United States

INTRODUCTION: Gestational diabetes mellitus (GDM) is an increasingly frequent complication of pregnancy and its presence is related to the development of adverse maternal and fetal outcomes. Pregnancy is deeply influenced by the circadian rhythm and the misalignment of the maternal rhythm can lead to disturbances in the temporal organization of physiological and metabolic functions. Previously, the association between circadian rhythm disorders and diabetes has been well described in pregnancy. OBJECTIVE: To investigate the influence of chronotype on the development of maternal and fetal complications during pregnancy in GDM. METHODOLOGY: Prospective cohort study of patients with GDM. Clinical data and behavior questionnaires were collected by face- to- face interview. Questionnaires evaluated the chronotype (Morning-Eveningness-Questionnaire- MEQ), sleep quality (Pittsburgh Sleep Quality Index- PSQI), daytime sleepiness (Epworth Sleepiness Scale -ESS), depressive symptoms (Edinburgh Postnatal Depression Scale-EDPS) and insomnia (Insomnia Severity Index- ISI). Sleep diaries (14 days) and actigraphic record (5 to 7 days) were obtained. RESULTS: Overall, 305 2nd to 3rd trimester GDM patients were evaluated. Eighty-nine GDM patients completed the sleep diary and 53 performed a valid actigraphic registry. Amongst these, 49.5% were identified as morning chronotypes (MEQ≥59), 43.6% intermediate types (MEQ from 42 to 58) and 6.9% had evening type preference (MEQ&lt;41). Evening preference patients were younger (p&lt; 0.005), had more unstable marital status (P = 0.04), had worse sleep quality (p = 0.02); latter sleep midpoint (p= 0.01), higher prevalence of insomnia (p&lt; 0.005) and depression (p= 0.004) before and during pregnancy. No differences were found in total sleep duration as recorded by sleep diary (p=0.4) or actigraphic measurements (p=0.83). Evening type preference in GDM was associated with pre-eclampsia (p= 0.02) and neonatal ICU admission (p= 0.03). CONCLUSION: This study suggests that the presence of the evening-type chronotype in pregnant women with GDM is associated with adverse pregnancy outcomes such as the development of hypertensive syndromes, insomnia and depressive symptoms. In addition, a higher prevalence of newborns admitted to the neonatal ICU was found in the offspring of GDM patients with evening preference.?? 

ENDO 2021

Maternal Chronotype and Pregnancy Outcomes in Gestational Diabetes

ENDO is the leading meeting for endocrinology research and clinical care worldwide. 

INTRODUCTION: Gestational diabetes mellitus (GDM) is an increasingly frequent complication of pregnancy and its presence is related to the development of adverse maternal and fetal outcomes. Pregnancy is deeply influenced by the circadian rhythm and the misalignment of the maternal rhythm can lead to disturbances in the temporal organization of physiological and metabolic functions. Previously, the association between circadian rhythm disorders and diabetes has been well described in pregnancy. OBJECTIVE: To investigate the influence of chronotype on the development of maternal and fetal complications during pregnancy in GDM. METHODOLOGY: Prospective cohort study of patients with GDM. Clinical data and behavior questionnaires were collected by face- to- face interview. Questionnaires evaluated the chronotype (Morning-Eveningness-Questionnaire- MEQ), sleep quality (Pittsburgh Sleep Quality Index- PSQI), daytime sleepiness (Epworth Sleepiness Scale -ESS), depressive symptoms (Edinburgh Postnatal Depression Scale-EDPS) and insomnia (Insomnia Severity Index- ISI). Sleep diaries (14 days) and actigraphic record (5 to 7 days) were obtained. RESULTS: Overall, 305 2nd to 3rd trimester GDM patients were evaluated. Eighty-nine GDM patients completed the sleep diary and 53 performed a valid actigraphic registry. Amongst these, 49.5% were identified as morning chronotypes (MEQ≥59), 43.6% intermediate types (MEQ from 42 to 58) and 6.9% had evening type preference (MEQ<41). Evening preference patients were younger (p< 0.005), had more unstable marital status (P = 0.04), had worse sleep quality (p = 0.02); latter sleep midpoint (p= 0.01), higher prevalence of insomnia (p< 0.005) and depression (p= 0.004) before and during pregnancy. No differences were found in total sleep duration as recorded by sleep diary (p=0.4) or actigraphic measurements (p=0.83). Evening type preference in GDM was associated with pre-eclampsia (p= 0.02) and neonatal ICU admission (p= 0.03). CONCLUSION: This study suggests that the presence of the evening-type chronotype in pregnant women with GDM is associated with adverse pregnancy outcomes such as the development of hypertensive syndromes, insomnia and depressive symptoms. In addition, a higher prevalence of newborns admitted to the neonatal ICU was found in the offspring of GDM patients with evening preference.?? 

technical paper

Introduction: The Homeostasis Model Assessment of insulin resistance (HOMA-IR) is a validated surrogate measure of insulin sensitivity and beta-cell function for epidemiological studies. Insulin resistance is closely associated with obesity and shares dysmetabolic state with other cardiometabolic risk factors. The aim of this study was to analyze the change in HOMA-IR levels in US youth from 1999 to 2018.?Methods: Serial cross-sectional data of fasting insulin and glucose levels measured in youth aged 12 to 18 years enrolled in National Health and Nutrition Examination Surveys representative of US population from 1999 to 2018 were used. Two-year survey cycles were combined to derive 4-year survey periods, and NHANES recommended adjustments were made for glucose and insulin levels for forward conversion to 2017-18 instrument levels. Median HOMA-IR was compared across the survey periods in subgroups by age, gender, race/ethnicity and levels of obesity. Association of HOMA-IR with selected cardiometabolic risk factors was assessed. Change of HOMA-IR levels were analyzed using design adjusted multivariable regression models, association of HOMA-IR with cardiometabolic risk factors was assessed and percentiles were derived.?Results: In an analysis of 5,541 youth, the unadjusted median HOMA-IR increased from 1.83 (95% CI 1.77-1.89) in 1999-02 to 2.41 (95% CI 2.29-2.54) in 2007-10 and has remained in this range till 2015-18. Youth with obesity (Body mass index > 95th percentile or waist circumference > 90th percentile), female gender, those of Hispanic and Black origin, and those aged 12-16 years had higher levels of HOMA-IR (p <.001). While adjusting for age category, sex, race/ethnicity, obesity represented by waist circumference z-score with its quadratic term and interaction with gender, HOMA-IR increased by 26% (95% CI 15-37) in 2015-18 as compared to 1999-02. For waist-circumference z-score (WCZ) > 1, the HOMA-IR levels were higher in boys compared to girls. The addition of total body fat from DXA scans in the model, when available, explained the variance related to gender, but not the interaction between gender and WCZ. No change was observed in the higher levels of HOMA-IR by ethnicity when high sensitivity CRP level as a measure of inflammation was added to the model. There was a positive association of HOMA-IR with triglycerides (29 %/unit, 95% CI 25-33), non-HDL cholesterol (8 %/unit, 95% CI 6-10), systolic blood pressure (2% /unit, 95% CI 1-2) and alanine transferase (11 %/unit, 95% CI 8-14) while controlling for age, sex, race/ethnicity, obesity and survey period. In youth with BMI between 5-85th percentile, HOMA-IR decreased with increase in age from 12 to 18 years.?Conclusions: HOMA-IR levels in US youth have increased since 1999-02 and may herald future cardiometabolic risks.?? 

HOMA-IR Levels in US Youth Ages 12-18 Years from 1999-2018

Chronic low-grade inflammation and mitochondrial dysfunction are hallmarks of insulin resistance. However, the mechanisms by which the immune system can propagate systemic insulin resistance remains poorly understood. IgG antibodies are a critical component of immunity and display paradoxical properties. IgG can propagate inflammation by crosslinking Fc receptors activating innate immune cells, and conversely, when given intravenously at high doses (1-2 g/kg intravenous immunoglobulin), actively suppress inflammation. Here, we demonstrate that IgG can exert similar paradoxical properties on glucose metabolism. IgG can elicit insulin resistance, and conversely, when given at high doses, promote insulin sensitivity in a diabetic mouse model. IgG, through its Fc-mediated interactions, suppresses insulin-induced mitochondrial function as well as insulin signaling. Modulation of insulin-dependent mitochondrial respiration by serum or purified IgG highly correlates (R2 = 0.70) with the quantitative measurement of insulin sensitivity accessed by the modified insulin suppression test. Our studies indicate that IgG antibody glycosylation is critically important to these conflicting actions. In mice and humans, the progression of insulin resistance is associated with reduced IgG Fc region sialylation, and administration of asialylated IgG is sufficient to cause insulin resistance in IgG null mice. On the other hand, a single administration of high-dose IgG significantly improved insulin and glucose tolerance as well as plasma glucose levels lasting over 72 days post-administration. These results demonstrate new insights into the systemic nature of insulin resistance, a novel mechanism of the disease, and an innovative therapeutic strategy for treating type 2 diabetes.?? 

The IgG Antibody Paradox in Insulin Resistance: Pathogenic and Therapeutic

BACKGROUND: Obesity is associated with increased prevalence of type 2 diabetes and cardiovascular disease (CVD). Adipose tissue (AT) contains a complex immune environment and is a central contributor to heightened systemic inflammation in obese persons. Increased chronic inflammation in obesity contributes to metabolic disease by increasing insulin resistance, and to CVD by causing an atherogenic dyslipidemia and increasing endothelial cell dysfunction and activation. Despite these links between inflammation and cardiometabolic disease in obesity, there are no current targeted therapies to prevent or reverse chronic inflammation in AT.?Epoxyeicosatrienoic acids (EETs) are lipid signaling molecules that act as potent vasodilators and promote sodium excretion in the kidney. Increasing EETs in rodents protects against hypertension and endothelial dysfunction. In humans, circulating EETs correlate with insulin sensitivity and are decreased in individuals with insulin resistance. EETs also decrease the inflammatory response to obesity in animal models, but the effect of EETs on inflammation in humans is currently unknown. EETs are hydrolyzed to less active forms by the enzyme soluble epoxide hydrolase (sEH), and we hypothesized that pharmacologic sEH inhibition with a specific inhibitor GSK2256294 (GSK) in obese patients would decrease AT inflammation.?METHODS: Thirty-four obese prediabetic individuals were treated with placebo and GSK in a crossover design (NCT03486223). Participants had a seven-week washout in between drugs, and the order of drug was randomized and blinded. In a subgroup of patients, we collected subcutaneous AT by liposuction and characterized T cell phenotypes by flow cytometry (N=7 paired samples).?RESULTS: GSK decreased sEH activity in plasma (47.3% vs placebo; P=0.008) and in AT (58.8% vs placebo; P=0.002). GSK also decreased serum F2-isoprostanes (P=0.03), which are markers of oxidative damage and inflammation. In seven paired AT samples, T helper (Th) 1 cells producing the pro-inflammatory cytokine IFNγ were reduced by treatment with GSK as compared with placebo (% of total lymphocytes: Placebo 13.6% ± 6.9, GSK 11.0% ± 5.6, P=0.03 Wilcoxon Signed Rank). In this small sample, we did not detect significant differences in the percentage of other IFNγ-producing cells (natural killer: Placebo 19.0% ± 9.0, GSK 13.3% ± 4.9, P=0.18; CD8: Placebo 12.0 ± 11.0, GSK 6.1 ± 4.6, P=0.61). In addition, we did not detect any change in Th17, Th2, or regulatory T cells.?CONCLUSIONS: In a pilot study of seven individuals treated with placebo or an sEH inhibitor, we found that the sEH inhibitor decreased pro-inflammatory Th1 cells as compared with placebo in matched AT samples. Understanding the contribution of the EET/sEH pathway to inflammation in obesity could lead to new strategies to modulate AT and systemic inflammation and reduce the risk of CVD.?? 

The Contribution of Arachidonic Acid Metabolites EETs to Inflammation in Obesity

PCOS is common in adolescents and includes excess testosterone concentrations. The clinical importance of 11-oxyandrogens in PCOS is unclear. We sought to determine if fasted morning 11-oxyandrogens: 1) better identify PCOS diagnosis compared to testosterone, 2) relate to clinical comorbidities of PCOS, 3) are altered with combined oral contraceptive pill (COCP) or metformin therapy. Data from 186 adolescent females aged 12-21 years, mostly obese, enrolled in one of 6 studies in our division were included: 115 untreated PCOS (13 lean, BMI 22.5±2.2 kg/m2; 102 overweight/obese, BMI 35.4±5.3), 9 obese PCOS treated with COCP (BMI 33.7±5.7), 6 obese PCOS treated with metformin (BMI 36.4±5.3) and 70 normally cycling controls (19 lean, BMI 19.9±1.4; 52 overweight/obese BMI 35.5±10.4). Three 11-oxyandrogens (11-hydroxyandrostenedione (11OHA), 11-hydroxytestosterone (11OHT), and 11-ketotestosterone (11KT) and total testosterone were analyzed via liquid chromatography tandem mass spectroscopy (LabCorp/Esoterix, Calabasas, CA). Data between 1) untreated PCOS and controls and 2) untreated PCOS and the 2 treatment groups were compared. ROC analysis was performed to evaluate accuracy of diagnosis of PCOS and Pearson’s correlation coefficient was calculated for 11-oxyandrogens and clinical measures. Untreated PCOS girls had higher 11OHA (129±77 ng/dL PCOS vs 97±52 control, p=0.003) and 11OHT (13.8±7.9 ng/dL PCOS vs 10.5±6.7 control, p=0.005) compared to controls, with no difference in 11KT (30.7±17.3 ng/dL PCOS vs 26.6±16.6 control, p=0.208). Only 11OHA remained significant after controlling for obesity via multiple linear regression. However, neither of these metabolites better predicted PCOS status compared to testosterone (ROC analysis: 11OHA AUC=0.620, p=0.006; 11OHT AUC=0.638, p=0.002; total testosterone AUC=0.840, p<0.001, free androgen index AUC 0.860, p<0.001). Among girls with PCOS, all three 11-oxyandrogens correlated with hirsutism severity as graded by Ferriman Gallwey score. COCP treatment decreased 11KT concentrations compared to untreated PCOS (32±17 untreated vs 17±8 ng/dL COCP; p=0.039) as well as total testosterone (46.9±21 untreated vs 28.1±12.9 ng/dL COCP; p<0.006). However, metformin treatment had no effect 11-oxyandrogens, although total testosterone was lower (46.9±21 untreated vs 26.5±5.9 metformin; p=0.01). Whereas 11-oxyandrogens would not aid in the diagnosis of PCOS, they relate to excess hair growth and 11KT decreases with COCP therapy. Further work is needed to determine the relationship with metabolic outcomes and the clinical utility of measuring these androgens in PCOS.

11-Oxyandrogens in Youth with Polycystic Ovary Syndrome

Bone geometry can be described in terms of periosteal and endocortical growth and is partly determined by sex steroids. Periosteal and endocortical apposition are thought to be regulated by testosterone and estrogen, respectively. Gender-affirming hormone (GAH) treatment with sex steroids in transgender people might affect bone geometry. However, in adult transgender people no change in bone geometry during GAH was observed. In this study, we investigated changes in bone geometry among transgender adolescents using a gonadotropin-releasing hormone agonist (GnRHa) and GAH prior to achieving peak bone mass. Transgender adolescents treated with GnRHa and subsequent GAH at our center before the age of 18 years were eligible for inclusion. Participants were grouped based on their Tanner stage at the start of GnRHa treatment and divided into early, mid, and late puberty groups. Hip Strength Analysis software calculating subperiosteal width (SPW) and endocortical diameter (ED) was applied to dual-energy X-ray absorptiometry scans performed at start of GnRHa and GAH treatments, and after ≥ 2 years of GAH treatment. Mixed model analyses were performed to study differences over time. Data were visually compared with reference values of the general population retrieved from the literature. A total of 322 participants were included, of whom 106 trans women and 216 trans men. In both trans women and trans men participants resembled the reference curve for SPW and ED of the experienced gender, but only when GnRHa was started during early puberty. Those who started during mid- and late puberty remained within the reference curve of the gender assigned at birth. A possible explanation might be sought in the phenomenon of programming, which conceptualizes that stimuli during critical windows of development can have major consequences throughout one’s lifespan. Therefore, this study adds insights into sex-specific bone geometry development during puberty of transgender adolescents treated with GnRHa, as well as the general population.

Development of Hip Bone Geometry During Gender-Affirming Hormone Therapy in Transgender Adolescents Resembles that of Experienced Gender When Pubertal Suspension Is Started Early in Puberty

Introduction and Purpose: Polycystic Ovary Syndrome (PCOS) is recognized as the most common endocrine disorder in women of reproductive age. Notably, PCOS women with hyperandrogenism have a pronounced increased risk for cardio-metabolic comorbidities compared with healthy individuals. Bile acids are endocrine signaling molecules that modulate hepatic lipid, glucose, and energy metabolism by aiding in absorption of lipids. Alteration of bile acid homeostasis affects overall metabolic homeostasis and contributes to pathogenesis of an array of metabolic diseases, although the molecular mechanisms of this have not been studied in PCOS. Methods: Four-week old C57BL/6N female mice were implanted subcutaneously with dihydrotestosterone (DHT, 8.0 mg) or vehicle silastic tubes (n=8/grp). Weekly body weight, food intake, and body composition was assessed. Fasting serum was obtained and the oral glucose tolerance test (OGTT) was performed in the last week of treatment. Animals were euthanized on treatment day 90 and livers were harvested. Expression levels of mRNA were assessed using RT-qPCR. Results: DHT treated females had significantly higher liver mass (1,387 ± 51 vs 1,197 ± 29 g, p<0.05), increased lean mass (21.25 ± 0.27 vs 19.58 ± 0.23 g, p<0.05) and increased fat mass (4.83 ± 0.47 vs 3.59 ± 0.36 g, p<0.05) compared to the vehicle counterparts. These hyperandrogenemic females additionally showed altered glucose homeostasis, having increased fasting glucose (201.10 ± 11.11 vs 152.80 ± 9.23 mg/dL, p<0.05) and an increased area under the curve (209.2± 11.0 vs 160.8± 3.5 mg.min/dL, p<0.05) following OGTT. Hepatic expression of both classic (Cyp8b1, 1.4 ± 0.1-fold, p<0.05) and alternative (Cyp7b1, 2.0 ± 0.3-fold, p<0.05) bile acid synthesis cytochrome P450 enzyme genes were significantly upregulated in DHT treated animals. Additionally, expression of sulfotransferase Sult2a2 was completely abolished in DHT treated animals compared with vehicle animals, indicating the possibility of androgen regulation of the sulfonation of bile acids marked for elimination. Liver expression of both the bile acid receptor G-protein coupled bile acid receptor 1 and the androgen receptor were both significantly downregulated (Gpbar1: 0.68 ± 0.08-fold, AR: 0.46 ± 0.04-fold, p<0.05) in DHT treated animals. Conclusions: Bile acid synthesis, transport, and elimination are tightly controlled processes in the liver to maintain a constant bile acid pool and limit reabsorption. Together, our results highlight the potential role of androgens in DHT-treated female mice in the dysregulation of bile acid homeostasis and its potential contribution to influence metabolic dysfunction. (Supported by NIH grants NIGMS P20GM-121334 to LLYC and DGR, and NIH NIDDK R21DK-113500 to DGR and the Mississippi Center of Excellence in Perinatal Research.)?? 

Hepatic Dysregulation of Bile Acid Homeostasis in Hyperandrogenemic Female Mouse Model of Polycystic Ovary Syndrome

Introduction: During the 22-week core LINC 2 study, the oral 11β-hydroxylase inhibitor osilodrostat normalized mean urinary free cortisol (mUFC) in 79% (15/19) of patients with Cushing’s disease. This report describes long-term LINC 2 efficacy and safety results following an optional extension. Methods: Patients receiving clinical benefit at week 22 could enter the extension (that ran until Oct 22, 2019), continuing the same osilodrostat dose; dose adjustments were permitted based on efficacy and safety. Response rate (mUFC ≤ULN [controlled] or mUFC >ULN but ≥50% decrease from baseline [BL; partially controlled]) was assessed over time. Efficacy/safety were assessed for all patients from core BL until study end. Results: Of 19 enrolled patients (female:male 14:5; mean [SD] age 36.8 years [8.4]), 16 entered the optional extension and 8 of them remained on treatment until study end. Median (range) osilodrostat exposure was 282 weeks (2-351). Mean mUFC decreased from BL (9.9 x ULN) to ≤ULN by week 4 and remained stable throughout the study. All 19 patients achieved mUFC ≤ULN at least once during the study. At each assessment up to month 70 of the extension phase, 50-88% of ongoing patients were controlled, and up to 18% were partially controlled. Mean percentage change in clinical signs from BL (mean [SD]) to last assessment were: fasting plasma glucose, -10.8% (22.1) (from BL: 105.6 mg/dL [49.2]); HbA1c, -2.1% (9.0) (from BL: 5.7% [0.7]); systolic BP, -3.3% (12.6) (from BL: 132.6 mmHg [11.6]); diastolic BP, -2.0% (10.4) (from BL: 85.0 mmHg [6.5]); BMI, -5.9% (8.8) (from BL: 30.7 kg/m2 [7.0]). Overall, 9 patients discontinued treatment (n=2 core and n=7 extension), mostly because of AEs or no longer requiring treatment (n=3 each). The most common AEs during the entire treatment period were nausea (n=10), adrenal insufficiency, and headache (both n=9). AEs related to hypocortisolism and adrenal hormone precursor accumulation occurred in 11 (mostly adrenal insufficiency, n=9) and 12 patients (mostly hypertension, n=4), respectively; most were grade 1/2 and managed with dose adjustment/interruption and/or concomitant medication. Mean (SD) plasma ACTH increased from 1.8 x ULN (0.9) at BL to 7.1 x ULN (12.3) at week 22 and 6.9 x ULN (12.6) at last assessment. Mean (SD) 11-deoxycortisol increased from 1.2 x ULN (1.3) at BL to 13.6 x ULN (12.2) at week 22 and 3.6 x ULN (4.2) at last assessment. In females, mean (SD) testosterone increased from 0.8 x ULN (0.4) at BL to 2.4 x ULN (2.1) at week 22 and 1.0 x ULN (0.9) at last assessment. Two patients, both female, reported an AE of hirsutism.?Conclusions: Rapid reductions in mUFC were sustained for up to 6 years of osilodrostat treatment and were accompanied by improvements in clinical signs of hypercortisolism. Osilodrostat was well tolerated, with no new safety signals during long-term treatment.?? 

Long-Term Control of Urinary Free Cortisol with Osilodrostat in Patients with Cushing's Disease: Final Results from the LINC 2 Study

Depot injection formulations of peptide somatostatin receptor ligands (SRLs) are routinely used to treat acromegaly and neuroendocrine tumors (NETs). Paltusotine (CRN00808), an orally administered small molecule nonpeptide selective somatostatin receptor 2 (SST2) agonist has been shown to maintain GH and IGF-1 levels in acromegaly patients previously on depot SRLs (ACROBAT Edge NCT03789656). In this study, a capsule formulation was used, which did not exhibit dose proportional pharmacokinetics (PK) at doses >40 mg, required a 2-hour post-dose fast in overnight fasted patients, and had the potential for reduced bioavailability when taken with proton-pump inhibitors (PPI). A spray-dried dispersion (SDD) tablet formulation was developed with improved solubility in the physiological pH range and its performance was evaluated in healthy volunteers. Male and female healthy volunteers who met inclusion/exclusion criteria were enrolled in a single-center Phase 1 study (ANZCTR registration ACTRN12619001562167). A Cohort of 12 subjects was administered a single dose of paltusotine in a four-period cross-over design. Periods 1 and 2 assessed the effect of lansoprazole (a PPI) on PK of 20 mg dose of paltusotine SDD tablets. In Period 3, 20 mg dose of paltusotine SDD tablets was co-administered with a high fat, high-calorie meal. In Period 4, a 60 mg dose of paltusotine SDD tablets was administered to assess dose proportionality. In a separate cohort of subjects (n=12; also, a 4-period cross-over design), the relative bioavailability of capsules and SDD tablets was assessed, and the effect of food administration 0.5, 1, and 2 hour post-dose was evaluated. Subsequently, in another cohort of 12 subjects (a 3-period cross-over design), dose proportionality of the SDD tablets was evaluated at 40 mg and 80 mg dose with a 1-hour post-dose fast. A 4 hours post-dose fast was also assessed for the 80 mg dose. Pharmacokinetics and safety of paltusotine were evaluated. Paltusotine was generally well tolerated in this study. SDD tablets exhibited dose proportional increase in total systemic exposure (AUC) up to a dose of 80 mg. Healthy volunteers pretreated with the PPI, lansoprazole (15 mg bid for 3 days), and co-administered with paltusotine SDD tablets exhibited a small decrease (approximately 25%) in systemic exposure to paltusotine compared with the same subjects that had washed out from the PPI-pretreatment. SDD tablets exhibited significant reduction in systemic exposure when co-administered with a high-fat, high-calorie meal. However, the SDD formulation was less sensitive to timing of post-dose food administration compared to the capsule formulation. These data suggest that the SDD tablet formulation of paltusotine improves flexibility in dose administration, can be co-administered with PPIs and other agents that increase stomach pH, and reduces the post-dose fasting requirement.

Pharmacokinetics and Safety of an Improved Oral Formulation of Paltusotine, a Selective, Nonpeptide Somatostatin Receptor 2 (SST2) Agonist for the Treatment of Acromegaly

Cushing disease (CD) is a serious endocrine disorder that is most often caused by an ACTH-secreting pituitary adenoma. Patients can be treated medically when surgery is not an option or was unsuccessful. However, currently used pituitary-targeting drugs are effective in only 40% of patients. To efficiently identify new pituitary-targeting treatment options, we need an in vitro system that closely mimics in vivo conditions. We therefore aimed to establish organoid cultures of normal anterior pituitary and corticotroph adenomas. Organoids or tumoroids are miniature three-dimensional (3D) structures grown from stem cells, that closely resemble the organ or tumor they originate from. Because CD is a thousand times more prevalent in dogs than in humans, and hypophysectomy is the treatment of choice, we used canine tissues. Normal anterior pituitary glands were collected from three healthy dogs that were euthanized for reasons unrelated to the current study. Corticotroph adenomas were collected from six dogs that underwent transsphenoidal hypophysectomy at our University Clinic. The dogs were diagnosed with CD based on clinical signs, endocrine testing, and CT scan imaging. Normal anterior pituitary and corticotroph adenoma cells were cultured in a 3D matrix (basement membrane extract) with anterior pituitary organoid medium containing specific growth factors and ligands, which was refreshed twice a week. The organoids and tumoroids were characterized with histopathology and RT-qPCR. Structures resembling organoids or tumoroids grew from all nine samples (3 normal, 6 adenoma) that were put in culture. Both cystic and dense structures were observed. The organoids and tumoroids expanded rapidly, and could be passaged once every week. The organoids and tumoroids were successfully cultured up until passage number 10, and were then frozen down. Histopathology showed that the organoid or tumoroid cells morphologically resembled healthy anterior pituitary or corticotroph adenoma cells. All organoids cultures expressed mRNA of pituitary stem cell markers SOX2 and SOX9. This study shows that corticotroph adenomas can be cultured as tumoroids in vitro, something not previously published in any species. Based on the many opportunities in organoid culture (e.g., high-throughput drug screenings, gene editing, studying developmental processes), we expect that this in vitro model will pave the way to efficiently and reliably identify new treatment options for CD. Not only for humans, but also for our best friends: dogs.?? 

Canine Pituitary Organoids as 3D in vitro model for Cushing Disease

Background: Cushing’s disease (CD) is caused by high levels of blood cortisol resulting from excess secretion of ACTH from a corticotroph adenoma in the anterior pituitary gland. Clinical features include hypertension, diabetes, osteoporosis, and depression. If untreated CD has an increased mortality of five-fold owing to cardiovascular comorbidities, stroke or raised vulnerability to infection. Transsphenoidal surgery is considered the first-line treatment but remission is achieved in only 65% of cases and the relapse rate is high. Furthermore, medical treatments are often accompanied by unpleasant side-effects. Antisense therapy is a technique for suppressing gene expression at the level of translation using antisense oligonucleotides (ASOs) against the mRNA of interest. Aims: To investigate antisense therapy as a treatment for CD by targeting ASOs against ACTH-encoding POMC mRNA thereby reducing secretion of the hormone. To transfect mouse AtT20 cells (cells that secrete high levels of ACTH) with ASOs against POMC at varying doses to determine which is the most effective at reducing ACTH secretion. Methods: AtT-20 cells that secrete high levels of ACTH were used as the model system. ASOs were designed to specifically target exon 3 of the POMC gene. Transfection of AtT-20 was carried out using Lipofectamine. FACS was used to determine transfection efficiency. ACTH levels secreted by AtT-20 cells were determined by immunoassay. Statistical analysis was done using ANOVA with P values < 0.05 considered significant. Results: ASOs that targeted POMC exon 3 (ASO-2 and ASO-3) were transfected into AtT-20 cells at 10 and 100 nM. Control ASOs were ASO-1 (matched to POMC sense strand) and ASO-4 (a scrambled version of ASO-3). Experiments included untreated AtT-20 cells and AtT-20 cells treated with transfection reagent or ASOs alone. The results of six experiments indicated that ACTH secretion from AtT-20 cells was reduced after transfection with ASO-2 and ASO-3 at 100 nM (ANOVA, P = < 0.05) and 10 nM (ANOVA, P < 0.05) when compared with untreated AtT20 cells. ASO-1 and ASO-4 had no effect on ACTH secretion by AtT-20 cells (ANOVA, P > 0.05). Conclusions: Initial experiments have shown that ASOs against POMC can reduce ACTH secretion from AtT-20 cells and may be useful as a novel therapy for CD.?? 

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