United States

Background: Functional Hypothalamic Amenorrhea (FHA) is characterized by suppression of central GnRH drive with secondary anovulation and hypoestrogenemia, hypercortisolism, and low leptin that results from a combination of psychosocial stress and metabolic imbalance due to excess physical activity and/or weight loss. Cardiovascular disease (CVD) is the leading killer of women at all ages and CVD mortality rates are unchanged or increasing in young women aged 35-44 years. We investigated relations between FHA and preclinical CVD measured by endothelial dysfunction in premenopausal women with and without FHA, and recently menopausal women not on hormone therapy.
Methods: We studied 31 women with FHA, 29 eumenorrheic controls, and 30 recently menopausal women not on hormone therapy. FHA was defined as amenorrhea ≥3 consecutive months, estradiol &lt;50 pg/mL, FSH &lt;10 mIU/L, and LH &lt;10 mIU/L, and exclusion of other etiologies including polycystic ovary syndrome, prolactinoma, thyroid dysfunction, and pregnancy. Eumenorrheic controls had self-reported monthly menstrual cycles, were not on hormones, with a day 22-24 progesterone level &gt;3 ng/ml to confirm ovulation. Preclinical CVD testing for eumenorrheic controls took place days 3-5 of menses. Recently menopausal women had natural menopause within 3 years, FSH &gt;30, and not on hormone therapy. Preclinical CVD was measured using Endo PAT 2000 (Itamar® Medical Ltd) to calculate reactive hyperemic index (RHI) stress to rest pulse amplitude. RHI ≤1.67 indicates endothelial dysfunction. Statistical analysis included analysis of variance for normal distributed variables and Kruskal Wallis test for non-parametric variables.
Results: The mean age, BMI and estradiol of FHA, no FHA and recent menopause was 26.4 ± 6.2 y, 30.3 ± 3.7 and 53.3 ± 2.6, respectively (p&lt;0.0001); BMI 21.6 ± 6.2 kg/m2, 21.8 ± 2.0, and 23.5 ± 4.2 (p=0.06); and estradiol 29.7±18.2, 46.4 ± 15.7, and 10.9 ± 14.4 pg/ml (p&lt;0.0001), respectively. The median months of FHA amenorrhea was 10.8 months. There were no differences in testosterone (p=0.48), however FHA women had lower leptin (p=0.012) and higher cortisol (p=0.0005) compared to both no FHA and recently menopausal women. Preclinical CVD measured by RHI was significantly lower in FHA compared to no FHA and recently menopausal women (1.8 + 0.5 vs 2.2 + 0.5 vs 2.2 +0.6;p=0.007, respectively). Further 35% of FHA women had RHI ≤1.67 indicating endothelial dysfunction.
Conclusion: Our results demonstrate endothelial dysfunction in young women with FHA women as compared to eumenorrheic women and older recently menopausal women not on hormone therapy. Our findings suggest that FHA may be an important contributor to CVD mortality in younger women and that preclinical CVD in FHA is not likely due to hypoestrogenemia alone. Future studies will focus on identifying potential treatment targets for CVD prevention.

ENDO 2022

Functional Hypothalamic Amenorrhea and Preclinical Cardiovascular Disease

**Program**
ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

This year’s education program features:
more than 100 in-person and virtual sessions with another 70+ in-person only sessions;
six plenary sessions including plenaries on Envisioning Future Therapies for Type 1 Diabetes and Addressing Challenges in Fertility and Reproduction;
over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

**What Programming Is Included in My Virtual Meeting Registration?**
As you build your schedule during the conference, you can select the sessions that are tagged with 'Livestreamed', so you can view virtually. These sessions are being livestreamed, so they are available in real time to our virtual audience. In addition, our in-person and livestreamed sessions will be recorded, so you can access them on demand after 72 hours. We will have over 200 hours available on demand for Standard and Premium pass registrants.

Product theaters and the Communications and Career Center will also include hybrid content delivery.

**Plenaries**
Spanning the spectrum of science, clinical care, and social implications, an outstanding slate of world-renowned speakers will showcase the most cutting-edge advances on research and medicine.

**Symposia**
Our ENDO 2022 lineup of sessions offers a unique opportunity to dive into topics from nearly every major therapeutic area of endocrinology and hormone science. Listen, learn, and be inspired by world-class faculty who are at the forefront of scientific discovery and high-quality health care.

**Basic Science Pathways**

These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
Diabetes and Metabolism
Neuroendocrinology
Nuclear Receptors and Signaling
Reproductive Endocrinology

**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

Background: Functional Hypothalamic Amenorrhea (FHA) is characterized by suppression of central GnRH drive with secondary anovulation and hypoestrogenemia, hypercortisolism, and low leptin that results from a combination of psychosocial stress and metabolic imbalance due to excess physical activity and/or weight loss. Cardiovascular disease (CVD) is the leading killer of women at all ages and CVD mortality rates are unchanged or increasing in young women aged 35-44 years. We investigated relations between FHA and preclinical CVD measured by endothelial dysfunction in premenopausal women with and without FHA, and recently menopausal women not on hormone therapy.
Methods: We studied 31 women with FHA, 29 eumenorrheic controls, and 30 recently menopausal women not on hormone therapy. FHA was defined as amenorrhea ≥3 consecutive months, estradiol <50 pg/mL, FSH <10 mIU/L, and LH <10 mIU/L, and exclusion of other etiologies including polycystic ovary syndrome, prolactinoma, thyroid dysfunction, and pregnancy. Eumenorrheic controls had self-reported monthly menstrual cycles, were not on hormones, with a day 22-24 progesterone level >3 ng/ml to confirm ovulation. Preclinical CVD testing for eumenorrheic controls took place days 3-5 of menses. Recently menopausal women had natural menopause within 3 years, FSH >30, and not on hormone therapy. Preclinical CVD was measured using Endo PAT 2000 (Itamar® Medical Ltd) to calculate reactive hyperemic index (RHI) stress to rest pulse amplitude. RHI ≤1.67 indicates endothelial dysfunction. Statistical analysis included analysis of variance for normal distributed variables and Kruskal Wallis test for non-parametric variables.
Results: The mean age, BMI and estradiol of FHA, no FHA and recent menopause was 26.4 ± 6.2 y, 30.3 ± 3.7 and 53.3 ± 2.6, respectively (p<0.0001); BMI 21.6 ± 6.2 kg/m2, 21.8 ± 2.0, and 23.5 ± 4.2 (p=0.06); and estradiol 29.7±18.2, 46.4 ± 15.7, and 10.9 ± 14.4 pg/ml (p<0.0001), respectively. The median months of FHA amenorrhea was 10.8 months. There were no differences in testosterone (p=0.48), however FHA women had lower leptin (p=0.012) and higher cortisol (p=0.0005) compared to both no FHA and recently menopausal women. Preclinical CVD measured by RHI was significantly lower in FHA compared to no FHA and recently menopausal women (1.8 + 0.5 vs 2.2 + 0.5 vs 2.2 +0.6;p=0.007, respectively). Further 35% of FHA women had RHI ≤1.67 indicating endothelial dysfunction.
Conclusion: Our results demonstrate endothelial dysfunction in young women with FHA women as compared to eumenorrheic women and older recently menopausal women not on hormone therapy. Our findings suggest that FHA may be an important contributor to CVD mortality in younger women and that preclinical CVD in FHA is not likely due to hypoestrogenemia alone. Future studies will focus on identifying potential treatment targets for CVD prevention.

technical paper

Background: The subtyping of primary aldosteronism (PA) requires interpretation of aldosterone and cortisol concentrations measured during adrenal vein sampling (AVS). The use of intravenous sedation during AVS is not standardized but may affect AVS outcomes.
Purpose: To characterize the impact of sedation on the outcomes of AVS, including cortisol and aldosterone concentrations, cannulation success and rates of lateralisation.
Materials and Methods: This is a retrospective cohort study based at a tertiary health centre. Consecutive patients who underwent AVS both before and after ACTH stimulation between June 2018 and July 2020 were included. AVS was performed without IV sedation in patient cohort 1 (n = 70), with sedation (midazolam and fentanyl) in cohort 2 (n = 34) and both before and after sedation in cohort 3 (n = 14). Comparisons were performed using the nonparametric (Mann Whitney U) test for continuous variables and the Chi square test for categorical variables.
Results. Overall, 118 patients (median age, 53 years; 67 men) were evaluated with no significant demographic differences between the three cohorts. Compared to cohort 1, patients in cohort 2 displayed significantly lower cortisol concentrations in the peripheral vein (median of 141 nmol/L vs 218 nmol/L), left adrenal vein (522 nmol/L vs 1284 nmol/L) and right adrenal vein (577 nmol/L vs 1578 nmol/L) in unstimulated AVS. Fewer patients were assessed to have successful right sided cannulation in cohort 2 (79% vs 93% in cohort 1, p = 0.047).  In cohort 3, cortisol concentrations were markedly lower after sedation, with the decrease being more marked in the adrenal veins compared to peripheral veins, hence leading to lower selectivity indices.  There were no clinically significant changes in aldosterone concentration or rates of lateralisation after sedation.
Conclusions. IV sedation reduces cortisol concentrations during unstimulated-AVS, leading to lower selectivity indices and fewer cannulations judged as successful. Importantly, radiologists are turning to point-of-care rapid cortisol tests to determine if the catheter is 'in' the adrenal vein in real-time.  Adrenal vein samples collected after sedation are more likely to be considered "unsuccessful" using these semi-quantitative assays. The need for IV sedation during AVS should be carefully assessed.

Impact of Intra-Procedural Sedation on Outcomes of Adrenal Vein Sampling

Background: Primary aldosteronism (PA) is broadly divided into unilateral and bilateral forms, and the most reliable subtyping tool available is adrenal vein sampling (AVS). Unilateral PA is generally diagnosed based on AVS lateralization indices (LI=[aldosterone/cortisol] ratio between the dominant and contralateral adrenal vein) ≥4, or even ≥2 when AVS is performed without cosyntopin stimulation. Long-term follow-up data from patients with PA treated with unilateral adrenalectomy have been minimal.
Objective: To assess the rates of residual and recurrent PA after AVS-guided unilateral adrenalectomy.
Methods: We enrolled patients with PA who underwent unilateral adrenalectomy between 09/2017 and 08/2021 in a single tertiary referral center and followed them longitudinally. Demographics, laboratory, imaging, and pathology data were collected. Aldosterone synthase (CYP11B2) immunohistochemistry and next generation sequencing were performed on available formalin-fixed paraffin-embedded adrenal tissue blocks.
Results: During the study period, 80 patients (52 men) with median age 53 (range, 19-76) years underwent unilateral adenectomy, and were followed for a median 80 days (range, 6-653 days). In total 14 (17.5%) patients had evidence of residual PA following surgery, including 3 who had complete biochemical failure, and 1 who had apparent cure of PA immediately post-operatively. All 14 patients had severe PA, with baseline plasma aldosterone concentrations between 23.4 and 496 ng/dL and suppressed renin. Baseline and post-cosyntropin stimulation LIs ranged from 2.7- 187.5 and 1.3-53.7. The LI was >4 only at baseline in 3 cases, and only after cosyntropin stimulation in 2 cases. Histology showed single adenomas, ranging from 0.7-5.3 cm in 7 (50%) patients, and multiple adenomas and/or nodular hyperplasia in 7 (50%) patients. Of the 7 cases analyzed for aldosterone-driver mutations so far, 4 harbored CACNA1D mutations, 2 ATP1A1 mutations, and 1 a KCNJ5 mutation.
Conclusions: Patients with severe PA and frank AVS aldosterone lateralization might have multifocal, bilateral disease. Long-term follow up and targeted medical therapy should be pursued after unilateral adrenalectomy for presumed unilateral PA.

Bilateral Primary Aldosteronism Is Not Excluded by High Adrenal Vein Sampling Lateralization Indices

Context: Accumulating evidence suggests that primary aldosteronism can manifest across a broad phenotypic spectrum and that its origins can at times be detected among normotensive individuals. Herein, we conducted deep-phenotyping studies to interrogate the pathophysiologic continuum of the primary aldosteronism phenotype among normotensive participants.

Methods: Normotensive participants with obesity, metabolic syndrome, and/or high-normal untreated blood pressure underwent dietary sodium loading and restriction protocols (to maximally suppress and stimulate angiotensin II-mediated aldosterone production), dexamethasone suppression and cosyntropin stimulation protocols (to maximally suppress and stimulate ACTH-mediated aldosterone production), and 24h ambulatory blood pressure monitoring. Measurement of aldosterone and 18-hybrid steroids via LC-MS/MS was conducted to quantify the degree of dysregulated CYP11B2 expression. Urinary extracellular vesicles (uEV) were isolated to measure renal tubular protein content associated with mineralocorticoid receptor activation. In order to characterize parameters across the severity spectrum of non-suppressible and renin-independent aldosterone production, participants with suppressed renin activity were categorized by tertile of 24h urinary aldosterone production following oral sodium suppression testing. Repeated measures linear mixed models were used to analyze trends for each parameter across tertiles of autonomous aldosterone production; for ease of presentation, the mean [SD] of the first vs. third tertile are presented for selected parameters.

Results: Following oral sodium loading, greater non-suppressible renin-independent aldosterone production was associated with greater kaliuresis, higher daytime ambulatory systolic blood pressure within the normal range, and a progressive impairment in the ability to suppress production of serum aldosterone (P-trend=0.009), 18-hydroxycorticosterone (9.3 [5.0] vs. 19.6 [6.5] ng/dL, P-trend=0.004), 18-hydroxycortisol (45.0 [25.4] vs. 70.9 [29.9] ng/dL, P-trend=0.03), and 18-oxocortisol (1.2 [0.03] vs. 3.4 [7.2], P-trend=0.01). Similarly, following dexamethasone suppression testing, there was a progressive inability to suppress 18-hydroxycorticosterone (8.3 [5.0] vs. 15.4 [8.2] ng/dL, P-trend=0.004), 18-hydroxycortisol (6.6 [3.8] vs. 10.2 [3.6] ng/dL, P-trend=0.03), and 18-oxocortisol (1.2 [0.01] vs. 1.4 [0.6] ng/dL, P-trend=0.01) in parallel with greater renin-independent aldosterone production. In contrast, following stimulation with cosyntropin, greater non-suppressible and renin-independent aldosterone production was associated with progressively enhanced production of 18-hydroxycorticosterone (77.6 [25.4] vs. 121.1 [31.1] ng/dL, P-trend=0.004), 18-hydroxycortisol (68.5 [28.4] vs. 86.2 [26.3] ng/dL, P-trend=0.03), and 18-oxocortisol (3.2 [2.8] vs. 7.3 [3.5] ng/dL, P-trend=0.01). Excretion of uEV's of pendrin (P=0.002) and aquaporin 2 (P=0.002) all increased with greater non-suppressible aldosterone production.

Conclusions: Dynamic phenotyping studies in normotensive individuals demonstrate a continuum of non-suppressible and renin-independent aldosterone production that parallels higher blood pressure, greater kaliuresis, increased 18-hybrid steroid production indicative of dysregulated CYP11B2 expression, and increased uEV excretion indicative of renal mineralocorticoid receptor activation. This pathophysiologic continuum of normotensive primary aldosteronism provides phenotypic support for histopathology studies showing ectopic CYP11B2 expression within adrenal glands of normotensive humans, and for epidemiologic studies demonstrating renin-independent aldosterone production in normotensive populations increases risk for developing hypertension.

Characterization of the Pathophysiologic Spectrum of Subclinical Primary Aldosteronism in Normotension

Background: Aldosterone-producing adenoma (APA) is a major cause of primary aldosteronism (PA) which is the most frequent form of secondary hypertension (1-2). The pathophysiology of PA is quite complex and still incompletely understood. Substance P (SP) encoded by the TAC1 gene belongs to a family of bioactive peptides named tachykinins. A recently published study indicated that SP, released by subcapsular nerve fibres, stimulates aldosterone production through activation of the neurokinin type 1 receptor (NK1R) in the human adrenal gland (3). The aim of our work was to investigate the presence of SP fibres and the NK1 receptor in a large series of APA in order to assess the potential role of this peptide in the pathophysiology of PA. Methods: APA tissues were studied by molecular and histological approaches. Expression of SP and NK1 receptor was searched for in a series of 51 APA by RT-Q-PCR and immunohistochemistry. Results: Quantitative RT-PCR data indicated that adenomas strongly express TAC1 mRNA. Immunohistochemistry showed the presence of SP-positive nerve fibres in APAs tissues. SP was also detected in a subpopulation of adenomatous cells. Adenomas strongly express mRNA encoding the NK1 receptor. The distribution of the NK1 receptor within APAs is similar to that of aldosterone synthase (CYP11B2), suggesting that SP may regulate aldosterone secretion by APAs. Conclusion: These results suggest that SP and its NK1 receptor may be involved in the pathophysiology of aldosterone hypersecretion by APAs. References: 1. Monticone S, Burrello J, Tizzani D, Bertello C, Viola A, Buffolo F, et al. Prevalence and Clinical Manifestations of Primary Aldosteronism Encountered in Primary Care Practice. J Am Coll Cardiol. 2017;69(14): 1811‑20. 2. Buffolo F, Monticone S, Tetti M, Mulatero P. Primary aldosteronism in the primary care setting. Curr Opin Endocrinol Diabetes Obes. 2018;25(3): 155‑9. 3. Wils J, Duparc C, Cailleux A-F, Lopez A-G, Guiheneuf C, Boutelet I, et al. The neuropeptide substance P regulates aldosterone secretion in human adrenals. Nat Commun. 2020;11(1): 2673.

Expression of the Neuropeptide Substance P and the NK1 Receptor in Aldosterone-Producing Adenomas

Background:
To promote ideal cardiovascular health, the American Heart Association recommends adhering to Life's Simple 7 (LS7)—achieving healthy targets for BMI, physical activity, dietary intake, blood pressure, fasting plasma glucose, and cholesterol, along with smoking abstinence. Retrospective data analyses have shown associations between poorer achievement of LS7 (lower score) and the development of hypertension and cardiovascular disease (CVD). However, less is known about the associations between LS7 and specific biomarkers linked to cardiovascular health.

Hypothesis:
We hypothesized that achievement of LS7 (higher score) would be associated with lower levels of aldosterone and inflammatory markers implicated in CVD in individuals with controlled dietary sodium intake.

Methodology:
The analysis included 379 normotensive and hypertensive individuals (age 18-66 years) from the HyperPATH (International Hypertensive Pathotype) consortium, who were maintained on at least 200 mEq of NaCl daily for one week and had a urinary salt balance of >150 mEq/24 hours at the time of sample collection. We calculated a 14-point summative LS7 score according to participants' baseline data. Based on the range of LS7 score in this population, we classified participants as 'inadequate' (3-6), 'average' (7-10), and 'optimal' (11-14). We then performed regression analyses (adjusted for age, sex, race, and serum creatinine) between the LS7 score groups (inadequate, average, or optimal) and biochemical measures, including levels of aldosterone, C-reactive protein (CRP), and interleukin 6 (IL-6).

Results:
When comparing across all three LS7 score groups, a higher LS7 score group was associated with lower aldosterone levels both in the serum (p=0.005, -0.97 [-1.63, -0.30]) and urine (p=0.003, -2.34 [-4.07, -0.825]). Additionally, a higher LS7 score group was associated with lower renin (p=0.008, -0.13 [-0.22, -0.03]) and a blunted increase in aldosterone with angiotensin-II infusion (p=0.019, -1.66 [-3.04, -0.27]). Serum potassium and serum cortisol were not associated with the LS7 scores. Being in the 'optimal' LS7 score group was associated with lower serum levels of CRP (p=0.001, -0.1 [-0.15, -0.04]) and IL-6 (p=0.026, -0.84 [-1.59, -0.10]). Notably, among the individual LS7 components, achieving target blood pressure and exercise goals most significantly impacted the associations between LS7 score and aldosterone levels.

Conclusions:
Achieving an optimal American Heart Association's LS7 score was associated with a lower activity of the renin-angiotensin-aldosterone system and lower levels of the inflammatory markers CRP and IL-6, which may help to explain previous reports of better cardiovascular outcomes in individuals with a higher LS7 score. These findings offer a possible link between ideal cardiovascular health targets and biomarkers known to play a central role in the development of CVD.

The Association between Life's Simple 7 Score and Three Biomarkers of Cardiovascular Disease: Aldosterone, CRP, and IL-6

Background: Primary aldosteronism (PA) is the most common cause of endocrine-related hypertension.  However, due to its rare incidence in younger patients, the molecular features of young-onset PA have not been well defined.  Recent advances in targeted mutation analysis have identified aldosterone-driver somatic mutations in aldosterone-producing lesions including aldosterone-producing adenomas (APAs) and aldosterone-producing nodules (APNs).
Objective: To determine the histologic and somatic mutation profile in young-onset PA.
Methods: Formalin-fixed paraffin-embedded adrenals from 73 patients with unilateral PA (53 women, 19 men, and one with unknown sex) under 35 years old were analyzed.  Aldosterone synthase (CYP11B2) immunohistochemistry was used to define the histopathologic classification of the aldosterone-producing lesions based on the HISTALDO consensus as well as guide DNA capture. Somatic mutations were identified by direct Sanger sequencing or Ion Torrent-based targeted next-generation sequencing.
Results: We identified 48 APAs, 20 APNs, 2 multiple aldosterone-producing nodules (MAPNs), and 3 non-functioning adenomas (NFAs).  Of the 45 APAs and 18 APNs with successful sequencing, 43 APAs (96%) and 17 (94%) APNs harbored somatic mutations.  The most frequent alterations in APAs were KCNJ5 mutations (35/45, 78%), while CACNA1D mutations were the most common in APNs (8/18, 44%).  The mutation prevalence for KCNJ5 mutations did not vary between men and women. In both MAPNs, multiple CYP11B2-expressing lesions shared an identical somatic KCNJ5 mutation (c.451G>A, p.G151R).  No somatic mutations were detected in the 3 NFAs.
Conclusion: APAs are the most common histologic feature of unilateral young-onset PA.  Somatic KCNJ5 mutations are more commonly seen in APAs in both men and women, while somatic CACNA1D mutations are frequently seen in APNs in these younger patients.

Histopathology and Genetic Causes of Primary Aldosteronism in Young Patients

Background:
Hypoparathyroidism is a rare disease characterized by deficiency of parathyroid hormone (PTH), resulting in abnormal mineral homeostasis. Conventional therapy (active vitamin D and calcium) targets short-term symptoms, but fails to restore normal PTH physiology, and may lead to complications such as nephrolithiasis, nephrocalcinosis, and renal insufficiency. TransCon PTH is an investigational long-acting prodrug of PTH(1-34) dosed once-daily for the treatment of adults with hypoparathyroidism. In the phase 2 PaTH Forward trial of TransCon PTH, 82% of participants achieved independence from conventional therapy at Week 4, a trend observed through Week 26 and 58. Here we report the Week 84 results from PaTH Forward trial.
Methods:
The phase 2 PaTH Forward trial is a multicenter, randomized, double-blind, placebo-controlled, parallel-group, 4-week clinical trial with an open-label extension (OLE) period planned through Week 214. Participants received 4 weeks fixed dose of TransCon PTH (15, 18, or 21 mcg/d) or placebo co-administered with conventional therapy, followed by an OLE period during which TransCon PTH dose was titrated to maintain normocalcemia. Efficacy assessments at Week 84 included levels of 24-hour urine calcium (uCa) and sCa and independence from conventional therapy (defined as taking no active vitamin D and ≤600 mg calcium). After Week 58, a subset of 12 participants had frequent blood and urine sampling over 24 hours for evaluation of pharmacodynamic effect on sCa and uCa level and active PTH pharmacokinetic assessment (PD/PK substudy).
Results:
Fifty-eight of 59 participants continued in the trial through Week 84. The average dose of TransCon PTH at Week 84 was 22.8 mcg/d. Fifty-four of 58 participants (93%) at Week 84 achieved independence from conventional therapy. Baseline mean 24-hour uCa was above normal (428 mg/24-hour) but normalized at Week 26 (173 mg/24-hour), and sustained through Week 58 (144 mg/24-hour), and Week 84 (131 mg/24-hour). Average values for sCa baseline were 8.8 mg/dL and remained in normal range at Weeks 26 (8.9 mg/dL), 58 (8.5 mg/dL) and 84 (8.5 mg/dL). The majority of adverse events (AE) were mild and unrelated to study drug; no serious AEs were reported. No AEs led to discontinuation of the study drug, nor from the trial.
In the PD/PK substudy, mean sCa and uCa levels were stable in the normal range at all time points. Active PTH levels reflected steady physiologic exposure throughout the 24-hour period.
Conclusion:
Week 84 results from the PaTH Forward trial investigating TransCon PTH for adults with hypoparathyroidism showed continued independence from conventional therapy for 93% of participants and maintenance of normal sCa and 24-hour uCa. Results from the PD/PK substudy demonstrated 24-hour stability of sCa and uCa levels and confirm infusion-like PK profile in a population of adults with hypoparathyroidism. TransCon PTH was generally well-tolerated through Week 84.

The PaTH Forward Trial: Efficacy and Safety of TransCon PTH Through Week 84 for Adults With Hypoparathyroidism

Autosomal dominant hypocalcemia type 1 (ADH1), caused by gain-of-function variants in the gene encoding the calcium-sensing receptor (CaSR), is characterized by hypocalcemia, hyperphosphatemia, hypomagnesemia, low parathyroid hormone (PTH), and hypercalciuria. Conventional therapy (calcium and activated Vitamin D) worsens hypercalciuria and can lead to renal morbidity. Calcilytics (negative allosteric modulators of the CaSR) decrease the sensitivity of hyperactive receptors to extracellular calcium and normalize blood and urine abnormalities in ADH1 rodent models. Encaleret is an oral calcilytic under investigation as a potential treatment for ADH1.
Thirteen adults (22-60y) with ADH1 due to 9 unique CASR variants participated in a 3-period, Phase 2b, open-label, dose-ranging study. Conventional therapy was discontinued prior to encaleret initiation. Period 1 (P1) was a 5-day inpatient dose-escalation course (n=6). Period 2 (P2) was a 5-day inpatient course (n=13) in which doses were individually titrated to normalize albumin-corrected blood calcium (cCa) and minimize hypercalciuria and hypophosphatemia. All 13 participants continued into Period 3 (P3), a 24-week outpatient maintenance period. Patients underwent serial 24hr blood and urine sampling in P1, P2, and in P3 at Weeks 8, 16, and 24, with additional outpatient laboratory timepoints.
The mean±SD encaleret dose at the end of P2, Day 5 (P2D5) was 94±64mg BID (range: 10-180 BID); by P3, Week 24 (P3W24), the mean was 78±67mg BID (5-190 BID). Encaleret was well-tolerated with no serious adverse events reported; there were no treatment discontinuations or study withdrawals.
Twenty-four hour mean±SD values from P2D5 (n=13) and P3W24 (n=12) compared to baseline are presented. Baseline PTH was low at 6.3±7.8 pg/mL (nl 10-65) and had normalized by P2D5 (40.5±37.5, p<0.01); this was sustained through P3W24 (31.3±20.8, p<0.01). Likewise, baseline hypocalcemia (cCa=7.1±0.4 mg/dL [nl 8.4-10.2]) corrected to 8.6±0.7 by P2D5 (p<0.01) and remained normal through P3W24 (9.0±0.6, p<0.01). Baseline urinary calcium was 395±216 mg/d (nl <250-300) and decreased to 179±108 by P2D5 (p<0.01) and 189±72 at P3W24 (p<0.05); urinary calcium excretion was normal in 10/12 patients at P3W24. Baseline phosphate decreased from 4.5±1.1 mg/dL (nl 2.3-4.7) to 3.2±0.7 on P2D5 (p<0.01) and was maintained through P3W24 (3.5±0.6, p<0.05). Magnesium increased from low- to mid-normal (baseline 1.7±0.2 mg/dL [nl 1.6-2.6]; P2D5 1.9±0.2 [p<0.01]; P3W24 2.0±0.2, [p<0.01]). 1,25-dihydroxy-Vitamin D increased from 19.5±4.4 pg/mL (nl 20-70) to 32.0±16.7 on P2D5 (p<0.05) and 30.2±14.0 on P3W24 (p<0.05). Bone turnover markers were not different between baseline (n=7, CTX=253±111 pg/mL; P1NP=34±10 mcg/L) and P2D5 (CTX=241±181, p=NS; P1NP=26±12, p=NS) but had increased by P3W24 (CTX=784±686, p<0.01; P1NP=102±87, p<0.01).
In conclusion, this study represents a molecularly targeted, precision medicine approach to the treatment of ADH1. The consistent and sustained results from all periods of this Phase 2 study establish a clinically meaningful efficacy, tolerability, and safety profile for encaleret as a potential treatment of adults with ADH1.

Encaleret (CLTX-305) Restored Mineral Homeostasis in a Phase 2 Study in Autosomal Dominant Hypocalcemia Type 1 (ADH1)

Skeletal development is orchestrated by the interaction of the parathyroid hormone related peptide (PTHrP) with the parathyroid hormone receptor type 1 (PTH1R). Knock-out or severe deficiency of either PTH1R or PTHrP by homozygous loss-of-function mutations results in premature hypertrophy of chondrocytes and accelerated bone mineralization, leading to neonatal death. In contrast, patients with a heterozygous gain-of-function PTH1R mutation (H223R, T410P/R or I458K/R) present with Jansen metaphyseal chondrodysplasia (JMC), characterized by delayed bone mineralization, hypercalcemia, hypercalciuria and hypophosphatemia. Eiken syndrome is also characterized by markedly delayed bone mineralization but lacks the biochemical abnormalities of JMC. Eiken syndrome has been described for three unrelated families, in which there are one of three homozygous PTH1R mutations: R485X, E35K or Y134S. As the functional properties of these PTH1R mutants have not been studied, we assessed their characteristics in cell-based assays.
GS22A cells (HEK293/Glosensor) were transiently transfected to express the WT or a mutant PTH1R. Cell surface expression levels assessed by extracellular HA tag-directed immunofluorescence flow cytometry and normalized to PTH1R-WT (100%) were 50% for PTH1R-R485X, 90% for PTH1R-E35K and 20% for PTH1R-Y134S. Dynamic cAMP measurements via the Glosensor reporter revealed that the basal activities of PTH1R-R485X and PTH1R-E35K were 7-fold and 1.5-fold higher than that of PTH1R-WT (p<0.005), while that of PTH1R-Y134S was comparable to PTH1R-WT. Stimulation with PTH(1-34) or PTHrP(1-36) yielded similar cAMP potencies for the three mutants and PTH1R-WT (EC50s ~ 0.5 nM, p >0.05). Intracellular calcium signaling assessed by changes in FURA2 fluorescence after PTHrP(1-36) (100 nM) addition was more robust with PTH1R-R485X than for PTH1R-WT (AUCs over 2.5 mins. = 606±9 vs 551±10, p=0.0074) and similar to PTH1R-WT with PTH1R-E35K and PTH1R-Y134S (p ≥0.2). Both basal and agonist-activated cAMP signaling by the R485X mutant were reduced by addition of a dTrp¹²-PTHrP(7-36) antagonist/inverse agonist analog.
Fluorescence microscopy of HEK293/βarrestin2YFP (GBR24) cells stimulated with TMRPTH(1-34) revealed a clear association of βarrestin2YFP with PTH1R-WT, as well as with PTH1R-E35K and PTH1R-Y134S in internalized complexes, but no such association with PTH1R-R485X. Nevertheless, over-expression of βarrestin2YFP in GS22A cells markedly reduced basal and ligand-stimulated cAMP generation by PTH1R-R485X, consistent with a residual capacity of PTH1R-485X to bind βarrestin2. Treatment with the endocytosis inhibitor Dyngo4A in GS22A cells transfected with PTH1R-R485X completely blocked TMRPTH(1-34) internalization assessed by fluorescent microscopy, and enhanced ~5-fold the cAMP response potency to PTH(1-34) vs DMSO treatment. In contrast, treatment with Dyngo4A did not increase PTH(1-34) cAMP potency in the PTH1R-WT. Collectively these results suggest that PTH1R-R485X signals via cAMP predominantly from the cell surface.
The data reveal distinct functional properties for the three PTH1R mutants associated with Eiken syndrome and suggest a novel correlation between excessive PTH1R cAMP signaling due to impaired βarrestin-mediated internalization and a delay in ossification.

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