Protein hydrolysates from boarfish (Capros aper) and Atlantic salmon (Salmo salar) skin gelatin improve metabolic control in genetically obese diabetic (ob/ob) mice

There is increasing interest in dietary protein for management of Type 2 diabetes mellitus (T2DM) and obesity. The effects of twice-daily oral administration of a salmon skin gelatin hydrolysate (SSGH, 50 mg/kg), boarfish protein hydrolysate (BPH, (50 mg/kg), metformin (200 mg/kg), or saline control, were investigated in ob/ob mice. Non-fasting blood glucose was significantly reduced with SSGH (p<0.01), BPH (p<0.001) and metformin (p<0.001), which were reflected in reductions in glycated haemoglobin (HbA1c) (p<0.001, p<0.01 and p<0.01, respectively). Responses to oral and intraperitoneal glucose tolerance were improved (p<0.05-0.01), as well as circulating plasma lipid profiles (p<0.05-0.001). Chronic BPH treatment increased circulating plasma insulin (p<0.01), whereas SSGH improved insulin sensitivity (p<0.05), versus respective controls. All treatments significantly reduced energy intake (p<0.05-<0.001) versus (ob/ob) controls, without affecting overall bodyweight. These findings suggest that fish hydrolysates mediate potent anti-diabetic actions similar to metformin and might be suitable for the management and prevention of T2DM.

A Monoclonal Antibody Targeting ALK2 As a Potential Therapeutic Agent for Anemia of Inflammation

Background: Iron homeostasis is primarily regulated by hepcidin, a hormone predominantly expressed in the liver. Hepcidin activates the degradation of the transmembrane iron exporter ferroportin, thereby downregulating the release of iron from cells. Hepcidin expression is, at least partly, regulated in response to signaling of the type I TGF-β receptor ALK2, via SMAD2/3 phosphorylation. IL-6, which is commonly elevated in chronic kidney disease (CKD) and other inflammatory conditions, upregulates hepcidin expression and reduces serum iron bioavailability. As a result, chronic inflammatory conditions are often accompanied by secondary anemia of inflammation (AI). We have previously demonstrated that ALK2 inhibition suppressed hepcidin expression in rodents, monkeys and healthy humans. We further described that administration of a selective small molecule ALK2 kinase inhibitor (KTI-2338) reversed changes in hepcidin and iron in a mouse model of CKD, supporting the potential benefit of ALK2 inhibition in AI. Another approach to targeting ALK2 signaling is use of a neutralizing antibody. KTI-018 is a neutralizing ALK2 antibody with high affinity and selectivity for ALK2. This biologic has been demonstrated to reduce serum hepcidin and increase serum iron in healthy non-human primates. Aims: To further elucidate the specific contribution of ALK2 signaling as a driver in AI, and to determine the therapeutic potential of the antibody in this type of anemia, we assessed the effect of KTI-018 in the CKD mouse model. Methods: The study was conducted with 6-week-old male C57Bl/6 mice. Mice in the CKD cohort (CKD) were treated with once daily oral administration of adenine, a compound that metabolizes to 2,8-dihydroxyadenine, forming crystals in the proximal tubular epithelia and causing inflammation and fibrosis in the kidneys. Mice in the control cohort (healthy) received once daily oral administration of vehicle. Upon confirmation of disease, the CKD cohort was subdivided into two groups. The treatment group received twice weekly intraperitoneal treatment with KTI-018 (CKD-KTI-018), and the control group received tris-buffered saline (CKD-TBS). Healthy mice received TBS only. All mice were maintained on their assigned daily adenine or vehicle regimen. At day 53, the study was terminated and hematologic parameters, serum hepcidin, iron, and IL-6 levels were assessed. Results: After 42 days of adenine or vehicle administration, serum hepcidin, serum iron, and hematologic parameters were assessed in representative cohorts of CKD-TBS and healthy mice. The CKD-TBS cohort experienced changes associated with anemia of inflammation as compared to the healthy mice, including increased hepcidin, decreased serum iron, and decreased hematologic parameters. The differences between the healthy and CKD-TBS groups were maintained through the duration of the study. At study termination, CKD-TBS mice had increased serum IL-6 levels (218%), elevated serum hepcidin (149%), and reduced serum iron (-30%) as compared to the healthy mice. Laboratory findings characteristic of anemia were present in the CKD-TBS group, including decreased red blood cells (-6.1%), hemoglobin (-13.2%), and reticulocyte hemoglobin content (-9.3%) as compared to healthy mice. In contrast, CKD-KTI-018 mice had decreased serum hepcidin (-25%) and increased serum iron (59%) as compared to CKD-TBS mice. This restoration of serum iron corresponded to improvements in red blood cells, hemoglobin, and reticulocyte hemoglobin content, which were increased by 7.6%, 9.6%, and 6.7%, respectively, in the CKD-KTI-018 mice as compared to the CKD-TBS mice. These results demonstrate that, by decreasing serum hepcidin, KTI-018 increased the bioavailability of iron, which led to the restoration of hematologic parameters and appeared to reverse AI in mice. Discussion: In this study, a neutralizing ALK2 antibody decreased serum hepcidin, increased serum iron and consequently reversed AI in a mouse model of CKD. These results support the role of ALK2 signaling in AI and suggest that inhibition of ALK2 may be a potential treatment approach for anemia resulting from CKD and other chronic inflammatory diseases. Future studies will explore if ALK2 inhibition may prevent or treat progression of CKD itself, and the role that ALK2 inhibition may play in other chronic inflammatory conditions associated with elevated hepcidin. Disclosures Medeiros: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Backus: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Materna: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Fisher: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Seehra: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Oral prodrug of remdesivir parent GS-441524 is efficacious against SARS-CoV-2 in ferrets

Remdesivir is an antiviral approved for COVID-19 treatment, but its wider use is limited by intravenous delivery. An orally bioavailable remdesivir analog may boost therapeutic benefit by facilitating early administration to non-hospitalized patients. This study characterizes the anti-SARS-CoV-2 efficacy of GS-621763, an oral prodrug of remdesivir parent nucleoside GS-441524. Both GS-621763 and GS-441524 inhibit SARS-CoV-2, including variants of concern (VOC) in cell culture and human airway epithelium organoids. Oral GS-621763 is efficiently converted to plasma metabolite GS-441524, and in lungs to the triphosphate metabolite identical to that generated by remdesivir, demonstrating a consistent mechanism of activity. Twice-daily oral administration of 10 mg/kg GS-621763 reduces SARS-CoV-2 burden to near-undetectable levels in ferrets. When dosed therapeutically against VOC P.1 gamma γ, oral GS-621763 blocks virus replication and prevents transmission to untreated contact animals. These results demonstrate therapeutic efficacy of a much-needed orally bioavailable analog of remdesivir in a relevant animal model of SARS-CoV-2 infection.

Combined Correction of Experimental Critical Ischemia of the Lower Extremities in a Rat Model

The purpose of our research was to study the effectiveness of correcting experimental critical ischemia (CI) of the lower extremities with a combination of udenafil, simvastatin, and autologous bone marrow mononuclear cells (ABMMC). Methods and Results: The experiments were carried out on 24-month-old Wistar rats, weighing 220–250g. The animals were randomized by sex and weight. Groups were formed according to the manipulations carried out during the operations. The animals were divided into 7 groups, each with 20 animals: Group 1 included intact animals; Group 2 - falsely operated animals; Group 3 (control group) - animals with simulated CI without treatment; Group 4- animals with CI and monotherapy with udenafil (daily oral administration of 8.6mg/kg for 28 days); Group 5 - animals with CI and simvastatin monotherapy (daily oral administration of 1.71mg/kg for 28 days); Group 6 - animals with CI and monotherapy with ABMMC (parenterally, once on Day 7 after modeling CI, 50μl at 4 points and, paravasally, above the inguinal ligament in the area where the lateral artery leaves the artery enveloping the femur from the internal iliac artery; in the area of the superficial artery that bends around the iliac bone under the inguinal ligament; into the area of origin of the muscular branch of the femoral artery r. muscularis, the place of attachment of the comb and long adductor muscles of the thigh; in the upper third of the gastrocnemius muscle]); Group 7 - animals with CI and combination therapy (udenafil and simvastatin drugs were administered intragastrically 0.86 mg/kg, once a day, for 7 days) and one-time parenteral administration of ABMMC, according to the same scheme as in Group 6. On Days 21 and 28 of the experiment, the level of blood microcirculation was determined in the muscles of the rat leg; for this, laser Doppler flowmetry was used. For further morphometric assessment of the leg muscles, they were removed. Preparations for morphometric analysis were prepared according to the standard technique with Van Gieson staining, as well as H&E. Our study demonstrated the effectiveness of combination therapy with udenafil, simvastatin, and ABMMC to correct critical lower limb ischemia in rats. The severity of morphological changes on the background of this combination was minimal, compared to the findings of other study groups, and the level of blood microcirculation in the ischemic zone on Day 28 was, significantly, 1.9 times higher than in animals of the control group. The results obtained allow us to recommend the use of the investigated combination (udenafil+simvastatin+ABMMC) for the treatment of patients with critical limb ischemia, both in outpatient and inpatient practice.

Oral prodrug of remdesivir parent GS-441524 is efficacious against SARS-CoV-2 and a variant of concern in ferrets

Remdesivir is the only small-molecule antiviral approved to date for COVID-19 treatment, but its wider use is limited by intravenous delivery. An orally bioavailable remdesivir analog may boost therapeutic benefit by facilitating early administration to non-hospitalized patients. This study characterized the anti-SARS-CoV-2 efficacy of GS-621763, an oral prodrug of remdesivir parent nucleoside GS-441524. Both GS-621763 and GS-441524 inhibited SARS-CoV-2, including variants of concern (VoC) in cell culture. Oral GS-621763 was efficiently converted to plasma metabolite GS-441524, and in lungs to the triphosphate metabolite identical to that generated by remdesivir, demonstrating a consistent mechanism of activity. Twice-daily oral administration of 10 mg/kg GS-621763 reduced SARS-CoV-2 burden to near-undetectable levels. When dosed therapeutically against VoC P.1 gamma (γ), oral GS-621763 blocked virus replication and prevented transmission to untreated contact animals. These results demonstrate therapeutic efficacy of a much-needed orally bioavailable analog of remdesivir in a relevant animal model of SARS-CoV-2 infection.

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)

Inhibition of CDK4/6 Overcomes Primary Resistance to PD-1 Blockade in Malignant Mesothelioma

ABSTRACTBackgroundDespite the profound number of malignant pleural mesothelioma (MPM) patients now treated with PD-1 blockade, insight into the underpinnings of rational therapeutic strategies to treat resistance to checkpoint immunotherapy remain unrealized. Our objective was to develop a novel therapeutic approach to overcome primary resistance to PD-1 blockade in MPM.MethodsWe generated a transcriptome signature of resistance to PD-1 blockade in MPM patients treated with nivolumab (4 responders and 4 non-responders). We used the TCGA MPM cohort (N=73) to determine what genomic alterations were associated with the resistance signature. We tested whether regulation of identified molecules could overcome resistance to PD-1 blockade in an immunocompetent mouse malignant mesothelioma model.ResultsImmunogenomic analysis by applying our anti-PD-1 resistance signature to the TCGA cohort revealed that deletion of CDKN2A was highly associated with primary resistance to PD-1 blockade. Under the hypothesis that resistance to PD-1 blockade can be overcome by CDK4/6 inhibition, we tested whether CDK4/6 inhibitors could overcome resistance to PD-1 blockade in subcutaneous tumors derived from Cdkn2a(−/−) AB1 malignant mesothelioma cells, which were resistant to PD-1 blockade. The combination of daily oral administration of CDK4/6 inhibitors (abemaciclib or palbociclib) and intraperitoneal anti-PD-1 treatment markedly suppressed tumor growth, compared with anti-PD-1 or CDK4/6 inhibitor alone.ConclusionsWe identified a novel therapeutic target, CDK4/6, to overcome primary resistance to PD-1 blockade through comprehensive immunogenomic approaches. These data provide a rationale for undertaking clinical trials of CDK4/6 inhibitors in the more than 40% of patients with MPM who demonstrate loss of CDKN2A.

Application of Ligilactobacillus salivarius CECT5713 to Achieve Term Pregnancies in Women with Repetitive Abortion or Infertility of Unknown Origin by Microbiological and Immunological Modulation of the Vaginal Ecosystem

In this study, the cervicovaginal environment of women with reproductive failure (repetitive abortion, infertility of unknown origin) was assessed and compared to that of healthy fertile women. Subsequently, the ability of Ligilactobacillus salivarius CECT5713 to increase pregnancy rates in women with reproductive failure was evaluated. Vaginal pH and Nugent score were higher in women with reproductive failure than in fertile women. The opposite was observed regarding the immune factors TGF-β 1, TFG-β 2, and VEFG. Lactobacilli were detected at a higher frequency and concentration in fertile women than in women with repetitive abortion or infertility. The metataxonomic study revealed that vaginal samples from fertile women were characterized by the high abundance of Lactobacillus sequences, while DNA from this genus was practically absent in one third of samples from women with reproductive failure. Daily oral administration of L. salivarius CECT5713 (~9 log10 CFU/day) to women with reproductive failure for a maximum of 6 months resulted in an overall successful pregnancy rate of 56%. The probiotic intervention modified key microbiological, biochemical, and immunological parameters in women who got pregnant. In conclusion, L. salivarius CECT5713 has proved to be a good candidate to improve reproductive success in women with reproductive failure.

Butterbur (Petasites hybridus) Extract Ameliorates Hepatic Damage Induced by Ovalbumin in Mice

The liver is the most vital organ that could be influenced by inducers of hypersensitivity such as ovalbumin. The current study was carried out to explore the effects of butterbur (Petasites hybridus) extract on the ovalbumin-induced liver hypersensitivity in Swiss albino male mice. Animals were divided into 4 groups, 1st group served as a control group, 2nd group treated with daily oral administration of 75 mg/kg of butterbur extract, 3rd group received single oral dose 100 mg/kg of ovalbumin to induce hypersensitivity, and 4th group treated with oral administration of butterbur extract one-day post to the hypersensitivity induction. Ovalbumin induces a significant increase in the activity of liver enzymes and MDA and decreased the activity of CAT after the ovalbumin treatment. Histopathological investigations revealed marked pathological alterations in liver tissues in the form of hyaline degeneration and fibrosis. Additionally, heavy immune response indicated by immunostaining of MDA and TNF-α could be observed. In contrast, posttreatment with butterbur extract after hypersensitivity induction resulted in a significant decrease of liver enzymes and oxidative stress and reduced the inflammation and fibrosis of liver tissues. These results suggest that butterbur extract is considered as anti-inflammatory and antioxidant therapeutic herb for hypersensitivity treatment of liver.

Anthocyanin Extract from Flowers of Lavender dentate Beats Oxidative Stress In vitro and In vivo

Free radicals, oxidative stress or antioxidants is more and more often used to explain different pathological disorders and their therapeutic approach. The aim of this study was to evaluate the protective effect of anthocyanin extract obtained by maceration of the flowers of Lavandula dentata in 0.1 % HCl/methanol (v/v) solution on oxidative stress. The antioxidant activity of anthocyanin extract in vitro was evaluated by reduction of iron (FRAP), DPPH and the ß-carotene tests. The in vivo oxidative stress was induced by intraperitoneal injection of 0.5 ml/kg of CCl4 and treated orally by 500mg kg/day of the extract. Anthocyanins extract inhibited the free radical DPPH (IC50:1.3 ± 0.23 mg/ml). Lavender extract prevented the oxidation of B carotene (28.34 ± 0.07%) and has an ability to reduce iron (0.736 ± 0.03). Intraperitoneal injection of CCl4 has increased biochemical parameters, which was evidence of oxidative stress in vivo. In contrast, daily oral administration of anthocyanin extract has restored the biochemical parameters. Histopathological examinations of liver stained with haematoxylin and eosin showed loss of hepatic architecture. These injuries observed have been improved by treatment with anthocyanin extract. The findings revealed that anthocyanin extract from lavender possesses a significant antioxidant activity. Dhaka Univ. J. Pharm. Sci. 19(2): 145-151, 2020 (December)