bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitophagy‐mediated NLRP3 inflammasome inhibition by andrographolide contributes to the prevention of colitis‐associated cancer (1052.3)
Authors: Sun Yang , Guo Wenjie , Xu Qiang
Published: June 2021

Intestinal nonresolving inflammation predisposes to the development of colitis‐associated cancer (CAC). Inflammasomes are believed to mediate intestine homeostasis, and their dysregulation contribute to inflammatory diseases and CAC. However, few agent capable of targeting inflammasomes to reduce CAC has been reported thus far. Here we report that small molecule andrographolide protects mice against CAC in the mouse model of azoxymethane combination of dextran sulfate sodium through inhibiting NLRP3 inflammasome. Oral administration of andrographolide significantly attenuated the colitis progression and tumors burden, which was accompanied by lessened body weight loss, lower disease activity index, decreased expression of cyclooxygenase‐2 and proinflammatory cytokines, lower activation of NF‐κB and STAT3, as well as reduced cell proliferation and angiogenesis in the colons of CAC mice. Further study showed that andrographolide inhibited NLRP3 inflammasome activation in macrophage in vivo and in vitro, as indicated by reduced expression of cleaved CASP1, disruption of NLRP3/PYCARD/CASP1 complex assembly and lower IL‐1β secretion. Moreover, andrographolide was found to trigger mitophagy in macrophage, leading to the less mitochondrial membrane potential collapse, and this in turn inactivated the NLRP3 inflammasome. Furthermore, inhibition of the PIK3CA‐AKT1‐MTOR‐RPS6KB1 pathway accounted for the induction of autophagy by andrographolide. Finally, andrographolide‐induced inhibition of IL‐1β production was blocked by the knockdown of BECN1 expression, or by autophagy inhibitor 3‐methyladenine. Taken together, our findings demonstrate that andrographolide‐driven mitophagy‐mediated NLRP3 inflammasome inhibition is responsible for the prevention of CAC. Our data may help to guide decisions regarding the use of andrographolide in colitis patients, which ultimately reduces the risk of CAC.

Grant Funding Source: supported by NSFC of China (Nos. 91229109, 90913023, 81121062, 91129728 and 81273528)
Suppression of colorectal cancer subcutaneous xenograft and experimental lung metastasis using nanoparticle‐mediated drug delivery to tumor neovasculature (1052.4)
Authors: Wang Chao , Zhao Mei , Liu Ya‐Rong , Luan Xin , Guan Ying‐Yun , Lu Qin , Chen Hong‐Zhuan , Fang Chao
Published: June 2021

Antiangiogenic therapy is a validated approach for colorectal cancer (CRC) treatment. However, diverse adverse effects inevitably appear due to the off‐target effect of the approved antiangiogenic inhibitors on the physiological functions and homeostasis. This study was to investigate a new tumor vessel targeting nanoparticulate drug delivery system, F56 peptide conjugated nanoparticles loading vincristine (F56‐VCR‐NP), for the effective treatment of CRC subcutaneous xenograft and experimental lung metastasis model. The controlled release behavior and in vivo pharmacokinetic profile of F56‐VCR‐NP were characterized. The tumor vessel targeting and antiangiogenic activity of F56‐VCR‐NP was evaluated in human umbilical vein endothelial cells (HUVEC, a classical cell model mimicking tumor vascular EC), subcutaneous human HCT‐15 xenograft in immunodeficient nude mice, and experimental CT‐26 lung metastasis model in immunocompetent mice. The therapeutic efficacy (animal survival and toxicity) was further investigated in the model of CT‐26 lung metastasis in mice. F56‐VCR‐NP could achieve 30‐day controlled drug release in PBS (pH 7.4) and exhibited favorable long‐circulating feature in vivo. F56‐VCR‐NP could accurately target the CRC neovasculature and elicit nanoparticle internalization in the tumor vascular EC, where the antiangiogenic VCR‐induced dramatic EC apoptosis and necrosis of CRC tissue. F56‐VCR‐NP significantly prolonged the mouse survival with no obvious toxicity (weight loss and anepithymia) in the CT‐26 lung metastasis mice model, and this pronounced antitumor effect was closely related with the decreased microvessel density in the metastases. The present nanoparticle‐based targeted antiangiogenic therapy may provide a new promising approach for the therapy of CRC and lung metastasis, which deserves further translational research.
Role of substance P and CGRP in gastric MALT lymphoma induced by Helicobacter heilmannii infection (1052.5)
Authors: Nakamura Masahiko , Matsui Hidenori , Takahashi Tetsufumi , Tsuchimoto Kanji
Published: June 2021

Recently, many neuroendocrine mediators have been reported to be related to the formation and enlargement of the neoplasm. From our electron microscopic analysis of the gastric MALT lymphoma, many unmyelinated nerve fiber‐like structures have been detected especially in the early stage of the tumor formation. Thus, the present study was undertaken to clarify the localization of choline acetyltransferase (ChAc), substance P, its receptor NK‐1R, CGRP and RAMP‐1, in the MALT lymphoma tissues. The effect of eradication of the bacilli as well as spantide II, a substance P antagonist, was also investigated. Materials and Methods The gastric, hepatic and pulmonary MALT lymphoma was formed by the per oral administration of homogenized gastric mucosa from Helicobacter heilmannii‐infected mice. The localization of ChAc, substance P, NK‐1R, CGRP and RAMP‐1 was observed by the indirect immunohistochemisty. The eradication treatment was performed by the combined administration of amoxicillin, chlarithromycin and lansoprazole for seven days. Results Substance P immunoreactivity (Ir) was observed in perivascular area within the tumor tissues, and the NK‐1R Ir was distributed mostly on the lymphoma cells. On the other hand, CGRP Ir was recognized in the area surrounding the MALT lymphoma. After the eradication treatment, the substance P Ir as well as CGRP Ir decreased in the reduced lymphoma tissues. Spantide II also induced the size of the tumor. In conclusion, the importance of substance P was shown in the maintenance and enlargement of the MALT lymphoma, having the clinical relevance.
Bioequivalence of alendronate and vitamin D3 in an alendronate/vitamin D3 combination tablet (1053.2)
Authors: Yeh Geng‐Chang , Brown Kevin , Mols Ramon , Woolf Eric , Wright Hamish , Maganti Lata , Zajic Stefan
Published: June 2021

FOSAMAX® PLUS D is a once‐weekly alendronate (ALN) 70‐mg / vitamin D3 (vit‐D3) 5600 IU combination tablet developed by Merck & Co., Inc. A single‐dose, open‐label, randomized, 2‐period, crossover study was conducted to test bioequivalence between FOSAMAX® PLUS D combination tablets and co‐administration of corresponding doses of ALN 70‐mg (FOSAMAX®) and vit‐D3 5600 IU (2x 2800 IU) as individual tablets in healthy male and female Asian subjects to support registration in Taiwan.

Results: the 90%CI of the GMR for ALN AUC0‐∞ fell within the pre‐specified bioequivalence bounds of 0.8 to 1.25, but slightly exceeded 1.25 for ALN AUC0‐last and Cmax. The 90% CI of the GMR for vit‐D3, unadjusted for endogenous vit‐D3, was also contained within the pre‐specified bioequivalence bounds. Thus, the combination tablet was bioequivalent based on ALN AUC0‐∞ and vit‐D3 unadjusted for endogenous levels. However, it was not bioequivalent to co‐administration with respect to ALN AUC0‐last and Cmax .

In conclusion, the small differences between the combination tablet and co‐administration of individual doses for ALN AUC0‐last and Cmax are not considered to be clinically significant, as slightly increased ALN concentrations are unlikely to meaningfully affect the safety profile. TFDA accepted these results and considered them supportive for registration of the combination tablet in Taiwan.
New antiplatelet and vasorelaxant agents based on the pyridazinone moiety (1054.6)
Authors: Besada Pedro , Costas Tamara , Costas‐Lago M. Carmen , Vila Noemi , Yañez Matilde , Acevedo Laura , Cano Ernesto , Teran Carmen
Published: June 2021

The pharmacological profile of pyridazinone derivatives includes interesting properties on cardiovascular system as platelet aggregation inhibition and antihypertensive activity. The presence of a 6‐aryl group in the pyridazinone ring was considered essential for the activity on cardiovascular system through the inhibition of phosphodiesterase III (PDE III). Recently, the removal / replacement of the aryl group at C6 has provided potent antiplatelet analogues whose activity does not rely on PDE III inhibition. Therefore, in the search of new structural modifications on the pyridazinone ring that could provide derivatives with novel mechanisms of action, we have designed and synthesized pyridazinones with a functionalized alkyl chain at C4, C5 and/or C6 as well as N2 substituted. Vasorelaxant activity was tested on noradrenaline treated intact rat aortic rings showing several derivatives moderate activity. The most promise results were obtained in the platelet aggregation studies performed following the Born's turbidimetric method and inducing aggregation with thrombine and collagen. Thus, the majority of the new analogues selectively inhibited aggregation induced by collagen in the µM range, being more potent than aspirin

Grant Funding Source: Supported by Xunta de Galicia (CN 2012/184)
Epigenetic regulation of IL‐6 by pro‐inflammatory cytokines in rheumatoid arthritis synovial fibroblasts: role of HDAC inhibitors (1054.8)
Authors: Riegsecker Sharayah , Wingerter Wylie , Singh Anil , Bhansali Pravin , Tillekeratne L. M. Viranga , Ahmed Salahuddin
Published: June 2021

Histone deacetylase inhibitors (HDACi) have gained significant attention in recent years due to their anti‐rheumatic activity and potential clinical application. However, the exact role of different HDAC classes in rheumatoid arthritis (RA) is not yet defined, which has limited our ability to target HDACs for therapeutic gain. In order to gain some insights on the epigenetic regulation of synovial inflammation and tissue destruction observed in RA, the present study was carried out to evaluate the role of HDACs in cytokine‐induced IL‐6 synthesis by RA synovial fibroblasts (RA‐FLS) and the influence of cytokine combination. Effect of class I HDACi (Largazole, LAR) or pan‐HDACi vorinostat (SAHA) was determined on TNF‐α (20 ng/ml), IFN‐γ (10 ng/ml), or IL‐1β (10 ng/ml)‐induced IL‐6 expression in RA‐FLS by Western blotting and qRT‐PCR. IL‐6 production under different treatment conditions was also compared with IL‐8 production. Results of the study showed that TNF‐α‐induced IL‐6 expression in RA‐FLS was further amplified in the presence of class I HDACi (LAR, 2‐5 μM) or pan‐HDACi (SAHA, 5 μM) (p<0.05). Surprisingly, we also observed that LAR downregulated TNF‐α/IL‐1β‐induced IL‐6 production by ~50%, but further enhanced TNF‐α/ IFN‐γ‐induced IL‐6 production by ~25%. Evaluation of the signaling pathways suggests that LAR effectively cooperated with TNF‐α to induce phosphorylation of AKT, which may partly be the reason for its IL‐6 increasing property in RA‐FLS. Results of the present study suggest that LAR may interact differently with TNF‐α, IL‐1β, and/or IFN‐γ to induce or inhibit IL‐6 production in RA‐FLS. Thus, our study highlights a need for validating the exact role of specific HDAC isoforms in RA pathogenesis before highly selective inhibitors could be designed and tested for the treatment of RA.

Grant Funding Source: Supported by NIH/NIAMS grants (AR063104 and AR55741) and University of Toledo Start‐up funds to SA.
Differential modulation of p‐4EBP1, p‐ERK and cyclin D1 by amuvatinib/erlotinib treatment in prostate cancer cells (1055.4)
Authors: Canatsey Ryan , Cai Wesley , Mastrandrea Nicholas , Gard Jaime , Nagle Ray , Monks Terrence , Lau Serrine
Published: June 2021

Cyclin D1 is a key regulator of cell cycle progression and a therapeutic endpoint for cancer treatment. The human prostate cancer cell lines, LNCaP (PTEN‐) and DU145 (PTEN+), were used to examine the differential regulation of cyclin D1 as a determinant of drug sensitivity to amuvatinib, a receptor tyrosine kinase inhibitor, and erlotinib, an epidermal growth factor inhibitor. 1D and 2D Western blot analysis revealed that combination drug treatment of LNCaP cells caused a decrease in 4EBP1 pSer65, pThr70, and pThr37/46, with concomitant decreases in cyclin D1 levels. Such combination treatment, however, did not modulate pERK status. Moreover, single drug treatment with amuvatinib, but not erlotinib, decreased p‐4EBP1 and cyclin D1 levels. In DU‐145 cells, combination drug treatment had no effect on p‐4EBP1 status, but decreased p‐ERK and cyclin D1 levels. Additionally, single drug treatment with erlotinib, but not amuvatinib, caused a loss of p‐ERK and cyclin D1. The data reveal the differential modulation of cyclin D1, and its upstream regulators, in prostate cancer cell lines by amuvatinib and erlotinib. Such differential responses likely contribute to specific anti‐tumor agent efficacy. An appreciation of factors that determine the cell‐dependent modulation of cyclin D1 will assist in the selection of appropriate biomarkers for directing patient‐targeted therapy.

Grant Funding Source: Supported by Ventana/Roche, P30 ES006694
Pentadecapeptide BPC 157 and anaphylactoid reaction in rats and mice after intravenous dextran and white egg administration (1056.7)
Authors: Duplancic Bozidar , Stambolija Vasilije , Katancic Holjevac Jadranka , Zemba Mladen , Balenovic Igor , Drmic Ivan , Suran Jelena , Radic Bozo , Filipovic Marinko , Boban Blagaic Alenka , Brcic Luka , Kolenc Danijela , Seiwerth Sven , Sikiric Predrag , Grabarevic Zeljko
Published: June 2021

The objective of this study was to evaluate the effect of pentadecapeptide BPC 157 in anaphylactoid reaction in rats and mice after intravenous dextran and white egg administration.

Anesthetized mice and rats received intravenously 6%, 10%, 20%, 40%, 60%, 80%, 90% dextran and/or white egg (1ml/rat or 0.15ml/mouse) into their tails. Medication (/kg b.w., 5 ml/kg) was given intraperitoneally (BPC 157 10 µg, 1 µg, 10 ng, 10 pg/kg, chloropyramine 20 mg/kg, cimetidine 10 mg/kg intraperitoneally, alone or in combination while controls received an equivolume of saline), immediately after challenge or, alternatively, at 5 min after or 24 or 48 h before challenge. The effect was assessed at 5, 10, 20 and 30 min after dextran and/or white egg challenge. We commonly noted prominent edema involving the face, upper and lower lip, snout, paws and scrotum (presented with extreme cyanosis), poor respiration and the number of fatalities after dextran and/or white egg application. Contrary, BPC 157 regimens (10 µg, 1 µg, 10 ng, 10 pg/kg) effectively, may both prevent anaphylactoid reactions that may arise from dextran and/or white egg application and furthermore, rescue already advanced reactions when given after the challenge. Chloropyramine and cimetidine given alone were only moderately effective. When given together with BPC 157, the observed effect correlates with the strong effect of BPC 157 given alone.

In conclusion, we advocate BPC 157 use in anaphylaxis therapy.

Grant Funding Source: Supported by Grant 108‐ 1083570‐3635, Croatia
PSD95 scaffolding of the Shaker‐type K+ channel enables PKA‐dependent phosphorylation and vasodilation of cerebral arteries (1057.7)
Authors: Moore Christopher , Nelson Piper , Parelkar Nikhil , Hanvey Hillary , Rusch Nancy , Rhee Sung
Published: June 2021

Postsynaptic density‐95 (PSD95) is a membrane‐associated scaffolding protein in rat cerebral vascular smooth muscle cells (cVSMC), which associates with voltage‐gated, Shaker‐type K+ (KV1) channels at the plasma membrane. Here, we used a membrane‐permeable peptide (KV1‐C) that competes with KV1.2 subunits for PSD95 binding to demonstrate the physiologic role of PSD95‐KV1.2 association in cVSMC. Application of 10 µM KV1‐C peptide to pressurized cerebral arteries (CA) induced vasoconstriction (23 ± 2%); the KV1 channel blocker, 5‐(4‐phenylalkoxypsoralen (100 nM psora4), did not further constrict these CA. Vasoconstriction by 1 µM linopirdine or 30 µM BaCl was unaffected by KV1‐C, suggesting that KV1‐C does not interfere with the dilator function of KV7 or KIR channels, respectively. cVSMCs in pressurized CA depolarized by 12 ± 2 mV in response to KV1‐C and psora4‐sensitive K+ current was reduced ~40% by KV1‐C peptide in cVSMCs. CA visualized in cranial windows in vivo also constricted (17 ± 3%) in response to KV1‐C. Finally, Western blots employing a phospho‐PKA substrate antibody revealed markedly less phosphorylation of KV1 channels in CA exposed to KV1‐C, and phosphatase inhibitors partially blunted KV1‐C mediated vasoconstriction. These results suggest that the PSD95 scaffold exerts a basal vasodilator influence in rat CA by enabling the phosphorylation ‐dependent and selective opening of KV1 channels.

Grant Funding Source: Supported by NIH R01 HL097107 (SWR) and American Heart Association 13PRE17070035 (CLM)
Construction of a common medication list for use in undergraduate and graduate nursing curricula: bivariate organization, pedagogical integration, and iterative redevelopment (1058.2)
Authors: Murphy Patrick
Published: June 2021

An essential element in pharmacology course development is the selection of exemplar medications, which are introduced to students as prototype drugs and intended to be representative of the mechanisms, uses, and effects of entire pharmacological categories seen in clinical practice. While physician‐directed medical education has benefited from consensus medical pharmacology knowledge objectives and drug lists that were compiled by the Association of Medical School Pharmacology Chairs, no such counterpart exists for nursing. The purpose of this project was to construct a clinically relevant and pedagogically sound exemplar drug list, with an emphasis on medications used in family practice and primary care, that could be incorporated across undergraduate and graduate nursing program curricula. Prototype medications referenced by authors of leading pharmacology and pharmacotherapy texts were analyzed and compared to the 2013 NCLEX‐RN and 2012 AANP Certification Program test plans, to the lists of 2010‐2013 most commonly prescribed and highest grossing medications, and to the Tier 1‐2 preferred drugs of several leading insurers. To accommodate students’ understanding of both molecular drug actions and therapeutic uses, a nested bivariate organization scheme was employed. This single exemplar drug list may serve as a college‐wide formulary, used across undergraduate and graduate nursing education.
Comparing the high affinity benzodiazepine binding site with the homologous “CGS 9895” site in GABA‐A receptors (1059.1)
Authors: Puthenkalam Roshan , Varagic Zdravko , Dereky Anna , Suwattanasophon Chonticha , Sarto‐Jackson Isabella , Scholze Petra , Sieghart Werner , Ernst Margot
Published: June 2021

GABA‐A receptors are the major inhibitory transmitter receptors in the brain and the site of action of many clinically important drugs. CGS 9895 and many other pyrazoloquinolinones are high affinity null modulators at the benzodiazepine (Bz) binding site and at higher concentrations also modulate the receptors via the α+β‐ interface. To gain insights into the interactions of CGS 9895 with both binding sites, we used our homology models to select γ2‐ side amino acid residues important for Bz binding and to incorporate them into the β3‐ side by site directed mutagenesis. These mutated β3 subunits were co‐expressed with α subunits. The recombinant receptors were investigated by the two electrode voltage clamp method in Xenopus laevis oocytes and by radioligand binding assays. With only four mutations we were able to introduce a high affinity binding site for CGS 9895 into αβ3 receptors. Furthermore, GABA currents of several recombinant αβ3(mut) receptors could be modulated by the tested pyrazoloquinolinones and by other ligands of the high affinity Bz binding site, but not by diazepam. We thus introduced a subsite of the high affinity Bz binding site into the α+β3‐ interface.

Grant Funding Source: Supported by FWF W1232 Molecular Drug Targets
Modulatory effects of 5‐HT2A and AMPA receptors on in vivo serotonin levels in two key areas of the brain related to depression (1059.12)
Authors: Zhou Zhu , Chan Patrick , Uchizono James
Published: June 2021

In this work, we explored two in vivo pharmacodynamic targets that alter serotonin (5‐HT): AMPA and 5‐HT2A receptors in the dorsal raphe nucleus (DRN) and prefrontal cortex (PFC). We hypothesized that agonism of PFC 5‐HT2A receptors would modulate DRN 5‐HT levels through DRN AMPA receptors. Two microdialysis probes were surgically placed into the DRN and PFC from which 5‐HT measurements were simultaneously recorded. DOI (5‐HT2A agonist), NBQX (AMPA antagonist) and s‐AMPA (AMPA agonist) were delivered via these probes. Three experimental protocols were tested, each with minimum of 14 time data points (n=3‐5) were obtained: A) baseline control (aCSF); DOI (100μΜ). B) aCSF; DOI (100μΜ) + NBQX (300μΜ); C) aCSF; (s)‐AMPA (10μΜ); (s)‐AMPA (50μΜ); (s)‐AMPA (85μΜ). In Protocol A, 5‐HT levels in the DRN and PFC both increased compare to controls. In Protocol B, DOI (100uM)+NBQX (300uM) showed decreased 5‐HT levels compared to a DOI infusion only (100uM). In Protocol C, as s‐AMPA concentration was increased, 5‐HT levels in DRN simultaneously increased. The results support targeting the PFC 5‐HT2A receptor or the DRN AMPA receptor directly to modulate DRN [5‐HT] for future potential anti‐depressant modalities.

Grant Funding Source: Supported by Formurex, Inc.
Interaction between hydrogen sulfide and nitric oxide in isolated bovine retina (1060.2)
Authors: Kulkarni Chitnis Madhura , Belford Remington , Robinson Jenaye , Mitchell Leah , Njie‐Mbye Ya Fatou , Opere Catherine , Ohia Sunny
Published: June 2021

Hydrogen sulfide (H2S) and nitric oxide (NO) are gaseous mediators that are endogenously produced in mammalian ocular tissues. We tested the hypothesis that NO can regulate the basal production of H2S in isolated bovine neural retina. Methods: H2S and NO concentrations in bovine retina was measured using well‐established colorimetric assays. Retina homogenates were incubated at 37°C for different time periods for determination of basal endogenous production of H2S and NO. In studies examining the effects of the NO donor (SNP) and NO synthase inhibitor (L‐NAME) on basal levels of H2S production, retina tissues were homogenized in the presence of the respective compounds before assays were performed. Results: L‐NAME (300 nM ‐ 500 µM) caused a concentration‐dependent decrease (p < 0.05) in the basal endogenous production of H2S reaching a maximum at 500 µM. On the other hand, SNP (10 ‐ 100 µM) enhanced significantly (p < 0.05) the endogenous H2S production. In the concentration range, 10 µM to 300 µM, L‐cysteine attenuated basal endogenous production of NO reaching a maximum at 300 µM. Conclusions: We conclude that NO can interact with the pathway/s involved in the production of H2S in the retina. Furthermore, endogenously produced H2S can regulate basal NO concentrations. Further studies are needed to understand the pharmacological/physiological basis of the interaction between these two gases in the retina.
Comparative pharmacological actions of N‐acetylcysteine and L‐cysteine on excitatory neurotransmission in bovine isolated retina (1060.4)
Authors: Opere Catherine , Bankhele Pratik , Salvi Ankita , Jamil Jamal , Munt Dan , Njie‐Mbye Ya Fatou , Chitnis Madhura , Ohia Sunny
Published: June 2021

There is evidence that L‐cysteine, the substrate for biosynthesis of hydrogen sulfide (H2S) can regulate potassium (K+)‐evoked glutamate release from bovine isolated retina. In the present study, we compared the pharmacological effects of the precursor for L‐cysteine, N‐acetyl cysteine (NAC) to that of L‐cysteine on K+‐evoked [3H]D‐aspartate release, and on glutamate‐induced neurotoxicity in bovine isolated retina. Isolated neural retina were incubated in oxygenated Krebs solution containing 200 nM of [3H]D‐aspartate and then prepared for studies of neurotransmitter release. The 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay was used to assess retinal neuron survival. Both L‐cysteine (0.1 µM to 10 µM) and NAC (10 µM to 1 mM) elicited a concentration‐dependent inhibition of K+‐induced [3H]D‐aspartate release. At an equimolar concentration of 10 µM, both L‐cysteine and NAC reduced [3H]D‐aspartate release by 54.3% (p < 0.001) and 8.3%, respectively. Interestingly, L‐cysteine (1 mM) and NAC (1 µM) attenuated glutamate (12 mM)‐induced neuron degeneration by 31.1% (p<0.05) and 18.4%, respectively. L‐cysteine was more potent in attenuating neurotransmitter release while NAC was more effective in preventing glutamate‐induced toxicity suggesting that these compounds mediate their pharmacological actions via different mechanisms.
The development of bifunctional ligands as novel therapeutics for chronic pain (1061.5)
Authors: LaVigne Justin , Edwards Katie , Bilsky Edward , Porreca Frank , Ananthan Sam , Roberts Edward , Hruby Victor , Streicher John
Published: June 2021

Opioid drugs can be efficacious in the treatment of chronic pain, but are plagued by the development of serious side effects such as tolerance and dependence that worsen with chronic use. Recent investigations into the pathology of chronic pain and the compensatory changes induced by chronic opioid treatment has revealed that upregulated receptor systems, including the cholecystokinin (CCK) and the kappa and delta opioid receptors (KOR, DOR), mediate many of the negative side effects of chronic opioid use, and may also dampen mu opioid (MOR) mediated analgesia. Based on these findings, we have developed three novel classes of bifunctional ligands with MOR agonist activity, and CCK, DOR, or KOR antagonist activity, respectively. Using CHO cell models containing the individual receptors and assays of receptor activity, including [35S]‐GTPγS coupling, ERK MAPK activation, and βarrestin2 recruitment, we present data characterizing these compounds and demonstrating that this approach yields compounds with the desired activity profiles at both receptor pairs. We also present in vivo data demonstrating that some of these compounds have an improved therapeutic index when compared to established opioids. Novel molecular strategies such as these which take advantage of a more complete biological understanding of the disease process hold much promise for the development of improved analgesics for human health.
Regulated ubiquitination and SUMOylation of pregnane X receptor (1062.10)
Authors: Sun Mengxi , Staudinger Jeff
Published: June 2021

Highly expressed in the entero‐hepatic system, pregnane X receptor (PXR, NR1I2) is a well‐characterized nuclear receptor that regulates the expression of genes encoding key drug metabolizing enzymes and drug transporter proteins. The net effect of PXR activation is to increase metabolism and clearance of drugs and xenobiotics from the body. Activation of PXR also plays a pivotal role in mediating adverse drug‐drug interactions. The complete understanding of PXR biology is thus important for the prevention of adverse drug‐drug interactions and the development of safe and effective therapeutic strategies. Recent research recognizes that post‐translational modification (PTM) of PXR (e.g; phosphorylation, ubiquitination, SUMOylation) significantly impacts its function; however, these studies are still in their infancy. Here we present a novel method of determining the extent to which ubiquitination and SUMOylation of PXR occurs. Our data indicate that PXR is the simultaneous target of the ubiquitin and SUMO signaling pathways, and that these PTMs modulate PXR‐biological activity.

Grant Funding Source: Supported by NIH‐090558
4‐Phenylbutyrate protects renal proximal tubular cells from palmitic acid‐induced endoplasmic reticulum stress and cell death (1063.2)
Authors: Munusamy Shankar , Alkhiyami Dania , Berzou Souad , Moeinzadeh Atefeh , Mohamed Hebatalla
Published: June 2021

Obesity is implicated as a significant risk factor for chronic kidney disease (CKD). High plasma free fatty acid levels observed with obesity impairs the endoplasmic reticulum (ER) and cause ER stress. Chemical chaperones, which relieve ER stress, such as 4‐phenylbutyrate (4‐PBA) have been shown to protect the liver and pancreas against obesity‐induced organ damage; however, their protective role in obesity‐induced renal damage is unknown. Thus, we investigated the nephroprotective role of 4‐PBA in an in vitro model of palmitic acid (PA)‐induced ER stress and renal injury using normal rat kidney cells (NRK‐52E). Cells were divided into four groups: Control, 250 μM PA‐treated, 5 mM 4‐PBA‐treated, and 4‐PBA+PA co‐treated. After 24 h of PA and/or 4‐PBA treatments, cell viability and caspase‐3/7 activity (a marker of apoptosis) were determined. Immunoblot analyses were performed to quantitate the levels of ER stress markers ‐ glucose‐regulated protein (GRP78) and C/EBP homologous protein (CHOP) ‐ in renal cells. Exposure to PA significantly reduced the viability (81.3% to that of control) in NRK‐52E cells, and markedly increased the activity of caspase‐3/7 activity (2‐fold increase over control) and the expression of ER stress markers GRP78 and CHOP (1.6 and 2 folds over control respectively) at P‐value < 0.05. Intriguingly, cells treated with 4‐PBA were protected from PA‐induced ER stress and apoptotic cell death. Our studies demonstrate that 4‐PBA acts as nephroprotectant and prevents fatty acid‐induced ER stress and apoptosis in renal cells. Further investigations in vivo are required to validate the therapeutic potential of 4‐PBA to protect the kidney and prevent the development of CKD during obesity.

Grant Funding Source: Supported by Qatar Univ. Student Research Grants # QUST‐CPH‐FALL‐12/13‐6 and QUST‐CPH‐SPR‐12/13‐3.
Pinitol ameliorates impaired pressure‐natriuresis in experimental diabetes (1063.5)
Authors: Nascimento Nilberto , Cortez Lorrainy Umbelina , Sousa Luis Gustavo , Fonteles Manassés , Santos Claudia
Published: June 2021

Pressure‐induced natriuresis is a physiological protective mechanism that counteracts increased blood pressure with increased diuresis and natriuresis. We evaluated whether diabetes would affect this mechanism and if pinitol, an antidiabetic compound , could prevent and/or reverse any alterations. We induced diabetes by a single injection of 70 mg/kg streptozotocin (i.p) and 2‐month and 4‐months diabetic rats (Diab) compared to time‐matched euglycemic rats (eugly). We employed the Roman‐Cowley model of pressure‐natriuresis (P‐N) in male Wistar rats. We measured fractional excretion of sodium (FENa) and glomerular filtration rate (GFR), filtration fraction (FF), renal blood flow (RBF) and renal vascular resistance (RVR). We have shown that STZ‐diabetes blunts pressure‐natriuresis, in animals submitted to similar increases in renal perfusion pressure, after 2‐months (eugly‐ FENa from 0.5± 0.4 to 3.8±1.5% during P‐N vs diab‐ 0.4 ±0.2% to 0.8±0.2%) and after 4 months pressure‐natriuresis is completely absent (FENa from 0.16±0.04% to 0.23±0.08%). In another set of experiments, two regimens of pinitol treatments were administered to different diabetic groups. One received 20 mg/kg/12h (p.o) during 2‐months beginning 2‐months after the initial diagnosis of diabetes. The other group received the same dosage after the first day of diagnosis and was kept on this regimen during two months. The firs regimen was able to ameliorate kidney function during the P‐N period (FENa from 0.2±0.08% to 0.48±0.2%). Remarkably, the second treatment regimen further improved renal function during the pressure‐natriuresis period (FENa from 0.8±0.03% to 2.34±0.9%). Diabetes profoundly decreases kidney ability to respond to increased perfusion pressure with increased sodium excretion and 2‐month pinitol treatment is able to both reverse and especially prevent this diabetic complication.

Grant Funding Source: CNPq/Brazil; FUNCAP‐CE/Brazil
Mitochondrial superoxide generation in the rat pup kidney during sepsis (1063.7)
Authors: Sims Clark , Patil Naeem , MacMillian‐Crow Lee Ann , Mayeux Philip
Published: June 2021

Sepsis is a leading cause of acute kidney injury (AKI) and mortality in children. In order to develop new therapies, it is critical to understand the development of pediatric sepsis and its effects. In this study, we used a clinically relevant cecal ligation and puncture (CLP) model of peritonitis in rat pups 17‐19 days old to characterize the development of sepsis‐induced AKI. CLP produced severe sepsis demonstrated by hypothermia and a time‐dependent renal microcirculatory failure. Analysis of the renal microcirculation using intravital video microscopy showed a decrease in the number of continuously flowing cortical capillaries and an increase in capillaries with no flow. Renal microcirculatory failure was associated with an increase in mitochondrial superoxide generation as measured using MitoSOX Red. These effects were observed as early as 6h after CLP and were sustained through 18h. To determine if mitochondria dysfunction leads to superoxide generation, high‐resolution respirometry (HRR) was used in fresh tissue biopsies to measure the activities of complexes I, II/III, and IV. HRR showed that CLP did not produce changes in any of the mitochondrial complexes at these early time points studied. Therefore, sepsis does not appear to cause severe mitochondrial dysfunction in rat pups despite an increase in mitochondrial superoxide generation.

Grant Funding Source: Supported by NIH/NIDDK R01 DK075991
Pharmacokinetic and pharmacodynamic properties of β‐hydroxyphospho‐carnitine (1064.1)
Authors: Mendoza‐Rivera Brissa , Reyes‐Esparza Jorge , De La Cruz Cordero Ricardo , Rodriguez‐Fragoso Lourdes
Published: June 2021

The aim of this investigation was to study the pharmacokinetics properties in two species and the pharmacodynamics effects of β‐HPC in obese Zucker fa/fa rats administered during a period of time. Lean and obese Zucker fa/fa rats were treated for 14 weeks with β‐HPC (100 mg/kg) to assess its impact on glucose, triglycerides and cholesterol in liver and blood samples, and on hepatic steatosis. Besides of that, we described the first study about the pharmacokinetic properties of β‐HPC after single oral administration in rats and rabbits. β‐HPC reduced the levels of glucose, triglycerides and cholesterol in the liver, the levels of triglycerides and cholesterol in serum, and improved the architecture and reduced steatosis in the liver in obese rats. After a single oral dose of β‐HPC (100 mg/kg) the elimination half‐life (t1/2), clearance and the area under the concentration vs time (AUC) in rats and rabbits were 23.7 vs 8.87 h, 13.9 vs 151.1 mL/h, and 2174.4 vs 3128 µg.h/mL, respectively. The values of Tmax, Cmax, and Ka in rats and rabbits were 0.58 vs 1.5 h, 62.4 vs 221.4 µg/mL, and 0.02 vs 2.40 h‐1, respectively. This study demonstrated that, although there exist differences between species, the β‐HPC administered orally is rapidly absorbed, highly distributed and efficiently cleared. According with the pharmacodynamics properties β‐HPC could be useful for treating some metabolic alterations present in metabolic syndrome.

Grant Funding Source: CONACYT
Gender comparison of recovery from intravenous and inhalational anaesthetics among adult patients in South‐West Nigeria (1064.3)
Authors: Okunoren‐Oyekenu Yewande , Sanusi Arinola , Gbotosho Grace
Published: June 2021

Gender is a known factor in recovery from general anaesthetics. This study reports such a difference in South‐West Nigerian patients, and proposes gender‐based differences in pharmacokinetic profiles as explanation. Two main strata (20 males and 20 females) were considered, and written informed consents were obtained for both strata. Intravenous general anaesthesia was induced with propofol or thiopental and maintained with inhalational agent halothane or isoflurane. Patient plasma samples were analyzed with high performance liquid chromatography (HPLC), for anaesthetic agents before induction; at 10, 30 60, 180 mins after induction of anaesthesia, and at recovery. The Elimination Half Life, Mean Resident Time, and Clearance measured in propofol patients maintained on halothane or isoflurane were 310.7 ± 138.4 min, 459.2 ± 199.4 min and 431.1 ± 154.1 ml/min, respectively for females versus 503.0 ± 312.2 min, 732.8 ± 448.3 min and 290.0 ± 157.8 ml/min for males. The corresponding values with thiopental induction were 148.0 ± 112.7 min, 220.9 ± 180.3 min and 544.6 ± 500.1 ml/min for females versus 76.8 ± 28.7 min, 115.4 ± 35.4 min and 533.7 ± 502.5 ml/min for males. Data was significantly different (p<0.05) across gender. Gender differences in recovery from anaesthetic agents in Southwest Nigerian patients is due to differences in pharmacokinetic profiles.

Anaesthesia, Recovery time, HPLC, Pharmacokinetics.

Grant Funding Source: Self‐Funded Research Student supported by Supervisors
Stromal interaction molecule 1 deletion in smooth muscle cells protects hypertension‐induced vascular dysfunction by endoplasmic reticulum stress‐dependent mechanism (1065.13)
Authors: Kassan Modar , Belmadani Souad , Trebak Mohamed , Matrougui Khalid
Published: June 2021

Background: stromal interacting molecule‐1 (STIM1) and endoplasmic reticulum (ER) regulate intracellular calcium for vascular function. Hypertension is associated with vascular dysfunction raising the question of whether STIM1 has any role in vascular dysfunction through ER stress and oxidative stress in hypertension.

Methods & results: We used male C57/BL6, homozygous/heterozygous STIM1 knockout specifically in smooth muscle cells (STIM1SMC‐/‐, STIM1SMC‐/+), and homozygous CHOP knockout (CHOP‐/‐) mice infused with and without angiotensin II (Ang II, 400 ng/Kg/min) for 4 weeks. Hypertension development was significantly delayed in STIM1SMC‐/‐ and CHOP‐/‐ mice infused with Ang II compared to control and STIM1SMC‐/+ with Ang II. The vasoconstriction of mesenteric resistance artery and thoracic aorta in response to phenylephrine was significantly reduced only in STIM1SMC‐/‐ mice infused with and without Ang II. However, vasoconstriction to thromboxane was identical in all groups and was augmented in hypertension with low effect in STIM1SMC‐/‐ and CHOP‐/‐. The endothelium‐dependent relaxation was similar in all groups but it was impaired in the control and STIMSMC‐/+ infused with Ang II, protected in STIM1SMC‐/‐ + Ang II and rescued in CHOP‐/‐ + Ang II. The NADPH oxidase activity was elevated, while eNOS phosphorylation, cGMP and nitrite levels were reduced in control with Ang II, compared to STIM1SMC‐/‐ and CHOP‐/‐ with and without Ang II. ER stress markers were only enhanced in control infused with Ang II. The expression of STIM1 was augmented in all groups of mice infused with Ang II.

Conclusions: We conclude that in hypertension, the aberrant increase in STIM1 expression causes vascular dysfunction through ER stress and increases oxidative stress‐dependent mechanisms.
A novel anti‐inflammatory signaling role for the deubiquitinase USP20 in vivo (1065.5)
Authors: Jean‐Charles Pierre‐Yves , Zhang Lisheng , Han Sang‐oh , Kommaddi Reddy , Wu Jiao‐Hui , Freedman Neil , Shenoy Sudha
Published: June 2021

The pro‐inflammatory Toll‐like receptor‐4 (TLR4) signals via Lys63‐linked autoubiquitination of TRAF family E3 ligases, which effect ubiquitin‐dependent activation of NFκB. In vascular smooth muscle cells (SMCs), we found TLR4 activation by LPS induced Lys63 polyubiquitination of the endocytic/signaling adaptor β‐arrestin2 (βarr2), which regulates NFκB. In co‐IP from intact cells, endogenous ubiquitin‐specific protease‐20 (USP20) associated with βarr2 and TRAF6. To determine if USP20 regulates these proteins in vivo, we created mice with SMC‐specific expression of WT‐USP20 or dominant‐negative (DN)‐USP20. In SMCs from these mice, LPS induced activation of NFκB (assessed by Ser536‐phosphorylation of p65) was 4‐fold greater in DN‐USP20‐Tg than in WT‐USP20‐Tg and non‐Tg control SMCs. Furthermore, Lys63 ubiquitination of both βarr2 and TRAF6 was augmented in DN‐USP20‐Tg SMCs. Congruently, 4 wk after endothelial denudation injury, carotid arteries of SMC‐DN‐USP20‐Tg mice showed 1.8‐fold greater NFκB activation (phospho‐p65Ser536) and 2.6‐fold greater neointimal hyperplasia (comprising proliferating SMCs) than non‐Tg or SMC‐WT‐USP20 mice (p<0.01)_even though all groups had equivalent carotids at baseline. Thus, USP20 seems to function as an anti‐inflammatory deubiquitinase by inhibiting ubiquitin‐dependent activation of β‐arrestin2, TRAF6 and NFκB in SMCs.

Grant Funding Source: Supported by T32 HL007101 (PJYC), HL77185 (NJF) and HL080525 (SKS)
Suramin sensitive P2 receptor is involved in α1‐adrenergic receptor mediated mesenteric arterial constriction in normotensive and DOCA‐salt hypertensive rats (1065.9)
Authors: Lian Mary , Xu Hui , Galligan James
Published: June 2021

Pannexin‐1 is an ATP permeable hemichannel that mediates ATP released from smooth muscle cells (SMCs) in response to stimulation of α1‐adrenergic receptors (AR) by norepinephrine. ATP is released and acts back on vascular SMCs to further enhance adrenergic constriction. It is unclear if the channel function is altered in hypertension. Our earlier studies have indicated that pannexin‐1 activation contributes to α1‐AR mediated mesenteric arterial (MA) constriction in normotensive (sham) and DOCA‐salt hypertensive rats, since the concentration‐response curves (CRCs) of phenylephrine (PE) were significantly right‐shifted by mefloquine (a pannexin‐1 blocker) in all MA. In this study, we determined the role of P2 receptors in α1‐AR mediated MA constriction in both rats. PE and ATP‐induced MA constrictions were recorded using a pressure myograph and video microscopy before and after adding P2 receptor (P2R) antagonists. The PE‐CRCs were similar in MA from sham and DOCA‐salt hypertensive rats with or without PPADs, a P2X/P2Y1 receptor antagonist. PPADs shifted ATP‐CRCs to the right in all MA. PE‐CRCs in all MA were significantly right‐shifted in a concentration‐dependent manner by suramin, a P2X/P2Y2 receptor antagonist. Our studies indicate that α1‐AR mediated constrictions of rat MA are not caused by ATP released from SMCs to acting on PPADs sensitive P2X or P2Y1 receptors. A suramin‐sensitive P2R may mediate the ATP‐induced MA constriction caused by α1‐AR activation of pannexin‐1 ATP permeable channels on SMCs.

Grant Funding Source: Supported by NIH 5‐R25‐HL108864‐01 and HL P01 HL070687
Fingerprinting of GPCR activity across multiple G protein substrates unveils complex profiles of functional selectivity (1066.12)
Authors: Masuho Ikuo , Jones Christopher , Martemyanov Kirill
Published: June 2021

Despite the tractability of G protein‐coupled receptors (GPCRs) as drug targets, there are substantial challenges in understanding the mechanisms of drug action at these receptors. Since more than 800 GPCRs and 16 Gα subunits are identified in human genome and are capable to generate enormous array of GPCR‐G protein combinations, G protein‐coupling profiles of GPCRs are a critical missing piece of the GPCR‐signaling puzzle. Here, we have undertaken an attempt to comprehensively characterize G protein‐coupling selectivity and its modulation by agonists and intracellular regulatory proteins. We employed a single‐platform BRET assay to evaluate GPCR responses via 14 different Gα subunits using dynamic association/dissociation of Venus‐tagged Gβγ with NanoLuc‐tagged GRK3‐derived sensor in living cells. Distinct G protein‐coupling profiles for different receptors in both maximal amplitudes and kinetics of responses were observed and used as a basis for modeling “fingerprints” for each of the examined receptors. These fingerprints in many cases differed between physiological agonists and synthetic ligands. We further found that members of Regulators of G protein Signaling (RGS) family shape GPCR fingerprints. These results reveal underappreciated complexity of GPCR coupling to different G protein substrates and suggest a new avenue for designing signaling pathway‐specific therapeutics.

Grant Funding Source: Supported by the NIH grants HL105550 and NS081282.
Inhibition of microRNA‐429 in the renal medulla increased salt sensitivity of arterial blood pressure in Sprague Dawley rats (1067.1)
Authors: Zhu Qing , Hu Junping , Wang Zhengchao , Li Pin‐Lan , Han Weiqing , Li Ningjun
Published: June 2021

We have reported that high salt intake (HS) inhibits HIF prolyl‐hydroxylase 2 (PHD2), an enzyme promoting the degradation of hypoxia inducible factor (HIF)‐1α, and upregulates HIF‐1α along with its target genes such as NOS2 and HO‐1 in the renal medulla, which is an important molecular adaptation to promote extra sodium excretion and maintain blood pressure. Our preliminary data showed that HS significantly increased the expression of miR429, a microRNA targeting PHD2 mRNA, in the renal medulla. The present study determined whether inhibition of miR429 would increase the levels of PHD2, decrease the levels of HIF‐1α target genes in the renal medulla and induce salt sensitive hypertension. Transfection of miR429 inhibitor into the renal medulla decreased miR429 level by 93%, increased PHD2 mRNA level by 2.5 fold, and reduced the expression of NOS2 and HO‐1 by 67% and 74%, respectively. Ten‐day high salt diet significantly increased mean arterial pressure (MAP) in miR429 inhibitor‐treated rats compared with controls (145±3.5 vs. 118±4.1 mmHg). MAP did not change in miR429 inhibitor‐treated rats that were fed a low salt diet. These results suggested that renal medullary miR429 is an important regulator in renal adaptation to HS and that deficiency in miR429‐mediated PHD2 inhibition in response to HS in the renal medulla may represent a pathogenic mechanism for salt‐sensitive hypertension.

Grant Funding Source: Supported by NIH grants HL089563 and HL106042
Inhibition of angiotensin‐converting enzyme‐2 (ACE‐2) activity and radioligand binding of a putative ACE‐2 inhibitor (1067.5)
Authors: Linares Andrea , Carrera Eduardo , Speth Robert
Published: June 2021

While closely related to angiotensin‐converting enzyme (ACE), ACE‐2 has very different effects on the renin‐angiotensin system. A potent inhibitor of ACE‐2 (MLN‐4760) was developed, but then abandoned when it became clear that ACE‐2 metabolizes angiotensin II (Ang II) to form Ang 1‐7. To develop a novel inhibitor of ACE‐2 to use as a tool to study ACE‐2, we developed an analog of MLN‐4760 (JFS101) in which a leucinyl side‐chain (designed to mimic the carboxy terminal amino acid of Ang I) was replaced with a tyrosyl side‐chain to more closely mimic the carboxy terminal amino acid of Ang II and allow for the inhibitor to be radioiodinated. Both the uniodinated and radioiodinated and the S,S versus the S,R analogs were tested for their ability to inhibit ACE‐2 metabolism of an artificial ACE‐2 substrate (MCA‐APK(Dnp) using recombinant human ACE‐2 (rhACE‐2) and for their ability to bind to ACE‐2 in rat lung and kidney membranes. The S,S isomer of JFS101 inhibited rhACE‐2 in the nanomolar range and was 5‐10% as potent as the S,S, isomer of MLN‐4760, which inhibited rhACE‐2 in the low nanomolar range. The S,R isomer of JFS101 inhibited rhACE‐2 activity in the low micromolar range. Radioligand binding assays using 125I‐JFS101 (S,S isomer) revealed a high level of binding to lung and kidney membranes 196 and 321 fmoles/mg wet weight at 50 nM 125I‐JFS101. However, less than 10 % of this binding was displaceable by 1 µM MLN‐4760. In contrast, 2 mM EDTA inhibited ~80 % of total binding at concentrations ranging from 3 ‐35 nM 125I‐JFS101. The EDTA displaceable 125I‐JFS101 binding was not saturable suggesting that the KD of 125I‐JFS101 for lung membranes is >> 35 nM. The inability of MLN4760 combined with the ability of EDTA to inhibit 125I‐JFS101 binding suggests that 125I‐JFS101 is not selective for ACE‐2, but that it does bind to another metallopeptidase.

Grant Funding Source: NIH‐HL113905