Protective effect of alamandine on doxorubicin‑induced nephrotoxicity in rats

Background This study aimed to evaluate the protective effects of alamandine, a new member of the angiotensin family, against doxorubicin (DOX)-induced nephrotoxicity in rats. Methods Rats were intraperitoneally injected with DOX (3.750 mg/kg/week) to reach a total cumulative dose of 15 mg/kg by day 35. Alamandine (50 µg/kg/day) was administered to the rats via mini-osmotic pumps for 42 days. At the end of the experiment, rats were placed in the metabolic cages for 24 h so that their water intake and urine output could be measured. After scarification, the rats’ serum and kidney tissues were collected, and biochemical, histopathological, and immunohistochemical studies were carried out. Results DOX administration yielded increases in pro-inflammatory cytokines, including interleukin (IL)-1β and IL-6, pro-fibrotic proteins transforming growth factor-β (TGF-β), pro-inflammatory transcription factor nuclear kappa B (NF-κB), kidney malondialdehyde (MDA), creatinine clearance, blood urea nitrogen (BUN), and water intake. On the other hand, the DOX-treated group exhibited decreased renal superoxide dismutase (SOD), renal glutathione peroxidase (GPx) activity, and urinary output. Alamandine co-therapy decreased these effects, as confirmed by histopathology and immunohistochemical analysis. Conclusions The results suggest that alamandine can prevent nephrotoxicity induced by DOX‎ in rats.

Effects of CRN04894, a Nonpeptide Orally Bioavailable ACTH Antagonist, on Corticosterone in Rodent Models of ACTH Excess

CRN04894 is an orally administered nonpeptide that is a potent and selective antagonist for adrenocorticotropic hormone (ACTH) acting at the melanocortin 2 receptor (MC2R) and is currently under development for the treatment of diseases of ACTH excess such as Cushing’s disease, congenital adrenal hyperplasia, and ectopic ACTH-secreting tumors. Cushing’s disease results from an adenoma derived from pituitary corticotropic cells that secrete excess ACTH, whereas ectopic ACTH syndrome arises from nonpituitary ACTH secreting tumors. Congenital adrenal hyperplasia is a genetic disease that results in cortisol deficiency leading to high levels of ACTH and adrenal androgens. Each of these indications is characterized by high ACTH levels that act on MC2R expressed in the adrenal cortex to drive pathological elevations of adrenally derived steroid hormones. CRN04894 blocks the action of ACTH at MC2R, providing a potential novel treatment for these diseases. Preclinical models of chronic hypercortisolemia include implantation of ACTH-secreting pituitary tumor cells in mice and continuous administration of ACTH via subcutaneously implanted osmotic pumps in rats. These models induce features consistent with human diseases of ACTH excess including hypercortisolemia and hypertrophy of the adrenal glands. We employed both rodent models to examine the pharmacodynamic effects of CRN04894 on corticosterone levels and adrenal gland morphology. In the mouse pituitary tumor model, subcutaneous inoculation of the ACTH-secreting mouse pituitary tumor cell line, AtT-20, into immunodeficient mice resulted in formation of tumors and increased plasma ACTH and corticosterone levels. Repeated daily oral administration of CRN04894 for 14 days dose-dependently and robustly suppressed plasma corticosterone levels in mice with AtT-20 tumors. In the rat model, subcutaneous implantation of osmotic pumps delivering ACTH resulted in increased corticosterone levels, reduction in body weight, and hypertrophy of the adrenal glands after 7 days. Daily oral administration of CRN04894 over 7 days dose-dependently suppressed corticosterone levels, mitigated the effect of ACTH excess on body weight, and rescued the adrenal gland hypertrophy. These findings provide evidence that CRN04894 functions as an effective ACTH antagonist at MC2R to suppress adrenal corticosterone secretion in both mouse and rat models of ACTH excess and hypercortisolemia, thus providing a strong rationale for its potential therapeutic utility in diseases of ACTH excess. This work was supported in part by an SBIR grant from the NIH awarded to Dr. Struthers (R43- DK115245)

Abstract P815: Decoding the Cross-Talk Between Grafted Neural Stem Cells and Host Brain to Predict the Molecular Mechanisms of Stem Cell-Induced Functional Recovery After Stroke

Stroke is a leading cause of long-term disability and death in the united states. The development of new therapies for stroke are sorely needed. There is great hope that stem cell therapy will create a paradigm shift in the treatment of stroke patients. A barrier to ensuring clinical success of stem cell therapy is the paucity of understanding of the mechanisms by which stem cells exert their beneficial effects. Using a novel mRNA purification method, we identified 50 genes encoding extracellular space proteins, expressed by human neural stem cells (hNSCs) whose expression positively correlated with functional recovery. In this study, we focus on one of the paracrine factors from grafted hNSCs that correlated best with functional recovery, to investigate its therapeutic potential in promoting recovery after stroke. Male nude rats underwent stroke using the distal middle cerebral artery occlusion (dMCAo) model. One week following stroke, osmotic pumps were prepared and loaded with recombinant MTN-2. The osmotic pumps were inserted into the peri-infarct area and infused recombinant MTN-2 for 5 days. Post-stroke, animals were assessed for functional recovery for 5 weeks using both the Montoya staircase test and the whisker-paw reflex test to assess for forelimb function, dexterity, side bias, and placing deficits. After 5 weeks, brain tissue was isolated to assess glial cell morphology. Brain sections were stained with GFAP and IBA1 to visualize astrocytes and microglia, respectively. Confocal images were processed and analyzed using the Bitplane Imaris image analysis software. Output measurements of number of cells/mm2, cell volume, cell branching, and process length and thickness were obtained to characterize the changes in astrocytic and microglial response to injury and paracrine factor treatment. By identifying paracrine factors that are responsible for the regeneration of brain tissue following implantation of hNSCs in stroke brain, this work will increase the likelihood of successful clinical translation of stem cell therapy for stroke. Moreover, elucidating these molecular pathways important for brain recovery may ultimately identify novel therapeutic targets and offer hope to millions of Americans who live with the devastating effects of stroke.

Trabecular and cortical bone are unaltered in response to chronic lipopolysaccharide exposure via osmotic pumps in male and female CD-1 mice

Chronic low-grade inflammation has been identified as an underlying cause of many diseases including osteoporosis. Lipopolysaccharide (LPS) is a potent inducer of the inflammatory response that can negatively affect bone outcomes by upregulating bone resorption and inhibiting bone formation. The objective of this study was to assess the longitudinal response of trabecular and cortical bone structure and bone mineral density to LPS continuously administered for 12 weeks in male and female CD-1 mice. Mice were assigned to one of four LPS groups at 8-weeks of age: placebo (0.0 μg/d), low (0.9 μg/d), mid (3.6 μg/d) and high (14.4 μg/d) dose. Trabecular and cortical bone outcomes were measured at 8, 12, 16, and 20 weeks of age using in vivo micro-computed tomography. The anticipated serum LPS dose-dependent response was not observed. Therefore, the low, mid, and high LPS groups were combined for analysis. Compared to the placebo group, endpoint serum LPS was elevated in both males (p < 0.05) and females (p < 0.05) when all LPS treatment groups were combined. However, there was no significant change in trabecular or cortical bone outcomes in the combined LPS groups compared to the placebo following the 12-week LPS intervention for either sex. This suggests that although serum LPS was elevated following the 12-week LPS intervention, the dosages administered using the osmotic pumps was not sufficient to negatively impact trabecular or cortical bone outcomes in either male or female CD-1 mice.

Protective Effect of Alamandine on Doxorubicin‑Induced Nephrotoxicity in Rats

Background: The objective of this study was to evaluate the protective effects of alamandine, a new member of the angiotensin family, against doxorubicin (DOX)-induced nephrotoxicity in rats.Methods: Rats, intraperitoneally injected with DOX (3.750 mg/kg/week) to reach total cumulative dose of 15 mg/kg on day 35. Alamandine (50µg/kg/day) was administered to the rats via mini-osmotic pumps for 42 days. At the end of experiment, rats were placed in the metabolic cages for 24 h for measurement of water intake and urine output. After scarification, Serum and kidney tissues were collected, and biochemical, histopathological and immunohistochemical studies were carried out.Results: Inflammatory cytokines (IL-1β, IL-6), pro-fibrotic mediator (TGF-β), pro-inflammatory transcription factor (NF-kB), renal MDA, creatinine clearance, BUN, and water intake were increased by DOX administration. On the other hand, renal SOD, renal GPx activity and urinary output were decreased in the DOX-treated group. Alamandine co-therapy decreased these effects, as confirmed by histopathology and immunohistochemical analysisConclusion: The results of this study suggest that alamandine has the potential in preventing the nephrotoxicity induced by DXR in rats.

Critical Factors for Metformin Osmotic Controlled Release Pump

Aim: Studied the critical factors in the design of an osmotic pump with metformin release rate constant at 4%/hr in diabetes mellitus within 24 hr. with the goal to reduce daily medications. Study Design: Experimental design 32 Methodology: In vitro drug release profiles for 24h. The effects of different formulation variables, that is, concentration of hydrophilic polymer, diameter of drug releasing orifice and coating thickness, on the drug release profile were investigated. Also, the impact of pH, osmotic pressure and morphology with stereo microscopy and scanning electronic microscopy of the osmotic pumps were investigated. At last, osmotic pumps surface was analyze with scanning electronic microscopy. Results: Metformin osmotic pump were successfully prepared in this study to overcome the weak point of multiple doses and great concentration fluctuation of metformin. The formulation determined finally have a release orifice of 700 mm and 3.0% of weight gain, achieved the desired effect which can realize the constant drug release rate at the first 24 hr. Conclusion: The developed osmotic systems have a linear release near 4%/hr. and demonstrated that the behavior was independent of the agitation intensity and the pH of the gastrointestinal apparatus.

Vasohibin-2 Aggravates Development of Ascending Aortic Aneurysms But Not Abdominal Aortic Aneurysms Nor Atherosclerosis in ApoE Deficient Mice

Background Vasohibin-2 (VASH2) has been isolated as a homologue of vasohibin-1 (VASH1) that promote angiogenesis counteracting with VASH1. Chronic angiotensin II (AngII) infusion promotes both ascending and abdominal aortic aneurysms (AAs) in mice. The present study aimed to investigate whether exogenous VASH2 influenced AngII-induced vascular pathology in apolipoprotein E deficient (ApoE -/-) mice. Methods Male, ApoE -/- mice (9 to 14 weeks old) were injected with Ad LacZ or Ad VASH2. After a week, saline or AngII (1,000 ng/kg/min) was infused into the mice subcutaneously via mini-osmotic pumps for 3 weeks. Consequently, all these mice were divided into 4 groups: saline + LacZ (n=5), saline + VASH2 (n=5), AngII + LacZ (n=18), and AngII + VASH2 (n=17). Results Exogenous VASH2 had no significant effect on ex vivo maximal diameters of abdominal aortas (AngII + LacZ; 1.67±0.17 mm, AngII + VASH2; 1.52±0.16 mm, n.s.) or elastin fragmentation and accumulation of inflammatory cells. Conversely, exogenous VASH2 significantly increased intima areas of aortic arches (AngII + LacZ; 16.6±0.27 mm 2, AngII + VASH2; 18.6±0.64 mm 2, p=0.006). VASH2 effect of AngII-induced ascending AAs was associated with increased cleaved caspase-3 abundance. AngII-induced atherosclerosis was not altered by VASH2. Conclusion The present study demonstrated that augmented VASH2 expression had no effect of AngII-induced abdominal AAs or atherosclerosis, while increasing dilation in the ascending aorta.

72 Endocrine and ovarian responses to combined estradiol benzoate-sulpiride in anestrous mares treated with kisspeptin

The objective of this study was to determine if incorporation of kisspeptin 10 (Kp10) into an estradiol benzoate (EB)-sulpiride treatment would result in greater endocrine responses, and a greater number of mares ovulating within 28 days of treatment compared to EB-sulpiride alone. Eighteen anestrous mares were blocked by horse type (light horse and pony crosses), body condition, and age, then randomly assigned to treatment or control. On day 0, all mares received 50 mg EB. On day 1, mini osmotic pumps containing saline (n = 9) or Kp10 (50 mg/hour; n = 9) were inserted subcutaneously in the neck and remained for 7 days. Serial blood sampling occurred for 24 hours after pump placement. On day 2, all mares received 3 g sulpiride. Serial blood sampling continued for 36 hours and daily for 28 days. Transrectal ultrasounds were performed regularly for detection of ovulation. Plasma was assayed for prolactin, luteinizing hormone (LH) and progesterone. Data were analyzed by ANOVA with repeated measures. Plasma prolactin increased (P &lt; .001) in response to sulpiride in all mares and remained stimulated for 7 days. Prolactin responses tended to be greater (P = .09) in Kp10- treated mares compared to controls. No differences were detected in plasma LH during the first 24 hours after pump placement; however, LH increased in all mares beginning 5 days after sulpiride and were greater (P &lt; .05) in Kp10-mares from day 7 to day 21. Eleven of 18 (61%) mares ovulated within 18 days of sulpiride treatment; however, no differences in ovulation dates were detected between Kp10 treated- and control- mares. No differences were detected in plasma progesterone during the first 5 days post ovulation. In conclusion, incorporation of Kp10 potentiated the prolactin and LH responses to EB-sulpiride, but did not further advance first ovulation in treated-mares.