bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of Dithiolethione 59 as Potential Neuroprotective Antiparkinsonian Agent
Authors: Betharia Swati , Brown Dennis , Fuentes Steven , Aoude Iman
Published: November 2023

Parkinson's disease (PD) is a chronic, progressive neurodegenerative disorder characterized by the disintegration of nigrostriatal dopaminergic neurons. The resulting loss of dopamine gives rise to the cardinal symptoms of the disease, including resting tremor, rigidity, and bradykinesia. Oxidative stress and depletion of the ubiquitous antioxidant glutathione (GSH) have been implicated in the pathophysiology of PD. Towards the development of novel neuroprotective antiparkinsonian agents, we report the pharmacological characterization of the sulfur‐containing dithiolethone (DTT) 59 in the dopaminergic neuroblastoma SH‐SY5Y cell line. DTTs are known activators of the transcription factor nuclear factor erythroid‐2 factor (Nrf2). Activation of Nrf2 is associated with the induction of various cytoprotective antioxidants such as GSH, the catalytic unit of enzyme glutamate cysteine ligase (GCLC), the cytosolic isoform of glutathione S‐transferase (GSTP1), the light chain subunit of the cysteine‐glutamate transporter (xCT), and the phase II enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1). Compound 59 was found to dose‐dependently increase cellular levels of total GSH. 59 also provided significant neuroprotection in the 6‐hydroxydopamine (6‐OHDA) model of neurotoxicity at concentrations as low as 10 μM. Mechanistic studies showed a concentration‐ and time‐dependent increase in NQO1 at both the message and protein levels; however, GCLC, GSTP1 and xCT levels remained unchanged. Pretreatment of SH‐SY5Y cells with the GCLC inhibitor buthionine sulfoximine (BSO) was found to block 59‐mediated GSH induction. BSO treatment also abolished the protective properties of 59, indicating that GSH induction is important for neuroprotection. While the Nrf2 pathway did not appear to be involved in this induction, these data suggest that 59 is a potential neuroprotective antiparkinsonian agent and warrants additional exploration.

Support or Funding Information

This research was supported by Manchester University College of Pharmacy, Natural and Health Sciences.
EFFECTS OF UCH‐L1 INHIBITION IN IMMORTALIZED DOPAMINERGIC NEURONS
Authors: Winner Brittany Michele , Zhang Harue , Goudreau John , Lookingland Keith
Published: November 2023

In Parkinson disease (PD), a debilitating neurodegenerative disorder characterized by a host of motor and non‐motor symptoms, neurons in the substantia nigra pars compacta (SN) progressively degenerate. The effect of this degeneration is the loss of striatal dopamine (DA), which famously results in bradykinesia, postural instability, shuffling gait, and resting tremor. Animal and cellular models only partially recapitulate the progression of the disease, since much is still not known about the etiology of PD. Using cell lines affords a method in which to manipulate parameters in a homogeneous population whereby we can study phenomena occurring specifically in dopaminergic neurons. The dopaminergic cell line, MN9D, was generated by fusing mouse neuroblastoma cells with primary mouse mesencephalon neurons. A key feature of these cells is their ability to be differentiated using sodium butyrate in the culture medium, which halts their cell cycle and makes them look morphologically like neurons. Expression of the dopamine transporter and tyrosine hydroxylase, markers of dopamine neurons, are also highly expressed after differentiation of MN9D cells. In these cells, we have studied components of the ubiquitin proteasome system (UPS) such as parkin and UCH‐L1, which are two genes found mutated in familial PD. The latter, UCH‐L1 (ubiquitin carboxy‐terminal hydrolase L1), is a neuron‐specific, highly expressed de‐ubiquitinating enzyme thought to play a role in the susceptibility of mouse SN neurons exposed to the toxicant MPTP (active metabolite is MPP+). UCH‐L1 acts to remove monomeric ubiquitin from protein substrates, regulating proteasome activity and replenishing the pool of monomeric ubiquitin to be tagged on to new substrates. In MN9D cells with MPP+ treatment, we have found that intracellular dopamine, intracellular DOPAC, and UCH‐L1 protein all decrease. In addition, when we treated cells with LDN‐57444 (a UCH‐L1 inhibitor), DA but not DOPAC decreased in these cells, highlighting a possible link between the loss of DA and loss of function of UCH‐L1. These studies will help shed light on the dynamics between UPS function and dopamine synthesis and turnover in MN9D cells as an in vitro model for isolated SN neurons.

Support or Funding Information

T32 Pre‐Doctoral Training Grant Institute for Integrative Toxicology; PIs Grant: 1R01 NS065338‐01A2
Loss of GPR37 Increases Susceptibility to Demyelination
Authors: Coleman Brilee M. , Li Fengqiao , Giddens Michelle M. , Hall Randy A.
Published: November 2023

GPR37 is an orphan G protein‐coupled receptor that is highly expressed in the brain. Previous work has shown that GPR37 is a receptor for the secreted neuroprotective and glioprotective factor prosaposin and that prosaposin acts through GPR37 to protect cultured astrocytes from oxidative stress‐induced cell death (Meyer et al., Proc. Natl. Acad. USA, 2013). Furthermore, it has previously been shown that prosaposin enhances the survival of oligodendroglia in vitro and also promotes myelination in vivo (Hiraiwa et al., Proc. Natl. Acad. Sci. USA, 1997; Hiraiwa et al., Glia, 1999). Interestingly, several reports indicate that GPR37 is expressed at particularly high levels in oligodendrocytes (Imai et al., Cell, 2001; Cahoy et al., J. Neurosci., 2008). However, despite the aforementioned evidence for GPR37 expression in oligodendroglia, the role(s) that GPR37 may play in oligodendroglial biology remain uncharacterized. Here we show that GPR37‐knockout (GPR37‐KO) mice display more rapid and extensive demyelination than their wild type (WT) counterparts in the cuprizone model of demyelination. Furthermore, we observed that GPR37‐KO mice do not show an increased loss of oligodendrocyte precursor cells or mature oligodendrocytes. Parallel studies on primary cultures of oligodendrocytes also revealed a protective role for GPR37. From these data, we conclude that loss of GPR37 alters oligodendrocyte physiology and increases susceptibility to demyelination, independent of effects on oligodendrocyte proliferation or differentiation. The present work identifies GPR37 as a potential drug target for the treatment of demyelinating diseases such as multiple sclerosis. Ongoing studies in this area seek to elucidate the signaling pathways downstream of GPR37 that regulate oligodendrocyte physiology and support myelination.

Support or Funding Information

This research was supported through the Fast Forward program of the National Multiple Sclerosis Society.
Stalk‐dependent and Stalk‐independent Activity of the Adhesion GPCR GPR56/ADGRG1
Authors: Kishore Ayush , Hall Randy A.
Published: November 2023

The adhesion G protein‐coupled receptors (aGPCRs) comprise an enigmatic family of 33 receptors in humans. A hallmark of the aGPCRs is the presence of a large extracellular N‐terminal (NT) domain that has been shown to inhibit receptor activity. Yet another defining characteristic of virtually all aGPCRs is the presence of a GPCR Autoproteolysis Inducing (GAIN) domain, which is found on the membrane‐proximal end of the NT and controls the autocatalytic proteolysis of the receptor into an NT protomer and seven‐transmembrane (7TM) protomer, which can stay non‐covalently associated for some time. Recently, great progress has been made to shed light upon a more detailed view of how exactly the NT regions inhibit aGPCR‐mediated signaling. Notably, two separate groups have espoused a “cryptic agonist” model of aGPCR activation wherein GAIN domain cleavage unveils a new NT stalk which serves as the receptor's agonist. In this model, the stalk is a requisite agonist for aGPCR activity and deletion of the stalk result in a signaling‐incompetent receptor. In the work presented here, we show that the stalk of the aGPCR GPR56/ADGRG1 (“G1”) is indeed necessary for activation of the certain downstream readouts (such as activation of SRF luciferase) but dispensable for others (such as the shedding of membrane‐bound TGFalpha and activation of NFAT luciferase). Moreover, a stalkless version of G1 also displays comparable activity to the highly‐active, GAIN‐cleavage‐mimetic form of G1 (G1‐deltaNT) in several measures of activity including beta‐arrestin recruitment, receptor ubiquitination, and association with Galpha13. These lines of evidence suggest that G1 is capable of both stalk‐dependent and stalk‐independent modes of signaling. Based upon our results, we propose a more nuanced model of aGPCR activation in which the NT inhibits activity of the 7TM region in two distinct ways: i) antagonizing stalk‐dependent signaling by masking the cryptic stalk peptide, and ii) directly antagonizing the inherent stalk‐independent activity of the 7TM protomer.

Support or Funding Information

This work was supported by NIH R01 award R01‐NS072394.
The BAI Sub‐Family of Adhesion GPCRs: New Insights into Regulation and Signaling
Authors: Purcell Ryan H. , Hall Randy A.
Published: November 2023

The three members of the BAI sub‐family of adhesion GPCRs (BAI1‐3/ADGRB1‐3) have been implicated in dendritic spine morphogenesis, macrophagic and microglial phagocytosis, and skeletal myoblast fusion, suggesting an important role for these receptors in directing cytoskeletal dynamics. However, the activation mechanisms, downstream signaling pathways, and regulation of these orphan receptors remain poorly understood. Adhesion receptors can be autoproteolytically cleaved by their conserved N‐terminal GAIN domains into non‐covalently attached N‐terminal and seven‐transmembrane protomers. Cleavage and removal of the NT is thought to be a critical step in receptor activation by relieving an inhibitory constraint imposed by the NT on the 7TM. Several other aGPCRs have recently been shown to then be activated by their post‐cleavage stalks in a manner reminiscent of the protease‐activated receptor (PAR) family. Surprisingly, we found that BAI1/ADGRB1 (B1) exhibits no stalk‐dependent activity because a truncated receptor lacking the entire N‐terminus (including the stalk) signals at least as strongly as a truncated version of B1 that mimics the GAIN‐cleaved form of the receptor and thus still possesses the N‐terminal stalk. In addition, ongoing studies have revealed B1 and B2 interactions with several C‐terminus‐binding proteins, which are involved in both the regulation of G protein‐dependent signaling by B1 as well as the mediation of G protein‐independent aspects of receptor signaling. These studies have also led to insights into cross‐talk between heterotrimeric G protein and small GTPase signaling downstream of B1, which may significantly improve our understanding of the diverse functions of the BAI receptors.

Support or Funding Information

Supported by NINDS R21NS094136
Modulation of the Facilitatory Effect of Dopamine at the Neuromuscular Junction by Adenosinergic and Purinergic Receptors
Authors: AlQot Hadir E. , Elnozahi Neveen A. , Mohy El‐Din Mahmoud M. , Bistawroos Azza E. , Abou Zeit‐Har Mohammed S.
Published: November 2023

The objective of this study is to investigate the potential interactions between dopamine and the endogenous neuromodulators; adenosine triphosphate (ATP) and adenosine, at the neuromuscular junction.

We have shown previously that dopamine, via the activation of dopamine D1‐like receptors, can enhance neuromuscular transmission. ATP and adenosine play a pivotal role in modulation of dopamine function in the central nervous system via purinergic and adenosinergic receptors, respectively. As such, we probed the potential existence of similar interactions in the periphery.

We employed in‐vitro experiments using the isolated rat phrenic hemi‐diaphragm preparation which was stimulated indirectly through the stimulation of the phrenic nerve at low frequencies. The parameter used to assess neuromuscular transmission was the percentage change in the amplitude of nerve‐evoked muscle twitches relative to control.

In presence of the selective adenosine A2A receptor antagonist (8‐(3‐Chlorostyryl) caffeine; CSC), the dopamine‐mediated facilitation of indirect muscle twitches was significantly reduced. This implies a positive interaction between dopamine and adenosine, via adenosine A2A receptors at the motor end‐plate.

The enhancement of neuromuscular transmission by dopamine was, unexpectedly, depressed by 1,3‐Dipropyl‐8‐cyclopentylxanthine (DPCPX), a selective adenosine A1 receptor antagonist. Unlike what would be expected from an inhibitory receptor, the adenosine A1 receptor appears to contribute a positive effect towards the facilitation mediated by dopamine at the neuromuscular junction.

On the other hand, a negative collaboration was established between dopamine and ATP as evidenced by the enhanced dopamine induced‐potentiation of indirect muscle twitches subsequent to the addition of suramin (purinergic antagonist). Hence suggesting that endogenous ATP, via purinergic P2 receptors, exerts an antagonistic influence on the dopamine effects.

In conclusion, these results provide new insights into how dopamine effects can be modulated by co‐transmitters at the neuromuscular junction. Clinically this study highlights potential therapeutic targets for the treatment of disorders characterized by neuromuscular deficits based on the conceived interactions at the motor end‐plate.
Cannabinoid Receptor 2 stimulation enhances bactericidal activity of human neutrophils
Authors: Wiley Mark B , Nizet Victor , Insel Paul A , Corriden Ross
Published: November 2023

Polymorphonuclear neutrophils (PMNs), the most abundant white blood cell in circulation, play an essential role in host defense against pathogenic bacteria. Central to this activity is the ability of PMNs to sense and rapidly respond to extracellular stimuli. Although GPCRs are the largest membrane receptor family in the human genome, only a limited number of GPCRs have been evaluated as regulators of PMN function. Here, we investigate the role of Cannabinoid Receptor 2 (CB2), typically expressed by immune cells in the periphery, in the regulation of PMN function. We find that activation of neutrophil CB2 receptors using the cannabinoid receptor agonist CB‐13 enhances killing by PMNs of methicillin‐resistant Staphylococcus aureus (MRSA) in an in vitro assay of bactericidal activity. However, CB‐13 does not affect PMN migration in a chemoattractant gradient (measured using a transwell assay), production of reactive oxygen species (ROS; determined using the fluorescent ROS scavenger DCF‐AM) or neutrophil extracellular trap (NET) formation (assayed via fluorescence‐based DNA quantification). Experiments using fluorescently‐labeled, pH‐sensitive S. aureus bioparticles suggest that enhancement of PMN killing by CB‐13 results from an increase in phagocytosis. Together these results lead us to conclude that CB2 activation may “prime” PMNs to detect and kill bacteria and thus, that CB2 may represent a novel therapeutic target for pharmacological modulation of neutrophil antimicrobial activity. As such, CB2 appears to be a previously unappreciated PMN‐expressed GPCR that enhances innate immunity and microbial surveillance.

Support or Funding Information

Research was supported by NIH grants HD071600, AI057153 and AI052453.
Investigation of signaling pathways activated by Cocaine‐ and Amphetamine‐Regulated Transcript peptide in PC‐12 cells
Authors: Prinster Steven , Kothari Arti , Wang Xia
Published: November 2023

The cocaine‐ and amphetamine‐regulated transcript (CART) was first isolated from rat striatum following the acute administration of cocaine or amphetamine. It was quickly determined that CART or CART peptide was expressed in a variety of tissues affecting a wide range of important processes. These functions include roles in feeding behavior, pleasure and reward, diabetes, ovarian follicle development, bone remodeling and anxiety. In spite of the relevance of these conditions as therapeutic targets, development of small molecules to modulate these systems has been impeded by the lack of an identified receptor for the CART peptide. These studies focused on the signaling pathways activated in PC‐12 cells by treatment with the CART peptide. When treated with CART peptide, an inhibition of protein kinase B (PKB) was observed, in the presence of the 6‐38 fragment of pituitary adenylate cyclase‐activating polypeptide (PACAP 6‐38), no change was observed, but in the presence of CART and PACAP 6‐38, PKB phosphorylation increased 2‐fold. The CART peptide has previously been found to stimulate ERK1/2 phosphorylation in a pertussis toxin‐sensitive manner, indicating that the receptor is Gi/o‐coupled receptor. The effect of CART peptide on cAMP production was also assessed using a real‐time luminescent biosensor. In these assays, the CART peptide led to an increase in cAMP, which may suggest the receptor is also able to couple to Gs. Additional work is required to confirm the characteristics of the CART receptor.
Experimentally‐ induced chronic kidney disease in rats, and the influence of curcumin thereon
Authors: Ali Badreldin H. , Al‐Salam Suhail , Al Za'abi Mohamed H. , Al Suleminai Yousif , Nemmar Abderrahim
Published: November 2023

Curcumin (a natural phenolic yellow pigment isolated from turmeric) has several pharmacological properties that include antioxidant, anti‐inflammatory and antiapoptotic activities. Here, we investigated some effects of three graded oral doses of curcumin (37.5, 75 and 150 mg/kg) for 35 days on kidney structure and function in rats with chronic kidney disease (CKD) induced by adenine (0.25% w/w in feed for 35 days), which is known to involve inflammation, oxidative stress and apoptosis. Using several indices in plasma, urine and kidney homogenates, adenine was found to lower creatinine clearance and increase plasma concentrations of creatinine, urea, neutrophil gelatinase‐associated lipocalin and N‐Acetyl‐beta‐D‐glucosaminidase activity. Furthermore, it raised plasma concentrations of the uremic toxin indoxyl sulfate, some inflammatory cytokines and urinary albumin concentration. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activities, total antioxidant capacity and reduced glutathione were all adversely affected. Most of these adenine ‐ induced actions were moderately and dose ‐dependently mitigated by curcumin, especially at the highest dose. Curcumin did not cause any overt adverse effect on the treated rats. The results suggest that different doses of curcumin produce variable salutary effects against adenine‐induced CKD in rats, and that, pending further pharmacological and toxicological studies, its usability as a possible ameliorative agent in human CKD should be considered.

Support or Funding Information

This work was funded by the Research Council, Oman (RC/MED/PHAR/13/01)
Development of a New Practical Approach to Perform Single‐Mouse Pharmacokinetic Study
Authors: Jilek Joseph Lionel , Tian Ye , Yu Ai‐Ming
Published: November 2023

Single‐animal pharmacokinetic (PK) study has largely been limited to rats and larger animals. However, given the increasingly large body of research using transgenic and immunocompromised mouse models, and an economical advantage, a single‐mouse PK model is highly warranted. Given a high sensitivity of LC‐MS/MS in quantification of drugs and metabolites, we have established a practical and standard protocol for series blood samplings from single mouse via tail vein at multiple time points towards a complete PK profile. CD‐1 Mice were given a cocktail of cytochrome P450 (CYP) isoform‐specific probe substrates per os, consisting of midazolam (MDZ), dextromethorphan (DXM), phenacetin (PHE), diclofenac (DIC), and chlorzoxazone (CHL). CYP inhibitors, ketoconazole (KTZ) and α‐napthoflavone (α‐NF), were administered by intraperitoneal injection 1 hour prior to dosing; CYP inducers, pregnenolone 16α‐carbonitrile (PCN) and 3‐methyl cholanthrene (3‐Me), were administered 3 days prior to dosing. Blood (10–20 μl per time point) was collected by tail vein puncture at 7–10 different time points over 3 hours and plasma was prepared for LC‐MS/MS quantification of MDZ, DXM, PHE, DIC, and CHL, and CYP isoform‐specific metabolites 1′‐OH‐MDZ, DXO, ACE, 4′‐OH‐DIC, and 6′‐OH‐CHL, respectively.

A small volume (3 μl) of plasma at each time point was found to be sufficient to produce a complete PK profile in a single mouse. Visual inspection and non‐compartmental PK modeling showed that formation of 1′‐OH‐MDZ and ACE was sharply and significantly reduced in mice pre‐treated with KTZ and α‐NF, respectively, suggesting that these compounds preferentially inhibit MDZ 1′‐hydroxylase and PHE O‐deethylase, respectively, in mice in vivo. Additionally, plasma MDZ concentrations and all PK parameters (Cmax, AUC, t1/2) were significantly altered in mice administered PCN. Interestingly, this was not reflected by an increase of 1′‐OH‐MDZ levels, yet by a decrease in plasma 1′‐OH‐MDZ and Cmax and AUC values, suggesting that formation of other metabolites may be induced. Furthermore, plasma PHE and all PK parameters were significantly reduced to near negligible concentrations in mice administered 3‐Me, but plasma ACE, Cmax, and AUC were also slightly attenuated relative to control, suggesting that other metabolic pathways may be induced. Lastly and surprisingly, plasma MDZ concentrations were also reduced in mice administered 3‐Me, but to a much lesser extent, suggesting that 3‐Me is a promiscuous inhibitor to MDZ 1′‐hydroxylation in mice.

This approach provides a useful and practical means to conduct single mouse PK research and may prove useful for studying drug metabolism in widely‐used laboratorial mouse models. Additionally, our data suggest alternative roles for classical human CYP inhibitors and inducers in a mouse model.

Support or Funding Information

J.L.J., Y.T., and A.‐M. Y. were supported by a National Institutes of Health National Cancer Institute grant [U01CA175315].
Absence of Age‐Related Differences in Internal Dosimetry of Deltamethrin (DLM) at Low Exposure Levels
Authors: Chen Chen
Published: November 2023

Most people are exposed to low levels of pyrethroids, the most widely‐used insecticides in the U.S. and Europe. Immature rats are more susceptible than adult animals to high doses of the pyrethroid DLM. This is attributed to the less efficient metabolic detoxification of the neurotoxic parent compound. Experiments were conducted to test the hypothesis that age differences in DLM internal dosimetry disappear when doses are low enough to be adequately metabolized. Postnatal day (PND) 15, 21 and 90 rats were gavaged with 100, 250 or 500 μg DLM/kg. Serial plasma, brain and liver samples were taken for 48 h post dosing and analyzed for their DLM content by GC‐MS. Age‐dependent differences in biological sample DLM content generally diminished with decrease in dose. PND 15 plasma AUC∞° values, for example, were 4.6‐, 2.4‐, and 1.5‐fold higher than corresponding adult values for DLM doses of 500, 250 and 100 μg/kg. Liver and brain Cmax values exhibited similar patterns. The 100 μg/kg dose is similar to EPA upper bound estimates of DLM environmental exposure levels. Our findings suggest that children and adults will experience comparable internal doses and, in turn, risks from current pyrethroid exposures.

Support or Funding Information

Council for the Advancement of Pyrethroid Human Health Risk Assessment (CAPHRA)
Pharmacokinetics and Pharmacodynamics of Bioengineered miR‐34a Prodrug
Authors: Ho Pui Yan , Wang Wei‐Peng , Chen Qiu‐Xia , Addepalli Balasubrahmanyam , Limbach Patrick A. , Li Mei‐Mei , Jilek Joseph Lionel , Qiu Jing‐Xin , Zhang Hong‐Jian , Li Tianhong , Wun Theodore , White Ralph DeVere , Lam Kit S , Yu Ai‐Ming
Published: November 2023

The complexity of cancer has been demonstrated to closely align with an alteration in gene expression profiles. Dysregulated miRNAs control many fundamental biological processes including tumor progression through the suppression of multiple target genes, suggesting the development of miRNA based therapy. Recently we have developed a novel approach to biosynthesize chimeric tRNA/mir‐34a agents in Escherichia coli on a large scale, which may act as a prodrug for miR‐34a replacement therapy. In this study, we aimed to define the pharmacokinetics and pharmacodynamics of bioengineered miR‐34a prodrug. We found that miR‐34a prodrug is susceptible to Dicer and other RNases (e.g., RNase A and Angiogenin), yet protected from degradation when formulated with in vivo‐jetPEI. Higher levels of accumulation were seen in mouse liver and lung for in vivo‐jetPEI formulated miR‐34a prodrug administered intravenously. We further monitored the metabolic fate of tRNA/mir‐34a in human carcinoma cells through deep sequencing and qPCR analyses, and our data demonstrated a precise production of mature miR‐34a from the prodrug. As a result, miR‐34a prodrug caused a significant downregulation of miR‐34a target genes (e.g., CDK6, MET, SIRT1) in human cancer cells and a sharp reduction of tumor growth in A549 xenograft mouse models. In addition, we examined the acute effects of biologic miR‐34a agents by analyzing blood chemistry profiles and IL‐6 levels in mouse models, which showed no signs of acute toxicity. Therefore, we illustrate the fate, efficacy and safety of recombinant miR‐34a prodrug that would exemplify the promise of bioengineered miRNA agents in research and development.
Use of Age‐dependent Carboxylesterase 1 Protein Expression to Predict in vitro Elimination and in vivo Pharmacokinetics of Oseltamivir in Infants
Authors: Boberg Mikael , Mehrotra Aanchal , Vrana Marc , Prasad Bhagwat
Published: November 2023

90% of drugs prescribed for neonates and infants are off‐label or unlicensed. Therefore it is imperative to develop alternate approaches to predict drug disposition in this special population. In this direction, the aim of this study is to test whether age‐dependent protein expression data of carboxylesterase 1 (CES1) can predict in vitro elimination and in vivo pharmacokinetics (PK) of oseltamivir in infants using physiologically based PK (PBPK) modeling.

CES1 protein expression data determined by LC‐MS/MS in pediatric (n=44, including infants (0 – 1 yr, n=14)) and adult (n=7) human liver microsome (HLM) samples, were used to compare relative hepatic metabolic clearance of oseltamivir in vitro and in vivo. CES1 mediated metabolic clearance of oseltamivir was quantified by incubating 100 nM of oseltamivir in 100 μg/ml of pediatric and adult HLM samples in phosphate buffer (pH 7.4) at 37ºC. For in vivo simulation study, the adult PBPK model was developed for oseltamivir by incorporating various reported observed and predicted drug‐ and system‐dependent parameters in Simcyp (Version 14, Sheffield, UK). The model was then fitted with the observed in vivo data [1, 2] for adult subjects and key PK parameters were compared.

An age‐dependent variability in oseltamivir metabolic stability and CES1 expression was observed. A significant correlation was observed between CES1 protein expression and oseltamivir metabolism in representative HLM samples (p<0.05). The CES1 activity measured as metabolic rate constant (k, min−1) was higher by 2.4‐fold (p<0.05) in the adults compared to the pediatric group. The k (min−1) for the active metabolite oseltamivir carboxylate (OC) formation was correlated with depletion of oseltamivir (p<0.001) suggesting that OC is the only metabolite formed. Significantly, the CES1 expression was found to be 3.7‐fold (p<0.001) higher in adults as compared to the infants. Oseltamivir PK in adult was predicted accurately with mean parameters (predicted vs. observed [2]) as AUC 314 vs. 297 ng/ml.h, Cmax 89 vs. 99 ng/ml and Tmax 1.04 vs. 0.95 h, respectively. All the observed in vivo data points for the adults were within the predicted 5th and 95th percentiles.

Once the data are available from our large number of pediatric and adult samples, we will incorporate the expression data into the already developed adult PBPK model. The adult model optimized in this study and difference in the protein expression will be applied to predict drug disposition in infants. The predicted oseltamivir concentration‐time profile will be compared to the reported infant in vivo data[3]. If successful, the neonatal/infant PBPK model of CES1 metabolism will be important in predicting efficacy of ester prodrugs, safety of ester drugs and toxicity of environmental toxins such as phthalates, benzoates and carbamates.

Support or Funding Information

Funded by NIH/NICHD1R01HD081299‐01A1
Toxicokinetics of Domoic Acid in Cynomolgus Monkeys
Authors: Jing Jing , Petroff Rebekah , Crouthamel Brenda , Grant Kimberly , Burbacher Thomas , Isoherranen Nina
Published: November 2023

Objectives

Domoic acid (DA), a potent neurotoxin, is produced by various marine algae and diatoms. DA acts as an analog of the neurotransmitter glutamate and is excitotoxic in the central nervous system and other glutamate receptor‐rich organs. Multiple DA intoxication cases have been reported worldwide in both marine animals and humans due to the increase of DA producing algal blooms. However, the toxicokinetics of DA has not yet been characterized in humans and it is unclear how well different animal models reproduce DA disposition in humans. In addition, due to the low assay sensitivity of DA, no toxicokinetic studies have been conducted at doses close to the tolerable daily intake (TDI) of DA in humans (0.075 ppm). The aim of this study was to determine the bioavailability and distribution and elimination kinetics of DA at doses close to the TDI in the cynomolgus monkey, a non‐human primate model for toxicokinetic studies.

Methods

Three healthy female adult cynomolgus monkeys (5.1±1.9 kg) were included in this study. DA was administered in a crossover design as an IV bolus of 0.005 mg/kg or as an oral solution in sugary water (0.075 mg/kg and 0.15 mg/kg, the TDI and 2* TDI in humans) with and without concomitant food. Plasma samples were collected before dosing and up to 48 hr post‐dosing. A novel LC‐MS/MS method was developed to measure DA concentrations in plasma.

Results

The elimination of DA from plasma after IV dosing was biphasic with alpha half‐life of 6.0 ± 4.3 min and beta half‐life of 59.6 ± 2.7 min. The clearance of DA was 2.7 ± 0.7 ml/min/kg. The clearance of DA is similar to the reported glomerular filtration rate in cynomolgus monkeys (3.1 ± 0.5 ml/min/kg) suggesting that the renal clearance of DA is primarily through filtration. The mean absolute bioavailability of DA was 13.7 % and food had no effect on DA absorption kinetics. After oral administration, the absorption of DA was slow with Tmax of 5.0 ± 4.3 hr and Cmax of 5.6 ± 1.1 ng /ml. The disposition of DA after oral dosing was characterized by flip‐flop kinetics with absorption half‐life of 16.7 ± 5.2 hr and elimination half‐life of 53.7 ± 17.4 min.

Conclusions

For the first time, our study fully characterized the toxicokinetics of DA in non‐human primates. The obtained toxicokinetic parameters could be further used to predict DA disposition in human and evaluate human exposure to DA.

Support or Funding Information

This research was funded by NIH grants P51OD010425 and 1R01ES023043‐01A1.
Distribution, metabolism and elimination of opicapone in the rats and non‐human primates
Authors: Loureiro Ana , Fernandes‐Lopes Carlos , Soares‐da‐Silva Patrício
Published: November 2023

Objectives

Pharmacokinetic evaluation of opicapone in Cynomolgus monkey and Wistar rat.

Methods

Opicapone disposition was evaluated in the Cynomolgus monkey and Wistar rat after intravenous (1 mg/kg) and oral administration (10 mg/kg) of [14C]‐opicapone.

Results

Five to seven days after administration of [14C]‐opicapone, more than 89.0% and more than 68% of opicapone related radioactivity has been excreted in rats and in monkeys, respectively. The radioactivity peaked in plasma at 4 and 0.25 h post‐dosing in rats and monkeys, respectively, followed by a quick decline and a very slow terminal phase. Opicapone related radioactivity were mainly recovered via the biliary route with a very low levels (less than 10%) excreted in urine in both species, rat and monkey. Whole body autoradiography studies in rat following single oral administration of [14C]‐opicapone (10 mg/kg), indicate that radioactivity was rapidly absorbed and distributed throughout the body. The high tissue to blood ratio of total radioactivity observed in liver and kidney at 48h post‐dosing suggests a slower elimination of opicapone related radioactivity from this tissues. Following oral administration of 1000 mg/kg opicapone, opicapone 3‐O‐sulfate, glucuronidation 3‐O‐glucuronide and the N‐oxide reduced form of opicapone were quantified in rat and monkey. With exception of N‐oxide reduced form for which the AUC0‐t was 13‐fold higher in monkeys, the exposure to opicapone and quantified metabolites were higher in rat.

Conclusions

Opicapone was found to be rapidly absorbed and primarily excreted via feaces in the Cynomolgus monkey and the Wistar rat. Differences in opicapone and metabolites exposure were noticed between species.
Cholic acid feeding increases CYP2D6 expression in CYP2D6‐humanized mice
Authors: Kent Rebecca , Pan Xian , Jeong Hyunyoung
Published: November 2023

Cytochrome P450 (CYP) 2D6 is a major drug‐metabolizing enzyme in the liver, but factors involved in the regulation of CYP2D6 expression remain poorly defined. Recent studies have shown that CYP2D6 expression is repressed by small heterodimer partner (SHP), a transcriptional repressor that is upregulated in cholestasis (1,2). The objective of this study was to determine whether cholestasis modulates CYP2D6 expression by upregulating SHP. To this end, CYP2D6‐humanized transgenic mice were fed normal chow (control) or chow supplemented with 1% (w/w) cholic acid (CA) for 2 weeks (n=4/group), and hepatic SHP and CYP2D6 expression was measured. SHP mRNA expression did not differ between the groups while SHP protein expression decreased by 2‐fold. CYP2D6 mRNA and protein expression levels were increased in the CA group by 1.5‐fold. To determine the mechanism for the disconnect between the changes in SHP mRNA and protein levels in CA‐fed mice, an in situ analysis of 3′‐untranslated region of SHP was performed, and a putative binding site for a microRNA, miR142‐3p, was identified. miR142‐3p expression was found to be increased by 5‐fold in the liver of CA‐fed mice. Promoter reporter assays using a luciferase vector harboring the 3′‐untranslated region of SHP revealed that miR142‐3p represses SHP expression by 2‐fold in HEK293T cells. Overall, these results indicate that CA feeding in mice leads to decreased SHP protein levels and increased CYP2D6 expression.

Support or Funding Information

This study was supported by NIH (GM112746 and HD065532).
c‐Jun N‐terminal kinase 2 contributes to the tumorigenesis of non‐small cell lung carcinoma through MKK4‐dependent and MKK7‐independent mechanism
Authors: Kaoud Tamer Saad , Du Lili , Tsai Kenneth Y. , Dalby Kevin N.
Published: November 2023

It had been reported previously that the JNKs require phosphorylation by both MKK4 and MKK7 to become activated. While the JNK2 isoform shows a unique propensity among the JNKs to autophosphorylate in vitro. Recent studies in glioblastoma and lung carcinoma have suggested important roles for constitutive activation of JNK2 (but not JNK1). Here we show that JNK2 autophosphorylation contributes to the proliferation of NSCLC cells under conditions of low serum through an MKK4‐dependent and MKK7‐independent mechanism. Using recombinant activated (MKK4 & MKK7) and inactivated (JNK1 & JNK2), we investigated the mechanism of JNK2 autophosphorylation and found that it has a strong preference to autophosphorylate on Thr‐183, while exhibiting a slower phosphorylation on Tyr‐185. This ability to efficiently autophosphorylate on Thr‐183 distinguishes it from other isoforms and facilitates its activation through a previously unknown mechanism involving MKK4. Phosphorylation of Tyr‐185 by MKK4 synergizes with Thr‐183 autophosphorylation to activate JNK2.

Evidence in multiple cell lines including mouse embryonic stem cells lacking MKK4 or MKK7 suggests that autophosphorylation alone activates JNK2 weakly in cells. However, JNK2 autophosphorylates on Thr‐183, to create a pool of JNK2 primed for activation by MKK4, which phosphorylates JNK2 on Tyr‐185 to activate it. Under conditions of low serum JNK2, but not JNK1 exhibits a dose‐dependent dephosphorylation upon treatment of NSCLC cells with the pan‐JNK ATP competitive inhibitors (JNK‐IN‐8 and SP600125), further supporting the notion that active JNK2 results primarily from autophosphorylation of Thr‐183. Under similar conditions of low serum the MKK4‐JNK2 pathway but not MKK7 or JNK1 promotes proliferation of NSCLC through STAT3. This was further investigated in vivo using A549 Xenografts in which either JNK2 or MKK4 were stably knocked down. Taken together the data suggest that JNK2 autophosphorylation primes it for activation by MKK4. This pathway promotes the activation of c‐jun and the proliferation of NSCLC in vivo.

Support or Funding Information

Support by NIH (GM059802, GM106137, and CA167505), the Welch Foundation (F‐1390 and F‐1691), CPRIT (RP101501, RP140648, and RP140649)
Ablation Of Neuropilin 1 in Glioma‐Associated Macrophages Slows Tumor Progression
Authors: Miyauchi Jeremy Tetsuo , Chen Danling , Nissen Jillian , Shroyer Kenneth , Selwood David , Tsirka Stella
Published: November 2023

Gliomas are the most commonly diagnosed primary tumors of the central nervous system (CNS). Median times of survival are dismal regardless of the treatment approach, underlying the need to develop more effective therapies. Modulation of the immune system is a promising strategy as innate and adaptive immunity play important roles in cancer progression. Microglia, the resident macrophages of the CNS, and peripheral macrophages can comprise over 30% of the cells in glioma biopsies. Gliomas secrete cytokines that suppress the anti‐tumorigenic properties of macrophages, causing them to secrete factors that support the tumor's spread and growth. Neuropilin 1 (Nrp1) is a transmembrane receptor that in mice both amplifies pro‐angiogenic signaling in the tumor microenvironment and affects behavior of innate immune cells; we show here that macrophages associated with gliomas in humans similarly express Nrp1. Using a Cre‐lox system, we generated mice that lack expression of Nrp1 in glioma‐associated microglia and macrophages (GAMs). We demonstrate, using an in vivo orthotopic glioma model, that tumors in mice with Nrp1‐deficient GAMs exhibit less vascularity, grow at a slower pace, and are populated by increased numbers of anti‐tumorigenic GAMs. Moreover, glioma survival times in mice with Nrp1‐deficient GAMs increase significantly. Treating wild‐type mice with a small molecule inhibitor of Nrp1's b1 domain, EG00229, which we show here is selective for Nrp1 over Nrp2, yielded an identical outcome. Nrp1‐deficient or EG00229‐treated wild‐type microglia exhibited a shift towards anti‐tumorigenicity as evidence by altered inflammatory marker profiles in vivo and decreased SMAD2/3 activation when conditioned with glioma‐derived factors. These results provide support for the proposal that pharmacological inhibition of Nrp1 constitutes a potential strategy for suppressing glioma progression.

Support or Funding Information

This work was supported by funding from the following grants: 2T32GM008444, 5T32GM007518, and NMSS PP1815.
Serum Starvation Induces a Rapid Increase of Akt Phosphorylation in Ovarian Cancer Cells
Authors: Dai Shuhang , Gocher Angela , Euscher Lindsey , Edelman Arthur
Published: November 2023

Background

Cellular stress responses can prevent cell death and help cells to survive in adverse environments. Such responses are important for cancer cells, which are often exposed to cellular stressors such as growth factor deprivation. Depletion of growth factor stimulation may occur in situations such as: metastasis when cancer cells encounter a poorly vascularized environment, angiogenesis inhibitor treatment to reduce blood supply to the tumor or solid tumors in which the central part is deprived of adequate nutrient supply. In vitro, serum starvation is commonly used to mimic growth factor deprivation to reduce activation of kinases under growth factor control, such as Akt.

Akt is an oncogene that is activated by the growth factor receptor/PI3K/PDK1 pathway via phosphorylation at Thr 308 to promote cell proliferation and inhibit apoptosis. Hyperactivation of Akt has been shown to promote resistance to first line platinum therapy in ovarian cancer. The canonical Akt activation pathway can be modulated by angiogenesis inhibitors, which are in clinical trials to treat platinum resistant ovarian cancer patients, by reducing tumor blood supply to decrease Akt activation.

Previous research suggests that calcium/calmodulin‐dependent protein kinase kinase 2 (CaMKK2) can directly phosphorylate Akt at Thr 308 in a PDK1 independent manner. This suggests that Akt can maintain its activity during serum starvation through multiple mechanisms helping cancer cells to survive stressful conditions. The objectives of the present studies were to investigate whether Akt was activated by serum deprivation and to evaluate the potential role of the PI3K/PDK1/Akt and CaMKK2/Akt pathway in this process.

Methods

OVCAR‐3 cells were serum starved for a series of times, then Akt phosphorylation at Thr 308 and expressions levels of CaMKK2 and PDK1 were assessed by Western Blotting. siRNA mediated knockdown of PDK1 and/or CaMKK2 were used to investigate the role of PI3K/PDK1/Akt and CaMKK2/Akt pathway respectively in serum starvation‐induced cellular responses.

Results

Serum starvation increased Akt phosphorylation up to fold at Thr 308 in a time dependent manner in OVCAR‐3 cells. Akt phosphorylation was associated with changes in the expression levels of CaMKK2 and PDK1. Current studies are in process using knockdown of CaMKK2 and/or PDK1 to assess their roles in serum deprivation‐induced Akt phosphorylation.

Conclusions

Serum deprivation in cell culture mimics the cellular stress faced by tumor cells in vivo. We used this model to evaluate the cellular stress response in ovarian cancer cells and observed an increase in Akt phosphorylation as well as changes in expression of its upstream activators CaMKK2 and PDK1. We conclude that Akt activation may be important for cancer cells including ovarian cancer cells to survive stressful environments through multiple activation pathways.

Support or Funding Information

University at Buffalo Foundation
Synergism of Aldehydes and Cytotoxic Chemotherapy
Authors: Miles Jana S , Flores‐Rozas Hernan
Published: November 2023

PURPOSE

Anthracyclines are frequently used to treat manycancers as single agents or in combination therapy. Triple negative breast cancer is commonly observed in African‐American women (AA) and tends to be more aggressive, carry worse prognoses and is harder to manage because it lacksmolecular targets required for therapies. This leaves anthracyclines as one of the few therapeutic alternatives. Although effective, the use of anthracyclinescan be limited by serious side‐effects and eventually the development of drug resistance. A genome‐wide genetic screening in S. cerevisiae identified mutants of HOM6 as hypersensitivity to doxorubicin. The sensitivity of this strain appears to result from the accumulation of the threonine intermediate L‐aspartate‐semialdehyde since it is abolished by the inactivation of a priorstep. We hypothesize that the accumulation of L‐aspartate‐semialdehydesensitizes the cells to anthracyclines.

METHODS

Using S. cerevisiae, survival assay was done to determine the sensitivity of yeast strains (wild type, hom6, hom3, hom3hom6, ydj1, siz1, and msh2)to aldehydes alone and combined with doxorubicin, cisplatin and etoposide. Statistical significance was set atp<0.05.

RESULTS

Combination of formaldehyde (2 mM) and doxorubicin was moste ffective at reducing cell survival by between 31 to 39‐fold (in wild type cells) relative to doxorubicin and formaldehyde alone. Combination of acetaldehyde (2 mM) and doxorubicin reduced cell survival by between 3 to5‐fold, 5 to 35‐fold, and 1 to 3‐fold in hom3, hom6, and hom3hom6 respectively. This effect was reliant on thedose‐dependent concentration of doxorubicin. Co‐treatment with formaldehyde and doxorubicin of anthracycline‐resistant strains siz1 and msh2 showed increased toxicity (albeit less pronounced) in these strains compared to the wild type.

CONCLUSION

The potential use of a combination of aldehydes andcytotoxic drugs could potentially lead to applications intended to enhanceanthracycline‐based therapy.

Support or Funding Information

Research supported by National Institutes of Health (NIH)under Award Number G12MD007582
Gender differences in the cardiac action of tyramine but not cathinone in the rat
Authors: Docherty James R , Alsufyani Hadeel A
Published: November 2023

Cathinone is the major active stimulant ingredient of Khat which is chewed by both males and females in approximately equal numbers in countries around the Horn of Africa. We have previously shown that the cardiac β‐adrenoceptor and vascular α‐adrenoceptor mediated actions of cathinone and, for comparison, methylenedioxymethamphetamine (MDMA) are largely indirect by release of noradrenaline (Alsufyani & Docherty, 2015). Until recently, the vast majority of pharmacological studies of stimulants have been carried out employing only male animals, despite the obvious use of stimulants by both males and females in the human population. We have now investigated gender differences in the cardiac actions of cathinone and MDMA in comparison with tyramine in Wistar rats. Animals were sympathectomised by treatment with 6‐hydroxydopamine or treated with vehicle. Residual responses following sympathectomy are likely to be largely direct. In male (230–300g) and female (190–230g) pentobarbitone anaesthetised rats, all three agonists given intravenously produced significant tachycardia but only tyramine produced a large pressor response, MDMA produced small pressor response, and cathinone produced mixed pressor and depressor responses. Sympathectomy virtually abolished the pressor responses to all three agents, and reduced the tachycardia to all three agents, but there were no differences between sympathectomised male and female rats in cardiac responses to any agonist. The tyramine tachycardia was less affected by sympathectomy, suggesting a direct component to the response. In vehicle treated rats, there was no gender difference in the tachycardia to MDMA or cathinone, but the tachycardia to tyramine, in terms of potency, was significantly greater in male than female rats, with a three fold difference in potency. Tyramine potency (−logED50, μg/kg) was 1.59±0.18 (n=8) in male and 2.08±0.14 (n=8) in female rats or 38.9μg/kg and 120μg/kg, respectively (Analysis of Variance and Bonferroni test: P<0.05). Since the mechanism of the tachycardia to tyramine may differ from those of cathinone and MDMA in terms of relative actions at the noradrenaline transporter and the vesicular transporter (Alsufyani & Docherty, 2015), it is concluded that gender differences occur in this system, and that the indirect tachycardia to tyramine is reduced in female rats. Responses to cathinone were unaffected by gender.

Support or Funding Information

HAA is funded by a scholarship from the Saudi Government Ministry of Higher Education, KAU.