Clozapine-N-oxide protects dopaminergic neurons against rotenone-induced neurotoxicity by preventing ferritinophagy-mediated ferroptosis.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, yet treatment options are limited. Clozapine (CLZ), an antipsychotic used for schizophrenia, has potential as a PD treatment. CLZ and its metabolite, Clozapine-N-Oxide (CNO), show neuroprotective effects on dopaminergic neurons, with mechanisms needing further investigation. This study aimed to confirm the neuroprotective effects of CLZ and CNO in a rotenone-induced mouse model and further explore the underlying mechanisms of CNO-afforded protection. Gait pattern and rotarod activity evaluations showed motor impairments in rotenone-exposed mice, with CLZ or CNO administration ameliorating behavioral deficits. Cell counts and biochemical analysis demonstrated CLZ and CNO's effectiveness in reducing rotenone-induced neurodegeneration of dopaminergic neurons in the nigrostriatal system in mice. Mechanistic investigations revealed that CNO suppressed rotenone-induced ferroptosis of dopaminergic neurons by rectifying iron imbalances, curtailing lipid peroxidation, and mitigating mitochondrial morphological changes. CNO also reversed autolysosome and ferritinophagic activation in rotenone-exposed mice. SH-SY5Y cell cultures validated these findings, indicating ferritinophage involvement, where CNO-afforded protection was diminished by ferritinophagy enhancers. Furthermore, knockdown of NCOA4, a crucial cargo receptor for ferritin degradation in ferritinophagy, hampered rotenone-induced ferroptosis and NCOA4 overexpression countered the anti-ferroptotic effects of CNO. Whereas, iron-chelating agents and ferroptosis enhancers had no effect on the anti-ferritinophagic effects of CNO in rotenone-treated cells. In summary, CNO shielded dopaminergic neurons in the rotenone-induced PD model by modulating NCOA4-mediated ferritinophagy, highlighting a potential therapeutic pathway for PD treatment. This research provided insights into the role of NCOA4 in ferroptosis and suggested new approaches for PD therapy.

Involvement of Extracellular Vesicles in the Proinflammatory Response to Clozapine: Implications for Clozapine-Induced Agranulocytosis.

Most idiosyncratic drug reactions (IDRs) appear to be immune-mediated, but mechanistic events preceding severe reaction onset remain poorly defined. Damage-associated molecular patterns (DAMPs) may contribute to both innate and adaptive immune phases of IDRs, and changes in extracellular vesicle (EV) cargo have been detected post-exposure to several IDR-associated drugs. To explore the hypothesis that EVs are also a source of DAMPs in the induction of the immune response preceding drug-induced agranulocytosis, the proteome and immunogenicity of clozapine- (agranulocytosis-associated drug) and olanzapine- (non-agranulocytosis-associated drug) exposed EVs were compared in two preclinical models: THP-1 macrophages and Sprague-Dawley rats. Compared with olanzapine, clozapine induced a greater increase in the concentration of EVs enriched from both cell culture media and rat serum. Moreover, treatment of drug-naïve THP-1 cells with clozapine-exposed EVs induced an inflammasome-dependent response, supporting a potential role for EVs in immune activation. Proteomic and bioinformatic analyses demonstrated an increased number of differentially expressed proteins with clozapine that were enriched in pathways related to inflammation, myeloid cell chemotaxis, wounding, transforming growth factor-ß signaling, and negative regulation of stimuli response. These data indicate that, although clozapine and olanzapine exposure both alter the protein cargo of EVs, clozapine-exposed EVs carry mediators that exhibit significantly greater immunogenicity. Ultimately, this supports the working hypothesis that drugs associated with a risk of IDRs induce cell stress, release of proinflammatory mediators, and early immune activation that precedes severe reaction onset. Further studies characterizing EVs may elucidate biomarkers that predict IDR risk during development of drug candidates. SIGNIFICANCE STATEMENT: This work demonstrates that clozapine, an idiosyncratic drug-induced agranulocytosis (IDIAG)-associated drug, but not olanzapine, a safer structural analogue, induces an acute proinflammatory response and increases extracellular vesicle (EV) release in two preclinical models. Moreover, clozapine-exposed EVs are more immunogenic, as measured by their ability to activate inflammasomes, and contain more differentially expressed proteins, highlighting a novel role for EVs during the early immune response to clozapine and enhancing our mechanistic understanding of IDIAG and other idiosyncratic reactions.

The cardiovascular toxicity of clozapine in embryonic zebrafish and RNA sequencing-based transcriptome analysis.

Clozapine (CLZ) is the most prescribed medication for treating refractory schizophrenia but is associated with significant cardiovascular toxicity. This study aimed to investigate the cardiovascular toxicity induced by CLZ using zebrafish as a model animal. For this purpose, zebrafish developed to 80-h post-fertilization were exposed to different CLZ concentration solutions for 24 h followed by cardiac morphological observations in yolk sac edema, pericardial edema, and blood coagulation, in addition to increased SV-BA distance, functionally manifested as bradycardia, and decreased cardiac ejection fraction using the untreated embryos as control. At the same time, RNA sequencing was used to study the possible molecular mechanism of CLZ-induced cardiovascular toxicity. The results indicated that compared to the control group, the experimental groups possessed a total of 5888 differentially expressed genes (DEGs), where gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment of analysis indicated that DEGs were mainly enriched in the pathways related to ion channels. These findings may provide new insights and directions for the subsequent in-depth study of the molecular mechanism of CLZ-induced cardiovascular toxicity.

Clozapine suppresses NADPH oxidase activation, counteracts cytosolic H2O2, and triggers early onset mitochondrial dysfunction during adipogenesis of human liposarcoma SW872 cells.

Long-term treatment of schizophrenia with clozapine (CLZ), an atypical antipsychotic drug, is associated with an increased incidence of metabolic disorders mediated by poorly understood mechanisms. We herein report that CLZ, while slowing down the morphological changes and lipid accumulation occurring during SW872 cell adipogenesis, also causes an early (day 3) inhibition of the expression/nuclear translocation of CAAT/enhancer-binding protein ß and peroxisome proliferator-activated receptor γ. Under the same conditions, CLZ blunts NADPH oxidase-derived reactive oxygen species (ROS) by a dual mechanism involving enzyme inhibition and ROS scavenging. These effects were accompanied by hampered activation of the nuclear factor (erythroid-derived2)-like 2 (Nrf2)-dependent antioxidant responses compared to controls, and by an aggravated formation of mitochondrial superoxide. CLZ failed to exert ROS scavenging activities in the mitochondrial compartment but appeared to actively scavenge cytosolic H2O2 derived from mitochondrial superoxide. The early formation of mitochondrial ROS promoted by CLZ was also associated with signs of mitochondrial dysfunction. Some of the above findings were recapitulated using mouse embryonic fibroblasts. We conclude that the NADPH oxidase inhibitory and cytosolic ROS scavenging activities of CLZ slow down SW872 cell adipogenesis and suppress their Nrf2 activation, an event apparently connected with increased mitochondrial ROS formation, which is associated with insulin resistance and metabolic syndrome. Thus, the cellular events characterised herein may help to shed light on the more detailed molecular mechanisms explaining some of the adverse metabolic effects of CLZ.

Exploring the mechanisms of clozapine-induced blood-brain barrier dysfunction using untargeted metabolomics and cellular metabolism analysis.

Brain microvascular endothelial cells (BMVECs) from the blood- brain barrier form a highly selective membrane that protects the brain from circulating blood and maintains a stable microenvironment for the central nervous system. BMVEC dysfunction has been implicated in a variety of neurological and psychiatric disorders. Clozapine, a widely used antipsychotics, has been demonstrated to alter the permeability of BMVECs, but the underlying mechanisms of this effect are not fully understood. In this study, we investigated the effects of clozapine in BMVECs using untargeted metabolomics analysis. Our results illustrated that treatment with clozapine led to significant changes in the metabolic profile of BMVECs, including alterations in amino acid and energy metabolism. These findings suggest that clozapine affects BMVEC permeability through its effects on cellular metabolism. Our study could inform the development of more targeted and effective treatments for understanding the relationships among clozapine, cellular metabolism, and BMVECs in more detail.

Self-Assembling Lecithin-Based Mixed Polymeric Micelles for Nose to Brain Delivery of Clozapine: In-vivo Assessment of Drug Efficacy via Radiobiological Evaluation.

Purpose: The research objective is to design intranasal brain targeted CLZ loaded lecithin based polymeric micelles (CLZ- LbPM) aiming to improve central systemic CLZ bioavailability. Methods: In our study, intranasal CLZ loaded lecithin based polymeric micelles (CLZ- LbPM) were formulated using soya phosphatidyl choline (SPC) and sodium deoxycholate (SDC) with different CLZ:SPC:SDC ratios via thin film hydration technique aiming to enhance drug solubility, bioavailability and nose to brain targeting efficiency. Optimization of the prepared CLZ-LbPM using Design-Expert® software was achieved showing that M6 which composed of (CLZ:SPC: SDC) in respective ratios of 1:3:10 was selected as the optimized formula. The optimized formula was subjected to further evaluation tests as, Differential Scanning Calorimetry (DSC), TEM, in vitro release profile, ex vivo intranasal permeation and in vivo biodistribution. Results: The optimized formula with the highest desirability exhibiting (0.845), small particle size (12.23±4.76 nm), Zeta potential of (-38 mV), percent entrapment efficiency of > 90% and percent drug loading of 6.47%. Ex vivo permeation test showed flux value of 27 µg/cm².h and the enhancement ratio was about 3 when compared to the drug suspension, without any histological alteration. The radioiodinated clozapine ([131I] iodo-CLZ) and radioiodinated optimized formula ([131I] iodo-CLZ-LbPM) were formulated in an excellent radioiodination yield more than 95%. In vivo biodistribution studies of [131I] iodo-CLZ-LbPM showed higher brain uptake (7.8%± 0.1%ID/g) for intranasal administration with rapid onset of action (at 0.25 h) than the intravenous formula. Its pharmacokinetic behavior showed relative bioavailability, direct transport percentage from nose to brain and drug targeting efficiency of 170.59%, 83.42% and 117% respectively. Conclusion: The intranasal self-assembling lecithin based mixed polymeric micelles could be an encouraging way for CLZ brain targeting.

Molecular dynamics and free energy calculations of clozapine bound to D2 and H1 receptors reveal a cardiometabolic mitigated derivative.

Most atypical antipsychotics derive from a high dropout of drug treatments due to adverse cardiometabolic side effects. These side effects are caused, in part, by the H1 receptor blockade. The current work sought a clozapine derivative with a reduced affinity for the H1 receptor while maintaining its therapeutic effect linked to D2 receptor binding. Explicit molecular dynamics simulations and end-point free energy calculations of clozapine in complex with the D2 and H1 receptors embedded in cholesterol-rich lipid bilayers were performed to analyze the intermolecular interactions and address the relevance of clozapine-functional groups. Based on that, free energy perturbation calculations were performed to measure the change in free energy of clozapine structural modifications. Our results indicate the best clozapine derivative is the iodine atom substitution for chlorine. The latter is mainly due to electrostatic interaction loss for the H1 receptor, while the halogen orientation out of the D2 active site reduces the impact on the affinity.Communicated by Ramaswamy H. Sarma.

Impact of clozapine on the expression of miR-675-3p in plasma exosomes derived from patients with schizophrenia.

OBJECTIVES: Recently, the expression changes of microRNAs (miRNAs) in the serum exosomes (EXO) of schizophrenia (SCZ) have been reported. The aim of this study was to investigate the global expression changes of miRNA derived from the plasma EXO of patients with treatment-resistant schizophrenia (TRS) and the effects of clozapine on miRNA expression. METHODS: Global miRNA expression changes in plasma EXO between TRS and controls were studied using microarray analysis. Then, miRNA expressions among TRS, non-TRS, and controls were confirmed with quantitative qPCR experiments. We also studied changes in EXO miRNA expression with in-vitro SH-SY5Y cells. RESULTS: A microarray for miRNA expression analysis (nine controls vs. nine patients with TRS) revealed 13 up- and 18 downregulated miRNAs that were relevant to neuronal and brain development based on gene ontology analysis. Of those, upregulated miR-675-3p expression was successfully validated in the same cohort by qPCR experiments. Conversely, miR-675-3p expression levels were significantly decreased in the non-TRS cohort (50 controls vs. 50 patients without TRS without clozapine treatment). CONCLUSIONS: We identified global miRNA changes in plasma EXO derived from patients with SCZ that were relevant to neuronal functions, among which, hsa-miR-675-3p expression was upregulated by clozapine treatment.

[A novel ligand for chemogenetic receptors, Deschloroclozapine, enables rapid and selective modulation of neuronal activity and behavior in living animals].

A brain function is manifested by harmonizing some brain regions responsible for the function. Since pathological conditions in neuropsychiatric disorders are induced by the failure of this mechanism, it is crucial to identify the affected function by manipulating the specific brain regions. Designer receptors exclusively activated by designer drugs (DREADDs) are one of the chemogenetic tools that offer a means to repeatedly reversible control of the activity of a target neural population expressing a "designer receptor" by systemic injection of "designer drug," which is biologically inert. The most widely used DREADDs are muscarinic-based receptors, such as hM3Dq (excitatory) and hM4Di (inhibitory), which can be activated by clozapine-N-oxide (CNO). However, CNO has some concerns. First, because CNO has a modest brain penetrability, the effect is slow. Second, since CNO is metabolized to clozapine, an antipsychotic drug that acts on numerous endogenous receptors, the systemic administration may produce off-target actions. Therefore, we developed a new compound, deschloroclozapine (DCZ), to solve these issues. DCZ has a higher affinity and greater agonist potency than CNO with reduced off-target actions and can rapidly modulate the neuronal activity and behavior with muscarinic-based DREADDs in living animals. Given the potential weak point of CNO, DCZ affords clear benefits to many users of muscarinic-based DREADD, with increased reliability by removing concerns about possible off-target responses.

Protective effects of salvianolic acid A on clozapine-induced cardiotoxicity in zebrafish.

The clinical use of clozapine (CLZ), an atypical antipsychotic drug, was affected by side effects, such as cardiotoxicity. We selected normally developing zebrafish embryos to explore the antagonism of salvianolic acid A (SAA) against clozapine-induced cardiotoxicity. Embryos were treated with CLZ and SAA, and zebrafish phenotypes were observed at 24 h, 48 h, 72 h, and 96 h after treatment. The observed phenotypes included heart shape, heart rate, and venous sinus-arterial bulb (SV-BA) interval. Real-time quantitative PCR was used to detect changes in the expression of genes involved in heart inflammation, oxidative stress, and apoptosis. The results showed that SAA relieved pericardial edema, increased heart rate, and reduced the SV-BA interval. The PCR results also showed that when the zebrafish embryos were incubated with SAA and CLZ for 96 h, the expression of il-1b and nfkb2 were significantly downregulated, the expression of sod1 and cat were significantly upregulated, and the expressions of mcl1a and mcl1b were significantly downregulated. In summary, SAA can antagonize clozapine-induced cardiotoxicity.

Evidence that GIRK Channels Mediate the DREADD-hM4Di Receptor Activation-Induced Reduction in Membrane Excitability of Striatal Medium Spiny Neurons.

The hM4Di receptor-based chemogenetic DREADD system has been widely used to suppress neuronal activities, which has contributed substantially to the identification of behavior-associated neuronal circuitries including those in the striatum. One major mechanism by which hM4Di receptor activation suppresses neuronal activity is that the activation reduces membrane excitability, which is thought to be mediated by the opening of GIRK channels. However, previous studies have suggested that GIRK channels are barely expressed in the striatum, which naturally raises the question whether the hM4Di receptor activation-induced reduction in membrane excitability found in striatal medium spiny neurons (MSNs, which constitute 95-98% of the striatal neuronal population) is truly mediated by the endogenous GIRK channels in such scarcity. This study aims to answer this question by applying a GIRK channel-selective blocker, tertiapin-Q (TPNQ), to striatal MSNs. This study first verified that application of clozapine (CZP), an hM4Di receptor agonist, to MSNs expressing the hM4Di receptors hyperpolarized the cell membrane, and reduced membrane excitability and input resistance. This study next revealed that TPNQ post-treatment completely canceled the above CZP-induced electrophysiological effects and that TPNQ pretreatment mostly prevented further expression of the above CZP-induced electrophysiological effects. In addition, confocal microscopy imaging also revealed significant above-background GIRK1 immunofluorescence signals in striatal MSNs. These data suggest that the TPNQ-sensitive GIRK channels, despite being expressed at low levels, are likely the major mediator downstream of hM4Di receptor activation to reduce membrane excitability in striatal MSNs. These results imply that the notion held by scientists in the field that GIRK channels are absent in the striatum or their expression level is not significant enough to exert any function might be oversimplified or incorrect.

The relationship of antipsychotic treatment with reduced brown adipose tissue activity in patients with schizophrenia.

BACKGROUND: Antipsychotic drug (APD) treatment has been associated with metabolic abnormalities. Brown adipose tissue (BAT) is the main site of adaptive thermogenesis and secretes various metabolism-improving factors known as batokines. We explored the association of BAT activity with APD treatment and metabolic abnormalities in patients with schizophrenia by measuring the blood levels of bone morphogenetic protein 8b (BMP8b), a batokine secreted by mature BAT. METHODS: BMP8b levels were compared among 50 drug-free, 32 aripiprazole-treated, and 91 clozapine-treated patients with schizophrenia. Regression analysis was used to explore factors, including APD types, that might be associated with BMP8b levels and the potential effect of BMP8b on metabolic syndrome (MS). RESULTS: APD-treated patients had decreased BMP8b levels relative to drug-free patients. The difference still existed after adjustment for body mass index and Brief Psychiatric Rating Scale scores. Among APD-treated group, clozapine was associated with even lower BMP8b levels than the less obesogenic APD, aripiprazole. Furthermore, higher BMP8b levels were associated with lower risks of MS after adjustment for BMI and APD treatment. CONCLUSION: Using drug-free patients as the comparison group to understand the effect of APDs, this is the first study to show APD treatment is associated with reduced BAT activity that is measured by BMP8b levels, with clozapine associated a more significant reduction than aripiprazole treatment. BMP8b might have a beneficial effect against metabolic abnormalities and this effect is independent of APD treatment. Future studies exploring the causal relationship between APD treatment and BMP8b levels and the underlying mechanisms are warranted.

Neutrophil Myeloperoxidase-Mediated N-Demethylation of Quetiapine Leads to N-Desalkylquetiapine, a Pharmacologically Active Cytochrome P450 Metabolite.

The atypical antipsychotic drugs, quetiapine and clozapine, are associated with idiosyncratic drug reactions (such as agranulocytosis or neutropenia) that are thought to involve reactive metabolites. Neutrophil myeloperoxidase (MPO) metabolism of quetiapine is not well-studied, but is metabolized by cytochrome P450. Based on structural similarity to clozapine, we hypothesized that quetiapine can be metabolized by MPO and that there is overlap between cytochrome P450 and MPO metabolism of quetiapine. The interaction of quetiapine and clozapine with MPO and MPO chlorination activity was studied using UV-vis spectrophotometry. The metabolites were characterized using liquid chromatography-mass spectrometry (LC-MS), and electron paramagnetic resonance (EPR) spectroscopy was used for detecting drug-catalyzed glutathione oxidation. In the presence of quetiapine, MPO compound II accumulated for about 7.5 min, whereas in the presence of clozapine, MPO compound II was not observed as it was rapidly reduced back to the resting state. Increasing quetiapine concentrations resulted in a decrease in MPO chlorination activity, while the opposite result was found in the case of clozapine. UV-vis spectral studies showed no change when quetiapine was oxidized in the absence and presence of chloride anion (Cl-, to catalyze chlorination reactions). Significant changes, however, were observed in the same assay with clozapine, where Cl- appeared to hinder the rate of clozapine metabolism. The MPO-catalyzed hydroxylated and dealkylated metabolites of quetiapine and hydroxylated metabolites of clozapine were observed from the LC-MS analyses, particularly when Cl- was included in the reaction. In addition, hydroxylated, dealkylated, and a proposed sulfoxide metabolite of quetiapine were also observed in the reaction catalyzed by human microsomes/NADPH. Lastly, compared to quetiapine, clozapine metabolism by MPO/H2O2 and glutathione produced more glutathionyl radicals using EPR spin trapping. In conclusion, MPO/H2O2/Cl- was shown to metabolize quetiapine to S-oxidation and P450-like dealkylation products, and quetiapine metabolites were generally less reactive than clozapine.

Clozapine induces astrocyte-dependent FDG-PET hypometabolism.

PURPOSE: Advances in functional imaging allowed us to visualize brain glucose metabolism in vivo and non-invasively with [18F]fluoro-2-deoxyglucose (FDG) positron emission tomography (PET) imaging. In the past decades, FDG-PET has been instrumental in the understanding of brain function in health and disease. The source of the FDG-PET signal has been attributed to neuronal uptake, with hypometabolism being considered as a direct index of neuronal dysfunction or death. However, other brain cells are also metabolically active, including astrocytes. Based on the astrocyte-neuron lactate shuttle hypothesis, the activation of the glutamate transporter 1 (GLT-1) acts as a trigger for glucose uptake by astrocytes. With this in mind, we investigated glucose utilization changes after pharmacologically downregulating GLT-1 with clozapine (CLO), an anti-psychotic drug. METHODS: Adult male Wistar rats (control, n = 14; CLO, n = 12) received CLO (25/35 mg kg-1) for 6 weeks. CLO effects were evaluated in vivo with FDG-PET and cortical tissue was used to evaluate glutamate uptake and GLT-1 and GLAST levels. CLO treatment effects were also assessed in cortical astrocyte cultures (glucose and glutamate uptake, GLT-1 and GLAST levels) and in cortical neuronal cultures (glucose uptake). RESULTS: CLO markedly reduced in vivo brain glucose metabolism in several brain areas, especially in the cortex. Ex vivo analyses demonstrated decreased cortical glutamate transport along with GLT-1 mRNA and protein downregulation. In astrocyte cultures, CLO decreased GLT-1 density as well as glutamate and glucose uptake. By contrast, in cortical neuronal cultures, CLO did not affect glucose uptake. CONCLUSION: This work provides in vivo demonstration that GLT-1 downregulation induces astrocyte-dependent cortical FDG-PET hypometabolism-mimicking the hypometabolic signature seen in people developing dementia-and adds further evidence that astrocytes are key contributors of the FDG-PET signal.

Chronic hM4Di-DREADD-Mediated Chemogenetic Inhibition of Forebrain Excitatory Neurons in Postnatal or Juvenile Life Does Not Alter Adult Mood-Related Behavior.

G-protein-coupled receptors (GPCRs) coupled to Gi signaling, in particular downstream of monoaminergic neurotransmission, are posited to play a key role during developmental epochs (postnatal and juvenile) in shaping the emergence of adult anxiodepressive behaviors and sensorimotor gating. To address the role of Gi signaling in these developmental windows, we used a CaMKIIα-tTA::TRE hM4Di bigenic mouse line to express the hM4Di-DREADD (designer receptor exclusively activated by designer drugs) in forebrain excitatory neurons and enhanced Gi signaling via chronic administration of the DREADD agonist, clozapine-N-oxide (CNO) in the postnatal window (postnatal days 2-14) or the juvenile window (postnatal days 28-40). We confirmed that the expression of the HA-tagged hM4Di-DREADD was restricted to CaMKIIα-positive neurons in the forebrain, and that the administration of CNO in postnatal or juvenile windows evoked inhibition in forebrain circuits of the hippocampus and cortex, as indicated by a decline in expression of the neuronal activity marker c-Fos. hM4Di-DREADD-mediated inhibition of CaMKIIα-positive forebrain excitatory neurons in postnatal or juvenile life did not impact the weight profile of mouse pups, and also did not influence the normal ontogeny of sensory reflexes. Further, postnatal or juvenile hM4Di-DREADD-mediated inhibition of CaMKIIα-positive forebrain excitatory neurons did not alter anxiety- or despair-like behaviors in adulthood and did not impact sensorimotor gating. Collectively, these results indicate that chemogenetic induction of Gi signaling in CaMKIIα-positive forebrain excitatory neurons in postnatal and juvenile temporal windows does not appear to impinge on the programming of anxiodepressive behaviors in adulthood.

Effects of a Novel UGT2B Haplotype and UGT1A4*3 Allele Variants on Glucuronidation of Clozapine In vivo.

BACKGROUND: Glucuronidation is an important metabolic pathway of clozapine (CLZ), but the impact of various uridine 5'diphospho-glucuronosyltransferases (UGT) polymorphisms on the exposure and metabolism of CLZ in vivo is unclear. OBJECTIVE: The objective of this study was to investigate the impact of UGT2B haplotype and UGT1A4*3 allele variants on the formation of CLZ glucuronide metabolites (5N- and N+-glucuronide) and CLZ exposure in patients' serum after adjusting for sex, age, and smoking habits. METHODS: The study was based on serum samples from CLZ-treated patients (n=79) subjected to routine therapeutic drug monitoring (TDM) at Diakonhjemmet Hospital, Oslo, Norway. From the same patients, the following UGT variants were genotyped using Real-Time PCR: UGT2B:GA haplotype (defined as UGT2B:GA; rs1513559A>G and rs416593T>A) and UGT1A4*3 (rs2011425T>G). Serum concentrations of CLZ 5N- and N+-glucuronide were measured by UPLC high-resolution mass spectrometry. RESULTS: None of the genotypes had significant impact on CLZ exposure (p>0.05). However, compared to UGT2B:AT/AT and UGT1A4*1/*1, the 5N-glucuronide exposure was reduced in UGT2B:GA/GA carriers (-75 %, p=0.03) while the exposure was non-significantly increased in UGT1A4*3 carriers (+100 %, p=0.14), respectively. The N+-glucuronide exposure was unchanged in UGT1A4*3 vs. noncarriers (p=0.28), but significantly reduced in heterozygous (-50 %, p=0.016) and homozygous carriers (-70 %, p=0.021) of UGT2B:GA compared to UGT2B:AT/AT carriers, respectively. CONCLUSION: The UGT2B:GA and UGT1A4*3 variants had no impact on CLZ exposure but were associated with differences and preferences in CLZ glucuronidation. The latter might be of potential relevance for CLZ tolerability since levels of the N+-glucuronide metabolite may reflect the generation and trapping of reactive metabolites involved in CLZ-induced toxicity.

Evidence in primates supporting the use of chemogenetics for the treatment of human refractory neuropsychiatric disorders.

Non-human primate (NHP) models are essential for developing and translating new treatments that target neural circuit dysfunction underlying human psychopathology. As a proof-of-concept for treating neuropsychiatric disorders, we used a NHP model of pathological anxiety to investigate the feasibility of decreasing anxiety by chemogenetically (DREADDs [designer receptors exclusively activated by designer drugs]) reducing amygdala neuronal activity. Intraoperative MRI surgery was used to infect dorsal amygdala neurons with AAV5-hSyn-HA-hM4Di in young rhesus monkeys. In vivo microPET studies with [11C]-deschloroclozapine and postmortem autoradiography with [3H]-clozapine demonstrated selective hM4Di binding in the amygdala, and neuronal expression of hM4Di was confirmed with immunohistochemistry. Additionally, because of its high affinity for DREADDs, and its approved use in humans, we developed an individualized, low-dose clozapine administration strategy to induce DREADD-mediated amygdala inhibition. Compared to controls, clozapine selectively decreased anxiety-related freezing behavior in the human intruder paradigm in hM4Di-expressing monkeys, while coo vocalizations and locomotion were unaffected. These results are an important step in establishing chemogenetic strategies for patients with refractory neuropsychiatric disorders in which amygdala alterations are central to disease pathophysiology.

Antipsychotic clozapine binding to alpha-2-macroglobulin protects interacting partners against oxidation and preserves the anti-proteinase activity of the protein.

In this study, the interaction between clozapine, an atypical antipsychotic drug, and alpha-2-macroglobulin (α2M), a multipurpose anti-proteinase, was investigated under simulated (patho) physiological conditions using multiple spectroscopic techniques and molecular modeling. It was found that α2M binds clozapine with a moderate affinity (the binding constant of 0.9 × 105 M-1 at 37 °C). The preferable binding site for both clozapine's atropisomers was revealed to be a large pocket at the interface of C and D monomer subunits of the protein. Hydrogen bonds and the hydrophobic effect were proposed as dominant forces in complex formation. The binding of clozapine did not induce significant conformational change of the protein, as confirmed by virtually unaltered α2M secondary structure and anti-proteinase activity. However, both clozapine and α2M shielded each other from the deleterious influence of strong oxidants: sodium hypochlorite and 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH). Moreover, clozapine in a concentration range that is usually targeted in the plasma during patients' treatment effectively protected the anti-proteinase activity of α2M under AAPH-induced free radical overproduction. Our results suggest that the cooperation between α2M and clozapine may be a path by which these two molecules synergistically protect neural tissue against injury caused by disturbed proteostasis or oxidative stress.

Effects of the co-administration of MK-801 and clozapine on MiRNA expression profiles in rats.

MiRNAs are small, non-coding RNAs that can silence the expression of various target genes by binding their mRNAs and thus regulate a wide range of crucial bodily functions. However, the miRNA expression profile of schizophrenia after antipsychotic mediation is largely unknown. Non-competitive N-methyl-D-aspartic acid (NMDA) receptor antagonists such as MK-801 have provided useful animal models to investigate the effects of schizophrenia-like symptoms in rodent animals. Herein, the hippocampal miRNA expression profiles of Sprague-Dawley rats pretreated with MK-801 were examined after antipsychotic clozapine (CLO) treatment. Total hippocampal RNAs from three groups were subjected to next-generation sequencing (NGS), and bioinformatics analyses, including differential expression and enrichment analyses, were performed. Eight miRNAs were differentially expressed between the MK-801 and vehicle (VEH) control groups. Interestingly, 14 miRNAs were significantly differentially expressed between the CLO + MK-801 and MK-801 groups, among which rno-miR-184 was the most upregulated. Further analyses suggested that these miRNAs modulate target genes that are involved in endocytosis regulation, ubiquitin-mediated proteolysis, and actin cytoskeleton regulation and thus might play important roles in the pathogenesis of schizophrenia. Our results suggest that differentially expressed miRNAs play important roles in the complex pathophysiology of schizophrenia and subsequently impact brain functions.

Development of a simultaneous analytical method for clozapine and its metabolites in human plasma using liquid chromatography/electrospray ionization tandem mass spectrometry with linear range adjusted by in-source collision-induced dissociation.

Clozapine (CLZ) is a key drug in treatment-resistant schizophrenia. Therapeutic drug monitoring (TDM) of CLZ and its metabolites, N-desmethylclozapine and clozapine N-oxide, is required to monitor and manage the risks of side effects. Although quantification methods for TDM have been developed for CLZ and its metabolites, they were not sufficiently accurate for the quantification of CLZ owing to the upper limits of the calibration curves. An analytical method using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry was developed and validated for the simultaneous measurement of CLZ and its metabolites in human plasma. To expand the concentration range of the calibration curves, we used a linear range shift technique using in-source collision-induced dissociation (CID). Using our approach, the linearity and quantitative range were improved compared to those reported by previous studies, and were sufficient for TDM in clinical practice. The intra- and inter-assay accuracy was 84.6%-114.8%, and the intra- and inter-assay precisions were ≤9.1% and ≤9.9%, respectively. Moreover, all samples from patients with treatment-resistant schizophrenia were successfully quantified. Therefore, our novel analytical method using in-source CID had the appropriate performance to measure the plasma concentrations of CLZ and its metabolites for TDM in clinical practice.