Although novel treatments for Alzheimer’s disease (AD) have begun to show modest therapeutic effects, agents that target hallmark AD pathology and offer neuroprotection are desired. Erythropoietin (EPO) is a glycoprotein hormone with neuroprotective effects but is faced with challenges including limited brain uptake and increased hematopoietic side effects with long-term dosing. Therefore, EPO has been modified and bound to a chimeric transferrin receptor monoclonal antibody (cTfRMAb); the latter shuttles EPO past the blood-brain barrier (BBB) into brain parenchyma and reduces its plasma exposure and potential for side effects. Our study sought to characterize the safety and pharmacokinetics (PK) of modified EPO following chronic dosing in healthy mice, and then utilize the optimized dose in mitigating hallmark AD pathologies in APP-SAA knock-in (KI) mice, a model that recapitulates Aβ pathology in the absence of APP overexpression, in vivo . For the PK and safety study, a multidose design was employed with 10-week-old C57BL/6 male mice (n = 4-5/dose) receiving doses ranging from 1- to 20-mg/kg SQ for 4 weeks, aimed to evaluate the dose-dependent plasma concentrations, and metabolic and hematologic safety of the modified EPO. The dose that resulted in the highest safety and sustained plasma exposure was dosed SQ to 5.5-month-old male APP-SAA KI mice (n = 6) for 14 weeks. Control APP-SAA KI mice (n = 5) received vehicle. The effect of modified EPO on Aβ load by immunoassays, and spatial memory via Y-maze test, were assessed. The 1mg/kg dose resulted in no adverse effects and sustained plasma exposure which are conducive to longitudinal dosing. APP-SAA KI mice treated with the modified EPO had a remarkable (70-80%, p<0.001) reduction in 6E10-positive-Aβ plaque area and number in the brain. Aggregated Aβ measured by ELISA was also significantly lower (p<0.05) with modified EPO treatment. Modified EPO increased the discrimination index for the novel arm (p<0.05), suggesting an improvement in spatial memory recall of the reinforced arm of the maze. These findings provide essential data for dose optimization for longitudinal studies using cTfRMAb-based therapeutics, and specifically modified EPO used herein, and illustrate the therapeutic potential of the brain-penetrating cTfRMAb-EPO in a novel AD mouse model devoid of APP overexpression.

Chronic heavy alcohol drinking may be a modifiable risk factor for Alzheimer’s disease (AD), but studies in rodent AD models more closely mimic chronic moderate alcohol drinking in humans and largely focus on the brain. The role of the liver, which is significantly impacted by chronic heavy alcohol intake, in driving brain changes in alcohol-dependent AD remains unexplored. Our study using intragastric-ethanol feeding, which mimics chronic heavy alcohol intake in humans, in C57BL/6J mice showed significant AD-relevant changes in the brain and liver. Therefore, we aimed to investigate how hepatic changes using this model of chronic heavy drinking drive AD pathology in AD mice, which has never been attempted. Eight-month-old male APP/PS1 mice were fed ethanol or control diet intragastrically for 5 weeks (n = 7-11/group). Brain and liver Aβ were assessed using immunoassays. Three important mechanisms of brain amyloidosis were investigated: hepatic LRP-1 (major peripheral Aβ regulator), blood-brain barrier (BBB) function (vascular Aβ regulator), and microglia (major brain Aβ regulator) using immunoassays. Hepatic LRP-1 expression was confirmed using Nanostring spatial transcriptomics. To elucidate the role of hepatic LRP-1 in brain amyloidosis, hepatic LRP-1 was silenced by injecting LRP-1 microRNA delivered by the adeno-associated virus 8 (AAV8) and the hepato-specific thyroxine-binding globulin promoter to 4-month-old male APP/PS1 mice (n = 6). Control APP/PS1 mice received control AAV8 (n = 6). Spatial memory was assessed 12 weeks after LRP-1 silencing using Y-maze, and brains and livers were harvested to detect Aβ. Alcohol feeding increased aggregated Aβ (p<0.05) by ELISA and 6E10-positive Aβ load (p<0.05) by immunostaining, and reduced plaque-associated microglia in APP/PS1 mice brains. Further, alcohol-fed APP/PS1 had liver steatosis and significantly downregulated hepatic LRP-1 (p<0.01) at the protein and transcript level, and brain and hepatic Aβ were positively correlated (p<0.05). Hepato-specific LRP-1 silencing significantly increased brain Aβ load (p<0.05) and reduced entries into the novel arm of the Y-maze (p<0.05) in APP/PS1 mice. Chronic heavy alcohol intake reduced hepatic LRP-1 expression, and hepato-specific LRP-1 silencing increased brain Aβ and spatial memory deficits in APP/PS1 mice. Our results place hepatic LRP-1 as a potential key driver of brain amyloidosis in alcohol-dependent AD.

Background: Hepatic lipoprotein receptor-related protein 1 (LRP-1) plays a central role in peripheral amyloid beta (A ss) clearance, but its importance in Alzheimer's disease (AD) pathology is understudied. Our previous work showed that intragastric alcohol feeding to C57BL/6 J mice reduced hepatic LRP-1 expression which correlated with significant AD-relevant brain changes. Herein, we examined the role of hepatic LRP-1 in AD pathogenesis in APP/PS1 AD mice using two approaches to modulate hepatic LRP-1, intragastric alcohol feeding to model chronic heavy drinking shown by us to reduce hepatic LRP-1, and hepato-specific LRP-1 silencing. Methods: Eight-month-old male APP/PS1 mice were fed ethanol or control diet intragastrically for 5 weeks (n = 7-11/group). Brain and liver A ss were assessed using immunoassays. Three important mechanisms of brain amyloidosis were investigated: hepatic LRP-1 (major peripheral A ss regulator), blood-brain barrier (BBB) function (vascular A ss regulator), and microglia (major brain A ss regulator) using immunoassays. Spatial LRP-1 gene expression in the periportal versus pericentral hepatic regions was confirmed using NanoString GeoMx Digital Spatial Profiler. Further, hepatic LRP-1 was silenced by injecting LRP-1 microRNA delivered by the adenoassociated virus 8 (AAV8) and the hepato-specific thyroxine-binding globulin (TBG) promoter to 4-month-old male APP/PS1 mice (n = 6). Control male APP/PS1 mice received control AAV8 (n = 6). Spatial memory and locomotion were assessed 12 weeks after LRP-1 silencing using Y-maze and open-field test, respectively, and brain and liver A ss were measured. Results: Alcohol feeding reduced plaque-associated microglia in APP/PS1 mice brains and increased aggregated A ss (p < 0.05) by ELISA and 6E10-positive A ss load by immunostaining (p < 0.05). Increased brain A ss corresponded with a significant downregulation of hepatic LRP-1 (p < 0.01) at the protein and transcript level, primarily in pericentral hepatocytes (zone 3) where alcohol-induced injury occurs. Hepato-specific LRP-1 silencing significantly increased brain A ss and locomotion hyperactivity (p < 0.05) in APP/PS1 mice.

Background Biologic TNF-α inhibitors (bTNFIs) can block cerebral TNF-α in Alzheimer’s disease (AD) if these macromolecules can cross the blood–brain barrier (BBB). Thus, a model bTNFI, the extracellular domain of type II TNF-α receptor (TNFR), which can bind to and sequester TNF-α, was fused with a mouse transferrin receptor antibody (TfRMAb) to enable brain delivery via BBB TfR-mediated transcytosis. Previously, we found TfRMAb-TNFR to be protective in a mouse model of amyloidosis (APP/PS1) and tauopathy (PS19), and herein we investigated its effects in mice that combine both amyloidosis and tauopathy (3xTg-AD). Methods Eight-month-old female 3xTg-AD mice were injected intraperitoneally with saline (n = 11) or TfRMAb-TNFR (3 mg/kg; n = 11) three days per week for 12 weeks. Age-matched wild-type (WT) mice (n = 9) were treated similarly with saline. Brains were processed for immunostaining and high-resolution multiplex NanoString GeoMx spatial proteomics. Results We observed regional differences in proteins relevant to Aβ, tau, and neuroinflammation in the hippocampus of 3xTg-AD mice compared with WT mice . From 64 target proteins studied using spatial proteomics, a comparison of the Aβ-plaque bearing vs. plaque-free regions in the 3xTg-AD mice yielded 39 differentially expressed proteins (DEP) largely related to neuroinflammation (39% of DEP) and Aβ and tau pathology combined (31% of DEP). Hippocampal spatial proteomics revealed that the majority of the proteins modulated by TfRMAb-TNFR in the 3xTg-AD mice were relevant to microglial function (⁓ 33%). TfRMAb-TNFR significantly reduced mature Aβ plaques and increased Aβ-associated microglia around larger Aβ deposits in the 3xTg-AD mice. Further, TfRMAb-TNFR increased mature Aβ plaque-associated microglial TREM2 in 3xTg-AD mice. Conclusion Overall, despite the low visual Aβ load in the 11-month-old female 3xTg-AD mice, our results highlight region-specific AD-relevant DEP in the hippocampus of these mice. Chronic TfRMAb-TNFR dosing modulated several DEP involved in AD pathology and showed a largely microglia-centric mechanism of action in the 3xTg-AD mice.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by cognitive impairment and memory loss. Epidemiological evidence suggests that heavy alcohol consumption aggravates AD pathology, whereas low alcohol intake may be protective. However, these observations have been inconsistent, and because of methodological discrepancies, the findings remain controversial. Alcohol-feeding studies in AD mice support the notion that high alcohol intake promotes AD, while also hinting that low alcohol doses may be protective against AD. Chronic alcohol feeding to AD mice that delivers alcohol doses sufficient to cause liver injury largely promotes and accelerates AD pathology. The mechanisms by which alcohol can modulate cerebral AD pathology include Toll-like receptors, protein kinase-B (Akt)/mammalian target of rapamycin (mTOR) pathway, cyclic adenosine monophosphate (cAMP) response element-binding protein phosphorylation pathway, glycogen synthase kinase 3-β, cyclin-dependent kinase-5, insulin-like growth factor type-1 receptor, modulation of β-amyloid (Aβ) synthesis and clearance, microglial mediated, and brain endothelial alterations. Besides these brain-centric pathways, alcohol-mediated liver injury may significantly affect brain Aβ levels through alterations in the peripheral-to-central Aβ homeostasis. This article reviews published experimental studies (cell culture and AD rodent models) to summarize the scientific evidence and probable mechanisms (both cerebral and hepatic) by which alcohol promotes or protects against AD progression.

The mechanisms of hepatic ischemia/reperfusion (I/R) injury, which occurs during liver transplantation or surgery, are poorly understood. The purpose of the current study was to generate and characterize a HepG2 cell line with a stable overexpression of CYP2E1 to investigate the role of the enzyme in hypoxia/reperfusion (H/R) injury in an ex vivo setting. GFP-tagged CYP2E1 and control clones were developed, and their gene expression and protein levels of GFP and CYP2E1 were determined using RT-PCR and ELISA/Western blot analysis, respectively. Additionally, the CYP2E1 catalytic activity was determined by UPLC-MS/MS analysis of 6-hydroxychlorzoxazone formed from the chlorzoxazone substrate. The CYP2E1 and control clones were subjected to hypoxia (10 h) and reoxygenation (0.5 h), and cell death and reactive oxygen species (ROS) generation were quantitated using LDH and flow cytometry, respectively. Compared with the control clone, the selected CYP2E1 clone showed a 720-fold increase in CYP2E1 expression and a prominent band in the western blot analysis, which was associated with a 150-fold increase in CYP2E1 catalytic activity. The CYP2E1 clone produced 2.3-fold more ROS and 1.9-fold more cell death in the H/R model. It is concluded that the constitutive CYP2E1 in the liver may play a detrimental role in hepatic I/R injury.

Rationale There is currently no treatment for spinocerebellar ataxias (SCAs), which are a group of genetic disorders that often cause a lack of coordination, difficulty walking, slurred speech, tremors, and eventually death. Activation of K Ca 2.2/K Ca 2.3 channels reportedly exerts beneficial effects in SCAs. Here, we report the development and validation of an analytical method for quantitating a recently developed positive allosteric modulator of K Ca 2.2/K Ca 2.3 channels (compound 2q) in mouse plasma. Methods Mouse plasma samples (10 μL) containing various concentrations of 2q were subjected to protein precipitation in the presence of a structurally similar internal standard (IS). Subsequently, the analytes were separated on a C 18 ultrahigh‐performance liquid chromatography column and detected by a tandem mass spectrometer. The method was validated using US Food and Drug Administration (FDA) guidelines. Finally, the validated assay was applied to the measurement of the plasma concentrations of 2q in plasma samples taken from mice after single intravenous doses of 2 mg/kg of 2q, and the pharmacokinetic parameters of 2q were determined. Results The calibration standards were linear ( r 2 ≥ 0.99) in the range of 1.56–200 nM of 2q with intra‐ and inter‐run accuracy and precision values within the FDA guidelines. The lower limit of quantitation of the assay was 1.56 nM (0.258 pg on the column). The recoveries of 2q and IS from plasma were >94%, with no appreciable matrix effect. The assay showed no significant carryover, and the plasma samples stored at −80°C or the processed samples stored in the autosampler at 10°C were stable for at least 3 weeks and 36 h, respectively. After intravenous injection, 2q showed a bi‐exponential decline pattern in the mouse plasma, with a clearance of 30 mL/min/kg, a terminal volume of distribution of 1.93 mL/kg, and a terminal half‐life of 45 min. Conclusions The developed assay is suitable for preclinical pharmacokinetic–pharmacodynamic studies of 2q as a potential drug candidate for ataxias.

Alzheimer’s disease (AD) is a chronic neuroinflammatory condition and the proinflammatory cytokine, tumor necrosis factor alpha (TNF-α), is a key mediator of neuroinflammation in AD. Biologic TNF-α inhibitors (bTNFIs) can block cerebral TNF-α if these large molecules cross the blood-brain barrier (BBB). Thus, a model bTNFI, the extracellular domain of type II TNF-α receptor (TNFR) which can bind to and sequester TNF-α, has been reengineered to enable brain delivery by fusion with a mouse transferrin receptor antibody (TfRMAb). TfRMAb binds to the BBB TfR to deliver the TNFR into the brain via receptor-mediated transcytosis. Previously, we found the TfRMAb-TNFR to be protective in a mouse model of amyloidosis (APP/PS1) and tauopathy (PS19), and the aim of the current study was to investigate its effects in a mouse model that combines both amyloidosis and tauopathy (3xTg-AD mice). Eight-month-old female triple transgenic 3xTg-AD mice were injected intraperitoneally with saline (n = 11) or TfRMAb-TNFR (3 mg/kg; n = 11) three days per week for 12 weeks. At the end of treatment, brains were processed for hippocampal immunohistochemistry and high-resolution multiplex NanoString spatial proteomics using the GeoMx protein assays for neuroscience. TfRMAb-TNFR treatment significantly reduced mature β-sheet rich Aβ plaques and increased plaque-associated microglial localization around larger plaques in the 3xTg mice. Further, TfRMAb-TNFR increased plaque-associated microglial TREM2 compared with saline-treated 3xTg mice. From a total of 64 target proteins studied using spatial proteomics (comparing protein expression in the Aβ-plaque bearing vs. plaque-free regions), 39 (61%) differentially expressed proteins (DEP) were identified in the 11-month-old 3xTg mice, comprising of proteins related to neurodegeneration (10% of DEP), neuroinflammation (41% of DEP), amyloid (21% of DEP) and tau (10% of DEP) pathology, autophagy (10% of DEP) and other pathways (8% of DEP). TfRMAb-TNFR treatment modulated proteins involved in Aβ clearance and processing, microglial function, RNA processing, and neurodegeneration. Majority of these proteins were relevant to microglial function (∼ 40%). The study highlights that TfRMAb-TNFR modulates several proteins involved in AD pathology and progression and largely suggests a microglia-centric mechanism of action of this treatment in the 3xTg mice.

Midazolam (MDZ), a benzodiazepine derivative, is metabolized to 1 '- and 4-hydroxylated metabolites (1 '-OH-MDZ and 4-OH-MDZ, respectively) by cytochrome P450 3A (CYP3A). The purpose of this study was to investigate the CYP3A-mediated hydroxylation of MDZ in the rat brain mitochondria (MT). Brain microsomes (MC) and MT fractions were prepared from rats (n = 8) using differential and density gradient centrifugations, and the purity of the fractions was evaluated using VDAC1 and calreticulin as markers of MT and MC, respectively. The formation rates of 1 '-OH-MDZ and 4-OH-MDZ in the rat brain MC and MT samples were determined using an LC-MS/MS method after validation. Subsequently, Michaelis-Menten kinetics of 1 '- and 4-hydroxylation of MDZ were estimated. Western blot (WB) analysis was used to determine the protein expression of CYP3A in the rat brain MC and MT. The MC fractions had 5.93% +/- 3.01% mitochondrial impurity, and the MT fractions had 19.3% +/- 7.8% microsomal impurity (mean +/- SD). The maximum velocity (V-max) values of the formation of the hydroxylated metabolites in the brain MT were 2.4-9-fold higher than those in MC. Further, the V-max values of 4-OH-MDZ in both MC and MT fractions were substantially higher than those of 1 '-OH-MDZ. The WB analysis showed that the intensity of the CYP3A immunoreactive band in MT was more than twofold higher than that in MC. It is concluded that compared with MC, rat brain MT contains substantial CYP3A, which may affect the pharmacology or toxicology of centrally acting xenobiotic and endogenous substrates of this enzyme.

Heavy alcohol consumption is a known risk factor for various forms of dementia and the development of Alzheimer’s disease (AD). In this work, we investigated how intragastric alcohol feeding may alter the liver-to-brain axis to induce and/or promote AD pathology. Four weeks of intragastric alcohol feeding to mice, which causes significant fatty liver (steatosis) and liver injury, caused no changes in AD pathology markers in the brain [amyloid precursor protein (APP), presenilin], except for a decrease in microglial cell number in the cortex of the brain. Interestingly, the decline in microglial numbers correlated with serum alanine transaminase (ALT) levels, suggesting a potential link between liver injury and microglial loss in the brain. Intragastric alcohol feeding significantly affected two hepatic proteins important in amyloid-beta (Aβ) processing by the liver: 1) alcohol feeding downregulated lipoprotein receptor-related protein 1 (LRP1, ∼46%), the major receptor in the liver that removes Aβ from blood and peripheral organs, and 2) alcohol significantly upregulated APP (∼2-fold), a potentially important source of Aβ in the periphery and brain. The decrease in hepatic LRP1 and increase in hepatic APP likely switches the liver from being a remover or low producer of Aβ to an important source of Aβ in the periphery, which can impact the brain. The downregulation of LRP1 and upregulation of APP in the liver was observed in the first week of intragastric alcohol feeding, and also occurred in other alcohol feeding models (NIAAA binge alcohol model and intragastric alcohol feeding to rats). Modulation of hepatic LRP1 and APP does not seem alcohol-specific, as ob/ob mice with significant steatosis also had declines in LRP1 and increases in APP expression in the liver. These findings suggest that liver steatosis rather than alcohol-induced liver injury is likely responsible for regulation of hepatic LRP1 and APP. Both obesity and alcohol intake have been linked to AD and our data suggests that liver steatosis associated with these two conditions modulates hepatic LRP1 and APP to disrupt Aβ processing by the liver to promote AD.

Chronic intraperitoneal injection of thioacetamide (TAA) in rats has been used as an animal model of human cirrhosis to study the effects of the disease on drug metabolism. However, TAA inhibits P450 enzymes directly and independently of cirrhosis. We investigated the effects of chronic cirrhosis in rats, induced by 10 weeks of intraperitoneal TAA, on the P450 enzymes after a 10-day washout period to eliminate TAA. Liver histology and serum biomarkers of hepatic function confirmed cirrhosis in all animals. Microsomal total P450 content, P450 reductase activity and ethoxycoumarin O-deethylase activity, a general marker of P450 activity, were significantly reduced by 30%-50% in cirrhotic animals. Additionally, the protein content and Michaelis-Menten kinetics of the activities of CYP2D, CYP2E1 and CYP3A were investigated. Whereas cirrhosis reduced the microsomal protein contents of CYP2D and CYP3A by 70% and 30%, respectively, the protein contents of CYP2E1 were not affected. However, the activities of all the tested isoenzymes were substantially lower in the cirrhotic livers. It is concluded that the TAA model of cirrhosis that incorporates a 10-day washout period after intraperitoneal injection of the chemical to rats produces isoenzyme-selective reductions in the P450 proteins or activities, which are independent of the direct inhibitory effects of TAA.

Tumor necrosis factor alpha (TNF-α) plays a vital role in Alzheimer’s disease (AD) pathology, and TNF-α inhibitors (TNFIs) modulate AD pathology. We fused the TNF-α receptor (TNFR), a biologic TNFI that sequesters TNF-α, to a transferrin receptor antibody (TfRMAb) to deliver the TNFI into the brain across the blood–brain barrier (BBB). TfRMAb-TNFR was protective in 6-month-old transgenic APP/PS1 mice in our previous work. However, the effects and safety following delayed chronic TfRMAb-TNFR treatment are unknown. Herein, we initiated the treatment when the male APP/PS1 mice were 10.7 months old (delayed treatment). Mice were injected intraperitoneally with saline, TfRMAb-TNFR, etanercept (non-BBB-penetrating TNFI), or TfRMAb for ten weeks. Biologic TNFIs did not alter hematology indices or tissue iron homeostasis; however, TfRMAb altered hematology indices, increased splenic iron transporter expression, and increased spleen and liver iron. TfRMAb-TNFR and etanercept reduced brain insoluble-amyloid beta (Aβ) 1-42, soluble-oligomeric Aβ, and microgliosis; however, only TfRMAb-TNFR reduced Aβ peptides, Thioflavin-S-positive Aβ plaques, and insoluble-oligomeric Aβ and increased plaque-associated phagocytic microglia. Accordingly, TfRMAb-TNFR improved spatial reference memory and increased BBB-tight junction protein expression, whereas etanercept did not. Overall, despite delayed treatment, TfRMAb-TNFR resulted in a better therapeutic response than etanercept without any TfRMAb-related hematology- or iron-dysregulation in aged APP/PS1 mice.

Midazolam (MDZ) is a short-acting benzodiazepine with rapid onset of action, which is metabolized by CYP3A isoenzymes to two hydroxylated metabolites, 1'-hydroxymidazolam and 4-hydroxymidazolam. The drug is also commonly used as a marker of CYP3A activity in the liver microsomes. However, the kinetics of CYP3A-mediated hydroxylation of MDZ in the brain, which contains much lower CYP content than the liver, have not been reported. In this study, UPLC-MS/MS and metabolic incubation methods were developed and validated for simultaneous measurement of low concentrations of both hydroxylated metabolites of MDZ in brain microsomes. Different concentrations of MDZ (1-500 mu M) were incubated with rat brain microsomes (6.25 mu g) and NADPH over a period of 10 min. After precipitation of the microsomal proteins with acetonitrile, which contained individual isotope-labeled internal standards for each metabolite, the analytes were separated on a C-18 UPLC column and detected by a tandem mass spectrometer. Accurate quantitation of MDZ metabolism in the brain microsomes presented several challenges unique to this tissue, which were resolved. The optimized method showed validation results in accordance with the FDA acceptance criteria, with a linearity ranging from 1 to 100 nM and a lower limit of quantitation of 0.4 pg on the column for each of the two metabolites. The method was successfully used to determine the Michaelis-Menten (MM) kinetics of MDZ 1'- and 4-hydroxylase activities in rat brain microsomes (n = 5) for the first time. The 4-hydroxylated metabolite had 2.4 fold higher maximum velocity < 0.01) and 1.9 fold higher (p < 0.05) MM constant values than the 1'-hydroxylated metabolite. However, intrinsic clearance values of the two metabolites were similar. The optimized analytical and metabolic incubation methods reported here may be used to study the effects of various pathophysiological and pharmacological factors on the CYP3A-mediated metabolism of MDZ in the brain.

Ethoxyresorufin (ER)-O-deethylation (EROD) activity has been widely used to assess cytochrome P450 1A (CYP1A) activity. The kinetics of CYP1A activity have been well characterized in the liver microsomes. However, studies in kidney microsomes are limited due to the much lower EROD activity in this organ. Here, we developed and validated a sensitive UPLC-MS/MS assay for the characterization of the EROD activity in the rat kidney microsomes. In a 50 µL reaction mixture, rat kidney microsomes (0.25 mg/mL) were incubated with ER (0.1-5 µM) and NADPH (1 mM) for 10 min. Acidic solvents, such as trichloroacetic acid or formic acid, used for quenching of the metabolic reactions and precipitation of the proteins, unexpectedly caused a spontaneous formation of resorufin (RES) from ER. Therefore, the metabolic reactions were terminated by adding acetonitrile, containing a deuterated internal standard (IS). Chromatographic separation was achieved on a C18 UPLC column, and the MS/MS ion transitions were 213.9/185.9 for RES and 220.0/192.0 for IS. The assay was validated in the linear range of 0.5 nM to 75 nM of RES and had a lower limit of quantitation of 0.5 nM. The overall recoveries of RES (90%-99%) and IS (85%-103%) were relatively high, with minimal matrix effect. The assay was successfully applied to the estimation of the Michaelis-Menten (MM) kinetics of EROD activity in the rat kidney microsomes (n = 3), which showed a maximum velocity of 2.68 ± 0.17 pmol/min/mg and a MM constant of 1.72 ± 0.24 µM (mean ± SD). It is concluded that our sensitive and specific analytical method, coupled with the optimized microsomal incubation conditions, provides a robust platform for further investigations of the effects of xenobiotics, environmental factors, or pathophysiologic conditions on the kinetics of EROD activity in the kidney microsomes.