Barrow Neurological Institute Biobank collection protocol PHXA-05TS038, and collection of outcomes data protocol PHXA-0004-72-29, and patient consent (protocol date of approval). Informed Consent Statement Written informed consent was obtained from all subjects involved in the study. Data Availability Statement The datasets generated during the analysis of the present study are available in the ReDATA repository, https://doi.org/10.25422/azu.data.19755244.v1. The datasets generated in the current study are also available from the corresponding author on reasonable request. All data generated or analyzed during this study are included in this published article (and its Supplementary Information Files). Acknowledgments We acknowledge the technical support of Maga Sanchez in the Tissue Acquisition and Cellular/Molecular Analysis Shared Resource (TACMASR University of Arizona Cancer Center) for assistance with embedding and sectioning of organoids. We would also like to acknowledge Patty Jansma (Marley Imaging Core, University Arizona) and, Douglas W Cromey (TACMASR imaging, University of Arizona Cancer Center) for assistance in microscopy. The authors thank the patients who consented to donate pituitary tumor tissues and blood for the development of the organoids. Without their willingness to participate in the study, this work would not be possible. Conflicts of Interest The authors declare no conflict of interest. References Cushing, H. Posterior Pituitary Activity from an Anatomical Standpoint. Am. J. Pathol. 1933, 9, 539–548.19. 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Recordati's Isturisa is expected to launch in the second or third quarter. (Getty) As part of a small 2019 deal, Italian drugmaker Recordati snagged a trio of underperforming Novartis endocrinology meds, including a late-stage candidate for Cushing's disease. Less than a year later, that drug is cleared for market after an FDA green light. The FDA on Friday approved Recordati's Isturisa (osilodrostat) to treat Cushing's disease—a rare disease in which patients' adrenal glands produce too much cortisol—in those who have undergone a prior pituitary gland surgery or are not eligible for one. Isturisa, a cortisol synthesis inhibitor, will come with the FDA's orphan drug designation, providing market exclusivity for seven years, Recordati said (PDF) in a release. The drug is expected to be commercially available in the second or third quarter. The FDA based its review on phase 3 data showing 86% of patients treated with Isturisa showed normal cortisol levels in their urine after eight weeks, compared with 29% of patients treated with placebo, the drugmaker said. Recordati is "actively building its commercial, medical, and market access teams" to accommodate Isturisa's launch through its recently created U.S. endocrinology business unit, it said. The drugmaker will launch the drug with a "comprehensive distribution model" through specialty pharmacies. Novartis, once the owner of Isturisa, turned the asset over to Recordati in 2019 as part of a $390 million offload of some of the Swiss drugmaker's endocrinology portfolio. Recordati received Signifor, long-acting sister Signifor LAR and Isturisa, positioned as a successor drug to Signifor. The purchase included milestone payments tied to Isturisa. Recordati talked up the buy of the Cushing's disease trio as a boon for its rare disease portfolio, calling it a "key and historical milestone" at the time. From https://www.fiercepharma.com/pharma/recordati-scores-fda-nod-for-cushing-s-disease-med-isturisa

More than 80% of adults with Cushing’s disease receiving osilodrostat had normalized mean urinary free cortisol levels at 72 weeks of treatment, according to findings from the LINC 3 study extension. “Cushing’s disease is a chronic condition, and many patients require prolonged pharmacological treatment. Therefore, evaluating long-term efficacy and safety of drug therapies in clinical trials is essential,” Maria Fleseriu, MD, FACE, professor of medicine and neurological surgery and director of the Pituitary Center at Oregon Health & Science University in Portland and a Healio | Endocrine Today co-editor, told Healio. “Our findings build on the positive results of the LINC 3 study core phase, and it was reassuring to see that continued treatment with osilodrostat for over 72 weeks provided long-term normalization of cortisol levels. Furthermore, continued treatment with osilodrostat also led to sustained improvements in clinical signs and physical manifestations of hypercortisolism, as well as health-related quality of life, which are all important factors in the management of these patients.” Fleseriu and colleagues enrolled 106 adults with Cushing’s disease who were responders to osilodrostat (Isturisa, Recordati) at 48 weeks during the LINC 3 core study to enter the extension phase of the trial. Participants continued to receive open-label osilodrostat until 72 weeks or treatment discontinuation. Mean urinary free cortisol was collected every 12 weeks. Physical manifestations of hypercortisolism were rated at 48 and 72 weeks. Participants completed the Cushing’s Quality of Life questionnaire and Beck Depression Inventory II at 48 and 72 weeks. Adults were deemed to have completely responded to treatment if mean urinary free cortisol was less than the upper limit of normal and partially responded to treatment if mean urinary free cortisol was above the upper limit of normal but decreased more than 50% from baseline. The findings were published in the European Journal of Endocrinology. Of the 106 participants in the extension study, 98 completed 72 weeks of treatment. At 72 weeks, 81.1% of participants were complete responders to treatment, and reductions in mean urinary free cortisol from the core phase were maintained during the extension. Improvements in most cardiovascular and metabolic-related parameters from the core study were maintained or improved in the extension phase. The cohort also had increases in quality of life score and improvements in Beck Depression Inventory II scores. The proportion of participants with improvements in physical manifestation of hypercortisolism were maintained or improved in all areas at 72 weeks. For hirsutism in women, 86.4% had an improved or stable severe score at 72 weeks. Improved scores were observed in participants with mild, moderate and severe physical manifestations at baseline with few adults experiencing worse manifestations at the end of the extension study. There were no new safety signals reported in the extension study. Of the extension study participants, 11.3% discontinued osilodrostat due to adverse events, a similar percentage to the 10.9% discontinuation rate during the core phase of the study. Several hormone concentrations, including mean adrenocorticotropic hormone, 11-deoxycortisol and plasma aldosterone, stabilized during the extension phase after changes were observed in the core study compared with baseline. Mean testosterone in women decreased from 2.6 nmol/L at 48 weeks to 2.1 nmol/L at 72 weeks. There were no changes observed in mean testosterone levels for men. “Patients should be regularly monitored and osilodrostat dose titrated as necessary, alongside adjustment of concomitant medications, to optimize outcomes,” the researchers wrote. “Taken together, these findings support osilodrostat as an effective and well-tolerated long-term treatment option for patients with Cushing’s disease.” For more information: Maria Fleseriu, MD, FACE, can be reached at fleseriu@ohsu.edu. From https://www.healio.com/news/endocrinology/20220914/osilodrostat-normalizes-urinary-free-cortisol-in-cushings-disease-for-most-at-72-weeks

Recordati Rare Diseases, a US biopharma that forms part of the wider Italian group, has presented multiple positive data sets on Isturisa (osilodrostat) at the annual ENDO 2022 meeting in Atlanta, Georgia. Isturisa is a cortisol synthesis inhibitor indicated for the treatment of adult patients with Cushing’s disease for whom pituitary surgery is not an option or has not been curative. Among the data presented, the Phase III LINC 4 study demonstrated that Isturisa maintained normal mean urinary free cortisol long-term in patients with Cushing’s disease while the Phase III LINC 3 study found adrenal hormone levels changed during early treatment with the drug while stabilizing during long-term treatment. The ILLUSTRATE study also showed patients treated with a prolonged titration interval tended to have greater persistence with therapy. Mohamed Ladha, president and general manager for North America, Recordati Rare Diseases, said: “The data from these studies reinforces the efficacy and safety of Isturisa as a treatment for patients with Cushing’s disease. “We are pleased to share these data with the endocrine community and are excited to provide patients with a much-needed step forward in the management of this rare, debilitating, and potentially life-threatening condition.” Cushing’s disease is a rare, serious illness caused by a pituitary tumor that leads to overproduction of cortisol by the adrenal glands. Excess cortisol can contribute to an increased risk of morbidity and mortality. Treatment for the condition seeks to lower cortisol levels to a normal range. Isturisa, which was approved by the US Food and Drug Administration in March 2020, works by inhibiting 11-beta-hydroxylase, an enzyme responsible for the final step of cortisol biosynthesis in the adrenal gland. From https://www.thepharmaletter.com/article/results-reinforce-efficacy-of-recordati-s-isturisa-in-cushing-s-disease

Dr. Friedman uses several medications to treat Cushing’s syndrome that are summarized in this table. Dr. Friedman especially recommends ketoconazole. An in-depth article on ketoconazole can be found on goodhormonehealth.com. Drug How it works Dosing Side effects Ketoconazole (Generic, not FDA approved in US) blocks several steps in cortisol biosynthesis Start 200 mg at 8 and 10 PM, can up titrate to 1200 mg/day • Transient increase in LFTs • Decreased testosterone levels • Adrenal insufficiency Levoketoconazole (Recorlev) L-isomer of Ketoconazole Start at 150 mg at 8 and 10 PM, can uptitrate up to 1200 mg nausea, vomiting, increased blood pressure, low potassium, fatigue, headache, abdominal pain, and unusual bleeding Isturisa (osilodrostat) blocks 11-hydroxylase 2 mg at bedtime, then go up to 2 mg at 8 and 10 pm, can go up to 30 mg Dr. Friedman often gives with spironolactone or ketoconazole. • high testosterone (extra facial hair, acne, hair loss, irregular periods) • low potassium • hypertension Cabergoline (generic, not FDA approved) D2-receptor agonist 0.5 to 7 mg • nausea, • headache • dizziness Korlym (Mifepristone) glucocorticoid receptor antagonist 300-1200 mg per day • cortisol insufficiency (fatigue, nausea, vomiting, arthralgias, and headache) • increased mineralocorticoid effects (hypertension, hypokalemia, and edema • antiprogesterone effects (endometrial thickening) Pasireotide (Signafor) somatostatin receptor ligand 600 μg or 900 μg twice a day Diabetes, hyperglycemia, gallbladder issues For more information or to schedule an appointment with Dr. Friedman, go to goodhormonehealth.com

Osilodrostat is associated with improvements in physical manifestations of hypercortisolism and reductions in mean body weight and BMI in adults with Cushing’s syndrome, according to a speaker. As Healio previously reported, in findings from the LINC 4 phase 3 trial, osilodrostat (Isturisa, Recordati) normalized mean urinary free cortisol level at 12 weeks in more than 75% of adults with Cushing’s disease. In new findings presented at the AACE Annual Scientific and Clinical Conference, most adults with Cushing’s syndrome participating in the LINC 3 phase 3 trial had improvements in physical manifestations of hypercortisolism 72 weeks after initiating osilodrostat, with more than 50% having no dorsal fat pad, supraclavicular fat pad, facial rubor, proximal muscle atrophy, striae, ecchymoses and hirsutism for women at 72 weeks. Source: Adobe Stock “Many patients with Cushing’s syndrome suffer from clinical manifestations related to hypercortisolism,” Albert M. Pedroncelli, MD, PhD, head of clinical development and medical affairs for Recordati AG in Basel, Switzerland, told Healio. “The treatment with osilodrostat induced a rapid normalization of cortisol secretion, and improvements in physical manifestations associated with hypercortisolism were observed soon after initiation of osilodrostat and were sustained throughout the study.” Albert M. Pedroncelli Pedroncelli and colleagues analyzed changes in the physical manifestations of hypercortisolism in 137 adults with Cushing’s syndrome (median age, 40 years; 77.4% women) assigned osilodrostat. Dose titration took place from baseline to 12 weeks, and therapeutic doses were administered from 12 to 48 weeks, with some participants randomly assigned to withdrawal between 26 and 34 weeks. An extension phase of the trial took place from 48 to 72 weeks. Investigators subjectively rated physical manifestations of hypercortisolism in participants as none, mild, moderate or severe. Participants were evaluated at baseline and 12, 24, 34, 48 and 72 weeks. At baseline, the majority of the study cohort had mild, moderate or severe physical manifestations of hypercortisolism in most individual categories, including dorsal fat pad, central obesity, supraclavicular fat pad, facial rubor, hirsutism in women and striae. Central obesity was the most frequent physical manifestation rated as severe. The percentage of participants with improvements in physical manifestations of hypercortisolism increased from week 12 on for all individual manifestations evaluated in the study, and improvements were maintained through week 72. At 72 weeks, the percentage of participants who had no individual physical manifestations was higher than 50% for each category except central obesity, where 30.6% of participants had no physical manifestations. In addition to improvement in physical manifestations, the study cohort had decreases in body weight, BMI and waist circumference at weeks 48 and 72 compared with baseline. “The main goal of treating patients with Cushing’s syndrome is to normalize cortisol secretion,” Pedroncelli said. “The rapid reduction and normalization of cortisol levels is accompanied by improvement in the associated clinical manifestations. This represents an important objective for patients.” From https://www.healio.com/news/endocrinology/20220512/osilodrostat-improves-physical-manifestations-of-hypercortisolism-for-most-adults

— More than half of patients saw physical manifestations fully resolve by week 72 by Kristen Monaco, Staff Writer, MedPage Today May 16, 2022 SAN DIEGO -- Osilodrostat (Isturisa) improved many physical features associated with Cushing's disease, according to additional findings from the phase III LINC-3 study. Among 137 adults with Cushing's disease, a 39.5% improvement in central obesity scores was observed from baseline to week 72 with osilodrostat, reported Alberto Pedroncelli, MD, PhD, of Recordati AG in Basel, Switzerland. Not only was central obesity the most common physical manifestation associated with hypercortisolism among these Cushing's disease patients, but it was also more frequently rated as severe at baseline, Pedroncelli explained during the American Association of Clinical Endocrinology (AACE) annual meeting. Osilodrostat treatment also led to a 34.9% improvement in proximal muscle atrophy at week 72, along with a 34.4% improvement in hirsutism scores. By week 72, nearly all physical manifestations of hypercortisolism saw significant improvement -- marked by more than 50% of patients scoring these physical traits as nonexistent: Dorsal fat pat: 50.6% Central obesity: 30.6% Supraclavicular fat pad: 51.8% Facial rubor: 64.7% Hirsutism in women: 53.1% Proximal muscle atrophy: 61.2% Striae: 63.5% Ecchymoses: 87.1% Most of these physical manifestation improvements were notable soon after treatment initiation with osilodrostat, Pedroncelli pointed out. When stratified according to testosterone levels, hirsutism scores remained either stable or improved in the majority of patients who had normal or above normal testosterone levels. More women with normal testosterone levels over time experienced improvements in hirsutism versus those with levels above the upper limit of normal, who mostly remained stable. Osilodrostat is an oral agent that was first FDA approved in March 2020 for adults with Cushing's disease who either cannot undergo pituitary gland surgery or have undergone the surgery but still have the disease. Available in 1 mg, 5 mg, and 10 mg film-coated tablets, the drug acts as a potent oral 11-beta-hydroxylase inhibitor -- the enzyme involved in the last step of cortisol synthesis. Osilodrostat is taken orally twice daily, once in the morning and once in the evening. Approval was based upon findings from the LINC-3 and LINC-4 trials, which found osilodrostat was able to normalize cortisol levels in 53% of patients, based on mean 24-hour urinary free cortisol (UFC) concentrations. During an initial 10-week randomization phase, 86% of patients maintained their complete cortisol response if they remained on osilodrostat versus only 29% of those who were switched to placebo. As expected, 77.4% of the 137 adults included in the trial were women. The median participant age was 40 and about 47 months had passed since their initial diagnosis. A total of 87.6% underwent previous pituitary surgery and 16.1% underwent previous pituitary irradiation. At baseline, median and mean 24-hour UFC levels were 3.5 nmol and 7.3 nmol, respectively, based on two or three urine samples. Participants had an average body weight of 176.4 lb, body mass index (BMI) of 30, and 41 in waist circumference at baseline. Throughout the trial, all measures dropped, reaching the nadir at week 72: body weight of 165 lb, BMI of 27, and 37.8 in waist circumference. The most common side effects reported with the agent include adrenal insufficiency, fatigue, nausea, headache, and edema. Kristen Monaco is a staff writer, focusing on endocrinology, psychiatry, and nephrology news. Based out of the New York City office, she’s worked at the company since 2015. Disclosures The study was supported by Recordati AG. Pedroncelli reported employment with Recordati. Primary Source American Association of Clinical Endocrinology Source Reference: Pedroncelli AM, et al "Osilodrostat therapy improves physical features associated with hypercortisolism in patients with Cushing's disease: findings from the phase III LINC 3 study" AACE 2022. From https://www.medpagetoday.com/meetingcoverage/aace/98745

Osilodrostat is associated with rapid normalization of mean urinary free cortisol (mUFC) excretion in patients with Cushing disease and has a favorable safety profile, according to the results of a study published in the Journal of Clinical Endocrinology & Metabolism. The phase 3 LINC-4 study (ClinicalTrials.gov Identifier: NCT02697734) evaluated the safety and efficacy of osilodrostat, a potent, orally available 11β­-hydroxylase inhibitor, compared with placebo in patients with Cushing disease. The trial, which was conducted at 40 centers in 14 countries, included a 12-week, randomized, double-blind, placebo-controlled period that was followed by a 36-week, open-label osilodrostat treatment period with an optional extension. Eligible patients were aged 18 to 75 years with a confirmed diagnosis of persistent or recurrent Cushing disease after pituitary surgery and/or irradiation or de novo disease, as well as an mUFC level greater than 1.3 times the upper limit of normal (ULN). The patients were randomly assigned 2:1 to osilodrostat 2 mg twice daily or matching placebo, stratified by prior pituitary irradiation. The primary endpoint was the proportion of patients who achieved mUFC ≤ULN at week 12. The key secondary endpoint was the proportion of patients who achieved mUFC ≤ULN at week 36. A total of 73 patients (median age, 39.0 years; 83.6% women) were randomly assigned to either osilodrostat (n=48) or placebo (n=25) and received at least 1 study drug dose from November 2016 to March 2019. The participants had a median (interquartile range [IQR]) time since diagnosis of Cushing disease of 67.4 (26.4-93.8) months. The median treatment duration in the randomized, placebo-controlled period was 12.0 weeks in both the osilodrostat group (IQR, 2.0-13.0 weeks) and the placebo group (IQR, 11.7-13.7 weeks). The proportion of patients who achieved mUFC ≤ULN (≤138 nmol/24 h) at week 12 was significantly increased in those who received osilodrostat (n=37, 77.1%) vs those who received placebo (n=2, 8.0%), with an estimated odds ratio of 43.4 (95% CI, 7.1-343.2) in favor of osilodrostat (P <.0001). A total of 59 patients (80.8%; 95% CI, 69.9-89.1) also achieved the key secondary endpoint of mUFC ≤ULN at week 36, after 24 weeks of open-label osilodrostat. The most frequently occurring adverse events in the placebo-controlled period in the osilodrostat and placebo groups, respectively, were decreased appetite (37.5% vs 16.0%), arthralgia (35.4% vs 8.0%), nausea (31.3% vs 12.0%), and fatigue (25.0% vs 16.0%). A potential study limitation is that although osilodrostat exposure was greater than 1 year among the participants, some adverse effects may take longer to be observed. “This randomized, placebo-controlled trial demonstrates that osilodrostat is a highly effective treatment for Cushing disease, normalizing UFC excretion in 77% of patients after 12 weeks’ treatment,” stated the investigators. “Cortisol reductions were maintained throughout 48 weeks of treatment and were accompanied by improvements in clinical signs of hypercortisolism and quality of life. The safety profile was favorable.” Disclosure: This study was funded by Novartis Pharma AG. Some of the study authors declared affiliations with biotech, pharmaceutical, and/or device companies. Please see the original reference for a full list of disclosures. Reference Gadelha M, Bex M, Feelders RA, et al. Randomized trial of osilodrostat for the treatment of Cushing’s disease. J Clin Endocrinol Metab. Published online March 23, 2022. doi:10.1210/clinem/dgac178 From https://www.endocrinologyadvisor.com/home/topics/general-endocrinology/osilodrostat-effective-for-cushing-disease/

More than three-quarters of adults with Cushing’s disease assigned osilodrostat had a normalized mean urinary free cortisol level at 12 weeks and maintained a normal level at 36 weeks, according to data from the LINC 4 phase 3 trial. In findings published in The Journal of Clinical Endocrinology & Metabolism, 77% of adults with Cushing’s disease randomly assigned to osilodrostat (Isturisa, Recordati) had mean urinary free cortisol (UFC) levels reduced to below the upper limit of normal at 12 weeks compared with 8% of adults assigned to placebo. Most adults with Cushing's disease taking 2 mg twice daily osilodrostat had normalized mean UFC levels at 12 weeks compared with placebo. Data were derived from Gadelha M, et al. J Clin Endocrinol Metab. 2022;doi:10.1210/clinem/dgac178. “Osilodrostat is a highly effective treatment for Cushing’s disease, normalizing urinary free cortisol excretion in 77% of patients after 12 weeks’ treatment,” Mônica Gadelha, MD, professor of endocrinology at The Federal University of Rio de Janeiro, and colleagues wrote. “Cortisol reductions were maintained throughout 48 weeks of treatment and were accompanied by improvements in clinical signs of hypercortisolism and quality of life.” Gadelha and colleagues enrolled 73 adults aged 18 to 75 years with Cushing’s disease from 40 centers in 14 countries into the LINC 4 phase 3 trial. Participants were randomly assigned to 2 mg osilodrostat twice daily (n = 48) or placebo (n = 25) for 12 weeks. Urinary samples were collected at weeks 2, 5 and 8 to measure mean UFC, and dosage was adjusted based on efficacy and tolerability. After 12 weeks, participants from both groups received osilodrostat in a 36-week open-label treatment period. All participants restarted the open-label portion of the trial at 2 mg osilodrostat unless they were on a lower dose at week 12. Dose adjustments in the open-label phase were made using the same guidelines in the randomized, double-blind, placebo-controlled trial. The primary endpoint was the efficacy of osilodrostat at achieving a mean UFC below the upper limit of normal of 138 nmol per 24 hours at 12 weeks vs. placebo; the key secondary endpoint was the percentage of participants achieving a normal mean UFC at 36 weeks. At 12 weeks, the percentage of adults with a normalized mean UFC level was higher in the osilodrostat group compared with placebo (77.1% vs. 8%; P < .0001). At 36 weeks, 80.8% of all participants had a normal mean UFC level. The overall response rate was 79.5% at 48 weeks. Median time to first controlled mean UFC response was 35 days for those randomly assigned to osilodrostat as well as those randomly assigned to placebo who crossed over to osilodrostat for the open-label phase. At 48 weeks, 84% of participants were receiving 10 mg or less of osilodrostat per day, including 56% receiving 4 mg or less daily. At 12 weeks, the osilodrostat group had several cardiovascular and metabolic-related improvements, including systolic and diastolic blood pressure, HbA1c, HDL cholesterol, body weight and waist circumference. No changes were observed in the placebo group. “The improvements in cardiovascular and metabolic parameters were sustained throughout osilodrostat treatment and have the potential to alleviate the burden of comorbidities in many patients with Cushing’s disease,” the researchers wrote. At 12 weeks, 52.5% of those receiving osilodrostat had a reduction in supraclavicular fat pad and 50% had a reduction in dorsal fat pad. At least 25% of participants also had improvements in facial redness, striae, proximal muscle atrophy and central obesity. Improvements were sustained through week 48. During the placebo-controlled trial, grade 3 and 4 adverse events occurred for about 20% of participants in both groups. For the entire study, 38.4% of adults reported grade 3 and 4 adverse events, with the most common being hypertension. Eight participants discontinued the study due to adverse events. From https://www.healio.com/news/endocrinology/20220408/osilodrostat-normalizes-urinary-free-cortisol-in-most-adults-with-cushings-disease

Cushing’s disease is a progressive pituitary disorder in which there is an excess of cortisol in the body. While the disease can be treated surgically, this option is not possible for all patients. This is one of the approved medications that assist in controlling cortisol levels in people with Cushing’s disease. sturisa was approved in 2020 to treat adults with Cushing’s disease for whom pituitary surgery is ineffective or not an option. The oral medication works by inhibiting an enzyme called 11-beta-hydroxylase, which is involved in cortisol production. Isturisa, also known as osilodrostat or LCI699, is an approved treatment originally developed by Novartis, but now acquired by Recordati to treat people with Cushing’s disease, a condition in which a pituitary tumor causes the body to produce excessive levels of the stress hormone cortisol. In 2020, the U.S. Food and Drug Administration (FDA) approved Isturisa to treat adults with Cushing’s disease for whom pituitary surgery was not an option, or ineffective. Earlier that same year, the European Commission (EC) approved Isturisa to treat people with endogenous Cushing’s syndrome. The medication also was approved for the same indication in Japan in 2021. How does Isturisa work? Isturisa is an oral medicine that inhibits an enzyme called 11-beta-hydroxylase, which is involved in cortisol production. Blocking the activity of this enzyme prevents excessive cortisol production, normalizing the levels of the hormone in the body and easing the symptoms of Cushing’s disease. Isturisa in clinical trials A Phase 2 clinical trial (NCT01331239) investigated the safety and efficacy of Isturisa as a Cushing’s disease treatment. The trial that concluded in October 2019 initially was named LINC-1, but, through a study protocol amendment, patients who completed the study could continue into a second phase called LINC-2. The company published findings that covered both patient groups in the journal Pituitary. Data showed that Isturisa reduced cortisol levels in the urine of all patients by week 22. Urine cortisol levels reached and remained within a normal range in 79% of the patients by then. Common adverse effects included nausea, diarrhea, lack of energy, and adrenal insufficiency — a condition in which the adrenal glands are unable to produce enough hormones. A Phase 3 clinical trial (NCT02180217) called LINC-3 also assessed the safety and efficacy of Isturisa in 137 patients with Cushing’s disease (77% female, median age 40 years). Participants were given Isturisa for 26 weeks, with efficacy-based dose adjustments during the first 12 weeks. Then, the 71 participants with a complete response (those whose urine cortisol levels were within normal limits) at week 26 and who did not require a dose increase after week 12, were assigned randomly to either continue treatment with Isturisa or switch to a placebo. After this 34-week period, 86% of Isturisa-treated patients had normal urinary cortisol levels, as compared to 29% of participants given placebo. All participants then were given Isturisa for an additional 12 weeks. At the end of the 48-week study, 66% of participants had normal urine cortisol levels. Results from LINC-3 formed the basis for regulatory approvals of Isturisa. Common adverse side effects in the trial included nausea, headache, fatigue, and adrenal insufficiency. A multi-center, randomized, double-blind, placebo-controlled Phase 3 trial (NCT02697734) called LINC-4 further confirmed the safety and efficacy of Isturisa as a Cushing’s disease therapy. During the trial, patients received Isturisa or a placebo through a 12-week period followed by treatment with Isturisa until week 48. Top-line results showed that 77% of patients on Isturisa experienced a complete response after the 12-week randomized period, as compared to 8% of those on placebo. No new safety data were noted. A roll-over, worldwide Phase 2 study (NCT03606408) is recruiting patients who have successfully completed any of the previous clinical trials. Patients can continue to take the dosage they received during the initial trial. The aim of this study is to assess the long-term effects of Isturisa for up to five years.

Abstract Summary Here, we describe a case of a patient presenting with adrenocorticotrophic hormone-independent Cushing’s syndrome in a context of primary bilateral macronodular adrenocortical hyperplasia. While initial levels of cortisol were not very high, we could not manage to control hypercortisolism with ketoconazole monotherapy, and could not increase the dose due to side effects. The same result was observed with another steroidogenesis inhibitor, osilodrostat. The patient was finally successfully treated with a well-tolerated synergitic combination of ketoconazole and osilodrostat. We believe this case provides timely and original insights to physicians, who should be aware that this strategy could be considered for any patients with uncontrolled hypercortisolism and delayed or unsuccessful surgery, especially in the context of the COVID-19 pandemic. Learning points Ketoconazole–osilodrostat combination therapy appears to be a safe, efficient and well-tolerated strategy to supress cortisol levels in Cushing syndrome. Ketoconazole and osilodrostat appear to act in a synergistic manner. This strategy could be considered for any patient with uncontrolled hypercortisolism and delayed or unsuccessful surgery, especially in the context of the COVID-19 pandemic. Considering the current cost of newly-released drugs, such a strategy could lower the financial costs for patients and/or society. Keywords: Adult; Male; White; France; Adrenal; Adrenal; Novel treatment; December; 2021 Background Untreated or inadequately treated Cushing’s syndrome (CS) is a morbid condition leading to numerous complications. The latter ultimately results in an increased mortality that is mainly due to cardiovascular events and infections. The goal of the treatment with steroidogenesis inhibitors is normalization of cortisol production allowing the improvement of comorbidities (1). Most studies dealing with currently available steroidogenesis inhibitors used as monotherapy reported an overall antisecretory efficacy of roughly 50% in CS. Steroidogenesis inhibitors can be combined to better control hypercortisolism. To the best of our knowledge, we report here for the first time a patient treated with a ketoconazole–osilodrostat combination therapy. Case presentation Here, we report the case of Mr D.M., 53-years old, diagnosed with adrenocorticotrophic hormone (ACTH)-independent CS 6 months earlier. At diagnosis, he presented with resistant hypertension, hypokalemia, diabetes mellitus, easy bruising, purple abdominal striae and major oedema of the lower limbs. Investigations A biological assessment was performed, and the serum cortisol levels are depicted in Table 1. ACTH levels were suppressed (mean levels 1 pg/mL). Mean late-night salivary cortisol showed a four-fold increase (Table 2), and mean 24 h-urinary cortisol showed a two-fold increase. Serum cortisol was 1000 nmol/L at 08:00 h after 1 mg dexamethasone dose at 23:00 h. The rest of the adrenal hormonal workup was within normal ranges (aldosterone: 275 pmol/L and renin: 15 mIU/L). An adrenal CT was performed (Fig. 1) and exhibited a 70-mm left adrenal mass (spontaneous density: 5 HU and relative washout: 65%) and a 45-mm right adrenal mass (spontaneous density: −2 HU and relative washout: 75%). The case was discussed in a multidisciplinary team meeting, which advised to perform 18F-FDG PET-CT and 123I-Iodocholesterol scintigraphy before considering surgery. A genetic screening was performed, testing for ARMC5 and PRKAR1A pathogenic variants. View Full Size Figure 1 Adrenal CT depicting the bilateral macronodular adrenocortical hyperplasia. Citation: Endocrinology, Diabetes & Metabolism Case Reports 2021, 1; 10.1530/EDM-21-0071 Download Figure Download figure as PowerPoint slide Table 1 Serum cortisol levels at diagnosis (A), using ketoconazole monotherapy (B), using osilodrostat monotherapy (C) and using osilodrostat–ketoconazole combination therapy (D). Serum cortisol (nmol/L) 08:00 h 24:00 h 16:00 h 20:00 h 12:00 h 16:00 h A. At diagnosis 660 615 716 566 541 561 B. Ketoconazole monotherapy 741 545 502 224 242 508 C. Osilodrostat monotherapy 658 637 588 672 486 692 D. Osilodrostat–ketoconazole combination 436 172 154 103 135 274 Table 2 Salivary cortisol levels at diagnosis (A), using ketoconazole monotherapy (B), using osilodrostat monotherapy (C) and using osilodrostat-ketoconazole combination therapy (D). Salivary cortisol (nmol/L) 23:00 h 12:00 h 13:00 h Mean A. At diagnosis 47 62 38 49 B. Ketoconazole monotherapy 20 15 21 18 C. Osilodrostat monotherapy 85 90 56 77 D. Osilodrostat–ketoconazole combination 10 14 9 11 Treatment As this condition occurred during the COVID-19 pandemic, it was decided to first initiate steroidogenesis inhibitors to lower the patient’s cortisol levels. Initially, ketoconazole was initiated and uptitrated up to 1000 mg per day based on close serum cortisol monitoring, with a three-fold increase of liver enzymes and poor control of cortisol levels (Table 1). In the absence of biological efficacy, ketoconazole was replaced by osilodrostat, which was gradually increased up to 30 mg per day (10 mg at 08:00 h and 20 mg at 20:00 h) without reaching normal cortisol levels (Table 1) and with slightly increased blood pressure levels. Considering the lack of efficacy of anticortisolic drugs used as monotherapy, we combined osilodrostat (30 mg per day) to ketoconazole (600 mg per day), that is, at the last maximal tolerated dose as monotherapy of each drug. Outcome This combination of steroidogenesis inhibitors achieved a good control in cortisol levels, mimicking a physiological circadian rhythm (Table 1D). The patient did not exhibit any side effect and the control of cortisol levels resulted in a rapid improvement of hypertension, kalemia, diabetes control and disappearance of lower limbs oedema. The patient underwent a 18F-FDG PET-CT that did not exhibit any increased uptake in both adrenal masses and a 123I-Iodocholesterol scintigraphy exhibiting a highly increased uptake in both adrenal masses, predominating in the left adrenal mass (70 mm). Unilateral adrenalectomy of the larger mass was then performed, and as the immediate post-operative serum cortisol level was 50 nmol/L, hydrocortisone was administered at a dose of 30 mg per day, with a stepwise decrease to 10 mg per day over 3 months. Pathological examination exhibited macronodular adrenal hyperplasia with a 70-mm adreno cortical adenoma (WEISS score: 1 and Ki67: 1%). The genetic screening exhibited a c.1908del p.(Phe637Leufs*6) variant of ARMC5 (pathogenic), located in exon 5. The patient has no offspring and is no longer in contact with the rest of his family. Discussion The goal of the treatment with steroidogenesis inhibitors is normalization of cortisol production allowing the improvement of comorbidities (1). Most studies dealing with currently available steroidogenesis inhibitors used as monotherapy reported an overall antisecretory efficacy of roughly 50% in CS. This rate of efficacy was probably underestimated in retrospective studies due to the lack of adequate uptitration of the dose; For example, the median dose reported in the French retrospective study on ketoconazole was only 800 mg/day, while 50% of the patients were uncontrolled at the last follow-up (2). Steroidogenesis inhibitors can be combined to better control hypercortisolism. Up to now, such combinations, mainly ketoconazole and metyrapone, were mainly reported in patients with severe CS (median urinary-free Cortisol (UFC) 30- to 40-fold upper-limit norm (ULN)) and life-threatening comorbidities (3, 4). Normal UFC was reported in up to 86% of these patients treated with high doses of ketoconazole and metyrapone. Expected side effects (such as increased liver enzymes for ketoconazole or worsened hypertension and hypokalemia for metyrapone) were reported in the majority of the patients. The fear of these side effects probably explains the lack of uptitration in previous reports. Combination of steroidogenesis inhibitors has previously been described by Daniel et al. in the largest study reported on the use of metyrapone in CS; 29 patients were treated with metyrapone and ketoconazole or mitotane, including 22 in whom the second drug was added to metyrapone monotherapy because of partial efficacy or adverse effects. The final median metyrapone dose in patients controlled with combination therapy was 1500 mg per day (5). Combination of adrenal steroidogenesis inhibitors should not be reserved to patients with severe hypercortisolism. In the case shown here, the association was highly effective in terms of secretion, using lower doses than those applied as a single treatment, but without the side effects previously observed with higher doses of each treatment used as a monotherapy. To our knowledge, the association of ketoconazole and osilodrostat had never been reported. Ketoconazole blocks several enzymes of the adrenal steroidogenesis such as CYP11A1, CYP17, CYP11B2 (aldosterone synthase) and CYP11B1 (11-hydroxylase), leading to decreased cortisol and occasionally testosterone concentrations. Though liver enzymes increase is not dose-dependent, it usually happens at doses exceeding 400–600 mg per day (2). Osilodrostat blocks CYP11B1 and CYP11B2; a combination should thus allow for a complete blockade of these enzymes that are necessary for cortisol secretion. Short-term side effects such as hypokalemia and hypertension are similar to those observed with metyrapone, due to increased levels of the precursor deoxycorticosterone, correlated with the dose of osilodrostat (6). As for our patient, the occurrence of side effects should not lead to immediately switch to another drug, but rather to decrease the dose and add another cortisol-lowering drug. Moreover, considering the current cost of newly-released drugs such a strategy could lower financial costs for patients and/or society. Another point to take into account is the current COVID-19 pandemic, for which, as recently detailed in experts’ opinion (7), the main aim is to reach eucortisolism, whatever the way. Indeed patients presenting with CS usually also present with comorbidities such as obesity, hypertension, diabetes mellitus and immunodeficiency (8). Surgery, which represents the gold standard strategy in the management of CS (1, 9), might be delayed to reduce the hospital-associated risk of COVID-19, with post-surgical immunodepression and thromboembolic risks (7). Because immunosuppression and thromboembolic diathesis are common CS features (9, 10), during the COVID-19 pandemic, the use of steroidogenesis inhibitors appears of great interest. In these patients, combing steroidogenesis inhibitors at intermediate doses might allow for a rapid control of hypercortisolism without risks of major side effects if a single uptitrated treatment is not sufficient. Obviously, the management of associated comorbidities would also be crucial in this situation (11). To conclude, we report for the first time a case of CS, in the context of primary bilateral macronodular adrenocortical hyperplasia successfully treated with a well-tolerated combination of ketoconazole and osilodrostat. While initial levels of cortisol were not very high, we could not manage to control hypercortisolism with ketoconazole monotherapy, and could not increase the dose due to side effects. The same result was observed with another steroidogenesis inhibitor, osilodrostat. This strategy could be considered for any patient with uncontrolled hypercortisolism and delayed or unsuccessful surgery, especially in the context of the COVID-19 pandemic. Declaration of interest F C and T B received research grants from Recordati Rare Disease and HRA Pharma Rare Diseases. Frederic Castinetti is on the editorial board of Endocrinology, Diabetes and Metabolism case reports. Frederic Castinetti was not involved in the review or editorial process for this paper, on which he is listed as an author. Funding This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. Patient consent Informed written consent has been obtained from the patient for publication of the case report. Author contribution statement V A was the patient’s physician involved in the clinical care and collected the data. T B and F C supervised the management of the patient. F C proposed the original idea of this case report. V A drafted the manuscript. F C critically reviewed the manuscript. T B revised the manuscript into its final version. References 1↑ Nieman LK, Biller BMK, Findling JW, Murad MH, Newell-Price J, Savage MO, Tabarin A & Endocrine Society. Treatment of Cushing’s syndrome: an Endocrine Society clinical practice guideline. Journal of Clinical Endocrinology and Metabolism 2015 100 2807–2831. (https://doi.org/10.1210/jc.2015-1818) Search Google Scholar Export Citation 2↑ Castinetti F, Guignat L, Giraud P, Muller M, Kamenicky P, Drui D, Caron P, Luca F, Donadille B & Vantyghem MC et al.Ketoconazole in Cushing’s disease: is it worth a try? Journal of Clinical Endocrinology and Metabolism 2014 99 1623–1630. (https://doi.org/10.1210/jc.2013-3628) Search Google Scholar Export Citation 3↑ Corcuff JB, Young J, Masquefa-Giraud P, Chanson P, Baudin E, Tabarin A. Rapid control of severe neoplastic hypercortisolism with metyrapone and ketoconazole. European Journal of Endocrinology 2015 172 473–481. (https://doi.org/10.1530/EJE-14-0913) Search Google Scholar Export Citation 4↑ Kamenický P, Droumaguet C, Salenave S, Blanchard A, Jublanc C, Gautier JF, Brailly-Tabard S, Leboulleux S, Schlumberger M & Baudin E et al.Mitotane, metyrapone, and ketoconazole combination therapy as an alternative to rescue adrenalectomy for severe ACTH-dependent Cushing’s syndrome. Journal of Clinical Endocrinology and Metabolism 2011 96 2796–2804. (https://doi.org/10.1210/jc.2011-0536) Search Google Scholar Export Citation 5↑ Daniel E, Aylwin S, Mustafa O, Ball S, Munir A, Boelaert K, Chortis V, Cuthbertson DJ, Daousi C & Rajeev SP et al.Effectiveness of metyrapone in treating Cushing’s syndrome: a retrospective multicenter study in 195 patients. Journal of Clinical Endocrinology and Metabolism 2015 100 4146–4154. (https://doi.org/10.1210/jc.2015-2616) Search Google Scholar Export Citation 6↑ Pivonello R, Fleseriu M, Newell-Price J, Bertagna X, Findling J, Shimatsu A, Gu F, Auchus R, Leelawattana R & Lee EJ et al.Efficacy and safety of osilodrostat in patients with Cushing’s disease (LINC 3): a multicentre phase III study with a double-blind, randomised withdrawal phase. Lancet: Diabetes and Endocrinology 2020 8 748–761. (https://doi.org/10.1016/S2213-8587(2030240-0) Search Google Scholar Export Citation 7↑ Newell-Price J, Nieman LK, Reincke M, Tabarin A. ENDOCRINOLOGY IN THE TIME OF COVID-19: Management of Cushing’s syndrome. European Journal of Endocrinology 2020 183 G1–G7. (https://doi.org/10.1530/EJE-20-0352) Search Google Scholar Export Citation 8↑ Kakodkar P, Kaka N, Baig MN. A comprehensive literature review on the clinical presentation, and management of the pandemic coronavirus disease 2019 (COVID-19). Cureus 2020 12 e7560. (https://doi.org/10.7759/cureus.7560) Search Google Scholar Export Citation 9↑ Pivonello R, De M, Cozzolino A, Colao A. The treatment of Cushing’s disease. Endocrine Reviews 2015 36 385–486. (https://doi.org/10.1210/er.2013-1048) Search Google Scholar Export Citation 10↑ Hasenmajer V, Sbardella E, Sciarra F, Minnetti M, Isidori AM, Venneri MA. The immune system in Cushing’s syndrome. Trends in Endocrinology and Metabolism 2020 31 655–669. (https://doi.org/10.1016/j.tem.2020.04.004) Search Google Scholar Export Citation 11↑ Pivonello R, Ferrigno R, Isidori AM, Biller BMK, Grossman AB, Colao A. 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Osilodrostat therapy was found to be effective in improving blood pressure parameters, health-related quality of life, depression, and other signs and symptoms in patients with Cushing disease, regardless of the degree of cortisol control, according to study results presented at the 30th Annual Scientific and Clinical Congress of the American Association of Clinical Endocrinologists (ENVISION 2021). Investigators of the LINC 3 study (ClinicalTrials.gov Identifier: NCT02180217), a phase 3, multicenter study with a double-blind, randomized withdrawal period, sought to assess the effects of twice-daily osilodrostat (2-30 mg) on signs, symptoms, and health-related quality of life in 137 patients with Cushing disease. Study endpoints included change in various parameters from baseline to week 48, including mean urinary free cortisol (mUFC) status, cardiovascular-related measures, physical features, Cushing Quality-of-Life score, and Beck Depression Inventory score. Participants were assessed every 2, 4, or 12 weeks depending on the study period, and eligible participants were randomly assigned 1:1 to withdrawal at week 24. The median age of participants was 40.0 years, and women made up 77.4% of the cohort. Of 137 participants, 132 (96%) achieved controlled mUFC at least once during the core study period. At week 24, patients with controlled or partially controlled mUFC showed improvements in blood pressure that were not seen in patients with uncontrolled mUFC; at week 48, improvement in blood pressure occurred regardless of mUFC status. Cushing Quality-of-Life and Beck Depression Inventory scores, along with other metabolic and cardiovascular risk factors, improved from baseline to week 24 and week 48 regardless of degree of mUFC control. Additionally, most participants reported improvements in physical features of hypercortisolism, including hirsutism, at week 24 and week 48. The researchers indicated that the high response rate with osilodrostat treatment was sustained during the 48 weeks of treatment, with 96% of patients achieving controlled mUFC levels; improvements in clinical signs, physical features, quality of life, and depression were reported even among patients without complete mUFC normalization. Disclosure: This study was sponsored by Novartis Pharma AG; however, as of July 12, 2019, osilodrostat is an asset of Recordati AG. Please see the original reference for a full list of authors’ disclosures. Visit Endocrinology Advisor‘s conference section for complete coverage from the AACE Annual Meeting 2021: ENVISION. Reference Pivonello R, Fleseriu M, Newell-Price J, et al. Effect of osilodrostat on clinical signs, physical features and health-related quality of life (HRQoL) by degree of mUFC control in patients with Cushing’s disease (CD): results from the LINC 3 study. Presented at: 2021 AACE Virtual Annual Meeting, May 26-29, 2021. From https://www.endocrinologyadvisor.com/home/conference-highlights/aace-2021/osilodrostat-improves-blood-pressure-hrqol-and-depression-in-patients-with-cushing-disease/

Data from LINC3 and LINC4 provide insight into the impact of dosing titration schedules on risk of hypocortisolism-related adverse events associated with osilodrostat use in patients with Cushing's disease. Data from a pair of phase 3 studies presented at the American Academy of Clinical Endocrinology’s 30th Annual Meeting (AACE 2021) is providing insight into the effect of dose titration schedules with use of osilodrostat (Isturisa) in patients with Cushing’s disease. Presented by Maria Fleseriu, MD, of Oregon Health and Science University, the analysis of the LINC3 and LINC4 demonstrated the more gradual titration occurring in LINC4 resulted in a lower proportion of hypocortisolism-related adverse events, suggesting up-titration every 3 weeks rather than every 2 weeks could help lower event risk without compromising mean urinary free cortisol (mUFC) control. “For patients with Cushing’s disease, osilodrostat should be initiated at the recommended starting dose with incremental dose increases, based on individual response/tolerability aimed at normalizing cortisol levels,” concluded investigators. With approval from the US Food and Drug Administration in March 2020 for patients not eligible for pituitary surgery or have undergone the surgery but still have the disease, osilodrostat became the first FDA-approved therapy address cortisol overproduction by blocking 11β-hydroxylase. Based on results of LINC3, data from the trial, and the subsequent LINC4 trial, provide the greatest available insight into use of the agent in this patient population. The study presented at AACE 2021 sought to assess whether slow dose up titration might affect rates of hypocortisolism-related adverse events by comparing titration schedules from both phase 3 trials. Median osilodrostat exposure was 75 (IQR, 48-117) weeks and 70 (IQR, 49-87) weeks in LINC3 and LINC4, respectively. The median time to first mUFC equal to or less than ULN was 41 (IQR, 30-42) days in LINC3 and 35 (IQR, 34-52) days in LINC4. Adverse events potentially related to hypocortisolism were more common among patients in LINC3 (51%, n=70) than LINC4 (27%, n=20). Upon analysis of adverse events, investigators found the most commonly reported type of adverse event was adrenal insufficiency, which included events of glucocorticoid deficiency, adrenocortical insufficiency, steroid withdrawal syndrome, and decreased urinary free cortisol. Results incited the majority of hypocortisolism-related adverse events occurred during the dos titration periods of each trial. In LINC3, 54 of the 70 (77%) hypocortisolism-related adverse events occurred by week 26. In comparison, 58% of hypocortisolism-related adverse events occurring in LINC4 occurred prior to week 12. Investigators noted most of events that occurred were mild or moderate and managed with dose interruption or reduction of osilodrostat or concomitant medications. This study, “Effect of Dosing and Titration of Osilodrostat on Efficacy and Safety in Patients with Cushing's Disease (CD): Results from Two Phase III Trials (LINC3 and LINC4),” was presented at AACE 2021. From https://www.endocrinologynetwork.com/view/fda-panels-votes-to-support-teplizumab-potential-for-delaying-type-1-diabetes

— Gradual dose escalation had fewer adverse events, same therapeutic benefit, as quicker increases by Kristen Monaco, Staff Writer, MedPage Today May 27, 2021 A more gradual increase in oral osilodrostat (Isturisa) dosing was better tolerated among patients with Cushing's disease, compared with those who had more accelerated increases, a researcher reported. Looking at outcomes from two phase III trials assessing osilodrostat, only 27% of patients had hypocortisolism-related adverse events if dosing was gradually increased every 3 weeks, said Maria Fleseriu, MD, of Oregon Health & Science University in Portland, in a presentation at the virtual meeting of the American Association of Clinical Endocrinology (AACE). On the other hand, 51% of patients experienced a hypocortisolism-related adverse event if osilodrostat dose was increased to once every 2 weeks. Acting as a potent oral 11-beta-hydroxylase inhibitor, osilodrostat was first approved by the FDA in March 2020 for adults with Cushing's disease who either cannot undergo pituitary gland surgery or have undergone the surgery but still have the disease. The drug is currently available in 1 mg, 5 mg, and 10 mg film-coated tablets. The approval came based off of the positive findings from the complementary LINC3 and LINC4 trials. The LINC3 trial included 137 adults with Cushing's disease with a mean 24-hour urinary free cortisol concentration (mUFC) over 1.5 times the upper limit of normal (50 μg/24 hours), along with morning plasma adrenocorticotropic hormone above the lower limit of normal (9 pg/mL). During the open-label, dose-escalation period, all the participants were given 2 mg of osilodrostat twice per day, 12 hours apart. Over this 12-week titration phase, dose escalations were allowed once every 2 weeks if there were no tolerability issues to achieve a maximum dose of 30 mg twice a day. After this 12-week dose-escalation schedule, additional bumps up in dose were permitted every 4 weeks. The median daily osilodrostat dose was 7.1 mg. The LINC4 trial included 73 patients with Cushing's disease with an mUFC over 1.3 times the upper limit of normal. The 48 patients randomized to receive treatment were likewise started on 2 mg bid of osilodrostat. However, this trial had a more gradual dose-escalation schedule, as doses were increased only every 3 weeks to achieve a 20 mg bid dose. After the 12-week dose-escalation phase, patients on a dose over 2 mg bid were restarted on 2 mg bid at week 12, where dose escalations were permitted once every 3 weeks thereafter to achieve a maximum 30 mg bid dose during this additional 36-week extension phase. Patients in this trial achieved a median daily osilodrostat dose of 5.0 mg. In both studies, patients' median age was about 40 years, the majority of patients were female, and about 88% had undergone a previous pituitary surgery. When comparing the adverse event profiles of both trials, Fleseriu and colleagues found that more than half of patients on the 2-week dose-escalation schedule experienced any grade of hypercortisolism-related adverse events. About 10.2% of these events were considered grade 3. About 28% of these patients had adrenal insufficiency -- the most common hypercortisolism-related adverse event reported. This was a catch-all term that include events like glucocorticoid deficiency, adrenocortical insufficiency, steroid withdrawal syndrome, and decreased cortisol, Fleseriu explained. Conversely, only 27.4% of patients on a 3-week dose escalation schedule experienced a hypercortisolism-related adverse event, and only 2.7% of these were grade 3. No grade 4 events occurred in either trial, and most events were considered mild or moderate in severity. "These adverse events were not associated with any specific osilodrostat dose of mean UFC level," Fleseriu said, adding that most of these events occurred during the initial dose-escalation periods. About 60% and 58% of all hypocortisolism-related adverse events occurred during the dose titration period in the 2-week and 3-week dose-escalation schedules, respectively. These events were managed via dose reduction, a temporary interruption in medication, and/or a concomitant medication. Very few patients in either trial permanently discontinued treatment due to these adverse events, Fleseriu noted. "Despite differences in the frequency of dose escalation, the time to first mUFC normalization was similar in the LINC3 and LINC4 studies," she said, adding that "gradual increases in osilodrostat dose from a starting dose of 2 mg bid can mitigate hypocortisolism-related adverse events without affecting mUFC control." "For patients with Cushing's disease, osilodrostat should be initiated at the recommended starting dose with incremental dose increases, based on individual response and tolerability aimed at normalizing cortisol levels," Fleseriu concluded. Kristen Monaco is a staff writer, focusing on endocrinology, psychiatry, and dermatology news. Based out of the New York City office, she’s worked at the company for nearly five years. Disclosures The LINC3 and LINC4 trials were funded by Novartis. Fleseriu reported relationships with Novartis, Recordati, and Strongbridge Biopharma. Primary Source American Association of Clinical Endocrinology Source Reference: Fleseriu M, et al "Effect of dosing and titration of osilodrostat on efficacy and safety in patients with Cushing's disease (CD): Results from two phase III trials (LINC3 and LINC4)" AACE 2021. From https://www.medpagetoday.com/meetingcoverage/aace/92824?xid=nl_mpt_DHE_2021-05-28&eun=g1406328d0r&utm_source=Sailthru&utm_medium=email&utm_campaign=Daily Headlines Top Cat HeC 2021-05-28&utm_term=NL_Daily_DHE_dual-gmail-definition

Osilodrostat treatment was found to be associated with a rapid and sustained reduction in mean concentration of urinary free cortisol (UFC) and improved clinical symptoms in patients with Cushing’s disease, according to the results of a prospective, multicenter, open-label, phase 3 study published in the Lancet Diabetes Endocrinology. Osilodrostat is an oral inhibitor of 11-β hydroxylase cytochrome P450. Adults aged 18 to 75 years of age with diagnosed persistent or recurrent Cushing’s disease were recruited between 2014 and 2017 at 66 hospitals in 19 countries. Cushing’s disease was defined by a mean UFC concentration over a 24-hour period >1.5 times greater than the upper limit of normal (ULN) and morning plasma adrenocorticotropic hormone level above normal limits. Participants (n=137) received 30 mg osilodrostat twice daily, dose which was adjusted every 2 weeks until week 12 on the basis of mean 24-hour UFC concentration. The determined maintenance dose was continued until week 24. At week 26, participants who had achieved 24-hour UFC concentration ≤ ULN and did not need titration after week 12 were randomly assigned in an equal ratio to maintain osilodrostat treatment or were switched to a placebo for 8 weeks. This 8-week period of the study was double-blinded. During weeks 35 to 48, all patients were returned to osilodrostat treatment. In this cohort, mean age was 40.0 years (range, 19.0-70.0 years), 77% of participants were women, the average time since diagnosis was 47.2 months (interquartile range [IQR], 19.0-88.3), 88% had previous pituitary surgery, 16% had pituitary radiation therapy, and 74% had medicinal therapy. At baseline, the mean 24-hour UFC concentration was 1006±1590 nmol/24 h. At week 24, 53% of participants achieved a mean 24-hour UFC concentration ≤ULN without increases in dose after week 12 and were eligible for randomization (osilodrostat, n=36; placebo, n=35). At week 34, more patients receiving osilodrostat vs placebo maintained a complete response (86% vs 29%, respectively; odds ratio [OR], 13.7; 95% CI, 3.7-53.4; P <.0001). Improvements in cardiovascular-related metabolic parameters associated with hypercortisolism and overall measures of well-being were observed. Levels of high-density lipoprotein decreased by week 48 (-0.3 mmol/L; 95% CI, .0.3 to -0.2), mean Cushing’s quality of life score increased by 52.4% (95% CI, 32.3-72.7), and Beck Depression Inventory score decreased by 31.8% (95% CI, -44.3 to -19.3). Adverse events were hypocortisolism (51%), adverse events related with adrenal hormone precursors (42%), nausea (42%), headache (34%), fatigue (28%), and adrenal insufficiency (28%). A total of 18% of participants dropped out of the study due to adverse events. The major limitation of this study was the short withdrawal period (8 weeks) which may not have permitted to observe symptoms of hypercortisolism. “Alongside careful dose adjustments and monitoring of known risks associated with osilodrostat, our findings indicate a positive benefit– risk consideration of treatment for most patients with Cushing’s disease,” concluded the study authors. Disclosure: Multiple authors declared affiliations with industry. Please refer to the original article for a full list of disclosures. Reference Pivonello R, Fleseriu M, Newell-Price J, et al. Efficacy and safety of osilodrostat in patients with Cushing’s disease (LINC 3): a multicentre phase III study with a doubleblind, randomised withdrawal phase. Lancet Diabetes Endocrinol. 2020;S2213-8587(20)30240-0. doi:10.1016/S2213-8587(29)30240-0 From https://www.endocrinologyadvisor.com/home/topics/general-endocrinology/osilodrostat-sustained-reduction-mean-ufc-concentration-cushings-disease/

Hypercortisolism Quickly Reversed With Oral Tx Oral osilodrostat (Isturisa) normalized cortisol levels in Cushing's disease patients who were ineligible for or not cured with pituitary surgery, according to the phase III LINC 3 trial. After 24 weeks of open-label treatment with twice-daily osilodrostat, 53% of patients (72 of 137; 95% CI 43.9-61.1) were able to maintain a complete response -- marked by mean 24-hour urinary free cortisol concentration of the upper limit of normal or below -- without any uptitration in dosage after the initial 12-week buildup phase, reported Rosario Pivonello, MD, of the Università Federico II di Napoli in Italy, and colleagues. As they explained in their study online in The Lancet Diabetes & Endocrinology, following the 24-week open-label period these complete responders to treatment were then randomized 1:1 to either remain on osilodrostat or be switched to placebo. During this 10-week randomization phase, 86% of patients maintained their complete cortisol response if they remained on osilodrostat versus only 29% of those who were switched to placebo (odds ratio 13.7, 95% CI 3.7-53.4, P<0.0001) -- meeting the trial's primary endpoint. As for adverse events, more than half of patients experienced hypocortisolism, and the most common adverse events included nausea (42%), headache (34%), fatigue (28%), and adrenal insufficiency (28%). "Alongside careful dose adjustments and monitoring of known risks associated with osilodrostat, our findings indicate a positive benefit-risk consideration of treatment for most patients with Cushing's disease," the researchers concluded. This oral inhibitor of 11β-­hydroxylase -- the enzyme involved in the last step of cortisol synthesis -- was FDA approved in March 2020 based on these findings, and is currently available in 1 mg, 5 mg, and 10 mg film-coated tablets. The prospective trial, consisting of four periods, included individuals between the ages of 18 and 75 with confirmed persistent or recurrent Cushing's disease -- marked by a mean 24-h urinary free cortisol concentration over 1.5 times the upper limit of normal (50 μg/24 hours), along with morning plasma adrenocorticotropic hormone above the lower limit of normal (9 pg/mL). All individuals had either undergone prior pituitary surgery or irradiation, were not deemed to be candidates for surgery, or had refused to have surgery. During the first open-label study period, all participants took 2 mg of oral osilodrostat twice daily, spaced 12 hours apart. This dose was then titrated up if the average of three 24-h urinary free cortisol concentration samples exceeded the upper limit of normal. During the second study period, which spanned weeks 12 through 24, all participants remained on their osilodrostat therapeutic dose. By week 24, about 62% of the participants were taking a therapeutic dose of 5 mg or less twice daily; only about 6% of patients needed a dose higher than 10 mg twice daily. In the third study period, which spanned weeks 26 through 34, "complete responders" who achieved normal cortisol levels were then randomized to continue treatment or be switched to placebo, while those who did not fully respond to treatment continued on osilodrostat. For the fourth study period, from weeks 24 through 48, all participants were switched back to active treatment with osilodrostat. Overall, 96% of participants were able to achieve a complete response at some point while on osilodrostat treatment, with two-thirds of these responders maintaining this normalized cortisol level for at least 6 months. The median time to first complete response was 41 days. Metabolic profiles also improved along with this reduction in cortisol levels. These included improvements in body weight, body mass index, fasting plasma glucose, both systolic and diastolic blood pressures, and total cholesterol levels. "Given the known clinical burden of cardiovascular risk associated with Cushing's disease, the improvement in clinical features shown here indicates important benefits of osilodrostat," the researchers said. "By improving multiple cardiovascular risk factors, our findings are likely to be clinically relevant." Along with metabolic improvements, patients also had "clinically meaningful improvements" in quality of life, as well as reductions in depressive symptoms measured by the Beck Depression Inventory score, the investigators reported. One limitation to the trial, they noted, was an inability to control for concomitant medications, since nearly all participants were taking other medications, particularly antihypertensive and antidiabetic therapies. "Further examination of the effects of osilodrostat on the clinical signs of Cushing's disease, and the reasons for changes in concomitant medications and the association between such medications and clinical outcomes would be valuable," Pivonello's group said. From https://www.medpagetoday.com/endocrinology/generalendocrinology/87827

Cushing syndrome, a rare endocrine disorder caused by abnormally excessive amounts of the hormone cortisol, has a new pharmaceutical treatment to treat cortisol overproduction. Osilodrostat (Isturisa) is the first FDA approved drug who either can’t undergo pituitary gland surgery or have undergone the surgery but still have the disease. The oral tablet functions by blocking the enzyme responsible for cortisol synthesis, 11-beta-hydroxylase. “Until now, patients in need of medications…have had few approved options, either with limited efficacy or with too many adverse effects. With this demonstrated effective oral treatment, we have a therapeutic option that will help address patients' needs in this underserved patient population," said Maria Fleseriu, MD, FACE, professor of medicine and neurological surgery and director of the Pituitary Center at Oregon Health Sciences University. Cushing disease is caused by a pituitary tumor that releases too much of the hormone that stimulates cortisol production, adrenocorticotropin. This causes excessive levels of cortisol, a hormone responsible for helping to maintain blood sugar levels, regulate metabolism, help reduce inflammation, assist in memory formulation, and support fetus development during pregnancy. The condition is most common among adults aged 30-50 and affects women 3 times more than men. Cushing disease can lead to a number of medical issues including high blood pressure, obesity, type 2 diabetes, blood clots in the arms and legs, bone loss and fractures, a weakened immune system, and depression. Patients with Cushing disease may also have thin arms and legs, a round red full face, increased fat around the neck, easy bruising, striae (purple stretch marks), or weak muscles. Side effects of osilodrostat occurring in more than 20% of patients are adrenal insufficiency, headache, nausea, fatigue, and edema. Other side effects can include vomiting, hypocortisolism (low cortisol levels), QTc prolongation (heart rhythm condition), elevations in adrenal hormone precursors (inactive substance converted into hormone), and androgens (hormone that regulated male characteristics). Osilodrostat’s safety and effectiveness was evaluated in a study consisting of 137 patients, of which about 75% were women. After a 24-week period, about half of patients had achieved normal cortisol levels; 71 successful cases then entered an 8-week, double-blind, randomized withdrawal study where 86% of patients receiving osilodrostat maintained normal cortisol levels, compared with 30% who were taking a placebo. In January 2020, the European Commission also granted marketing authorization for osilodrostat. From https://www.ajmc.com/newsroom/patients-with-cushing-have-new-nonsurgical-treatment-option

NEW ORLEANS — The investigational drug osilodrostat (Novartis) continues to show promise for treating Cushing's disease, now with new phase 3 trial data. The data from the phase 3, multicenter, double-blind randomized withdrawal study (LINC-3) of osilodrostat in 137 patients with Cushing's disease were presented here at ENDO 2019: The Endocrine Society Annual Meeting by Beverly M.K. Biller, MD, of the Neuroendocrine & Pituitary Tumor Center at Massachusetts General Hospital, Boston. "Osilodrostat was effective and shows promise for the treatment of patients with Cushing's disease," Biller said. Osilodrostat is an oral 11β-hydroxylase inhibitor, the enzyme that catalyzes the last step of cortisol biosynthesis in the adrenal cortex. Its mechanism of action is similar to that of the older Cushing's drug metyrapone, but osilodrostat has a longer plasma half-life and is more potent against 11β-hydroxylase. Significantly more patients randomized to osilodrostat maintained a mean urinary free cortisol (mUFC) response versus placebo at 34 weeks following a 24-week open-label period plus 8-week randomized phase, with rapid and sustained mUFC reduction in most patients. Patients also experienced improvements in clinical signs of hypercortisolism and quality of life. The drug was generally well-tolerated and had no unexpected side effects. Asked to comment, session comoderator Julia Kharlip, MD, associate medical director of the Pituitary Center at the University of Pennsylvania, Philadelphia, told Medscape Medical News, "This drug is incredibly exciting because over 80% of people were controlled fairly rapidly. People could get symptom relief but also a reliable response. You don't have to wonder when you're treating a severely affected patient if it's going to work. It's likely going to work." However, Kharlip cautioned that it remains to be seen whether osilodrostat continues to work long-term, given that the older drug metyrapone — which must be given four times a day versus twice daily for osilodrostat — is known to become ineffective over time because the pituitary tumor eventually overrides the enzyme blockade. "Based on how osilodrostat is so much more effective at smaller doses, there's more hope that it will be effective long term...If the effectiveness and safety profile that we're observing now continues to show the same performance years in a row, then we've got our drug." Osilodrostat Potentially Addresses an Unmet Medical Need Cushing's disease is a rare disorder of chronic hypercortisolism with significant burden, increased mortality, and decreased quality of life. Pituitary surgery is the recommended first-line treatment for most patients, but not all patients remit with surgery and some require additional treatment. Pasireotide (Signifor, Novartis), an orphan drug approved in the United States and Europe for the treatment of Cushing's disease in patients who fail or are ineligible for surgical therapy, is also only effective in a minority of patients. "There hasn't been a medicine effective for long-term treatment, so a lot of patients end up getting bilateral adrenalectomy, thereby exchanging one chronic medical disease for another," Kharlip explained. Biller commented during the question-and-answer period, "I think because not all patients are placed in remission with surgery initially and because other patients subsequently recur — a problem that is more common than we used to believe — we do need medical therapies." She continued, "I think it's important to have a large choice of medical therapies that work in different places in the hypothalamic-pituitary-adrenal axis. "Even though surgery is the right initial therapy for everyone, I think in terms of subsequent medical therapy we have to tailor that to the individual circumstances of the patient in terms of the goals of treatment, and perhaps what other medicines they're on, the degree of cortisol excess [and other factors]." Highly Significant Normalization in Mean UFC Versus Placebo In a prior 22-week phase 2 study (LINC-2), osilodrostat normalized mUFC in most patients. Results of the extension phase were reported by Medscape Medical News 2 years ago. The current phase 3 study, LINC-3, was conducted on the basis of that proof-of-concept study, Biller said. The trial was conducted in 19 countries across four continents in patients with persistent or recurrent Cushing's disease screened for mUFC > 1.5 times the upper limit of normal and other entry criteria. In total, 137 patients were enrolled and randomized. Participants were a median age of 40 years, 77% were female, and 88% had undergone prior pituitary surgery. Nearly all (96%) had received at least one previous treatment for Cushing's. At baseline, patients' mean mUFC (364 µg/24 hours) was 7.3 times the upper limit of normal, which is "quite significant hypercortisolemia," Biller noted. All patients initially received osilodrostat, with a rapid dose uptitration every 2 weeks from 2 to 30 mg orally twice daily until they achieved a normal UFC. They continued on open-label medication until week 24, when urine samples were collected. Patients who had an mUFC less than the upper limit of normal and had not had a dose increase in the prior 12 weeks were eligible for the double-blind phase. Those who were ineligible continued taking open-label drug. The 70 eligible patients were randomized to continue taking osilodrostat (n = 36) or were switched to placebo (n = 34) for another 8 weeks. After that, the patients taking placebo were switched back to osilodrostat until week 48. A total of 113 patients completed the 48 weeks. The primary efficacy endpoint was mUFC at 34 weeks (the end of the 8-week randomized phase). For those randomized to continue on the drug, mUFC remained in the normal range in 86.1% of patients versus just 29.4% of those who had been switched to placebo for the 8 weeks. The difference was highly significant (odds ratio, 13.7; P < .001), Biller reported. A key secondary endpoint, proportion of patients with an mUFC at or below the ULN at 24 weeks without up-titration after week 12, was achieved in 53%. The mean dose at 48 weeks was 11.0 mg/day, "a fairly low dose," she noted. Clinical features were also improved at week 48, including systolic and diastolic blood pressure (percentage change –6.8 and –6.6, respectively), weight (–4.6), waist circumference (–4.2), fasting plasma glucose (–7.1), and HbA1c (–5.4). Scores on the Cushing Quality of Life scale improved by 52.4 points, and Beck Depression Inventory scores dropped by 31.8 points. Most Adverse Events Temporary, Manageable The most commonly reported adverse events were nausea (41.6%), headache (33.6%), fatigue (28.5%), and adrenal insufficiency (27.7%), and 10.9% of patients overall discontinued because of an adverse event. Adverse events related to hypocortisolism occurred in 51.1% of patients overall, with 10.2% being grade 3 or 4. However, most of these were single episodes of mild-to-moderate intensity and mainly occurred during the initial 12-week titration period. Most patients responded to dose reduction or glucocorticoid supplementation. Adverse events related to accumulation of adrenal hormone precursors occurred in 42.3% of patients overall, with the most common being hypokalemia (13.1%) and hypertension (12.4%). No male patients had signs or symptoms related to increased androgens or estrogens. However, 12 female patients experienced hirsutism, most of those patients also had acne, and one had hypertrichosis. None discontinued because of those symptoms. Kharlip commented, "What's really inspiring was that even though half of the patients had symptoms related to adrenal insufficiency, it sounded as if they were quickly resolved with treatment and none discontinued because of it." "And it may have been related to study design where the medication was titrated very rapidly. There is probably a way to do this more gently and get the good results without the side effects." Kharlip also praised the international consortium that devised the protocol and collaborated in the research effort. "It's incredibly exciting and gratifying to see the world come together to get these data. It's such a rare disease. To be able to have something like that in the field is a dream, to have a working consortium. The protocol was effective in demonstrating efficacy. It's just a win on so many levels for a disease that currently doesn't have a good therapy...I struggle with these patients all the time so I'm thrilled that there is hope." An ongoing confirmatory phase 3 study, LINC-4, is evaluating patients up to 48 weeks. Biller is a consultant for and has received grants from Novartis and Strongbridge. Kharlip has reported no relevant financial relationships. For more diabetes and endocrinology news, follow us on Twitter and on Facebook. From https://www.medscape.com/viewarticle/910864#vp_1

CLCI699C2302: A Phase III, Multi-center, Randomized, Double-blind, 48 Week Study with an Initial 12 Week Placebo-controlled Period to Evaluate the Safety and Efficacy of Osilodrostat in Patients with Cushing’s Disease Purpose In people with a disorder known as Cushing’s disease, levels of the hormone cortisol are very high in the urine and blood. Lowering cortisol levels relieves the symptoms of Cushing’s disease. Osilodrostat is an investigational drug that inhibits an enzyme needed for cortisol to be made. In this study, researchers are assessing the safety and effectiveness of osilodrostat in patients with Cushing¿s disease and observing its ability to reduce cortisol levels. In the first 12 weeks of the study, patients will receive osilodrostat or a placebo (inactive drug). After week 12 and continuing through week 48, all patients will receive osilodrostat. Patients will then have the option to continue taking osilodrostat for up to 100 weeks into the study, if they wish. Osilodrostat is taken orally (by mouth). Eligibility To be eligible for this study, patients must meet several criteria, including but not limited to the following: Patients must have Cushing¿s disease with elevated levels of cortisol in the urine. An acceptable amount of time must have passed between the completion of prior therapies and entry into the study, to allow for a sufficient “washout” period. This study is for patients ages 18 to 75. For more information about this study and to inquire about eligibility, please contact Dr. Eliza Geer at 646-888-2627. Protocol 17-351 Phase III Investigator Eliza B. Geer Co-Investigators Monica Girotra Diseases Pituitary Tumor Locations Memorial Sloan Kettering Memorial Hospital From https://www.mskcc.org/cancer-care/clinical-trials/17-351