Daily Sc Injections Research Articles

Oil-extracted from flaxseeds protects against endothelial dysfunction and oxidative stress by modulating nitric oxide, thiols contents and improving redox status in ouabain-induced hypertensive rats

ObjectivesThe purpose of this study was to evaluate the antihypertensive and antioxidant effects of flaxseed oil in ouabain-induced hypertensive in normal rats Wistar. MethodsExperimental hypertension was induced by a daily subcutaneous (sc) injection of 10μg/kg/day/rat of ouabain diluted in 9g/L of saline solution (NaCl) for 21 days (n=16). Animals were then divided into two groups fed an experimental diet containing 20% casein supplemented (Ouab-Oil-Lu) or not (Ouab) with 1% flaxseed oil whereas control rats (n=8) received only a daily sc injection of NaCl. ResultsThe results showed that supplementing hypertensive rats with 1% flaxseed oil decreased significantly (P<0.05) the systolic, diastolic, and mean arterial pressure, ameliorated plasma and liver lipid profile and reduced glucose levels in plasma. Similarly, flaxseed oil decreased TBARS levels in plasma and tissues, as well as carbonyl levels in the heart. Interestingly, this supplementation increased GSH, thiols and nitric oxide levels and improved AchE and antioxidant enzyme activities (SOD and GSH-Px) in the tissues. ConclusionsOur findings suggested that flaxseed oil may provide a rationale for antihypertensive use in traditional medicine.

Effect of melanocortin-4 receptor antagonist TCMCB07 on chemotherapy-induced anorexia and weight loss in rats.

e15196 Background: We previously reported that peripheral administration of TCMCB07, a synthetic antagonist of the type 4 melanocortin receptor (MC4R), ameliorates cancer- and chronic kidney disease-associated cachexia in rats. In this study, we used a rat model of cisplatin and 5-FU to evaluate the efficacy of TCMCB07 in alleviating chemotherapy-induced anorexia, weight loss and muscle wasting. Methods: Two separate experiments were performed in a total of 58 rats. 1) Cisplatin (Cis)/TCMCB07 experiment with 3 groups: Cis/saline (n = 11), Cis/TCMCB07 (n = 11), Saline/saline (n = 6). 2) 5-FU/TCMCB07 experiment with 3 groups: 5-FU/saline (n = 12), 5-FU/TCMCB07 (n = 12), Saline/saline (n = 6). Rats were treated with Cis (2.5mg/kg), 5-FU (70 mg/kg), or saline via IP injection once weekly for 3 weeks. Rats were also treated with TCMCB07 (3 mg/kg/day) or saline via daily SC injection for 21 days. Food intake and body weight were measured daily. Body composition was analyzed pre- and post-treatment by MRI. Heart and gastrocnemius were dissected and weighed after euthanasia. Results: Total food intake over 21 days was significantly increased in Cis/TCMCB07 vs Cis/saline group (p = 0.0006) and 5-FU/TCMCB07 vs 5-FU/saline group (p = 0.0134), and decreased in Cis/saline vs Saline/saline group (p = 0.001), but was no significant change in Cis/TCMCB07 vs Saline/saline group and 5-FU/saline or 5-FU/TCMCB07 vs Saline/saline group. Body weight was robustly increased in Cis/TCMCB07 vs Cis/saline group (p = 0.0007) and 5-FU/TCMCB07 vs 5-FU/saline group (p = 0.0013), and decreased in Cis/saline vs Saline/saline group (p = 0.0001) and 5-FU/saline vs Saline/saline group (p = 0.0041), but was no significant change in Cis/TCMCB07 vs Saline/saline group and 5-FU/TCMCB07 vs Saline/saline group. Cardiac and skeletal muscle loss was markedly attenuated in Cis/TCMCB07 vs Cis/saline (cardiac: 3.12% vs -11.95%, p &lt; 0.0001; skeletal: -3.88% vs -9.02%, p = 0.0259). Cardiac muscle loss was significantly attenuated in 5-FU/TCMCB07 vs 5-FU/saline group (1.40% vs -6.09%, p = 0.0131) and skeletal muscle was modestly changed (-4.17% vs -7.24%, p = 0.1782). While Cis/saline and 5-FU/saline groups lost 91.33% and 52.61% of fat mass respectively compared to Saline/saline group, Cis/TCMCB07 and 5-FU/TCMCB07 groups gained 37.68% (p = 0.0002) and 70.19% (p &lt; 0.0001). Conclusions: This preclinical trial provides evidence supporting peripheral TCMCB07 antagonism of central melanocortin signaling, increasing food intake and anabolism, ultimately alleviating anorexia, weight loss and muscle wasting over multiple cycles of cisplatin or 5-FU chemotherapy.

OR21-6 AZP-3601, a Long-Acting Parathyroid Hormone (PTH) Analog, Normalizes Blood Calcium in Thyroparathyroidectomized (TPTX) Rats Without Causing Deleterious Changes in Bone

Abstract PTH peptides can be effective in normalizing blood calcium (Ca++) levels in cases of hypoparathyroidism (HP), but long-term effects on bone are not well understood. AZP-3601 is a long-acting PTH/PTHrP(1-36) analog currently being investigated as a potential treatment option for HP. As compared to conventional PTH1-34, AZP-3601 binds with higher affinity to the R0 conformation of the PTH receptor and thereby induces more sustained signaling responses in cells as well as more prolonged calcemic responses in vivo, despite having a very short circulating half-life. Using the TPTX rat model of HP, we investigated whether the distinct mode-of-action used by AZP-3601 results in different down-stream effects in bone than those induced by PTH1-34, when the peptides are administered long-term (31 days) either by daily sc injection (AZP-3601 and PTH1-34) or by continuous infusion (PTH1-34), and at doses aimed to optimally normalize blood Ca++ levels. Methods Male TPTX rats (strain S-D, age 9 weeks, and 2 weeks post TPTX surgery) were injected daily with either AZP-3601 at a dose of 0.7 nmol/kg or PTH1-34 at a dose of 50 nmol/kg, or were continuously infused via Alzet mini-pump with PTH1-34 at a dose of 3.0 nmol/kg/day. Vehicle treatments and sham surgery rats were used as controls. Tail-vein blood was collected on days 1, 7 14, 21 and 28 at 6 hours post-injection and analyzed for total Ca++. Rats were euthanized on day 31 (24 hours after last injection) and blood and femurs collected for analysis. Results All peptide-treatments increased serum Ca++ levels on days 7-21 to within or near the normal range (8.8-11.7 mg/dL versus 10.8-11.9 mg/dL in sham-operated rats) and to levels significantly higher than those in vehicle-treated TPTX rats (6.9-7.9 mg/dL, P&amp;lt;0.01). Bone mCT analysis revealed that relative to TPTX-vehicle controls, PTH1-34 continuous infusion significantly decreased (p&amp;lt;0.001) distal femur trabecular bone volume relative to tissue volume (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th), while PTH1-34 daily injection significantly increased (P&amp;lt;0.001) distal femur BV/TV, Tb.Th and Tb.N, as well as mid-femur cortical thickness (Ct.Th, P&amp;lt;0.001). In contrast, AZP-3601 daily injection caused no change in trabecular or cortical bone parameters. Serum levels of the bone formation marker P1NP were significantly increased by daily injection of PTH1-34 (P&amp;lt;0.001 vs. TPTX-vehicle) but were not changed by other treatments. Conclusion With long-term (31 days) treatment and at doses that similarly normalized serum Ca++ in TPTX rats, effects on bone were catabolic for PTH1-34 by continuous infusion, anabolic for PTH1-34 by daily injection, and neutral for AZP-3601 by daily injection. The distinct MOA used by AZP-3601 may thus result in less of an impact on bone than either daily injection or sustained, continuous delivery of PTH(1-34), when used as a long-term treatment for HP. Presentation: Monday, June 13, 2022 12:15 p.m. - 12:30 p.m.

Abstract P345: Inhibition Of Nuclear Receptor Retinoid-related Orphan Receptor γt Attenuates Interleukin-17A Production And Ameliorates Angiotensin II-induced Neuroinflammation And Hypertension In Rats

Interleukin (IL)-17A, a key inflammatory mediator produced primarily by T helper (Th) 17 cells, has been implicated in the pathogenesis of cardiovascular diseases including hypertension and heart failure. IL-17A can access the brain by disrupting blood-brain barrier (BBB) integrity to induce neuroinflammation. We previously reported that IL-17A contributes to angiotensin (ANG) II-induced hypertension via promoting neuroinflammation and sympathetic excitation. The nuclear receptor retinoid-related orphan receptor γt (RORγt) is a master transcription factor regulating Th17 cell differentiation. Here, we sought to determine whether systemic inhibition of RORγt attenuates IL-17A production and diminishes ANG II-induced neuroinflammation and hypertension. Sprague-Dawley rats received a 2-week subcutaneous (sc) infusion of ANG II (150 ng/kg/min) combined with daily sc injection of a RORγt inhibitor digoxin or vehicle (VEH). Compared with the control animals, blood pressure (162±5* vs 118±3 mmHg, *P&lt;0.05) and sympathetic tone as indicated by blood pressure change in response to ganglionic blockade (55±5* vs 23±3 mmHg) were increased in ANG II+VEH rats, which were reduced (38-41%*) in ANG II+digoxin rats. ANG II+VEH rats also had elevated RORγt mRNA in peripheral blood mononuclear cells (PBMCs: 3.38±0.70* vs 1.12±0.22) and higher IL-17A levels in plasma (23.22±5.85 * vs 4.30±0.66 pg/mL), along with increased IL-17A levels in cerebrospinal fluid (CSF: 11.89±1.91* vs 2.01±0.55 pg/mL), mRNA of IL-17A (3.02±0.48* vs 1.07±0.17), cytokines tumor necrosis factor (TNF)-α (2.78±0.45* vs 1.04±0.12) and IL-1β (2.57±0.53* vs 1.03±0.14) in the hypothalamic paraventricular nucleus (PVN), a key cardiovascular-related center in the brain. Although RORγt mRNA was unchanged in PBMCs, levels of IL-17A in plasma and CSF, mRNA of IL-17A, TNF-α and IL-1β in the PVN was reduced (41-62 %*) in ANG II+digoxin rats. Additionally, mRNA of caveolin-1 (2.26±0.39* vs 1.06±0.17), a marker of the BBB permeability, was increased in the PVN in ANG II+VEH rats and decreased (43%*) in ANG II+digoxin rats. These data suggest that RORγt inhibition improves ANG II-induced hypertension and sympathetic activation probably by reducing IL-17A production and neuroinflammation.

Optimization of Renal Delivery of an MMP2‐inhibitory Peptide Delivered Using the Elastin‐like Polypeptide Carrier

Chronic kidney disease (CKD) is a global health issue associated with the development of renal fibrosis. Renal fibrosis is caused by the excessive deposition of extracellular matrix (ECM) leading to organ failure and is the final common pathway in CKD. ECM homeostasis and degradation are mediated by matrix metalloproteinases (MMPs). Imbalances in MMP activity exacerbate renal damage and fibrosis. While increased expression of some MMPs can be anti‐fibrotic, other MMPs, such as MMP2, activate pro‐inflammatory and fibrotic chemokines and cytokines. Our group has developed an MMP2 inhibitory biologic by fusing an MMP2 inhibitory peptide to a protein biopolymer (ELP‐MMP2i). We hypothesize that ELP‐MMP2i will accumulate in the kidney after systemic administration and reduce or reverse the effects of renal fibrosis. ELP‐MMP2i has potent MMP2 inhibitory activity in vitro (IC50 = 1.1 mM). A pilot study utilizing continuous infusion of ELP‐MMP2i demonstrated reduced proteinuria and albuminuria in a hypertension induced glomerulosclerosis rat model (Dahl Salt Sensitive Rat). The aim of the current study was to optimize the renal delivery of ELP‐MMP2i. Three ELP‐MMP2i proteins using ELP domains of varying molecular weights (26 kDa, 50 kDa, and 99 kDa) were constructed. Pharmacokinetics and biodistribution of these proteins were determined following iv or sc injections (600 nmol/kg) in Sprague Dawley Rats (n= 4/group). The plasma clearance following an iv injection was inversely proportional to the molecular weight, and half‐lives were 0.4, 0.6, and 1.1 hours for the 26, 50, and 99 kDa proteins respectively (Figure A3). Similar results were observed with sc administration, with the smallest protein exhibiting the fastest absorption and the highest peak plasma levels (400 nM, 240 nM, and 180 nM peak plasma, respectively; Figure B2). Bioavailability was 55.3, 25.1, and 16.7% for 26, 50, and 99 kDa proteins respectively following sc administration. Both iv and sc injections exhibited significant deposition in the kidney (Figures A1 &amp; B1), and an inverse correlation between protein size and renal tissue concentrations of 6.2 ± 1.9, 3.6 ± 0.2, and 2.5 ± 0.4 mM renal concentrations for the 26, 50, and 99 kDa proteins, respectively, following iv administration (two‐way ANOVA p&lt;0.0001 Figure A2), and 2.0 ± 0.7, 0.6 ± 0.1, and 0.5 ± 0.1 mM renal concentrations following sc injections (two‐way ANOVA p&lt;0.0001, Figure B2). To further elucidate the pharmacokinetic properties of the protein in a model with renal injury and to test the effects of various doses, Dahl Salt Sensitive rats on an 8% NaCl diet were given a daily sc injections of 300 nmol/kg, 600 nmol/kg or 1,200 nmol/kg of the smallest ELP‐MMP2i peptide (n=3/group) for 7 days. ELP‐MMP2i accumulated almost exclusively in the kidneys with renal concentrations of 7.1 ± 0.3, 12.9 ± 1.2, and 25.0 ± 2.8 mM (p&lt;0.0001, two‐way ANOVA) following doses of 300, 600, and 1,200 nmol/kg/day respectively). These studies revealed that the smallest 26 kDa ELP‐MMP2i protein achieved the best balance of bioavailability and renal deposition. The results indicate that therapeutic doses of ELP‐MMP2i above the IC50can be achieved by daily sc administration.

DHT Differentially Regulates T Helper Cell Related Cytokines and MicroRNAs In Visceral and Subcutaneous Adipose Tissue of Female Mice

Hyperandrogenemic, insulin resistant polycystic ovarian syndrome (PCOS) patients often have low-grade inflammation due to elevated circulating pro-inflammatory markers. As up to 60% of PCOS patients are obese, whether this low-grade inflammatory state is due to increased adiposity or other factors such as hyperandrogenemia is unknown. Moreover, the systemic inflammation of obesity is correlated with recruitment of pro-inflammatory immune cell populations to WAT. We hypothesized that short-term administration of the potent androgen, dihydrotestosterone (DHT), to female mice would increase pro-inflammatory cytokines and microRNA (miR) associated with pro-inflammatory cytokines and immune cell populations in WAT. Sexually mature, normally-cycling female C57/Bl6 mice received a daily sc injection of oil (0 g; n=7) or DHT (27.5 g; n=7) beginning at estrus. Females had vaginal cytology daily. After three cycles or 12-16 days if mice became acyclic, mice were euthanized for collection of blood and WAT. Serum was analyzed for DHT and testosterone (TEST) by LC-MS/MS. TaqManTM Array Mouse Immune Response PCR assays (Thermofisher Scientific) were used to measure transcript expression levels in vWAT and scWAT. Ingenuity Pathway Analysis (IPA) (Qiagen) was used to analyze relationships between different transcript levels in each treatment group for each tissue. DHT mice had 17 fold higher serum DHT levels than oil mice but there was no difference in serum TEST between treatment groups. DHT mice had a significantly longer estrous cycle length then oil mice. Short-term administration of DHT significantly upregulated 23% (21 of 92) of transcripts in scWAT and downregulated 49% (45 of 92) of transcripts in vWAT. The top four canonical pathways identified by IPA in WAT were: T helper cell 1 (Th1), Th1 & T helper 2 activation, Helper T cell differentiation, and Altered B & T cell signaling. Based on the Th1 pathway derived from IPA, the following miRs (both -3p and 5p) downstream of Th1 activation targets were selected for qPCR in vWAT and scWAT: miR21, miR146a, miR29a, and miR155. Interestingly, miR-21a-5p, miR-146a-5p, and miR-155-5p were significantly upregulated in scWAT from DHT mice. No miRs were different between treatment groups in vWAT. We demonstrate for the first time that short-term DHT administration may cause immunosuppression in vWAT and inflammation, possibly mediated by miRs, in scWAT of female mice.

The ability of hesperidin compared to that of insulin for preventing osteoporosis induced by type I diabetes in young male albino rats: A histological and biochemical study

BackgroundPatients with type I diabetes are at increased risk of osteoporosis even after insulin therapy in adult stage. This study was conducted to compare the efficacy of hesperidin (hesp) therapy versus that of insulin alone in the alleviation of osteoporosis arising from type I diabetes mellitus (T1DM) in young rats. Materials and methodsHesperidin was administered orally to STZ-induced diabetes. The animals were evaluated morphologically and biochemically and compared with that received daily SC injections of long-acting insulin. ResultsHistologically, we observed the degeneration of osteoblasts and osteocytes, decreased collagen fibers, and disturbed bone turn over markers in untreated DM rats. Hesperidin+ insulin supplementation to diabetic rats caused significant improvement of most of the bone histological and morphometric parameters compared with the insulin-treated group. Furthermore, hesp treatment significantly reduced pro-inflammatory mediators TNFα and NF-κB and increased serum biochemical markers of bone turnover, including osteopontin (OPN), osteocalcin (OC) and decreased serum alkaline phosphatase (ALP). ConclusionThese data demonstrated that hesp could be considered to be a beneficial drug for preventing diabetic osteoporosis in growing age.

Effect of placental restriction and neonatal exendin-4 treatment on postnatal growth, adult body composition, and in vivo glucose metabolism in the sheep.

Intrauterine growth restriction (IUGR) increases the risk of adult type 2 diabetes (T2D) and obesity. Neonatal exendin-4 treatment can prevent diabetes in the IUGR rat, but whether this will be effective in a species where the pancreas is more mature at birth is unknown. Therefore, we evaluated the effects of neonatal exendin-4 administration after experimental restriction of placental and fetal growth on growth and adult metabolic outcomes in sheep. Body composition, glucose tolerance, and insulin secretion and sensitivity were assessed in singleton-born adult sheep from control (CON; n = 6 females and 4 males) and placentally restricted pregnancies (PR; n = 13 females and 7 males) and in sheep from PR pregnancies that were treated with exendin-4 as neonates (daily sc injections of 1 nmol/kg exendin-4; PR + exendin-4; n = 11 females and 7 males). Placental restriction reduced birth weight (by 29%) and impaired glucose tolerance in the adult but did not affect adult adiposity, insulin secretion, or insulin sensitivity. Neonatal exendin-4 suppressed growth during treatment, followed by delayed catchup growth and unchanged adult adiposity. Neonatal exendin-4 partially restored glucose tolerance in PR progeny but did not affect insulin secretion or sensitivity. Although the effects on glucose tolerance are promising, the lack of effects on adult body composition, insulin secretion, and insulin sensitivity suggest that the neonatal period may be too late to fully reprogram the metabolic consequences of IUGR in species that are more mature at birth than rodents.

Effects of abaloparatide, a human parathyroid hormone-related peptide analog, on bone mineral density in postmenopausal women with osteoporosis.

Abaloparatide is a novel synthetic peptide analog of parathyroid hormone-related protein (PTHrP) that is currently being developed as a potential anabolic agent in the treatment of postmenopausal osteoporosis. This study sought to assess the effects of abaloparatide on bone mineral density (BMD) at the lumbar spine, total hip, and femoral neck in postmenopausal women with osteoporosis. Multi-center, multi-national, double-blind placebo controlled trial in which postmenopausal women were randomly assigned to receive 24 weeks of treatment with daily sc injections of placebo, abaloparatide, 20, 40, or 80 μg, or teriparatide, 20 μg. A 24-week extension was also performed in a subset of subjects. Postmenopausal women with osteoporosis (n = 222). BMD by dual-x-ray absorptiometry and biochemical markers of bone turnover. At 24 weeks, lumbar spine BMD increased by 2.9, 5.2, and 6.7% in the abaloparatide, 20-, 40-, and 80-μg groups, respectively, and 5.5% in the teriparatide group. The increases in the 40- and 80-μg abaloparatide groups and the teriparatide group were significantly greater than placebo (1.6%). Femoral neck BMD increased by 2.7, 2.2, and 3.1% in abaloparatide, 20-, 40-, and 80-μg groups, respectively, and 1.1% in the teriparatide group. The increase in femoral neck BMD with abaloparatide, 80 μg was significantly greater than placebo (0.8%). Total hip BMD increased by 1.4, 2.0, and 2.6% in the abaloparatide, 20-, 40-, and 80-μg groups, respectively. The total hip increases in the 40- and 80-μg abaloparatide groups were greater than both placebo (0.4%) and teriparatide (0.5%). Compared with placebo, 24 weeks of daily sc abaloparatide increases BMD of the lumbar spine, femoral neck, and total hip in a dose-dependent fashion. Moreover, the abaloparatide-induced BMD increases at the total hip are greater than with the marketed dose of teriparatide. These results support the further investigation of abaloparatide as an anabolic therapy in postmenopausal osteoporosis.

Genome-wide identification of aberrantly methylated promoters in ovarian tissue of prenatally androgenized rats

To identify aberrantly methylated candidate genes that are involved in the development of polycystic ovary syndrome (PCOS). Animal model. University-affiliated laboratory. Sprague-Dawley rats. The prenatally androgenized (PNA) rat model was established. Pregnant rats were treated with daily SC injections of T propionate during late gestation, and their female offspring were studied as adults. Serum glucose and hormone levels, ovary morphology and cell apoptosis, genome-wide CpG methylation, and expression of caspase-3 protein were measured. In the PNA group, the levels of serum glucose, 17-hydroxyprogesterone, and T were significantly higher when compared with the control group. Ovarian morphology showed increased atretic follicles and cystic follicles. Using the MeDIP-chip approach, we identified 528 genes that were hypermethylated in PNA ovaries. Gene ontology analyses revealed that these genes are involved in a variety of reproductive development and biological processes. The methylation enrichments of Bcl2l1 and Scr5a1 observed in the PNA group by MeDIP-quantitative polymerase chain reaction assay were significantly higher than those obtained from the control group. Furthermore, the mRNA level of the Bcl2l1 gene was significantly decreased in the PNA group. The percentage of caspase-3-positive cells in the PNA group was obviously higher compared with the control group, by terminal deoxynucleotidyl transferase dUTP nick end labeling detection as well. DNA methylation alteration may be an important factor affecting the genes involved in the pathophysiological processes that result in the phenotype of PCOS.

A reversible albumin-binding growth hormone derivative is well tolerated and possesses a potential once-weekly treatment profile.

Human growth hormone (hGH) replacement therapy currently requires daily sc injections for years/lifetime, which may be both inconvenient and distressing for patients. NNC0195-0092 is a novel hGH derivative intended for once-weekly treatment of GH deficiency. A noncovalent albumin binding moiety is attached to the hGH backbone. Clearance is reduced as a consequence of a reversible binding to circulating serum albumin, which prolongs the pharmacodynamic (PD) effect. To evaluate safety, local tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of a single dose (SD) and multiple doses (MD) of NNC0195-0092. Randomized, single-center, placebo-controlled, double-blind, SD/MD, dose-escalation trial of 105 healthy male subjects. NNC0195-0092 sc administration: Five cohorts of eight subjects received one dose of NNC0195-0092 (0.01-0.32 mg/kg) (n = 6) or placebo (n = 2). Sixteen subjects (equal numbers of Japanese and non-Asian) received once-weekly doses of NNC0195-0092 (0.02-0.24 mg/kg; n=12) or placebo (n=4) for 4 weeks. Blood samples were drawn for assessment of safety, PK, IGF-1, and IGF binding protein 3 profiles and anti-drug antibodies. SD and MD of NNC0195-0092 were well tolerated at all dose levels. No safety concerns or local tolerability issues were identified. A dose-dependent IGF-1 response was observed. IGF-1 profiles suggest that NNC0195-0092 may be suitable for once-weekly dosing, with a clinically relevant dose ≤0.08 mg/kg/week. No differences in PK and PD were observed between Japanese and non-Asian subjects. SD and MD of NNC0195-0092 administered to healthy Japanese and non-Asian male subjects were well tolerated at all doses. The present trial suggests that NNC0195-0092 has the potential for an efficacious, well-tolerated, once-weekly GH treatment.

Estrogen, Predominantly via Estrogen Receptor α, Attenuates Postpartum-Induced Anxiety- and Depression-Like Behaviors in Female Rats

Contributions from estrogen receptor (ER) subtypes (ERα and ERβ) to postpartum anxiogenic and depressive responses remain unresolved in rats. Using the elevated-plus maze (EPM) and forced swim (FS) tests, we confirmed that primiparous rats exhibited anxiogenic and depressive responses 3 weeks postpartum, improved 5 weeks postpartum (EPM), and recovered at 5 (FS) or 10 weeks postpartum (EPM) compared with diestrus nulliparous females. Immunohistochemistry suggested that these behavioral changes were temporally associated with decreased ERα but not ERβ expression in the medial amygdala (MEA). Additionally, ERα expression in the medial preoptic area (MPOA) significantly increased 10 weeks postpartum. Brain-derived neurotrophic factor (BDNF) expression was significantly elevated in the MEA 3 weeks postpartum. BDNF receptor tropomyosin-related kinase expression was significantly elevated in the MEA at 3 and 10 weeks but not at 5 weeks postpartum. The phosphorylation of ERK (pERK)-2 in the MEA, MPOA, and hippocampal CA1 region was significantly elevated 3 and 5 weeks postpartum. The effects of single daily sc injections of the ERα-selective agonist, propyl pyrazoletriol (PPT); ERβ-selective agonist, diarylpropionitrile; 17β-estradiol (E₂); and vehicle for 6 days in primiparous rats were assessed. PPT and E₂ significantly produced anxiolytic and antidepressant actions in the EPM and FS tests but PPT to a lesser degree than E₂ in the EPM test. Diarylpropionitrile affected the EPM test but was not significantly different from vehicle. BDNF expression was significantly increased 3 weeks postpartum by all treatments in the MPOA but not the CA1 and MEA. E₂ and PPT treatment significantly increased tropomyosin-related kinase and pERK1/2 expression in the MEA and MPOA and increased pERK1/2 expression in the CA1. The onset of anxiety- and depression-like behaviors in postpartum rats may be partly caused by a complex estrogen-mediated mechanism; nevertheless, changes in the ERα-related system, likely in the MEA, are predominantly involved.

GH Treatment to Final Height Produces Similar Height Gains in Patients With SHOX Deficiency and Turner Syndrome: Results of a Multicenter Trial

Growth impairment in short stature homeobox-containing gene (SHOX) deficiency and Turner syndrome share a similar etiology. Because of the established effect of GH treatment on height in patients with Turner syndrome, we hypothesized that GH therapy would also stimulate growth in patients with SHOX deficiency. Our objectives were to evaluate long-term efficacy of GH treatment in short patients with SHOX deficiency and to compare the effect on final (adult) height (FH) in patients with SHOX deficiency and Turner syndrome. A prospective, multinational, open-label, randomized 3-arm study consisting of a 2-year control period and a subsequent extension period to FH. The treatment groups were 1) SHOX-D-C/GH (untreated during the control period, GH-treated during the extension), 2) SHOX-D-GH/GH, and 3) Turner-GH/GH (GH-treated during both study periods). Short-statured prepubertal patients with genetically confirmed SHOX deficiency (n = 49) or Turner syndrome (n = 24) who participated in the extension. Depending on the study arm, patients received a daily sc injection of 0.05 mg/kg recombinant human GH from start of the study or start of the extension until attainment of FH or study closure. Height SD score gain from start of GH treatment to FH was similar between the combined SHOX-deficient groups (n = 28, 1.34 ± 0.18 [least-squares mean ± SE]) and the Turner group (n = 19, 1.32 ± 0.22). In this FH population, 57% of the patients with SHOX deficiency and 32% of the patients with Turner syndrome achieved a FH greater than -2 SD score. GH treatment in short children with SHOX deficiency showed similar long-term efficacy as seen in girls with Turner syndrome.

Peripheral oxytocin treatment ameliorates obesity by reducing food intake and visceral fat mass

Recent studies suggest that oxytocin (Oxt) is implicated in energy metabolism. We aimed to explore acute and sub-chronic effects of peripheral Oxt treatment via different routes on food intake and energy balance. Intraperitoneal (ip) injection of Oxt concentration-dependently decreased food intake in mice. Ip Oxt injection induced c-Fos expression in the hypothalamus and brain stem including arcuate nucleus (ARC), paraventricular nucleus (PVN) and nucleus tractus solitarius (NTS). Subcutaneous (sc) injection of Oxt suppressed food intake in normal and high fat diet-induced obese (DIO) mice. Daily sc injection of Oxt for 17 days in DIO mice reduced food intake for 6 days and body weight for the entire treatment period and additional 9 days after terminating Oxt. Oxt infusion by sc implanted osmotic minipumps for 13 days in DIO mice reduced food intake, body weight, and visceral fat mass and adipocyte size. Oxt infusion also decreased respiratory quotient specifically in light phase, ameliorated fatty liver and glucose intolerance, without affecting normal blood pressure in DIO mice. These results demonstrate that peripheral Oxt treatment reduces food intake and visceral fat mass, and ameliorates obesity, fatty liver and glucose intolerance. Peripheral Oxt treatment provides a new therapeutic avenue for treating obesity and hyperphagia.

Three-Year Efficacy and Safety of LB03002, a Once-Weekly Sustained-Release Growth Hormone (GH) Preparation, in Prepubertal Children with GH Deficiency (GHD)

GH treatment currently requires daily sc injections, resulting in suboptimal compliance. A GH regimen with fewer injections may offer patients and caregivers a less arduous option. LB03002 is a novel sustained-release GH formulation for once-weekly dosing. GH-deficient, GH-naive prepubertal children were randomized to four groups who received 0.2 mg/kg/wk LB03002 for 12 months, followed by 0.5 mg/kg/wk for another 24 months (n=13); 0.5 mg/kg/wk LB03002 for 36 months (n=13); 0.7 mg/kg/wk LB03002 for 12 months, followed by 0.5 mg/kg/wk for another 24 months (n=13); or daily GH 0.03 mg/kg/d for 24 months, switched to 0.5 mg/kg/wk LB03002 for 12 months (n = 12). Height velocity increased in all groups; the increase was less for the 0.2 mg/kg/wk LB03002 group at 12 (P = 0.008) and 24 months (P = 0.030), with no statistically significant differences at any time for the 0.5 mg/kg/wk and 0.7 mg/kg/wk LB03002 groups, vs. daily GH. Height sd score gain at 12 months was significantly (P = 0.023) less for the 0.2 mg/kg/wk group (1.05 ± 0.38) than daily GH (1.47 ± 0.29), but with no statistically significant difference for the 0.5 mg/kg/wk (1.37 ± 0.39) and 0.7 mg/kg/wk (1.50 ± 0.44) LB03002 groups vs. daily GH. There were no significant differences in height sd score gain between any groups at 24 and 36 months. Bone maturation did not differ for any LB03002 dose compared with daily GH. Serum IGF-I concentrations increased as expected, with no long-term differences between groups. Mean fasting glucose and glycosylated hemoglobin concentrations did not exceed normal ranges for any treatment group at any time. LB03002 at doses of 0.5 mg/kg/wk and 0.7 mg/kg/wk was shown to be effective and safe with once-weekly dosing in GH-deficient children, and 0.5 mg/kg/wk LB03002 was chosen as the optimal dose for long-term assessment.

GH, But Not GHRH, Plays a Role in the Development of Experimental Autoimmune Encephalomyelitis

GH has been suggested to influence the function of the immune system in several species. Experimental autoimmune encephalomyelitis (EAE) (an animal model for multiple sclerosis) has been reported not to occur in GH-deficient (GHD) mice. The aim of this study was to elucidate the effects of GH and GHRH replacement on development of EAE in a mouse model of isolated GHD due to removal of the GHRH gene [GHRH knockout (GHRHKO)]. We studied two groups of adult female mice: 12 GH-sufficient animals (control) and 36 GHRHKO animals. All mice were immunized with myelin oligodendrocyte glycoprotein peptide, a peptide known to induce EAE. GHRHKO mice were left untreated or were treated for 4 wk with daily sc injections of recombinant GH or of a GHRH super agonist JI-38 (JI38-GHD). Evaluation of EAE symptoms was carried out daily, and T-proliferative assay and histopathological analysis of the spinal cord were performed. GHRHKO mice were less prone to develop EAE when compared with control mice. GH (but not JI-38) restored the original susceptibility of mice to the disease, despite lack of complete serum IGF-I normalization. GH treatment was also associated with a markedly increase in spleen size and T-cell proliferation specific to myelin oligodendrocyte glycoprotein peptide. GH (but not GHRH) plays an important role in the development of EAE.

Long-Acting Growth Hormone for Replacement Therapy

GH replacement therapy has shown important beneficial effects on body composition, bone metabolism, lipid metabolism, and quality of life in adults with GH deficiency with a good safety profile (1, 2). Current management is based on daily sc injections before bedtime that are often experienced as cumbersome by the patient and may therefore affect adherence to treatment. Sustainedrelease GH formulations or long-acting GH for less frequent injections have therefore been developed. The paper by Biller et al. (3) published in this issue of the JCEM reports the results from a large 6-month study of adults with GH deficiency demonstrating efficacy and safety of a sustained-release formulation of GH with once weekly sc administration. The study concluded that the treatment was well tolerated and all clinical beneficial end-points were met, such as reduction in fat mass. GH is a powerful metabolic hormone with lipolytic actions, protein anabolic effects, and insulin antagonistic effects (4). The plasma pattern of GH, including pulse frequency, basal levels, and total integrated levels is influenced by several factors, including gender, age, and adiposity (5). Therefore, pattern of GH delivery and GH exposure may have an impact on the effects obtained by treatment. The importance of the mode of GH administration is supported by a number of animal studies demonstrating that a changed pattern of GH exposure has different effects on growth and metabolism (6). The GH secretion is suppressed by food and augmented by fasting. It is therefore a fasting hormone mobilizing energy from fat stores by lipolysis, inducing gluconeogenesis in the liver, and preserving protein mass during fasting through its protein anabolic action, i.e. in many aspects the reverse action to insulin in the postprandial state (7). Moreover, the gene expression and activity of some important regulatory enzymes in the liver are dependent on the pattern of GH exposure (8, 9). The clinical importance for safety and efficacy of not delivering GH in a physiological pulsatile manner is therefore something to consider in the pharmaceutical development of GH products for treatment. Current practice of GH treatment both in children and in adults is to administer GH as a bedtime sc injection. The basis for this practice comes from early observations that daily sc injections gave a better growth response in GHdeficient children than three injections per week (10). Moreover, the closest similarity to a normal diurnal metabolite pattern was achieved with evening sc injections of GH to GH-deficient adults (11). The diurnal plasma GH profile following daily sc evening injections is, however, an intermediate between a pulsatile and a continuous GH exposure time profile (12) and is therefore not physiological. The bedtime administration of GH means that most of the GH action occurs during the night in the fasting state. This is, however, the regime that has proved its safety and efficacy of modern GH therapy in children and in adults (1). Having a more continuous exposure, such as during treatment with long-acting GH, means that GH exerts its action both in fasting and in the fed state during the day, meaning that the normal interaction and balance with insulin is further set aside. The clinical importance of this is not clear. Whether the same efficacy and safety can be obtained by moving the GH exposure toward a more continuous pattern has been studied mainly in adults. In one small pediatric study, however, GH delivered either as daily sc injections or as a continuous GH administration using a sc pump demonstrated the same growth rate by the two treatment modes, but the serum IGF-I response was higher in the group receiving continuous GH (13). Studies in adults comparing the same dose of GH administered as a daily sc injection or as a continuous infusion have, in agreement with the pediatric experience, shown that the IGF-I and ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/jc.2011-0689 Received April 20, 2011. Accepted April 22, 2011.

Insulin and growth hormone-releasing peptide-6 (GHRP-6) have differential beneficial effects on cell turnover in the pituitary, hypothalamus and cerebellum of streptozotocin (STZ)-induced diabetic rats

Poorly controlled type1 diabetes is associated with hormonal imbalances and increased cell death in different tissues, including the pituitary, hypothalamus and cerebellum. In the pituitary, lactotrophs are the cell population with the greatest increase in cell death, whereas in the hypothalamus and cerebellum astrocytes are most highly affected. Insulin treatment can delay, but does not prevent, diabetic complications. As ghrelin and growth hormone (GH) secretagogues are reported to prevent apoptosis in different tissues, and to modulate glucose homeostasis, a combined hormonal treatment may be beneficial. Hence, we analyzed the effect of insulin and GH-releasing peptide 6 (GHRP-6) on diabetes-induced apoptosis in the pituitary, hypothalamus and cerebellum of diabetic rats. Adult male Wistar rats were made diabetic by streptozotocin injection (65 mg/kg ip) and divided into four groups from diabetes onset: those receiving a daily sc injection of saline (1 ml/kg/day), GHRP-6 (150 μg/kg/day), insulin (1–8 U/day) or insulin plus GHRP-6 for 8 weeks. Control non-diabetic rats received saline (1 ml/kg/day). Diabetes increased cell death in the pituitary, hypothalamus and cerebellum ( P < 0.05). In the pituitary, insulin treatment prevented diabetes-induced apoptosis ( P < 0.01), as well as the decline in prolactin and GH mRNA levels ( P < 0.05). In the hypothalamus, neither insulin nor GHRP-6 decreased diabetes-induced cell death. However, the combined treatment of insulin + GHRP-6 prevented the diabetes induced-decrease in glial fibrillary acidic protein (GFAP) levels ( P < 0.05). In the cerebellum, although insulin treatment increased GFAP levels ( P < 0.01), only the combined treatment of insulin + GHRP-6 decreased diabetes-induced apoptosis ( P < 0.05). In conclusion, insulin and GHRP-6 exert tissue specific effects in STZ-diabetic rats and act synergistically on some processes. Indeed, insulin treatment does not seem to be effective on preventing some of the diabetes-induced alterations in the central nervous system.

Pegylated Long-Acting Human Growth Hormone Possesses a Promising Once-Weekly Treatment Profile, and Multiple Dosing Is Well Tolerated in Adult Patients with Growth Hormone Deficiency

Recombinant human GH (rhGH) replacement therapy in children and adults currently requires daily sc injections for several years or lifelong, which may be both inconvenient and distressing for patients. NNC126-0083 is a pegylated rhGH developed for once-weekly administration. Our objective was to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple doses of NNC126-0083 in adult patients with GH deficiency (GHD). Thirty-three adult patients with GHD, age 20-65 yr, body mass index 18.5-35.0 kg/m(2), and glycated hemoglobin of 8.0% or below. Fourteen days before randomization, subjects discontinued daily rhGH. NNC126-0083 (0.01, 0.02, 0.04, and 0.08 mg/kg) was given sc once weekly for 3 wk (NNC126-0083 for six subjects and placebo for two subjects). Blood samples were collected up to 168 h after the first and up to 240 h after the third dosing. Physical examination, antibodies, and local tolerability were assessed. NNC126-0083 was well tolerated with no difference in local tolerability compared with placebo and with no signs of lipoatrophy. A more than dose-proportional exposure was observed at the highest NNC126-0083 dose (0.16 mg protein/kg). Steady-state pharmacokinetics seemed achieved after the second dosing. A clear dose-dependent pharmacodynamic response in circulating IGF-I levels was observed [from a predose mean (SD) IGF-I SD score of -3.2 (1.7) to peak plasma concentration of -0.5 (1.3), 1.6 (1.3), 2.1 (0.5), and 4.4 (0.9) in the four dose groups, respectively]. After multiple dosing of NNC126-0083, a sustained pharmacodynamic response was observed. NNC126-0083 has the potential to serve as an efficacious, safe, and well-tolerated once-weekly treatment of adult patients with GHD.

N-Glycosylation Increases the Circulatory Half-Life of Human Growth Hormone

Therapeutic use of recombinant GH typically involves daily sc injections. We examined the possibilities for prolonging the in vivo circulation of GH by introducing N-glycans. Human GH variants with a single potential N-glycosylation site (N-X-S/T) introduced by site-directed mutagenesis were expressed in HEK293 cells. In a scan of 15 different positions for N-glycosylation sites, four positions (amino acids 93, 98, 99, and 101) were efficiently utilized and did not influence GH in vitro activity. A GH variant (3N-GH) with all these sites was produced in CHOK1SV cells and contained up to three N-glycans. Two pools of 3N-GH were purified and separated according to their charge by anion-exchange chromatography. Anion-exchange HPLC revealed that the N-glycans in the two pools were very similar except for the extent of sialylation. Both 3N-GH pools circulated longer in rats than wild-type GH. The terminal half-life of 3N-GH after iv injection was 24-fold prolonged compared with wild-type GH for the pool with the most pronounced sialylation, 13-fold prolonged for the less sialylated pool, and similar to the wild-type for desialylated 3N-GH. The less sialylated 3N-GH pool exhibited a profound pharmacodynamic effect in GH-deficient rats. Over a 4-d period, a single injection of 3N-GH induced a stronger IGF-I response and a larger increase in body weight than daily injections with wild-type GH. Thus, N-glycans can prolong the in vivo circulation and enhance the pharmacodynamic effect of GH. Sialic acids seem to play a pivotal role for the properties of glycosylated GH.