Efficacy of Vesicular Monoamine Transporter 2 Inhibition and Synergy with Antipsychotics in Animal Models of Schizophrenia.

Antipsychotic medications function by blocking postsynaptic dopaminergic signaling in the central nervous system. Dopamine transmission can also be modulated presynaptically by inhibitors of vesicular monoamine transporter 2 (VMAT2), which inhibit loading of dopamine into presynaptic vesicles. Here we investigated the combination of these mechanisms in animal models of schizophrenia and weight gain (a primary side effect of antipsychotics). When dosed alone, the highly selective VMAT2 inhibitor RRR-dihydrotetrabenazine (RRR-DHTBZ, also known as [+]-α-HTBZ) elicited efficacy comparable to conventional antipsychotics in prepulse inhibition and conditioned avoidance models without eliciting weight gain. In combination experiments, synergy was observed: subthreshold doses of RRR-DHTBZ and risperidone or olanzapine produced robust efficacy, and in dose response experiments, RRR-DHTBZ increased the antipsychotic potency in the efficacy models but did not affect weight gain. The combinations did not affect plasma compound concentrations. The synergy is consistent with VMAT2 inhibition blocking the counterproductive presynaptic stimulation of dopamine by antipsychotics. These results suggest a therapeutic strategy of adding a VMAT2 inhibitor to lower the antipsychotic dose and reduce the side effect burden of the antipsychotic while maintaining and potentially enhancing its therapeutic effects. SIGNIFICANCE STATEMENT: Antipsychotics are often necessary and life-changing medications that reduce psychotic symptoms; however, these benefits come with a high side effect burden. This study shows that combining these postsynaptic dopaminergic modulators with a presynaptic dopamine modulator (vesicular monoamine transporter 2 [VMAT2] inhibitor) potentiates efficacy synergistically in animal models of schizophrenia without potentiating weight gain. Our data suggest that adding a VMAT2 inhibitor may be a viable therapeutic strategy for reducing antipsychotic side effects by lowering antipsychotic dose while maintaining therapeutic efficacy.

Discovery of 4-(3-aminopyrrolidinyl)-3-aryl-5-(benzimidazol-2-yl)-pyridines as potent and selective SST5 agonists for the treatment of congenital hyperinsulinism.

SST5 receptor activation potently inhibits insulin secretion from pancreatic ß-cells, and an orally available nonpeptide selective SST5 agonist may be used to effectively manage the blood glucose levels of congenital HI patients to avoid severe hypoglycemia. Our medicinal chemistry efforts have led to the discovery of 4-(3-aminopyrrolidinyl)-3-aryl-5-(benzimidazol-2-yl)-pyridine analogs as potent SST5 agonists. This class of molecules exhibits excellent human SST5 potency and selectivity against SST1, SST2, SST3 and SST4 receptors. Leading compound 3-{4-[(3S)-3-aminopyrrolidin-1-yl]-5-(4-methyl-1H-1,3-benzodiazol-2-yl)pyridin-3-yl-5-fluorobenzonitrile (28, CRN02481) showed limited off-target activity and good pharmacokinetic profiles in both male Sprague Dawley rats and Beagle dogs to advance into further preclinical evaluations.

Paltusotine, a novel oral once-daily nonpeptide SST2 receptor agonist, suppresses GH and IGF-1 in healthy volunteers.

PURPOSE: Evaluate the pharmacodynamics, pharmacokinetics, and safety of paltusotine, an orally bioavailable, nonpeptide, somatostatin receptor subtype 2 (SST2) agonist being developed for the treatment of acromegaly and neuroendocrine tumors. METHODS: A randomized, double-blind, placebo-controlled, single center, single and multiple ascending dose phase 1 study was conducted in healthy male volunteers who received (i) single-dose of oral paltusotine 1.25, 2.5, 5, 10, and 20 mg (solution); and 40 and 60 mg (capsules) or (ii) multiple-dose oral paltusotine capsules once daily 5 mg (× 7 days), 10, 20, and 30 mg (× 10 days). Main outcome measures were pharmacodynamics (changes in growth hormone-releasing hormone [GHRH] stimulated growth hormone [GH] and insulin-like growth factor 1 [IGF-1]), pharmacokinetics, safety, and tolerability. RESULTS: Single-dose cohorts: n = 41 active, n = 14 placebo. Multiple-dose cohorts: n = 24 active, n = 12 placebo. Paltusotine was well tolerated, orally bioavailable, associated with increased plasma concentrations to doses up to 40 mg, and was eliminated with a half-life of approximately 30 h. Single-dose paltusotine 1.25 to 20 mg suppressed GHRH-stimulated GH secretion by 44% to 93% compared to 15% with placebo. Multiple-dose paltusotine 5 to 30 mg administered once daily for 10 days suppressed IGF-1 by 19% to 37% compared to an increase of 2.4% with placebo. CONCLUSIONS: Paltusotine suppresses GH and IGF-1 in a dose-dependent fashion, with a safety profile similar to currently approved SST2 receptor ligands. Paltusotine is a promising once-daily oral nonpeptide SST2 agonist candidate for managing acromegaly and neuroendocrine tumors. TRIAL REGISTRATION: NCT03276858, registered September 8, 2017, retrospectively registered.

A hybrid IA-LC-MS/MS method for adrenocorticotropic hormone(1-24) to support interpretation of low-dose cosyntropin-stimulation test.

Adrenocorticotropic hormone 1-24 (ACTH[1-24]) has a similar effect as endogenous ACTH(1-39) to generate cortisol by targeting the MC2R receptor on the adrenal gland. A new investigational ACTH receptor antagonist drug is being developed to treat diseases of ACTH excess (e.g., Cushing's disease) by binding to the MC2R receptor. Administration of ACTH(1-24) was used in a Phase I clinical study to assess the ability of this drug candidate to suppress the cortisol response to ACTH stimulation. A hybrid immunoaffinity-LCMS assay measuring ACTH(1-24) with a concentration range of 10 to 400 pg/ml was developed to support the study. Consistent and acceptable A&P results were achieved. The assay development and qualification will be discussed.

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Discovery of CRN04894: A Novel Potent Selective MC2R Antagonist.

A novel class of nonpeptide melanocortin type 2 receptor (MC2R) antagonists was discovered through modification of known nonpeptide MC4R ligands. Structure-activity relationship (SAR) studies led to the discovery of 17h (CRN04894), a highly potent and subtype-selective first-in-class MC2R antagonist, which demonstrated remarkable efficacy in a rat model of adrenocorticotrophic hormone (ACTH)-stimulated corticosterone secretion. Oral administration of 17h suppressed ACTH-stimulated corticosterone secretion in a dose-dependent manner at doses ≥3 mg/kg. With its satisfactory pharmaceutical properties, 17h was advanced to Phase 1 human clinical trials in healthy volunteers with the goal of moving into patient trials to evaluate CRN04894 for the treatment of ACTH-dependent diseases, including congenital adrenal hyperplasia (CAH) and Cushing's disease (CD).

ACROBAT Edge: Safety and Efficacy of Switching Injected SRLs to Oral Paltusotine in Patients With Acromegaly.

CONTEXT: Paltusotine is a once-daily, oral, nonpeptide small-molecule somatostatin receptor type 2 (SST2) agonist in clinical development for treatment of acromegaly. OBJECTIVE: This work aimed to evaluate change in insulin-like growth factor I (IGF-I) levels in patients switched from octreotide long-acting release or lanreotide depot monotherapy to paltusotine. METHODS: A phase 2, open-label, prospective, multicenter, multinational, nonrandomized, single-arm exploratory study was conducted in which dosage uptitrations were performed in a double-blinded manner. At 26 global sites, patients with acromegaly switched to paltusotine from injected somatostatin receptor ligand (SRL)-based therapy. Patients received 13-week treatment with once-daily oral paltusotine (10-40 mg/d). The primary end point was change from baseline to week 13 in IGF-I for patients who switched from long-acting octreotide or lanreotide depot monotherapy to paltusotine (group 1). All patients underwent a 4-week paltusotine washout at end of treatment period (wk 13-17). IGF-I, growth hormone (GH), patient-reported outcome, and safety data were collected. RESULTS: Forty-seven patients enrolled. In group 1 (n = 25), IGF-I and GH showed no significant change between SRL baseline and end of paltusotine treatment at week 13 (median change in IGF-I = -0.03×upper limit of normal [ULN]; P = .6285; GH = -0.05 ng/mL; P = .6285). IGF-I and GH rose significantly in the 4 weeks after withdrawing paltusotine (median change in IGF-I = 0.55×ULN; P < .0001 [median increase 39%]; GH = 0.72 ng/mL; P < .0001 [109.1% increase]). No patients discontinued because of adverse events (AE); no treatment-related serious AEs were reported. CONCLUSION: These results suggest once-daily oral paltusotine was effective in maintaining IGF-I values in patients with acromegaly who switched from injected SRLs. Paltusotine was well tolerated with a safety profile consistent with other SRLs.

Discovery of Paltusotine (CRN00808), a Potent, Selective, and Orally Bioavailable Non-peptide SST2 Agonist.

The discovery of a novel 4-(4-aminopiperidinyl)-3,6-diarylquinoline series of potent SST2 agonists is described. This class of molecules exhibit excellent selectivity over SST1, SST3, SST4, and SST5 receptors. The compound 3-[4-(4-aminopiperidin-1-yl)-3-(3,5-difluorophenyl)quinolin-6-yl]-2-hydroxybenzonitrile (22, paltusotine, formerly known as CRN00808) showed no direct inhibition of major cytochrome P450 enzymes or the hERG ion channel and had sufficient exposure in rats and excellent exposure in dogs upon oral dosing. In pharmacodynamic studies, compound 22 dose-dependently suppressed growth hormone (GH) secretion induced by an exogenous growth-hormone-releasing hormone (GHRH) challenge in both male and female rats following a single oral dose and suppressed IGF-1 levels with repeated oral administration in both rats and dogs. To the best of our knowledge, compound 22 is the first non-peptide SST2 agonist to advance to human clinical trials and is currently in Phase 3 trials in acromegaly patients and a Phase 2 trial in neuroendocrine tumor patients suffering from carcinoid syndrome.

2022 White Paper on Recent Issues in Bioanalysis: Enzyme Assay Validation, BAV for Primary End Points, Vaccine Functional Assays, Cytometry in Tissue, LBA in Rare Matrices, Complex NAb Assays, Spectral Cytometry, Endogenous Analytes, Extracellular Vesicles Part 2 - Recommendations on Biomarkers/CDx, Flow Cytometry, Ligand-Binding Assays Development & Validation; Emerging Technologies; Critical Reagents Deep Characterization.

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on LBA, Biomarkers/CDx and Cytometry. Part 1 (Mass Spectrometry and ICH M10) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 16 and 14 (2023), respectively.

Single Dose and Repeat Once-Daily Dose Safety, Tolerability and Pharmacokinetics of Valbenazine in Healthy Male Subjects.

Valbenazine (VBZ) is a vesicular monoamine transporter 2 (VMAT2) inhibitor approved for the treatment of tardive dyskinesia. The safety, tolerability and pharmacokinetics of VBZ following single and repeat once-daily (QD) dosing were evaluated in 2 randomized, single-center, double-blind studies in healthy male subjects. In the first study, 2 cohorts of 8 subjects were administered single doses (SD) of placebo (PBO; N = 2/period) or VBZ (N = 6/period; 1, 2, 5, or 12.5 mg for Cohort 1 and 12.5, 25, 50, or 75 mg for Cohort 2) using a sequential escalation scheme. The second study consisted of 2 phases. In the initial phase, subjects were administered SD PBO (N = 2/period) or VBZ (N = 6/period; 75, 100, 125 or 150 mg) with sequential escalation. In the second phase, subjects received PBO, or 50 or 100 mg VBZ (N = 4:8:8) QD for 8 days (Cohort 1) or PBO or 50 mg VBZ (N = 6:6) QD for 8 days (Cohort 2). For both studies, plasma concentrations of VBZ and its active metabolite, NBI-98782, were determined. Safety was assessed throughout the studies. PK parameters were determined using noncompartmental methods. In both studies, VBZ was rapidly absorbed with peak concentrations typically observed within 1.5 hours. Peak NBI-98782 concentrations were typically observed at 4.0 to 9.0 hours. Terminal elimination half-life for both VBZ and NBI-98782 was ~20 hours. Across the 1 to 150 mg SD range evaluated across the studies, VBZ and NBI-98782 Cmax and AUC increased dose-proportionally from 50 to 150 mg and more than dose-proportionally from 1 to 50 mg. QD VBZ and NBI-98782 Cmax and AUC parameters were also dose-proportional between the 50 and 100 mg doses. Steady-state for both analytes appeared to be achieved by Day 8. The accumulation index was ~1.5 for VBZ and ~2.5 for NBI-98782. Peak to trough fluctuation was approximately 250% for VBZ and 70% for NBI-98782. Across both studies, NBI-98782 exposure was approximately 20%-30% that of VBZ based on molar ratios. In the first study, the maximum-tolerated dose was not achieved; headache (2 events) was the only treatment-emergent adverse event (TEAE) reported by more than one subject. In the second study, fatigue (4 events) was the only TEAE reported by more than one subject following SD VBZ. Following QD VBZ, the TEAEs of fatigue, insomnia, disturbance in attention, and nervousness were dose-dependent; the latter three TEAEs were considered dose-limiting. Subject withdrawals due to TEAEs were 1 each for PBO and 50 mg VBZ QD, and 3 for 100 mg VBZ QD. Clinically relevant effects on laboratory parameters, vital signs or ECGs were limited to increased CPK (SD: 1 each for 5 mg VBZ and PBO), ALT (QD: 1 each for 50 and 100 mg VBZ and PBO), and triglycerides (QD: 1 each for 50 mg VBZ and PBO). VBZ has an acceptable safety profile and predictable pharmacokinetics that result in stable concentrations of active compounds with low peak-to-trough fluctuation following once-daily dosing.

Single-Dose Study of a Corticotropin-Releasing Factor Receptor-1 Antagonist in Women With 21-Hydroxylase Deficiency.

CONTEXT: Treatment of 21-hydroxylase deficiency (21OHD) is difficult to optimize. Normalization of excessive ACTH and adrenal steroid production commonly requires supraphysiologic doses of glucocorticoids. OBJECTIVES: We evaluated the safety and tolerability of the selective corticotropin releasing factor type 1 (CRF1) receptor antagonist NBI-77860 in women with classic 21OHD and tested the hypothesis that CRF1 receptor blockade decreases early-morning ACTH and 17α-hydroxyprogesterone (17OHP) in these patients. PARTICIPANTS: The study enrolled eight classic 21OHD females, ages 18-58 years, seen at a single tertiary referral university setting. DESIGN: This was a phase Ib, single-blind, placebo-controlled, fixed-sequence, single-dose trial. During three treatment periods separated by 3-week washout intervals, patients sequentially received placebo, NBI-77860 300 mg, and NBI-77860 600 mg at 10 pm; glucocorticoid therapy was withheld for 20 hours. We evaluated ACTH, 17OHP, androstenedione, and testosterone as well as NBI-77860 pharmacokinetics over 24 hours. RESULTS: Dose-dependent reductions of ACTH and/or 17OHP were observed in six of eight subjects. Relative to placebo, NBI-77860 led to an ACTH and 17OHP reduction by a mean of 43% and 0.7% for the 300 mg dose, respectively, and by 41% and 27% for the 600 mg dose, respectively. Both NBI-77860 doses were well tolerated. CONCLUSION: The meaningful reductions in ACTH and 17OHP following NBI-77860 dosing in 21OHD patients demonstrate target engagement and proof of principle in this disorder. These promising data provide a rationale for additional investigations of CRF1 receptor antagonists added to physiologic doses of hydrocortisone and fludrocortisone acetate for the treatment of classic 21OHD.

Vascular effects of urocortins 2 and 3 in healthy volunteers.

BACKGROUND: Urocortin 2 and urocortin 3 are endogenous peptides with an emerging role in cardiovascular pathophysiology. We assessed their pharmacodynamic profile and examined the role of the endothelium in mediating their vasomotor effects in vivo in man. METHODS AND RESULTS: Eighteen healthy male volunteers (23±4 years) were recruited into a series of double-blind, randomized crossover studies using bilateral forearm venous occlusion plethysmography during intra-arterial urocortin 2 (3.6 to 120 pmol/min), urocortin 3 (1.2 to 36 nmol/min), and substance P (2 to 8 pmol/min) in the presence or absence of inhibitors of cyclooxygenase (aspirin), cytochrome P450 metabolites of arachidonic acid (fluconazole), and nitric oxide synthase (L-NMMA). Urocortins 2 and 3 evoked arterial vasodilatation (P<0.0001) without tachyphylaxis but with a slow onset and offset of action. Inhibition of nitric oxide synthase with L-NMMA reduced vasodilatation to substance P and urocortin 2 (P≤0.001 for both) but had little effect on urocortin 3 (P>0.05). Neither aspirin nor fluconazole affected vasodilatation induced by any of the infusions (P>0.05 for all). In the presence of all 3 inhibitors, urocortin 2- and urocortin 3-induced vasodilatation was attenuated (P<0.001 for all) to a greater extent than with L-NMMA alone (P≤0.005). CONCLUSIONS: Urocortins 2 and 3 cause potent and prolonged arterial vasodilatation without tachyphylaxis. These vasomotor responses are at least partly mediated by endothelial nitric oxide and cytochrome P450 metabolites of arachidonic acid. The role of urocortins 2 and 3 remains to be explored in the setting of human heart failure, but they have the potential to have major therapeutic benefits. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov//. Unique identifier: NCT01096706 and NCT01296607.

Validation and application of a liquid chromatography-tandem mass spectrometric method for the simultaneous determination of testosterone and dihydrotestosterone in rat prostatic tissue using a 96-well format.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to extract and quantify the androgen concentration in the rat prostate. This method introduced a novel 96-well plate format for the extraction and derivatization of testosterone (T) and dihydrotestosterone (DHT) from rat prostatic tissue that greatly simplified the sample preparation procedure. Due to the difficulty to obtain reproducible specimens with non-detectable level of androgen, a matrix-free standard solution was used for method validation. Both T and DHT calibration curves were linear over the calibration range (12.5-2500 pg) with correlation coefficient values greater than 0.9900. The intra-day and inter-day accuracy, reported as %bias, and precision, reported as %CV, of T and DHT were within +/-10%. The lower limit of detection (LLOD) and lower limits of quantification (LLOQ) for both T and DHT were determined to be 5 and 12.5 pg. The validation results demonstrated the selectivity, sensitivity, accuracy, precision, linearity and ruggedness of the method, as well as the suitability of the method for simultaneous detection of T and DHT in rat prostatic tissues. The validated method was successfully applied to determine the physiological T and DHT level in rat prostatic tissues. Similarly to the serum concentration profile pattern, T and DHT intraprostatic levels peaked 2 h after lights-on and decreased after lights-off with DHT level approximately 4-fold greater than T.

A novel on-line solid-phase extraction approach integrated with a monolithic column and tandem mass spectrometry for direct plasma analysis of multiple drugs and metabolites.

An on-line solid-phase extraction liquid chromatography/tandem mass spectrometry (SPE LC/MS/MS) assay using a newly developed SPE column and a monolithic column was developed and validated for direct analysis of plasma samples containing multiple analytes. This assay was developed in an effort to increase bioanalysis throughput and reduce the complexity of on-line SPE LC/MS/MS systems. A simple column-switching configuration that requires only one six-port valve and one HPLC pumping system was employed for on-line plasma sample preparation and subsequent gradient chromatographic separation. The resulting analytical method couples the desired sensitivity with ease of use. The method was found to perform satisfactorily for direct plasma analysis with respect to assay linearity, specificity, sensitivity, precision, accuracy, carryover, and short-term stability of an eight-analyte mixture in plasma. A gradient LC condition was applied to separate the eight analytes that cannot be distinctly differentiated by MS/MS. With a run time for every injection of 2.8 min, a minimum of 300 direct plasma injections were made on one on-line SPE column without noticeable changes in system performance. Due to the ruggedness and simplicity of this system, generic methods can be easily developed and applied to analyze a wide variety of compounds in a high-throughput manner without laborious off-line sample preparation.