bluuebunny/crossref_metadata_2025 huggingface dataset:
What Do Academic Investigators Want and Get from Preclinical Collaboration with the Pharmaceutical Industry?
Authors: Amiri Marjan , Michel Martin
Published: June 2021

Objective

As research collaboration between academia and the pharmaceutical industry is important, we explored expectations and experience of investigators with preclinical collaborations.

Methods

Academic investigators from Western Europe, North America and Japan with preclinical publications in the obstructed airways or diabetes field were invited to anonymously participate in a web‐based survey.

Results

Results were similar in the obstructed airways and diabetes field (134 investigators total, 28% of invitees). Obtaining funding, freedom to publish and obtaining compounds were the most important goals, cultivating professional relationships, getting external scientific input, direct relationship to disease treatment and involvement with drug development were less important. Among eight characteristics of the primary contact person in the company, trustworthiness ranked highest, followed by collaborative spirit and transparent information sharing; supportiveness, scientific qualification, accessibility and timeliness of responses ranked lower, and friendliness lowest. Overall satisfaction in their latest collaborations was typically rated as very good. However, the process of reaching a contract was often considered too long and difficult, for which both university and company legal departments were reported as culprits.

Conclusions

While generally satisfied in their preclinical collaboration with the pharmaceutical industry, academic investigators look for improved contracting procedures.
Exploring a competitive method of bias analysis on the kappa opioid receptor
Authors: Stahl Edward , Zhou Lei , Ehlert Frederick , Bohn Laura
Published: June 2021

It has been shown that G protein‐coupled receptors may adopt multiple active states and that some agonists are able to distinguish between different active receptor states. This ability is described as the bias of an agonist for one signaling pathway over another. The recognition that this potential was available to agonists has led to a search for agonists that exhibit bias. As this search continues, it is likely that different degrees of bias will be uncovered. One example of this is extreme bias where an agonist produces the selective stimulation of a subset of signaling pathways while not stimulating other signaling pathways know to be downstream of the same receptor. Although this outcome would be expected as more biased ligands are discovered, the currently available methods of bias analysis are not able to accommodate extreme bias. For this reason, we have constructed a competitive mechanism of bias analysis. In the studies presented here, we employ our competitive method to analyze both simulated studies and experimental evidence from the kappa opioid receptor. Our results demonstrate that this method produces highly accurate estimates of the bias of several test ligands. From these studies, we conclude that our competitive method provides a useful tool for studying biased agonists. Specifically, this method is well suited for the specific subset of cases where an agonist exhibits extreme bias and for cases where the partial nature of an agonist in a system is not sufficient to achieve robust stimulation.

Grant Funding Source: Supported by NIDA grant R01 DA031927(LMB)
Supraspinal Actions of N/OFQ, Morphine and Substance P in Regulating Pain and Itch in Nonhuman Primates
Authors: Ding Huiping , Hayashida Ken‐ichiro , Suto Takashi , Sukhtankar Devki , Kimura Masafumi , Mendenhall Vince , Ko Mei‐Chuan
Published: June 2021

NOP (nociceptin/orphanin FQ (N/OFQ) peptide) receptor agonists display a promising analgesic profile in preclinical studies. However, supraspinal N/OFQ produced hyperalgesia in rodents and such an effect has not been addressed in primates. The aim of this study was to investigate the behavioral effects of centrally administered ligands (N/OFQ, morphine and substance P) on regulating pain and itch in nonhuman primates. An intrathecal catheterization was established to enable intracisternal and lumbar intrathecal administration in awake and unanesthetized rhesus monkeys. Intracisternal morphine 100 nmol elicited more head scratches as compared to those of intrathecal morphine. Distinct dermatomal scratching locations between the two routes suggest a corresponding activation of supraspinal and spinal mu opioid receptors. Unlike hyperalgesia produced by intracisternal substance P, intracisternal N/OFQ 100 nmol produced NOP receptor‐mediated antinociceptive effects; both peptides did not increase scratching responses. Collectively, these results demonstrate differential actions of ligands in the primate supraspinal region in regulating pain and itch. Future pharmacological studies can be advanced to define the roles of supraspinal neuropeptides in regulating diverse behavioral and physiological functions in unanesthetized nonhuman primates (Supported by US‐PHS grants AR‐059193 and AR‐064456, and the US‐DOD grant W81XWH‐13‐2‐0045).
Lorcaserin (Belviq®) Reduces the Discriminative Stimulus and Reinforcing Effects of Cocaine in Rhesus Monkeys
Authors: Collins Gregory , Gerak Lisa , France Charles
Published: June 2021

Drug abuse and obesity are serious public health problems, and mounting evidence suggests that dopamine and serotonin systems are similarly involved in regulating the consumption of food and drugs. Lorcaserin, a serotonin 2C receptor agonist, was recently approved by the US FDA to help obese individuals lose weight and it might also be effective at reducing cocaine abuse. The current studies sought to (1) characterize the pharmacokinetic and behavioral profile of lorcaserin (I.G.), and (2) determine the effectiveness of lorcaserin to alter the discriminative stimulus and reinforcing effects of cocaine (I.V.) in rhesus monkeys. Administered acutely, lorcaserin dose‐dependently increased the occurrence of yawning and decreased spontaneous activity and operant responding for food. These effects occurred within 30‐60 min of administration and began to dissipate by 240 min, which closely matched plasma levels of lorcaserin. In monkeys discriminating cocaine from saline, lorcaserin alone did not increase cocaine‐appropriate responding. When administered prior to cocaine, lorcaserin shifted the cocaine dose‐response curve to the right and down. Lorcaserin also dose‐dependently decreased the rate of cocaine self‐administration with a dose of 3.2 mg/kg lorcaserin decreasing responding by 50% for the duration of the 14 day treatment. Together, these results provide clear evidence that lorcaserin effectively blunts the discriminative stimulus and reinforcing effects of cocaine following acute and repeated administration suggesting that it might have clinical utility for treating cocaine abuse.

Supported by NIH/NIDA grants U01DA034992 and K05DA017918 (CPF).
The Discriminative‐Stimulus Effects of Mephedrone in Comparison with Other Drugs of Abuse
Authors: DeLarge Alyssa , Winsauer Peter
Published: June 2021

Mephedrone (4‐methylmethcathinone) is one of the major constituents of the recreational drug, “bath salts”. It is a synthetic cathinone and potent psychostimulant that has been reported to cause auditory and visual hallucinations. The purpose of this study was to compare the discriminative stimulus effects of mephedrone alone with those of other prototypical drugs of abuse, such as cocaine, delta‐9‐tetrahydrocannabinol (THC), phencyclidine (PCP), and 2,5‐dimethoxy‐4‐iodoamphetamine (DOI). Rats were trained to discriminate an intraperitoneal injection of mephedrone from saline under a fixed‐ratio 20 schedule of food presentation. The final training dose of mephedrone was 3.2 mg/kg and it was reliably discriminated from saline by all subjects. Following training, substitution tests indicated that cocaine (0.56‐32 mg/kg) fully substituted for mephedrone (0.32‐10 mg/kg) by producing over 80% mephedrone‐lever responding, whereas THC (0.56‐10 mg/kg), PCP (1‐5.6 mg/kg) and DOI(0.1‐1 mg/kg) produced a maximum of 73%, 30% and 50% mephedrone‐lever responding, respectively. Only THC produced dose‐dependent rate‐decreasing effects (蠅 20% decrease in response rate compared to THC vehicle). These results indicate that the discriminative stimulus effects of mephedrone most closely approximate those for the stimulant drug, cocaine, and are only modestly similar to THC.
Pharmacology of Self‐Administration of a Non‐Selective Sigma1/2 Receptor Agonist, 1,3‐di‐o‐tolylguanidine (DTG), and its Induction of Sigma1‐Mediated Reinforcement in Rats
Authors: Hiranita Takato , Katz Jonathan
Published: June 2021

Previous studies showed that sigma1 receptor (σ1R) agonists (e.g. PRE‐084, (+)‐pentazocine) were reinforcing in rats with cocaine or d‐methamphetamine self‐administration (SA) experience, but not in experimentally naïve rats. Further studies demonstrated that the induction of σ1R agonist SA specifically occurred with SA of drugs acting at the dopamine transporter (DAT) but not with other abused drugs (e.g. heroin, ketamine). In contrast to effects with selective σ1R agonists, a recent study showed reinforcing effects of the non‐selective σ1/2R agonist DTG (1.0 mg/kg/inj) in naïve rats. The present study assessed the pharmacology of DTG SA, and whether experience with its SA would induce SA of a selective σ1R agonist, PRE‐084. Responding of rats trained with SA of DTG (0.1‐3.2 mg/kg/inj) under an FR 5‐response schedule of reinforcement was insensitive to antagonism by dopamine (DA) receptor antagonists (SCH 39166, L‐741626) which dose‐dependently right‐shifted cocaine (0.03‐1.0 mg/kg/inj) SA dose‐effect curves in a separate group of rats trained with SA of cocaine. Further, σR antagonists (haloperidol, BD 1063) blocked SA of DTG, but of these only haloperidol, which is also a DA antagonist, blocked cocaine SA. Pretreatments with PRE‐084 and the DAT inhibitor WIN 35,428 dose‐dependently left‐shifted dose‐effect curves for DTG and cocaine SA. The present results suggest that DA‐uptake inhibition while sufficient is not necessary for induction of reinforcement by σ1R agonists, and that once induced σR‐mediated reinforcement is independent of traditional DA receptor mechanisms.
2‐Substituted Aryltropane Cocaine Analogs Produce Atypical DAT Inhibitor Effects without Inducing Inward‐Facing DAT Conformations
Authors: Hong Weimin , Kopajtic Theresa , Xu Lifen , Lomenzo Stacey , Trudell Mark , Katz Jonathan
Published: June 2021

Previous structure‐activity studies indicated that a series of cocaine analogs (3ß‐aryltropanes with various 2ß‐diarylmethoxy substitutions) selectively bind the dopamine transporter (DAT) with nM affinity, and with corresponding 2α‐enantiomers having 10‐fold lower affinity. The present study assessed the similarity to cocaine of behavioral effects of several of these compounds by examining effects on locomotion in mice and substitution for cocaine (10 mg/kg, ip) in rats discriminating cocaine from saline. Despite DAT affinity only the 2β‐Ph2COCH2‐3ß‐4‐Cl‐Ph analog fully substituted for cocaine's discriminative effects. Whereas all of the 2β compounds increased locomotion, only the 2β‐(4‐ClPh)PhCOCH2‐3ß‐4‐Cl‐Ph analog had cocaine‐like efficacy. None of the 2α‐substituted compounds produced either of these cocaine‐like effects. To explore the molecular mechanisms of these drugs, effects of the analogs on DAT conformation were assessed using a cysteine‐accessibility assay. Previous results suggest that cocaine induces an outward‐facing DAT conformation, whereas atypical DAT‐inhibitors, such as benztropine, produce behavioral effects distinct from those of cocaine presumably by inducing an inward‐facing conformation. Most of the 2β‐ and 2α‐substituted compounds induced an outward‐facing DAT conformation similar to that induced by cocaine. Consistent with a previous study of phenyltropane analogs, these behavioral and biochemical results show that phenyltropane analogs can bind to the DAT and induce outward‐facing DAT conformations like cocaine and still produce effects that differ from those of cocaine.
Increasing Angiotensin Converting Enzyme 2 Activity in the Brain Is Anxiolytic and Dampens Activation of Hypothalamic‐Pituitary‐Adrenal Axis in Male Mice
Authors: Wang Lei , de Kloet Annette , Pati Dipanwita , Hiller Helmut , Smith Justin , Pioquinto David , Oh S. , Katovich Michael , Raizada Mohan , Frazier Charles , Krause Eric
Published: June 2021

This study evaluates whether driving the counter‐regulatory limb of the renin‐angiotensin‐system influences anxiety and activation of the hypothalamic‐pituitary‐adrenal (HPA) axis. Using the Cre/Lox system, we generated male mice overexpressing angiotensin converting enzyme 2 (ACE2 KI) and wild‐type littermate controls (WT). Relative to WT, ACE2 KI mice had reduced anxiety‐like behavior and decreased stress‐induced HPA activation. These effects were abolished by central antagonism of the Mas receptor (MasR) but delivery of an ACE2 activator into the brains of WT mice was anxiolytic. To investigate underlying mechanisms we examined Fos expression subsequent to an anxiogenic stimulus. Relative to WT, ACE2 KI mice had increased Fos in the basolateral amygdala (BLA) but decreased Fos in the bed nucleus of the stria terminalis. Anatomical studies determined that GABA neurons in the BLA robustly express the MasR and electrophysiological studies revealed that ACE2 overexpression increased the frequency of spontaneous inhibitory postsynaptic currents in BLA principal neurons, an effect abolished by MasR blockade. To determine whether ACE2 attenuates HPA activation through interactions with corticotropin‐releasing‐hormone (CRH) we generated mice with ACE2 expression driven by the CRH gene. Indeed, ACE2 overexpression in CRH cells decreased activation of HPA axis. Collectively, the results suggest that ACE2 acts in the brain to decrease anxiety and HPA activation, in part, by stimulating the MasR.
CO‐ULTRAMICRONIZED PEALUT: A NEW THERAPEUTIC TREATMENT IN AUTISM SPECTRUM DISORDER
Authors: Crupi Rosalia , impellizzeri Daniela , Bruschetta Giuseppe , Cordaro Marika , Esposito Emanuela , Cuzzocrea Salvatore
Published: June 2021

Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by neurological deficits, especially as related to cognitive function. Brain inflammation can be a key element in the pathogenesis of neuropsychiatric disorders, such as ASD. N‐palmitoylethanolamide (PEA) is a parent molecule of ALIAmides, known for its anti‐inflammatory, analgesic and neuroprotective properties. Flavonoids, such as luteolin, possess neuroprotective actions in central nervous pathophysiological conditions. Association of PEA with Luteolin (co‐ultra PEALut) displays neuroprotective and anti‐inflammatory actions in CNS pathologies which are superior when compared to each molecule alone.

Our goal was to evaluate the effects evoked by PEALut in the management of inflammatory events associated with ASD using established experimental model. A multidisciplinary approach was employed to study: behavioral, neuroinflammatory, neurogenesis and neuroplasticity alterations. On P14, C57BL/6 mice were injected with 400 mg/kg sodium valproate. Two different set of experiments were conducted. In the first, on P15 mice were administered with PEALut and on P30 the behavioral and neuroinflammatory studies were assessed. In the second, at P120, mice started treatment with PEALut (2 weeks) and then were sacrified for neurogenesis and neuroplasticity investigations. Our study showed that PEALut ameliorated aggressive behavior and improved spatial learning memory in the autistic mice. Moreover PEALut increases neurogenesis and neuroplasticity in mice hippocampus.
Maternal Protein Deficiency Causes Region‐Specific and Sex‐Dependent Changes in 5‐HT1A Receptor Function in Adult Offspring
Authors: Ye Wenrui , Thompson Brent , Hensler Julie
Published: June 2021

Protein insufficiency, rather than caloric restriction, during pregnancy leads to abnormalities in emotion and cognition in adult offspring. However, the underlying neural mechanisms are unclear. In rats, we examined serotonin1A (5‐HT1A) receptor function in adult offspring of mothers that were free‐fed standard (20%) or low (10%) protein isocaloric chow during pregnancy. Importantly, the maternal low protein diet did not affect the body weight of mothers or offspring. At P110‐117, 5‐HT1A receptor agonist DPAT (0.01‐1mg/kg, ip) induced stereotypic behaviors were measured. The potency of DPAT to induce flat body posture and lower lip retraction was reduced in female offspring, as indicated by rightward shifts (~3 fold) in the dose‐response curves. In littermates, we used quantitative autoradiography to measure the capacity of 5‐HT1A receptors to activate G proteins. The maternal low protein diet resulted in a 50% decrease in DPAT (1μM)‐stimulated [35S]GTPΥS binding in hippocampus of female offspring, with no change in frontal cortex, suggesting a region‐specific down‐regulation of postsynaptic 5‐HT1A receptor function. DPAT (1μM)‐stimulated [35S]GTPΥS binding was increased (54%) in dorsal raphe of female offspring, suggesting enhanced inhibition of serotonin neuronal activity. By contrast, no significant change in 5‐HT1A receptor function was observed in male offspring. Both in vivo and ex vivo data indicate that maternal protein deficiency causes profound region‐specific and sex‐dependent changes in 5‐HT1A receptor function, which may underlie abnormal emotion and cognition in adult offspring. Supported by Morrison Trust
Age‐Dependent Effects of Nicotine or Fluoxetine on 5‐HT1A Receptor Activity
Authors: Yuan Menglu , Larry‐Sar Daryl , Phan Michael , Hanna Peter , Leslie Frances
Published: June 2021

Initiation of smoking and onset of psychiatric diseases often emerge during adolescence, a sensitive developmental period. Clinical data indicates that teen smokers are more likely to develop psychiatric disorders and substance abuse. Preclinical data reveals that nicotine, the main psychoactive component of tobacco, elicits lasting changes in adolescent brain development, neuronal signaling, and motivated behavior. Our lab has shown that nicotine enhances behavioral sensitivity to cocaine reward in adolescents but not adults (Dao et al., 2011). This effect is mimicked by endogenous activation of the 5‐HT system with fluoxetine, a selective serotonin reuptake inhibitor commonly prescribed for teen depression. Furthermore, both co‐administration of 5‐HT1AR antagonist WAY‐100,635 (during drug exposure) and acute administration of WAY‐100,635 (after drug exposure) blocked nicotine and fluoxetine's enhancement effects, indicating that these drugs increase adolescent 5‐HT1AR signaling. Thus, this study examines whether nicotine or fluoxetine age‐dependently alters 5‐HT1AR activity, particularly in brain areas regulating drug reward. Adolescent or adult rats received nicotine (60µg/kg, i.v.) or fluoxetine (1mg/kg, i.v.) for 4 consecutive days. Next day, brains were collected and processed via agonist‐stimulated [35S]GTPγS binding to determine 5‐HT1AR activity. Preliminary data indicate that adolescent nicotine or fluoxetine exposure may increase 5‐HT1AR activity in brain regions mediating executive function and addiction‐related behaviors. ​These results provide region‐specific understanding of nicotine and fluoxetine's effects on brain development and clarify underlying mechanisms in which 5‐HT1ARs mediate sensitization of cocaine reward during adolescence.
A Novel Approach to Cholinergic Signaling Modulation: Development and Characterization of ML352, a Novel, Noncompetitive Inhibitor of the Presynaptic Choline Transporter
Authors: Ennis Elizabeth , Wright Jane , Tarr James , Locuson Charles , Hopkins Corey , Daniels J , Lindsley Craig , Blakely Randy
Published: June 2021

The choline transporter (CHT) is a critical determinant of acetylcholine (ACh) synthesis and signaling. Despite the medicinal uses of anticholinergics, CHT remains a largely unexplored target for such endeavors, with only one competitive inhibitor, hemicholinium‐3 (HC‐3), in common use. Thus, we initiated a high‐throughput screen to expand CHT pharmacology, biasing our search toward agents with allosteric modes action. Our efforts identified 5 novel classes of CHT antagonists, with one class subjected to further chemical diversification to improve potency, selectivity and pharmacokinetics. Among these compounds, ML352, was identified as a potent, noncompetitive inhibitor of CHT activity in both transfected cells and nerve terminal preparations. ML352 exhibits little or no inhibitory activity at the ACh homeostatic enzymes or against many other ion channels, transporters and receptors. DMPK studies revealed a high intravenous plasma clearance, a combination of the oral bioavailability and total brain concentrations indicate ML352 to be a suitable probe for investigations of altered in vivo ACh signaling and behavioral effects in rodents. Our efforts reveal ML352 and its derivatives as the first potent and selective CHT inhibitors developed since the identification of HC‐3 more than 50 years ago, providing a novel platform for the treatment of disorders characterized by excess ACh signaling.
Cadmium‐induces Cell Cycle Progression in Triple‐negative Breast Cancer Cells Through EGFR
Authors: Wei Zhengxi , Song Xiulong , Shaikh Zahir
Published: June 2021

Cadmium (Cd), a known endocrine disruptor, has been shown to promote the growth of estrogen receptor positive breast cancer cells. The objective of this study was to investigate whether Cd can also promote the growth of triple‐negative breast cancer cells, and if so, whether it is mediated by EGFR. Human breast cancer HCC 1937 cells were treated with 0.05–1 µM CdCl2. Cell proliferation was assessed by MTT assay, cell cycle analysis and expression of cyclins and CDKs. EGFR's role in Cd‐induced cell cycle progression was studied by siRNA knockdown and inhibition of phosphorylation of EGFR and its downstream targets. The results showed that indeed Cd treatment promoted HCC 1937 cell cycle progression and it did so by increasing the levels of cyclins A, B and E, and CDKs 1 and 2. The cell cycle progression was suppressed by EGFR inhibitor AG1478. Cd treatment caused phosphorylation of EGFR and its downstream targets, Src, AKT and ERK1/2. Both EGFR siRNA and EGFR inhibitor decreased the phosphorylation of ERK1/2. Furthermore, inhibitors of ERK1/2 and AKT phosphorylation decreased the Cd‐induced expression of cyclins and CDKs. Based on these results, it is concluded that low concentrations of Cd induce proliferation of HCC 1937 cells through EGFR‐mediated cell cycle progression. This study provides a novel mechanism by which Cd promotes cell growth in triple‐negative breast cancer cells.
A Pre‐Assembled GPCR‐Signalosome Reveals Unappreciated Ligand Sensitivity and Mediates Unique Whole Cell Responses
Authors: Halls Michelle
Published: June 2021

Cells endogenously express many different receptors that activate the same signaling pathways but with diverse physiological outcomes. This suggests considerable organization and regulation of intracellular signaling, achieved by the assembly of multimeric protein complexes with diverse functions. The use of high‐resolution technologies has opened a new window of sensitivity to measure sub‐cellular signal organization by G protein‐coupled receptors (GPCRs). We previously discovered a novel signaling complex (signalosome) organized around the relaxin receptor RXFP1 that facilitates responses to femtomolar (fM) relaxin (Halls & Cooper 2010 EMBO J). We now report that this is a previously undetected phenomenon common to many GPCRs including the prototypical β2‐adrenoceptor.

We show that constitutive assembly of the β2‐signalosome is dependent on C‐terminal helix 8 and involves multiple signaling and scaffolding proteins. Activation by fM concentrations of ligand causes a sustained increase in cAMP, which is abolished following mutation of the orthosteric binding site. β2‐signalosome activation is dependent on constitutive receptor recycling, and prolonged stimulation increases receptors at the cell membrane and decreases trafficking to lysosomes, suggesting a role in sensing the extracellular environment. In addition to sustained cAMP, activation of the β2‐signalosome generates sustained nuclear ERK, increases global gene transcription and has unique effects at the proteomic level.

The discovery of GPCR‐signalosomes that respond to very low concentrations of ligand has immediate relevance to our current understanding of GPCR function, and opens a new window for the therapeutic targeting of GPCRs.
Death Effects of Reveromycin A in Normal and Diseased Synoviocytes
Authors: Jones Dan , Svrcina Haley , Steinke Ellen , Davitt Kelsey , Greer Austin , Williams Calli
Published: June 2021

Reveromycin A (Rev A) is a natural compound that specifically causes apoptosis in osteoclasts and hence may have potential as an osteoporosis therapeutic. Earlier work in our laboratory demonstrated that Rev A did not cause significant cell death in chondrocytes. To date no studies have been conducted on Rev A activity in synoviocytes or the disease‐causing cells of rheumatoid arthritis, human fibroblast‐like synoviocytes (HFLS‐RA). LDH release assays were used to measure necrosis levels and caspase 3 was quantitated from cell extracts. Rev A‐induced cell death in both cell lines was characterized under the more acidic pH of 6.8, which mimics the pH of an inflamed joint and provides a pH believed to be important for Rev A action.10 microM Rev A‐treated normal synoviocytes showed distinct apoptotic changes with a 2‐fold increase in caspase 3 activity compared to untreated. At 0.1 and 1.0 microM Rev A, caspase 3 activity did not increase. Significant necrosis was not observed at any of the concentrations. These preliminary results suggest that 10 microM Rev A may have undesirable apoptotic effects in normal synoviocytes of the joint under acidic conditions. However at lower, potentially relevant therapeutic doses, apoptotic events do not appear to occur. Untreated and 10 microM Rev A‐treated HFLS‐RA at both 7.4 and 6.8 pH's demonstrated normal morphology under the phase contrast microscope and did not demonstrate significantly different levels of apoptosis or necrosis compared to untreated, suggesting that the typically apoptosis‐resistant HFLS‐RA behave in a similar fashion when exposed to Rev A. Funding provided by an Indiana Academy of Science senior research grant, the Hodson Summer Research Institute, and the Indiana Wesleyan University Division of Natural Sciences.
Efficacy of Novel Pygeum Extract (CR002) on Testosterone‐Induced Benign Prostatic Hyperplasia (BPH) in Male Wistar Rats
Authors: Swaroop Anand , Kumar Pawan , BAGCHI DEBASIS , Bagchi Manashi
Published: June 2021

The efficacy of a novel Prunus domestica extract (pygeum extract, CR002) was investigated on testosterone propionate (TP)‐induced BPH in male Wistar rats. BPH was induced by subcutaneous administration of TP (3.0 g/kg) over a period of 15 days (interim sacrifice group) and for 21 days (terminal sacrifice group). We evaluated the dose‐dependent efficacy (0, 50, 100 and 200 mg/kg body weight/day) of novel Prunus domestica extract (CR002) and a control group against BPH, and compared with a reference standard Prunus africana extract (CR001). Extensive clinical examinations were carried out on days 1, 7, 14, 21, 28 and 35 of treatment period to determine the onset, duration and severity of clinical signs. Clinical pathology, hematology, biochemistry and histopathology were performed on days 15 and 35, prior to necropsy. Animals were fasted overnight prior to blood collection. Prostate glands and tissues were examined. On day 36, histopathology of ventral prostrate of control rats demonstrate single layer of columnar mucin secreting epithelial cells and lumen was occupied with eosinophilic secretion. In contrast, CR002 and CR001 groups (100 and 200 mg/kg/day) exhibited no hyperplasia and proliferation of epithelial cells. Prostate histopathology of these treated groups was comparable with control rats. The hyperplasia and hypertrophy of prostrate was reduced to single layered cell indicating the efficacy of CR002 and CR001. Overall, results demonstrate that CR002 exhibits therapeutic efficacy/activity in TP‐induced BPH in rats, which is comparable to CR001.
Additive Renoprotective Effects Of Pioglitazone And Fenofibrate Against Cisplatin‐Induced Renal Failure: PPARs/TNF‐α Modulation
Authors: El‐Mas Mahmoud , Helmy Maged , Helmy Mai
Published: June 2021

We employed biochemical, pharmacological, and molecular studies to investigate the role of PPARs in cisplatin nephrotoxicity. Rats received cisplatin (5 mg/kg i.p.) alone or combined with oral pioglitazone (PPARγ agonist), fenofibrate (PPARα agonist), pioglitazone plus fenofibrate, or thalidomide (TNF‐α inhibitor). Cisplatin nephrotoxicity was evidenced by rises in renal indices of functional (serum BUN and creatinine), inflammatory (TNF‐α, IL‐6), apoptotic (caspase 3), oxidative (increased malondialdehyde and decreased superoxide dismutase and nitric oxide metabolites), and histological (glomerular atrophy, acute tubular necrosis and vacuolation) profiles. Cisplatin effects were (i) partly abolished upon concurrent exposure to pioglitazone, fenofibrate, or thalidomide and (ii) completely disappeared upon co‐treatment with the pioglitazaone/fenofibrate regimen. Immunohistochemical analyses showed that the renal protein expressions of PPARα and PPARγ were reduced by cisplatin and increased to near‐control values after simultaneous treatment with pioglitazone or fenofibrate. Blockade of PPARα (GW6471) or PPARγ (GW9662) failed to alter fenofibrate and pioglitazone effects, respectively. Overall, the combined use of pioglitazone and fenofibrate additively improved manifestations of cisplatin nephrotoxicity perhaps through GW6471/GW9662‐insensitive mechanisms.
Oxidative Stress Induced Following Exposure to 3,5‐Dichloroaniline (3,5‐DCA) In Vitro: Role in Nephrotoxicity
Authors: Racine Christopher , Anestis Dianne , Ball John , Valentovic Monica , Rankin Gary
Published: June 2021

Anilines and their chlorinated derivatives are used in the production of a wide variety of agricultural products, drugs and dyes. Studies have shown that chlorinated aniline exposure leads to nephrotoxicity in both in vitro and in vivo models. Of the mono‐ and dichloroanilines studied, 3,5‐dichloroaniline (3,5‐DCA) proved to posses the most nephrotoxic potential. Exposure to 1.0 mM 3,5‐DCA for 90 min significantly increased LDH release in isolated renal cortical cells (IRCC) from male Fischer 344 rats. Pretreatment of IRCC with antioxidants significantly attenuated LDH release, suggesting that free radical/oxidative stress mechanisms may play a role in 3,5‐DCA induced nephrotoxicity. The purpose of the current study was to explore the potential role of oxidative stress in 3,5‐DCA nephrotoxicity in vitro. IRCC (4 x 106 cells/ml; 3 ml) were exposed to 3,5‐DCA (0, 0.5, or 1.0 mM) for either 60 or 90 min. Aliquots were then taken for LDH release assays, protein carbonyl detection using Oxyblot kits from Millipore, and glutathione (GSH and GSSG) determination assays. Protein carbonyls were significantly increased following exposure to 1.0 mM 3,5‐DCA after 90 min only, while a shift in the ratio of GSSG/GSH was seen at exposure to 1.0 mM 3,5‐DCA for 60 min. However, in both cases the increase in oxidative stress markers occurred after there was a significant increase in LDH release. These findings suggest that despite 3,5‐DCA's ability to induce oxidative stress, oxidative stress is not an early initiator of 3,5‐DCA nephrotoxicity in IRCC. These studies were supported, in part, by NIH grant P20GM103434.
Delavirdine is Substrate of Bcrp and P‐gp
Authors: Kefelegn Adey , Bednarczyk Dallas
Published: June 2021

Introduction

Delavirdine is an antiretroviral drug characterized by a brain/plasma pharmacokinetic disequilibrium where the cerebrospinal fluid concentration, a surrogate for unbound brain concentration, in humans is approximately 5‐fold lower than corresponding unbound plasma concentrations (Rescriptor package insert). A previous report using Caco‐2 cells suggested that delavirdine is not a P‐gp substrate (Störmer, et al., 2002). Breast cancer resistance protein (Bcrp) was therefore investigated as a mechanism that could limit penetration of delavirdine into the CNS.

Material and Methods

MDCK‐mBcrp and MDCK‐MDR1 cells were used to assess transport of delavirdine by each respective transporter. The bidirectional permeability of delavirdine was investigated as a function of time and in the absence or presence of inhibitor. Assay samples were then subject to liquid chromatography/mass spectrometry.

Results

Delavirdine demonstrated asymmetric flux by the MDCK‐mBcrp cells that was time dependent. Interestingly, time dependent asymmetric flux of delavirdine was also observed in the MDCK‐MDR1 cells. The asymmetric flux of delavirdine in both cell lines could further be brought to near unity in the presence of inhibitors of Bcrp and MDR1 (Ko143 and LY335979, respectively).

Conclusion

The results established the asymmetric transport of delavirdine by P‐gp and mBcrp. The asymmetric transport of delavirdine by P‐gp and mBcrp was time dependent and inhibitable. Collectively, both transporters likely limit the CNS penetration of delavirdine observed in vivo.

Störmer E, et al. Differential modulation of P‐glycoprotein expression and activity by non‐nucleoside HIV‐1 reverse transcriptase inhibitors in cell culture. Pharm Res. 19(7):1038‐45, 2002.
The Selection and Assessment of a Low‐Efflux MDCK Cell Line for use in a High‐Throughput Permeability Screening Assay
Authors: Boiselle Carri , Bednarczyk Dallas
Published: June 2021

Objectives

To establish a low‐efflux Madin‐Darby canine kidney cell line for potential use in a high throughput permeability screening assay.

Methods

MDCK cells were subcloned by limiting dilution. Individual MDCK colonies were isolated and clones were expanded to cell culture plates and assessed for efflux potential. The permeability characteristics and corresponding efflux ratios were measured using a standard bidirectional permeability assay conducted using 96‐well permeable support plates.

Results

72 clones were initially screened for efflux potential. 8 clones demonstrating the lowest level of efflux were further cultured for continued evaluation. The 8 clones were further reduced in number to 2 by comparison of efflux between clones, where clones demonstrating the lowest efflux were maintained. One clone, whose efflux did not increase with longitudinal culture time, was selected for use in a high‐throughput permeability screening assay. The final clone showed limited efflux of common P‐gp substrates: loperamide, quinidine, dipyridamole; BCRP substrates: prazosin, topotecan, imatinib; and an MRP‐2 substrate: valsartan. Despite the lack of transport of multiple probe substrates efflux was observed for some compounds such as vinblastine and saquinavir. The final clone was further tested using a diverse set of molecules.

Conclusions

A low efflux MDCK subclone (MDCK‐LE) was identified. The MDCK‐LE cells demonstrate low efflux for many representative substrates of efflux transporters, though the cells are not entirely a “no efflux” cell line as demonstrated by the efflux of vinblastine and saquinavir, they do delineate the permeability of structurally diverse molecules over a broad permeability range.
Protein‐Protein Interactions Between Organic Anion Transporting Polypeptide 1B3 (OATP1B3) and Organic Cation Transporter 1 (OCT1)
Authors: Shoop Jessica , Ruggiero Melissa , Zhang Yuchen , Hagenbuch Bruno
Published: June 2021

The two drug transporters OATP1B3 and OCT1 are expressed at the basolateral membrane of human hepatocytes. In a search for potential interacting proteins a modified yeast‐two‐hybrid screen (DUALmembrane screen) identified OCT1 as an interaction partner for OATP1B3. In the present study we characterized the functional consequences of co‐expressing OATP1B3 with OCT1. We used transient transfection of OATP1B3 and OCT1 in HEK293 cells and measured uptake of model substrates for the two transporters. We performed co‐immunoprecipitation and a Proximity Ligation Assay (Duolink PLA) to detect interactions between OATP1B3 and OCT1. Using an anti‐OATP1B3 antibody we were able to co‐immunoprecipitate OCT1 from freshly isolated human hepatocytes, confirming the proposed interactions. When OCT1 was co‐expressed in HEK293 cells with OATP1B3, the uptake of the OATP1B3‐selective substrate cholecystokinin‐8 (CCK‐8) was reduced by about 40% while the uptake of the OCT1 substrate MPP+ was not affected. Co‐expression of OATP1B3 with either OATP1B1 or NTCP did not result in a decreased CCK‐8 uptake, suggesting that the effect on OATP1B3 function is specific for OCT1. Total OATP1B3 protein as well as surface expressed OATP1B3 was dramatically reduced, however, no changes were observed at the mRNA level supporting protein‐protein interactions between OATP1B3 and OCT1. Using the Duolink PLA technique, we could confirm co‐localization of His‐tagged OATP1B3 with OCT1 in transfected HEK293 cells. These findings indicate that OATP1B3 and OCT1 can form oligomers in human hepatocytes and that regulation of expression of OCT1 might affect OATP1B3‐mediated drug disposition.
Pharmacogenetic Analysis of Nucleotide Kinases that Metabolize the Antiviral Drug Tenofovir
Authors: Lade Julie , Hendrix Craig , Bumpus Namandjé
Published: June 2021

Tenofovir (TFV) is a nucleotide analog used in HIV pre‐exposure prophylaxis, requiring two phosphorylation events for pharmacological activity. We aimed to determine the nucleotide kinases responsible for TFV activation in peripheral blood mononuclear cells (PBMC), vaginal tissue and colorectal tissue as this is currently unknown. In order to identify relevant kinases, siRNA knockdown was performed in PBMC and tissues, incubated with 10µM TFV and metabolites were measured using mass spectrometry. Adenylate kinase 2 (AK2) knockdown decreased TFV‐monophosphate production to 17 ± 1.4%, 9.5 ± 1.1% and 12 ± 1.5% of control in PBMC, vaginal tissue and colorectal tissue, respectively. Knockdown of muscle pyruvate kinase (PKM) decreased TFV‐diphosphate (TFV‐DP) production to 33 ± 5.8% and 27 ± 4.3% and knockdown of liver and red blood cell pyruvate kinase (PKLR) decreased TFV‐DP production to 78 ± 6.6% and 81 ± 7.4% of control in PBMC and vaginal tissue, respectively. Muscle creatine kinase (CKM) knockdown in colorectal tissue decreased TFV‐DP production to 8 ± 2.9% of control. Subsequently, clinical trial participants taking oral TFV were genotyped for variants in these 4 kinases and results were compared to pharmacokinetic data. Of the 148 previously unreported single nucleotide variants and deletions identified, bioinformatics tools predicted 10 AK2 variants (8% of 142 participants), 12 PKLR variants (7%) and 8 CKM variants (6%) to be deleterious. PKM variants were of unknown consequence. In conclusion, these data demonstrate AK2 to be a key contributor to TFV activation and genetic variation within the kinases AK2, PKM, PKLR and CKM may have a potential impact on TFV pharmacokinetics.

Funding: R01 GM103853, UM1 AI068613 and UM1 AI106707
Prediction of the Magnitude of Induction of CYP3A Activity during Early Human Pregnancy
Authors: Zhang Zufei , Farooq Muhammad , Grepper Sue , Unadkat Jashvant
Published: June 2021

Background

In pregnant women, CYP3A activity increases by ~2 fold during the third trimester (T3) [1]. Due to logistical and ethical constraints, the magnitude of CYP3A induction during the first and second trimesters (T1 and T2, respectively) is unknown. We have shown that the combination of five pregnancy‐related hormones (PRHs), cortisol(C), estradiol (E2), progesterone (P), growth hormone (GH), and placental growth hormone (PGH), at their observed T3 plasma concentrations, induced CYP3A activity in human hepatocytes to an extent comparable to that observed in vivo. Amongst these PRHs, only C consistently and significantly induced CYP3A activity [2].

Objectives

Using human hepatocytes: 1) confirm C is the major CYP3A inducer during T1 and T2; 2) predict the magnitude of induction of CYP3A activity during T1 and T2 .

Methods

Sandwich cultured human hepatocytes from 3 premenopausal donors were incubated for 72h with 1X or 10X T1, T2, or T3 plasma concentrations of PRHs (adjusted for depletion). CYP3A activity was measured by 1'‐OH‐midazolam formation.

Results

At 1X T1, T2, or T3 concentrations: 1) C consistently and significantly induced CYP3A activity; 2) induction of CYP3A activity by C or PRHs was not significantly different.

Conclusions

C is the major inducer of CYP3A activity during T1‐T3. Based on these data, we predict that the magnitude of CYP3A induction during T1 and T2 will be similar to that observed during T3. This prediction is consistent with the similar increase in oral clearance of indinavir (CYP3A cleared drug) observed during T2 and T3 [3].

Acknowledgement

Supported by NIH grant P01DA032507
Therapeutic activity of N‐(2‐hydroxy phenyl) acetamide: a novel anti‐arthritic agent on pro‐inflammatory cytokines production and suppression of Toll‐like receptors (TLR‐2 and TLR‐4) in adjuvant‐induced arthritic rats
Authors: Simjee Shabana , Perveen kahkashan , Hanif Farina , Jawed Huma , Jamall Siddiqua
Published: June 2021

Toll‐like receptors (TLRs) are key recognition structures of immune system and emerged as potential contributors to the inflammation observed in human and rodent models of arthritis. In addition, the pro‐inflammatory cytokines IL‐1β and TNF‐α play an important role in aggravating the disease process. Present study aims to investigate the effect of N‐(2‐hydroxy phenyl)‐acetamide (NA‐2) on modulation of TLRs in the development of adjuvant‐induced arthritis. Arthritis was induced by heat‐killed MT H37Ra. Treatment of NA‐2 (5 mg/kg) was started on the day of arthritis induction. Body weights, paw volume and nociception measurements were done to monitor the progression of the disease. TLR‐2 and TLR‐4 mRNAs were analyzed in 48‐h cultured splenocytes and the supernatants were used to determine IL‐1β and TNF‐α. A significant increase in body weights with a parallel decrease in paw edema and nociception was observed in NA‐2 treated animals. Remission of the clinical signs was associated with improved histology. NA‐2 treatment significantly decreased the level of IL‐1β (p < 0.003) and TNF‐α (p < 0.001). Likewise, NA‐2 also reduced the expression of TLRs mRNA. Our findings suggest that NA‐2 affects AIA in a pleiotropic manner, suppressing TLRs‐mediated joint inflammation and thus it might have a therapeutic potential for arthritis
Differential Regulation of Adaptive and Innate Immunity activated Human Mast Cell by Osteopontin
Authors: Ng Chun Wai , Lau Hang Yung Alaster
Published: June 2021

Osteopontin (OPN), a RGD‐containing protein which has been shown to enhance inflammatory responses by modulating different functions of different cell types such as macrophages, dendritic cells and natural killer cells. However, OPN has also been shown to suppress inflammatory responses and is important in the healing process. To understand more about OPN, anti‐IgE and PGN activated human mast cells developed from human CD34+ stem cells were employed to study the role of this protein in the inflammatory responses. The release of histamine and cytokines (IL‐5, IL‐8 and TNF‐α) releases as well as cell adhesion were studied by spectro‐fluorescence assay, ELISA and CyQUANT assay respectively. OPN induced adhesion of human mast cells after anti‐IgE and PGN stimulation, and the adhesion was able to inhibit anti‐IgE‐induced IL‐5, IL‐8 and TNF‐α release while only PGN‐induced IL‐5 was enhanced by OPN. To investigate the interaction between OPN and human mast cells, cyclic RGD (cRGD) and neutralizing antibodies against integrin αVβ3 and CD44 were employed. cRGD was able to inhibit both anti‐IgE and PGN‐activated adhesion and adhesion‐mediated regulation of cytokines release. While only antibody against Integrin αVβ3 was able to reverse the anti‐IgE‐induced adhesion and the inhibition of anti‐IgE‐mediated cytokines release by OPN. In conclusion, OPN suppresses anti‐IgE‐induced inflammatory response by inhibiting the synthesis of chemokine (IL‐8) and both Th1 (TNF‐α) and Th2 (IL‐5) cytokine and the interaction is through the RGD domain in OPN and integrin αVβ3 expressed on human mast cells. On the other hand, OPN enhances Th2 (IL‐5) synthesis when human mast cells are stimulated by PGN.

Research Support: RGC Grant 469310
Discovery of a Novel Enhydrazinone Ester (CEE‐1) That Inhibits the Degranulation of Both Mast Cells and Eosinophils
Authors: Ezeamuzie Charles , Edafiogho Ivan
Published: June 2021

Allergen‐dependent activation of mast cells and eosinophils is a fundamental process in the pathophysiology of allergic diseases. We have previously reported that the novel enhydrazinone ester CEE‐1 (ethyl 4‐phenylhydrazinocyclohex‐3‐en‐2‐oxo‐6‐phenyl‐1‐oate) – possess potent anti‐inflammatory activity. We have now tested whether the compound also possess anti‐allergic effects using IgE‐dependent degranulation of rat mast cell line (RBL‐2H3) and the degranulation of isolated human blood eosinophils. RBL‐2H3 cells were passively sensitized with anti‐DNP IgE, pre‐treated with CEE‐1 for 10 min before being stimulated with the antigen, DNP‐BSA. Released β‐hexoseaminidase was determined as index of degranulation. Aliquots of purified human eosinophils, were similarly pre‐treated with CEE‐1 before stimulation with human C5a (30 nM) plus cytochalasin B (5 µg/ml) for 30 min. The amount of released granular eosinophil peroxidase (EPO) was determined by the O‐phenylenediamine method. Results showed that pre‐treatment of sensitized mast cells with CEE‐1 resulted in a strong concentration‐dependent inhibition of β‐hexoseaminidase release. The IC50 value was 4.5 µM, and at 30 μM inhibition was more than 80%. The compound also inhibited C5a‐stimulated EPO release from human eosinophils with even greater potency (IC50 = 0.1 μM). These results show that CEE‐1 possesses strong anti‐allergic properties exerted on both mast cells and eosinophils, and therefore has the potential to be a useful therapy for allergic diseases.

Sponsored by Research Sector, Kuwait University, Grant # MR 03/09.
Paroxetine‐mediated GRK2 Inhibition Reverses Cardiac Dysfunction and Remodeling Post‐Myocardial Infarction
Authors: Schumacher Sarah , Gao Erhe , Zhu Weizhong , Chen Xiongwen , Chuprun J Kurt , Feldman Arthur , Tesmer John , Koch Walter
Published: June 2021

Heart failure (HF) is a disease of epidemic proportion associated with exceedingly high healthcare costs. Data shows that G protein‐coupled receptor (GPCR) kinase 2 (GRK2) plays a critical role in HF progression, promoting dysfunctional adrenergic signaling and myocyte death. We found that the selective serotonin reuptake inhibitor, paroxetine, could selectively inhibit GRK2. In this study wild‐type mice underwent myocardial infarction (MI) and following 2 weeks of injury, underwent 4 weeks of treatment with paroxetine or control SSRI, fluoxetine. All post‐MI mice exhibited similar left ventricular (LV) dysfunction prior to treatment; however, in contrast to continuing degradation of function, paroxetine produced significant improvement in LV function and structure. Paroxetine enhanced hemodynamic function, blocking the ~2.5 fold increase in LV end diastolic pressure (LVEDP). Paroxetine restored βAR membrane density, returned serum catecholamine and GRK2 levels to baseline, and prevented induction of the fetal gene program. Importantly, the beneficial effects persisted after termination of treatment and were markedly greater than β‐blocker therapy, a current standard of care. These data demonstrate that paroxetine‐mediated inhibition of GRK2 improves cardiac function post‐MI and potentially represents novel re‐purposing as well as a starting point for innovative small molecule inhibitor development.