Comparative Effectiveness of Dupilumab and Omalizumab on Asthma Exacerbations and Systemic Corticosteroid Prescriptions: Real-World US ADVANTAGE Study.

BACKGROUND: In the US, dupilumab is approved for moderate-to-severe eosinophilic or oral corticosteroid-dependent asthma, while omalizumab is approved for managing moderate-to-severe allergic asthma uncontrolled by inhaled corticosteroids. However, limited comparative effectiveness data exist for these biologics due to differing patient characteristics and treatment histories. OBJECTIVE: This analysis assessed the real-world effectiveness of dupilumab and omalizumab for asthma among patients in the US. METHODS: In this retrospective observational study, TriNetX Dataworks electronic medical record data were used to identify asthma patients (age: ≥12 years) who initiated (index) dupilumab or omalizumab between November 2018 and September 2020, and who had at least 12 months of pre- and post-index clinical information. Inverse probability of treatment weighting (IPTW) was applied to balance potential confounding in treatment groups. Asthma exacerbation rates and systemic corticosteroid (SCS) prescriptions were compared using a doubly robust negative binomial regression model, adjusting for baseline exacerbation/SCS rates and patient characteristics with ≥10% standardized differences after IPTW. RESULTS: Overall, 2,138 patients in dupilumab and 1,313 in omalizumab treatment groups met all inclusion and exclusion criteria. After weighting, the majority of baseline characteristics were balanced (standard difference <10%) between the two groups. Dupilumab was associated with a 44% lower asthma exacerbation rate (p<0.0001) than omalizumab. Additionally, dupilumab treatment significantly (p<0.05) reduced SCS prescriptions by 28% during the follow-up period compared to omalizumab treatment. CONCLUSION: The US ADVANTAGE real-world study demonstrated a significant reduction in severe asthma exacerbations and SCS prescriptions for patients prescribed dupilumab compared to those prescribed omalizumab during 12 months of follow-up.

Real-world effectiveness of dupilumab versus benralizumab and mepolizumab.

Introduction: In the United States, this real-world study compared the effectiveness of dupilumab, benralizumab, and mepolizumab in reducing exacerbations and systemic corticosteroid (SCS) prescriptions among patients with asthma. Methods: Patients (≥12 years old) who initiated dupilumab, benralizumab, or mepolizumab (index) between November 2018 and September 2020 were identified by using electronic medical record data. Subjects were included if they had ≥ 12 months of data before and after the index date and two or more severe asthma-related exacerbations before the index date. Differences in baseline characteristics were addressed by using inverse probability treatment weighting (IPTW). Pairwise comparisons between dupilumab and benralizumab, or mepolizumab were conducted by using negative binomial regression, adjusting for baseline rates and unbalance characteristics (≥10% standardized differences) after IPTW. Results: Overall, a total of 1737 subjects met all criteria: 825 dupilumab, 461 benralizumab, and 451 mepolizumab initiators. In the postindex period, dupilumab was associated with a 24% and 28% significant reduction in the risk of severe asthma exacerbations versus benralizumab (incidence rate ratio [IRR] 0.76 [95% confidence interval {CI}, 0.67-0.86)] and mepolizumab (IRR 0.72 [95% CI, 0.63-0.82]), respectively. In addition, dupilumab treatment significantly reduced SCS prescriptions by 16% and 25% versus benralizumab and mepolizumab, respectively (p < 0.05). Conclusion: This study represents one of the largest real-world comparisons of biologics (dupilumab, benralizumab, and mepolizumab) for asthma in the United States to date. This analysis shows that the use of dupilumab was associated with a significantly greater reduction in both severe asthma exacerbations and SCS prescriptions compared with benralizumab and mepolizumab.

Urocortin 2 Gene Transfer Improves Heart Function in Aged Mice.

Prevalence of left ventricular (LV) systolic and diastolic dysfunction increases with aging. We previously reported that urocortin 2 (Ucn2) gene transfer increases heart function in mice with heart failure with reduced ejection fraction. Here, we test the hypotheses that (1) Ucn2 gene transfer will increase LV function in aged mice and that (2) Ucn2 gene transfer given in early life will prevent age-related LV dysfunction. Nineteen-month-old (treatment study) and 3-month-old (prevention study) mice received Ucn2 gene transfer or saline. LV function was examined 3-4 months (treatment study) or 20 months (prevention study) after Ucn2 gene transfer or saline injection. In both the treatment and prevention strategies, Ucn2 gene transfer increased ejection fraction, reduced LV volume, increased LV peak -dP/dt and peak +dP/dt, and reduced global longitudinal strain. Ucn2 gene transfer-in both treatment and prevention strategies-was associated with higher levels of LV SERCA2a protein, reduced phosphorylation of LV CaMKIIa, and reduced LV α-skeletal actin mRNA expression (reflecting reduced cardiac stress). In conclusion, Ucn2 gene transfer restores normal cardiac function in mice with age-related LV dysfunction and prevents development of LV dysfunction.

Treatment Patterns and Clinical Outcomes Among Patients With Metastatic Prostate Cancer Harboring Homologous Recombination Repair Mutations.

BACKGROUND: There is currently limited literature assessing the real-world treatment patterns and clinical outcomes of patients with metastatic castration-resistant prostate cancer (mCRPC) and homologous recombination repair (HRR) mutations. METHODS: Medical charts were abstracted for mCRPC patients with ≥ 1 of 12 HRR somatic gene alterations treated at US oncology centers participating in the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange. Treatment patterns and clinical outcomes were assessed from the initiation of first-line or later (1L+) mCRPC therapy received on or after July 1, 2014. RESULTS: Among 138 patients included in the study, the most common somatic HRR mutations were CDK12 (47.8%), BRCA2 (22.5%), and ATM (21.0%). Novel hormonal therapy and taxane chemotherapy were most commonly used in 1L; taxane use increased in later lines. Median overall survival (95% confidence interval [CI]) was 36.3 (30.7-47.8) months from initiation of 1L therapy and decreased for subsequent lines. Similarly, there was a trend of decreasing progression-free survival and prostate-specific antigen response from 1L to 4L+ therapy. CONCLUSIONS: Treatment patterns identified in this study were similar to those among patients with mCRPC regardless of tumor HRR mutation status in the literature.

Adenylyl cyclase 6 deletion increases mortality during sustained ß-adrenergic receptor stimulation.

Sustained ß-adrenergic receptor stimulation is associated with cardiomyopathy, an affect thought to result from cAMP-associated cardiac injury. Using a murine line with adenylyl cyclase 6 gene deletion (AC6KO), we tested the hypothesis that AC6 deletion, by limiting cAMP production, would attenuate cardiomyopathy in the setting of sustained ß-adrenergic receptor stimulation. During 7d isoproterenol infusion, there was unexpected higher mortality in AC6KO mice compared to wild type control mice (p<0.0001). However, left ventricular function was similarly impaired in isoproterenol-infused control and AC6KO mice. There were no group differences in left ventricular hypertrophy, apoptosis, and fibrosis. Telemetric electrocardiography showed progressive prolongation of PR interval (p<0.0001), QRS duration (p<0.0005), and QTc (p<0.0001), as well as reduction in heart rate (p<0.0001), in AC6KO mice during isoproterenol infusion. These defective electrophysiological properties in isoproterenol-infused AC6KO mice were associated with decreased longitudinal ventricular conduction velocity (p<0.05) and reduced phosphorylation of connexin 43 at S368 in left ventricular samples (p=0.006). Taken together, these data demonstrate that limiting cAMP production does not prevent sustained ß-adrenergic receptor stimulation-induced cardiomyopathy. Moreover, AC6 deletion impairs electrophysiological properties and increases mortality during sustained ß-adrenergic receptor stimulation. Decreased connexin 43 phosphorylation and impaired ventricular conduction may be of mechanistic importance for the defective electrophysiological properties.

Treatment Patterns and Healthcare Resource Use in Medicare Beneficiaries with Parkinson's Disease.

Background: Studies on real-world treatment patterns and long-term economic burden of Parkinson's disease (PD) have been limited. Objective: To assess treatment patterns, healthcare resource utilization (HRU), and costs associated with PD symptoms and treatment-related adverse events (AEs) among Medicare beneficiaries in the United States. Methods: A 100% Medicare Fee-For-Service data (2006-2020) of patients with PD were analyzed. PD treatment patterns were described for the subset of patients who had no previously observed PD treatments or diagnoses (ie, the incident cohort). HRU and healthcare costs associated with PD symptoms were assessed for all patients with PD (ie, the overall cohort) and that associated with treatment-related AEs were assessed for the subset of patients who received PD treatments after PD diagnosis (ie, the active treatment cohort), using longitudinal models with repeated measures. Results: Overall, 318,582 patients were included (mean age at PD diagnosis: 77.4 years; 53.3% female). Among patients in the incident cohort (N=214,829), 51.1% initiated levodopa monotherapy and 5.9% initiated dopamine agonists (DAs) monotherapy as first-line treatment. The proportion of incident patients treated with DAs and other PD therapies generally increased from post-diagnosis years 1 to 10. The median time from diagnosis to PD treatment initiation was 2.0 months; the median time to treatment discontinuation was the longest with levodopa (18.7 months), followed by DAs (9.5 months). In the overall cohort, PD symptoms, especially motor symptoms and severe motor symptoms, were associated with significantly higher rates of HRU and costs. In the active treatment cohort (N=234,298), treatment-related AEs were associated with significantly higher rates of HRU and medical costs. Conclusion: While levodopa is still the mainstay of PD management, considerable heterogeneity exists in real-world treatment patterns. Overall, PD symptoms and AEs were associated with significantly higher HRU and healthcare costs, suggesting unmet medical needs for PD treatments with better tolerability profiles.

Effects of Urocortin 2 Gene Transfer on Glucose Disposal in Insulin-Resistant db/db Mice on Metformin.

The study was designed to determine whether urocortin 2 (Ucn2) gene transfer is as safe and effective as metformin in insulin-resistant mice. Four groups of insulin-resistant db/db mice and a nondiabetic group were studied: (1) metformin; (2) Ucn2 gene transfer; (3) metformin + Ucn2 gene transfer; (4) saline; and (5) nondiabetic mice. After completion of the 15-week protocol, glucose disposal was quantified, safety assessed, and gene expression documented. Ucn2 gene transfer was superior to metformin, providing reductions in fasting glucose and glycated hemoglobin and enhanced glucose tolerance. The combination of metformin + Ucn2 gene transfer provided no better glucose control than Ucn2 gene transfer alone and was not associated with hypoglycemia. Metformin alone, Ucn2 gene transfer alone, and metformin + Ucn2 gene transfer together reduced fatty infiltration of the liver. Serum alanine transaminase concentration was elevated in all db/db groups (vs. nondiabetic controls), but the metformin + Ucn2 gene transfer combined group had the lowest alanine transaminase levels. No group differences in fibrosis were detected. In a hepatoma cell line, activation of AMP kinase showed a rank order of combined metformin + Ucn2 peptide > Ucn2 peptide > metformin. We conclude (1) The combination of metformin + Ucn2 gene transfer does not result in hypoglycemia. (2) Ucn2 gene transfer alone provides superior glucose disposal versus metformin alone. (3) The combination of Ucn2 gene transfer and metformin is safe and has additive effects in reducing serum alanine transaminase concentration, activating AMP kinase activity, and increasing Ucn2 expression, but is no more efficacious than Ucn2 gene transfer alone in reducing hyperglycemia. These data indicate that Ucn2 gene transfer is more effective than metformin in the db/db model of insulin resistance and combined treatment with metformin + Ucn2 gene transfer appears to have favorable effects on liver function and Ucn2 expression.

Pressure overload-induced cardiac remodeling and dysfunction in the absence of interleukin 6 in mice.

Congestive heart failure is associated with increased expression of pro-inflammatory cytokines, and the levels of these cytokines correlate with heart failure severity and prognosis. Chronic interleukin 6 (IL-6) stimulation leads to left ventricular (LV) hypertrophy and dysfunction, and deletion of IL-6 reduces LV hypertrophy after angiotensin II infusion. In this study, we tested the hypothesis that IL-6 deletion has favorable effects on pressure-overloaded hearts. We performed transverse aortic constriction on IL-6-deleted (IL6KO) mice and C57BL/6J mice (CON) to induce pressure overload. Pressure overload was associated with similar LV hypertrophy, dilation, and dysfunction in CON and IL6KO mice. Re-activation of the fetal gene program was also similar in pressure-overloaded CON and IL6KO mice. There were no differences between CON and IL6KO mice in LV fibrosis or expression of extracellular matrix proteins after pressure overload. In addition, no group differences in apoptosis or autophagy were seen. These data indicate that IL-6 deletion does not block LV remodeling and dysfunction induced by pressure overload. Attenuated content of IL-11 appears to be a compensatory mechanism for IL-6 deletion in pressure-overloaded hearts. We infer from these data that limiting availability of IL-6 alone is not sufficient to attenuate LV remodeling and dysfunction in failing hearts.

A fly GWAS for purine metabolites identifies human FAM214 homolog medusa, which acts in a conserved manner to enhance hyperuricemia-driven pathologies by modulating purine metabolism and the inflammatory response.

Elevated serum urate (hyperuricemia) promotes crystalline monosodium urate tissue deposits and gout, with associated inflammation and increased mortality. To identify modifiers of uric acid pathologies, we performed a fly Genome-Wide Association Study (GWAS) on purine metabolites using the Drosophila Genetic Reference Panel strains. We tested the candidate genes using the Drosophila melanogaster model of hyperuricemia and uric acid crystallization ("concretion formation") in the kidney-like Malpighian tubule. Medusa (mda) activity increased urate levels and inflammatory response programming. Conversely, whole-body mda knockdown decreased purine synthesis precursor phosphoribosyl pyrophosphate, uric acid, and guanosine levels; limited formation of aggregated uric acid concretions; and was sufficient to rescue lifespan reduction in the fly hyperuricemia and gout model. Levels of mda homolog FAM214A were elevated in inflammatory M1- and reduced in anti-inflammatory M2-differentiated mouse bone marrow macrophages, and influenced intracellular uric acid levels in human HepG2 transformed hepatocytes. In conclusion, mda/FAM214A acts in a conserved manner to regulate purine metabolism, promotes disease driven by hyperuricemia and associated tissue inflammation, and provides a potential novel target for uric acid-driven pathologies.

Beneficial effects of adenylyl cyclase type 6 (AC6) expression persist using a catalytically inactive AC6 mutant.

Cardiac-directed expression of AC6 has pronounced favorable effects on cardiac function possibly not linked with cAMP production. To determine rigorously whether cAMP generation is required for the beneficial effects of increased AC6 expression, we generated a catalytically inactive AC6 mutant (AC6mut) that has markedly diminished cAMP generating capacity by replacing aspartic acid with alanine at position 426 in the C1 domain (catalytic region) of AC6. Gene transfer of AC6 or AC6mut (adenovirus-mediated) in adult rat cardiac myocytes resulted in similar expression levels and intracellular distribution, but AC6mut expression was associated with marked reduction in cAMP production. Despite marked reduction in cAMP generation, AC6mut influenced intracellular signaling events similarly to that observed after expression of catalytically intact AC6. For example, both AC6 and AC6mut reduced phenylephrine-induced cardiac myocyte hypertrophy and apoptosis (p < 0.001), expression of cardiac ankyrin repeat protein (p < 0.01), and phospholamban (p < 0.05). AC6mut expression, similar to its catalytically intact cohort, was associated with increased Ca2+ transients in cardiac myocytes after isoproterenol stimulation. Many of the biological effects of AC6 expression are replicated by a catalytically inactive AC6 mutant, indicating that the mechanisms for these effects do not require increased cAMP generation.

Urocortin 2 Gene Transfer Improves Glycemic Control and Reduces Retinopathy and Mortality in Murine Insulin Deficiency.

Type 1 diabetes affects 20 million patients worldwide. Insulin is the primary and commonly the sole therapy for type 1 diabetes. However, only a minority of patients attain the targeted glucose control and reduced adverse events. We tested urocortin 2 gene transfer as single-agent therapy for insulin deficiency using two mouse models. Urocortin 2 gene transfer reduced blood glucose for months after a single intravenous injection, through increased skeletal muscle insulin sensitivity, increased insulin release in response to glucose stimulation, and increased plasma insulin levels before and during euglycemic clamp. The combined increases in both insulin availability and sensitivity resulted in improved glycemic indices-events that were not anticipated in these insulin-deficient models. In addition, urocortin 2 gene transfer reduced ocular manifestations of long-standing insulin deficiency such as vascular leak and improved retinal function. Finally, mortality was reduced by urocortin 2 gene transfer. The mechanisms for these beneficial effects included increased activities of AMP-activated protein kinase and Akt (protein kinase B) in skeletal muscle, increased skeletal muscle glucose uptake, and increased insulin release. These data suggest that urocortin 2 gene transfer may be a viable therapy for new onset type 1 diabetes and might reduce insulin needs in later stage disease.

Adenylyl cyclase type 6 deletion decreases left ventricular function via impaired calcium handling.

BACKGROUND: Adenylyl cyclases (ACs) are a family of effector molecules for G-protein-coupled receptors. The 2 ACs most abundantly expressed in cardiac myocytes are types 5 (AC5) and 6 (AC6), which have 65% amino acid homology. It has been speculated that coexpression of 2 AC types in cardiac myocytes represents redundancy, but the specific role of AC6 in cardiac physiology and its differences from AC5 remain to be defined. METHODS AND RESULTS: We generated transgenic mice with targeted deletion of AC6. Deletion of AC6 was associated with reduced left ventricular contractile function (P=0.026) and relaxation (P=0.041). The absence of AC6 was associated with a 48% decay in beta-adrenergic receptor-stimulated cAMP production in cardiac myocytes (P=0.003) and reduced protein kinase A activity (P=0.015). In addition, phospholamban phosphorylation was reduced (P=0.015), sarcoplasmic reticulum Ca2+-ATPase activity was impaired (P<0.0001), and cardiac myocytes showed marked abnormalities in calcium transient formation (P=0.001). CONCLUSIONS: The combination of impaired cardiac cAMP generation and calcium handling that result from AC6 deletion underlies abnormalities in left ventricular function. The biochemical and physiological consequences of AC6 deletion reveal it to be an important effector molecule in the adult heart, serving unique biological functions not replicated by AC5.

Significant alteration of liver metabolites by AAV8.Urocortin 2 gene transfer in mice with insulin resistance.

INTRODUCTION: Urocortin 2 (Ucn2) is a 38-amino acid peptide of the corticotropin-releasing factor family. Intravenous (IV) delivery of an adeno-associated virus vector serotype 8 encoding Ucn2 (AAV8.Ucn2) increases insulin sensitivity and glucose disposal in mice with insulin resistance. OBJECTIVE: To determine the effects of Ucn2 on liver metabolome. METHODS: Six-week-old C57BL6 mice were divided into normal chow (CHOW)-fed and high fat diet (HFD)-fed groups. The animals received saline, AAV8 encoding no gene (AAV8.Empt) or AAV8.Ucn2 (2x1013 genome copy/kg, IV injection). Livers were isolated from CHOW-fed and HFD-fed mice and analyzed by untargeted metabolomics. Group differences were statistically analyzed. RESULTS: In CHOW-fed mice, AAV8.Ucn2 gene transfer (vs. saline) altered the metabolites in glycolysis, pentose phosphate, glycogen synthesis, glycogenolysis, and choline-folate-methionine signaling pathways. In addition, AAV8.Ucn2 gene transfer increased amino acids and peptides, which were associated with reduced protein synthesis. In insulin resistant (HFD-induced) mice, HFD (vs CHOW) altered 448 (112 increased and 336 decreased) metabolites and AAV8.Ucn2 altered 239 metabolites (124 increased and 115 reduced) in multiple pathways. There are 61 metabolites in 5 super pathways showed interactions between diet and AAV8.Ucn2 treatment. Among them, AAV8.Ucn2 gene transfer reversed HFD effects on 13 metabolites. Finally, plasma Ucn2 effects were determined using a 3-group comparison of HFD-fed mice that received AAV8.Ucn2, AAV.Empt or saline, where 18 metabolites that altered by HFD (15 increased and 3 decreased), but restored levels to that seen in CHOW-fed mice by increased plasma Ucn2. CONCLUSIONS: Metabolomics study revealed that AAV8.Ucn2 gene transfer, through increased plasma Ucn2, provided counter-HFD effects in restoring hepatic metabolites to normal levels, which could be the underlying mechanisms for Ucn2 effects on increasing glucose disposal and reducing insulin assistance.

Cardiac-Directed Expression of Adenylyl Cyclase Catalytic Domain (C1C2) Attenuates Deleterious Effects of Pressure Overload.

A fusion protein (C1C2) constructed by fusing the intracellular C1 and C2 segments of adenylyl cyclase type 6 (AC6) retains beneficial effects of AC6 expression, without increasing cyclic adenosine monophosphate generation. The effects of cardiac-directed C1C2 expression in pressure overload is unknown. Left ventricular (LV) pressure overload was induced by transverse aortic constriction (TAC) in C1C2 mice and in transgene negative (TG-) mice. Four weeks after TAC, LV systolic function and diastolic function were measured, and Ca2+ handling was assessed. Four weeks after TAC, TG- animals showed reduced LV peak +dP/dt. LV peak +dP/dt in C1C2 mice was statistically indistinguishable from that of normal mice and was higher than that seen in TG- mice 4 weeks after TAC (p = 0.02), despite similar and substantial cardiac hypertrophy. In addition to higher LV peak +dP/dt in vivo, cardiac myocytes from C1C2 mice showed shorter time-to-peak Ca2+ transient amplitude (p = 0.002) and a reduced time constant of cytosolic Ca2+ decline (Tau; p = 0.003). Sarcomere shortening fraction (p < 0.03) and the rate of sarcomere shortening (p < 0.02) increased in C1C2 cardiac myocytes. Myofilament sensitivity to Ca2+ was increased in systole (p = 0.02) and diastole (p = 0.04) in C1C2 myocytes. These findings indicate enhanced Ca2+ handling associated with C1C2 expression. Favorable effects on Ca2+ handling and LV function were associated with increased LV SERCA2a protein content (p = 0.015) and reduced LV fibrosis (p = 0.008). Cardiac-directed C1C2 expression improves Ca2+ handling and increases LV contractile function in pressure overload. These data provide a rationale for further exploration of C1C2 gene transfer as a potential treatment for heart failure.

Urocortin 2 Gene Transfer Reduces the Adverse Effects of a Western Diet on Cardiac Function in Mice.

Diabetes mellitus is associated with increased risk of heart failure. It has been previously demonstrated in mice that a single injection of adeno-associated virus 8 encoding urocortin 2 (AAV8.UCn2) increases glucose disposal in models of insulin resistance and improves the function of the failing heart. The present study tested the hypothesis that UCn2 gene transfer would reduce diabetes-related left ventricular (LV) dysfunction. Eight-week-old C57BL6 male mice were fed a Western diet (WD; 45% fat, 35% carbohydrate) for 40 weeks. At week 30, they received saline or AAV8.UCn2 (2 × 1013 genome copies/kg) via intravenous injection. Ten weeks after gene transfer, fasting blood glucose, glucose tolerance, and cardiac function were measured via echocardiography and in vivo measurement of LV contractile function, and the results were compared to those of mice fed normal chow (NC; 10% fat; 70% carbohydrate). The contents of key LV signaling proteins were also measured to probe mechanisms. WD increased 12 h fasting glucose (WD: 190 ± 11 mg/dL, n = 8; NC: 105 ± 12 mg/dL, n = 7; p = 0.0004). WD tended to reduce LV peak +dP/dt (p = 0.08) and LV peak -dP/dt (p = 0.05). LV ejection fraction was unchanged. Among WD-fed mice, UCn2 gene transfer reduced 12 h fasting glucose (WD-UCn2: 149 ± 6 mg/dL, n = 8; WD-Saline: 190 ± 11 mg/dL, n = 8; p = 0.012), increased LV peak +dP/dt (p < 0.001) and LV peak -dP/dt (p = 0.013), and reduced Tau (p < 0.02), indicating beneficial effects on systolic and diastolic LV function. In addition, among WD-fed mice, UCn2 gene transfer increased LV ejection fraction (p < 0.005) and the velocity of circumferential fiber shortening (p = 0.0005). Finally, a reduction was seen in fatty infiltration of the liver in WD-fed mice that had received UCn2 gene transfer. LV samples from WD-UCn2 mice showed increased phosphorylation of the protein kinase A catalytic domain (p = 0.03). In conclusion, UCn2 gene transfer increased LV systolic and diastolic function and reduced blood glucose in mice with diabetes-related LV dysfunction, indicating that UCn2 gene transfer may be of potential therapeutic benefit.

Urocortin 3 Gene Transfer Increases Function of the Failing Murine Heart.

Peptide infusions of peptides the corticotropin releasing factor family, including urocortin 2, stresscopin, and urocortin 3 (UCn3), have favorable acute effects in clinical heart failure (HF), but their short half-lives make them unsuitable for chronic therapy. This study asked whether UCn3 gene transfer, which provides sustained elevation of plasma UCn3 levels, increases the function of the failing heart. HF was induced by transmural left ventricular (LV) cryoinjury in mice. LV function was assessed 3 weeks later by echocardiography. Those with ejection fractions (EF) <40% received intravenous saline or intravenous adeno-associated virus type-8 encoding murine UCn3 (AAV8.mUCn3; 1.9 × 1013 genome copies/kg). Five weeks after randomization, repeat echocardiography, assessment of LV function (+dP/dt, -dP/dt), and quantification of Ca2+ transients and sarcomere shortening in isolated cardiac myocytes were conducted, and assessment of LV Ca2+ handling and stress proteins was performed. Three weeks after myocardial infarction, prior to treatment, EFs were reduced (mean 31%, from 63% in sham-operated animals). Mice randomized to receive UCn3 gene transfer showed increased plasma UCn3 (from 0.1 ± 0.01 ng/mL in the saline group to 5.6 ± 1.1 ng/mL; n = 12 each group; p < 0.0001). Compared to mice that received saline, UCn3 gene transfer was associated with higher values for EF (p = 0.0006); LV +dP/dt (p < 0.0001), and LV -dP/dt (p < 0.0001). Cardiac myocytes from mice that received UCn3 gene transfer showed higher peak Ca2+ transients (p = 0.0005), lower time constant of cytosolic Ca2+ decline (tau, p < 0.0001), and higher rates of sarcomere shortening (+dL/dt, p = 0.03) and lengthening (-dL/dt, p = 0.04). LV samples from mice that received UCn3 gene transfer contained higher levels of SERCA2a (p = 0.0004 vs. HF) and increased amounts of phosphorylated troponin I (p = 0.04 vs. HF). UCn3 gene transfer is associated with improved Ca2+ handling and LV function in mice with HF and reduced EF.

Effects of Urocortin 2 Versus Urocortin 3 Gene Transfer on Left Ventricular Function and Glucose Disposal.

UCn2 and UCn3 peptides have recently been infused to treat patients with heart failure (HF) but are limited by their short half-lives. A 1-time intravenous injection of virus vectors encoding UCn2 or UCn3 provided sustained increases in plasma concentrations of the peptides. This was associated with increases in both systolic and diastolic left ventricular (LV) function, mediated by increased LV SERCA2a expression and Ca2+ handling. UCn2, but not UCn3, gene transfer reduced fasting glucose and increased glucose disposal. These findings support UCn2 and UCn3 gene transfer as potential treatments for HF and indicate that UCn2 may be an optimal selection in patients with diabetes and HF.

Development of Novel Microparticles for Effective Delivery of Thymol and Lauric Acid to Pig Intestinal Tract.

Antibiotics have been widely supplemented in feeds at subtherapeutic concentrations to prevent postweaning diarrhea and increase the overall productivity of pigs. However, the emergence of antimicrobial-resistant bacteria worldwide has made it urgent to minimize the use of in-feed antibiotics. The development of promising alternatives to in-feed antibiotics is crucial for maintaining the sustainability of swine production. Both medium-chain fatty acids (MCFA) and essential oils exhibit great potential to postweaning diarrhea; however, their direct inclusion has compromised efficacy because of several factors including low stability, poor palatability, and low availability in the lower gut. Therefore, the objective of this study was to develop a formulation of microparticles to deliver a model of essential oil (thymol) and MCFA (lauric acid). The composite microparticles were produced by the incorporation of starch and alginate through a melt-granulation process. The release of thymol and lauric acid from the microparticles was in vitro determined using simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF), consecutively. The microparticles prepared with 2% alginate solution displayed a slow release of thymol and lauric acid in the SSF (21.2 ± 2.3%; 36 ± 1.1%), SGF (73.7 ± 6.9%; 54.8 ± 1.7%), and SIF (99.1 ± 1.2%; 99.1 ± 0.6%), respectively, whereas, the microparticles without alginate showed a rapid release of thymol and lauric acid from the SSF (79.9 ± 11.8%; 84.9 ± 9.4%), SGF (92.5 ± 3.5%; 75.8 ± 5.9%), and SIF (93.3 ± 9.4%; 93.3 ± 4.6%), respectively. The thymol and lauric acid in the developed microparticles with or without alginate both exhibited excellent stabilities (>90%) during being stored at 4 °C for 12 weeks and after being stored at room temperature for 2 weeks. These results evidenced that the approach developed in the present study could be potentially employed to deliver thymol and lauric acid to the lower gut of pigs, although further in vivo investigations are necessary to validate the efficacy of the microparticles.

Cardiac-directed expression of a catalytically inactive adenylyl cyclase 6 protects the heart from sustained ß-adrenergic stimulation.

OBJECTIVES: Increased expression of adenylyl cyclase type 6 (AC6) has beneficial effects on the heart through cyclic adenosine monophosphate (cAMP)-dependent and cAMP-independent pathways. We previously generated a catalytically inactive mutant of AC6 (AC6mut) that has an attenuated response to ß-adrenergic receptor stimulation, and, consequently, exhibits reduced myocardial cAMP generation. In the current study we test the hypothesis that cardiac-directed expression of AC6mut would protect the heart from sustained ß-adrenergic receptor stimulation, a condition frequently encountered in patients with heart failure. METHODS AND RESULTS: AC6mut mice and transgene negative siblings received osmotic mini-pumps to provide continuous isoproterenol infusion for seven days. Isoproterenol infusion caused deleterious effects that were attenuated by cardiac-directed AC6mut expression. Both groups showed reduced left ventricular (LV) ejection fraction, but the reduction was less in AC6mut mice (p = 0.047). In addition, AC6mut mice showed superior left ventricular function, manifested by higher values for LV peak +dP/dt (p = 0.03), LV peak -dP/dt (p = 0.008), end-systolic pressure-volume relationship (p = 0.003) and cardiac output (p<0.03). LV samples of AC6mut mice had more sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) protein (p<0.01), which likely contributed to better LV function. AC6mut mice had lower rates of cardiac myocyte apoptosis (p = 0.016), reduced caspase 3/7 activity (p = 0.012) and increased B-cell lymphoma 2 (Bcl2) expression (p = 0.0001). CONCLUSION: Mice with cardiac-directed AC6mut expression weathered the deleterious effects of continuous isoproterenol infusion better than control mice, indicating cardiac protection.

Cardiac-Directed Expression of Adenylyl Cyclase Catalytic Domain Reverses Cardiac Dysfunction Caused by Sustained Beta-Adrenergic Receptor Stimulation.

OBJECTIVE: To test the hypothesis that cardiac-directed expression of the cytoplasmic domains of adenylyl cyclase-6 (AC6) would have beneficial effects on the heart. BACKGROUND: Eliminating the two transmembrane domains of AC6 yields a protein with an intact catalytic domain that is disengaged from membrane-associated ß-adrenergic receptor stimulation, but with enhanced propensity for intracellular interactions. METHODS: We constructed a peptide of the C1 and C2 segments of AC6 (C1C2), expressed C1C2 in an adenovirus vector and generated transgenic lines with cardiac-directed C1C2 expression, which underwent sustained isoproterenol (Iso) infusion. RESULTS: Gene transfer of C1C2 in cardiac myocytes showed reduced cAMP generation in response to Iso-stimulation. C1C2 transgenic mice had normal left ventricular (LV) structure and function. LV samples from C1C2 mice showed diminished Iso-stimulated cAMP generation but normal LV contractile responses, suggesting a compensatory mechanism. Cardiac myocytes from C1C2 mice showed increased Iso-stimulated Ca2+ release and reduced time to peak Ca2+ release. After 7 days Iso infusion, control mice tended to show reduced LV function, but C1C2 mice showed increases in both LV peak +dP/dt and peak -dP/dt indicating enhanced LV systolic and diastolic function. LV from C1C2 mice showed a 2.6-fold increase in SERCA2a protein, and cardiac myocytes showed increased Ca2+ release, reduced time to peak Ca2+ release and reduced Tau. CONCLUSIONS: In C1C2 mice, sustained isoproterenol infusion increases rather than decreases LV function. Reduced cAMP generation and resistance to catecholamine cardiomyopathy are attractive features of this novel AC-related protein.