Synthesis and biological evaluation of xanthine derivatives with phenacyl group as tryptophan hydroxylase 1 (TPH1) inhibitors for obesity and fatty liver disease.

Tryptophan hydroxylase 1 (TPH1) has emerged as a target for the treatment of metabolic diseases including obesity and fatty liver disease. A series of xanthine derivatives were synthesized and evaluated for their TPH1 inhibition. Among the synthesized compounds, compound 40 showed good in vitro activity and liver microsomal stability. Docking studies revealed that compound 40 showed better binding to TPH1 via key intermolecular interactions involving the xanthine scaffold, imidazo-thiazolyl ring, and hydroxyl-containing phenacyl moiety. In addition, compound 40 effectively suppressed the adipocyte differentiation of 3 T3-L1 cells.

Neurotoxicological Profiling of Paraquat in Zebrafish Model.

Paraquat is a polar herbicide protecting plant products against invasive species, it requires careful manipulation and restricted usage because of its harmful potentials. Exposure to paraquat triggers oxidative damage in dopaminergic neurons and subsequently causes a behavioral defect in vivo. Thereby, persistent exposure to paraquat is known to increase Parkinson's disease risk by dysregulating dopaminergic systems in humans. Therefore, most studies have focused on the dopaminergic systems to elucidate the neurotoxicological mechanism of paraquat poisoning, and more comprehensive neurochemistry including histaminergic, serotonergic, cholinergic, and GABAergic systems has remained unclear. Therefore, in this study, we investigated the toxicological potential of paraquat poisoning using a variety of approaches such as toxicokinetic profiles, behavioral effects, neural activity, and broad-spectrum neurochemistry in zebrafish larvae after short-term exposure to paraquat and we performed the molecular modeling approach. Our results showed that paraquat was slowly absorbed in the brain of zebrafish after oral administration of paraquat. In addition, paraquat toxicity resulted in behavioral impairments, namely, reduced motor activity and led to abnormal neural activities in zebrafish larvae. This locomotor deficit came with a dysregulation of dopamine synthesis induced by the inhibition of tyrosine hydroxylase activity, which was also indirectly confirmed by molecular modeling studies. Furthermore, short-term exposure to paraquat also caused simultaneous dysregulation of other neurochemistry including cholinergic and serotonergic systems in zebrafish larvae. The present study suggests that this neurotoxicological profiling could be a useful tool for understanding the brain neurochemistry of neurotoxic agents that might be a potential risk to human and environmental health.

Size-Dependent Effects of Polystyrene Nanoparticles (PS-NPs) on Behaviors and Endogenous Neurochemicals in Zebrafish Larvae.

Microplastics, small pieces of plastic derived from polystyrene, have recently become an ecological hazard due to their toxicity and widespread occurrence in aquatic ecosystems. In this study, we exposed zebrafish larvae to two types of fluorescent polystyrene nanoparticles (PS-NPs) to identify their size-dependent effects. PS-NPs of 50 nm, unlike 100 nm PS-NPs, were found to circulate in the blood vessels and accumulate in the brains of zebrafish larvae. Behavioral and electroencephalogram (EEG) analysis showed that 50 nm PS-NPs induce abnormal behavioral patterns and changes in EEG power spectral densities in zebrafish larvae. In addition, the quantification of endogenous neurochemicals in zebrafish larvae showed that 50 nm PS-NPs disturb dopaminergic metabolites, whereas 100 nm PS-NPs do not. Finally, we assessed the effect of PS-NPs on the permeability of the blood-brain barrier (BBB) using a microfluidic system. The results revealed that 50 nm PS-NPs have high BBB penetration compared with 100 nm PS-NPs. Taken together, we concluded that small nanoparticles disturb the nervous system, especially dopaminergic metabolites.

Neurochemical Effects of 4-(2Chloro-4-Fluorobenzyl)-3-(2-Thienyl)-1,2,4-Oxadiazol-5(4H)-One in the Pentylenetetrazole (PTZ)-Induced Epileptic Seizure Zebrafish Model.

Epilepsy is one of the most common neurological disorders, and it is characterized by spontaneous seizures. In a previous study, we identified 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one (GM-90432) as a novel anti-epileptic agent in chemically- or genetically-induced epileptic zebrafish and mouse models. In this study, we investigated the anti-epileptic effects of GM-90432 through neurochemical profiling-based approach to understand the neuroprotective mechanism in a pentylenetetrazole (PTZ)-induced epileptic seizure zebrafish model. GM-90432 effectively improved PTZ-induced epileptic behaviors via upregulation of 5-hydroxytryptamine, 17-ß-estradiol, dihydrotestosterone, progesterone, 5α -dihydroprogesterone, and allopregnanolone levels, and downregulation of normetanephrine, gamma-aminobutyric acid, and cortisol levels in brain tissue. GM-90432 also had a protective effect against PTZ-induced oxidative stress and zebrafish death, suggesting that it exhibits biphasic neuroprotective effects via scavenging of reactive oxygen species and anti-epileptic activities in a zebrafish model. In conclusion, our results suggest that neurochemical profiling study could be used to better understand of anti-epileptic mechanism of GM-90432, potentially leading to new drug discovery and development of anti-seizure agents.

Identification of new arylsulfide derivatives as anti-melanogenic agents in a zebrafish model.

A series of aryl sulfide derivatives was synthesized and evaluated for their anti-melanogenic activities. Several compounds, including 3e, 3i and 3q exhibited good anti-melanogenic activities. Among the derivatives, compound 3i showed good inhibitory effects against melanin synthesis and showed no toxicity in reconstituted human eye and skin tissues.

Computational determination of hERG-related cardiotoxicity of drug candidates.

BACKGROUND: Drug candidates often cause an unwanted blockage of the potassium ion channel of the human ether-a-go-go-related gene (hERG). The blockage leads to long QT syndrome (LQTS), which is a severe life-threatening cardiac side effect. Therefore, a virtual screening method to predict drug-induced hERG-related cardiotoxicity could facilitate drug discovery by filtering out toxic drug candidates. RESULT: In this study, we generated a reliable hERG-related cardiotoxicity dataset composed of 2130 compounds, which were carried out under constant conditions. Based on our dataset, we developed a computational hERG-related cardiotoxicity prediction model. The neural network model achieved an area under the receiver operating characteristic curve (AUC) of 0.764, with an accuracy of 90.1%, a Matthews correlation coefficient (MCC) of 0.368, a sensitivity of 0.321, and a specificity of 0.967, when ten-fold cross-validation was performed. The model was further evaluated using ten drug compounds tested on guinea pigs and showed an accuracy of 80.0%, an MCC of 0.655, a sensitivity of 0.600, and a specificity of 1.000, which were better than the performances of existing hERG-toxicity prediction models. CONCLUSION: The neural network model can predict hERG-related cardiotoxicity of chemical compounds with a high accuracy. Therefore, the model can be applied to virtual high-throughput screening for drug candidates that do not cause cardiotoxicity. The prediction tool is available as a web-tool at http://ssbio.cau.ac.kr/CardPred .

Anti-inflammatory effect of a novel synthetic compound 1-((4-fluorophenyl)thio)isoquinoline in RAW264.7 macrophages and a zebrafish model.

The compound, 1-((4-fluorophenyl)thio)isoquinoline (FPTQ), is a synthetic isoquinoline derivative. To test the anti-inflammatory effect of FPTQ, we used neutrophil-specific transgenic zebrafish Tg(mpx::EGFP)i114 line and lipopolysaccharide (LPS)-stimulated RAW264.7 cells. We also used two different methods, involving tail transection and LPS stimulation in the zebrafish model. Neutrophils translocation in the zebrafish tail-transected model was inhibited by FPTQ. Neutrophil aggregation was also inhibited by FPTQ in the LPS-stimulated zebrafish model. Decreased mRNA expression of the pro-inflammatory cytokine genes, interleukin-1ß (il-1ß) and interleukin-6 (il-6), was found in zebrafish larvae injected with FPTQ. Additionally, production of nitric oxide was inhibited by FPTQ in RAW264.7 macrophage cells treated with LPS. Moreover, the mRNA expression of Il-1ß and Il-6 suppressed by FPTQ treatment in RAW264.7 macrophage cells, and an enzyme immunoassay showed that FPTQ suppressed the secretion of IL-1ß and IL-6 in RAW264.7 cells. These results demonstrate that FPTQ reduced inflammatory responses and, therefore, suggest that it may be effective as an anti-inflammatory agent.

High risk of obstructive sleep apnea, insomnia, and daytime sleepiness among commercial motor vehicle drivers.

PURPOSE: We investigated the prevalence of sleep problems, such as obstructive sleep apnea (OSA), insomnia, and daytime sleepiness in commercial motor vehicle (CMV) drivers compared with that in the general population. METHODS: This is a cross-sectional study comparing sleep habits and sleep problems in 110 truck drivers with 1001 matched controls from the general population. The assessment was based on self-administered questionnaires that included the Berlin questionnaire, the insomnia severity index, and the Epworth sleepiness scale (ESS). Multivariate regression analysis was performed to determine whether CMV drivers were independently associated with these sleep problems compared with controls. RESULTS: The prevalence of a high risk of OSA and insomnia was 35.5% and 15.2%, respectively, in CMV drivers, which was significantly higher than in controls with a prevalence of 12.2% and 4.1%, respectively (P < 0.001 for both). Although CMV drivers showed higher ESS scores than controls, the prevalence of daytime sleepiness did not differ between the two groups (19.1% vs. 16.8%, P = 0.54). After adjusting for covariates, CMV drivers had 3.68 times higher odds (95% CI 2.29-5.84) of OSA and 2.97 times higher odds (95% CI, 1.46-6.06) of insomnia compared with controls. However, the degree of daytime sleepiness was not independently associated with CMV drivers. CONCLUSIONS: The prevalence of OSA and insomnia in CMV drivers was higher than that in the general population. Daytime sleepiness was associated with increased BMI, depression, OSA, and short sleep duration, regardless of CMV driving as an occupational factor.

Quantitative Evaluation of Cytochrome P450 3A4 Inhibition and Hepatotoxicity in HepaRG 3-D Spheroids.

Predicting drug-drug interactions (DDIs) is an important step during drug development to avoid unexpected side effects. Cytochrome P450 (CYP) 3A4 is the most abundant human hepatic phase I enzyme, which metabolizes >50% of therapeutic drugs. Therefore, it is essential to test the potential of a drug candidate to induce CYP3A4 expression or inhibit its activity. Recently, 3-dimensional (3-D) mammalian cell culture models have been adopted in drug discovery research to assess toxicity, DDIs, and pharmacokinetics. In this study, we applied a human 3-D spheroid culture protocol using HepaRG cells combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess its ability to predict CYP3A4 inhibition. Levels of midazolam, a specific substrate of CYP3A4, were used to determine the long-term metabolic capacity of CYP3A4. Midazolam was decreased in the 3-D HepaRG culture system by ∼80% over 7 days, whereas its primary metabolite, 1-hydroxymidazolam, increased by ∼40%. Next, we assessed hepatotoxicity by determining the cytotoxicity of known hepatotoxicants in HepaRG spheroids, HepG2 cells, and primary human hepatocytes. Significant differences in cytotoxicity were detected in the system using 3-D HepaRG spheroids. These results suggest that 3-D HepaRG spheroids are a good model for prediction of CYP inhibition and hepatotoxicity in screening of early drug candidates.

A novel assessment of nefazodone-induced hERG inhibition by electrophysiological and stereochemical method.

Nefazodone was used widely as an antidepressant until it was withdrawn from the U.S. market in 2004 due to hepatotoxicity. We have investigated methods to predict various toxic effects of drug candidates to reduce the failure rate of drug discovery. An electrophysiological method was used to assess the cardiotoxicity of drug candidates. Small molecules, including withdrawn drugs, were evaluated using a patch-clamp method to establish a database of hERG inhibition. Nefazodone inhibited hERG channel activity in our system. However, nefazodone-induced hERG inhibition indicated only a theoretical risk of cardiotoxicity. Nefazodone inhibited the hERG channel in a concentration-dependent manner with an IC50 of 45.3nM in HEK-293 cells. Nefazodone accelerated both the recovery from inactivation and its onset. Nefazodone also accelerated steady-state inactivation, although it did not modify the voltage-dependent character. Alanine mutants of hERG S6 and pore region residues were used to identify the nefazodone-binding site on hERG. The hERG S6 point mutants Y652A and F656A largely abolished the inhibition by nefazodone. The pore region mutant S624A mildly reduced the inhibition by nefazodone but T623A had little effect. A docking study showed that the aromatic rings of nefazodone interact with Y652 and F656 via π-π interactions, while an amine interacted with the S624 residue in the pore region. In conclusion, Y652 and F656 in the S6 domain play critical roles in nefazodone binding.

Evaluation of Drug Blood-Brain-Barrier Permeability Using a Microfluidic Chip.

The blood-brain-barrier (BBB) is made up of blood vessels whose permeability enables the passage of some compounds. A predictive model of BBB permeability is important in the early stages of drug development. The predicted BBB permeabilities of drugs have been confirmed using a variety of in vitro methods to reduce the quantities of drug candidates needed in preclinical and clinical trials. Most prior studies have relied on animal or cell-culture models, which do not fully recapitulate the human BBB. The development of microfluidic models of human-derived BBB cells could address this issue. We analyzed a model for predicting BBB permeability using the Emulate BBB-on-a-chip machine. Ten compounds were evaluated, and their permeabilities were estimated. Our study demonstrated that the permeability trends of ten compounds in our microfluidic-based system resembled those observed in previous animal and cell-based experiments. Furthermore, we established a general correlation between the partition coefficient (Kp) and the apparent permeability (Papp). In conclusion, we introduced a new paradigm for predicting BBB permeability using microfluidic-based systems.

Successive development of ischemic malignant strokes in a patient with multiple fusiform aneurysms: A case report.

BACKGROUND: Intracranial fusiform aneurysms are rare, spindle-shaped, and nonsaccular arterial dilatations that may be caused by dissection. CASE SUMMARY: A 48-year-old man complained of wake-up onset of dysarthria and left-sided weakness. Diffusion-weighted magnetic resonance imaging of the brain revealed an infarction in the territories of the right middle and posterior cerebral arteries. Computed tomography angiography showed fusiform aneurysms in the right vertebral artery and bilateral petrous segments of the internal carotid arteries (ICAs). Despite conservative management, malignant ischemic stroke recurred in the contralateral ICA territory within a day of the onset of the index stroke. CONCLUSION: We report a rare case of successive malignant strokes in a patient with multiple fusiform aneurysms. Herein, we emphasize that clinicians should consider aggressive treatment for patients with ischemic stroke and multiple fusiform aneurysms.

A case of disorder of arousal with prolonged postarousal hypersynchronous delta activity.

Disorder of arousal (DOA) is a form of non-rapid eye movement sleep parasomnia caused by partial or incomplete arousal from deep sleep. Most previous studies of patients with DOA analyzed prearousal hypersynchronous delta activity (HSDA), but few studies have described postarousal HSDA. Herein, we report a 23-year-old man with a history of abrupt arousal during sleep and confused behavior and speech since he was 14 years old. During video electroencephalography monitoring, he had 9 arousal events of getting up, sitting on the bed, looking around, or simple arousal, including eyes open, looking at the ceiling, or head flexion. During all arousal events, the postarousal electroencephalography pattern was prolonged HSDA for approximately 40 seconds. The patient was treated unsuccessfully for more than 2 years with an antiseizure medication (lacosamide); eventually, he responded to clonazepam that was administered for the possibility of DOA. Prolonged rhythmic HSDA without spatiotemporal evolution can appear as a postarousal electroencephalography pattern of DOA. When diagnosing DOA, it is important to recognize that postarousal HSDA can appear as a characteristic electroencephalography pattern of DOA. CITATION: Kang M, Shin D, Lee HC, Provini F, Jung KY. A case of disorder of arousal with prolonged postarousal hypersynchronous delta activity. J Clin Sleep Med. 2023;19(7):1365-1368.

Lesion Detection Through MRI Postprocessing in Pathology-Proven Focal Cortical Dysplasia: Experience at a Single Institution in the Republic of Korea.

BACKGROUND AND PURPOSE: Focal cortical dysplasia (FCD) is one of the most common causes of drug-resistant epilepsy, and necessitates a multimodal evaluation to ensure optimal surgical treatment. This study aimed to determine the supportive value of the morphometric analysis program (MAP) in detecting FCD using data from a single institution in Korea. METHODS: To develop a standard reference for the MAP, normal-looking MRIs by two scanners that are frequently used in this center were chosen. Patients with drug-resistant epilepsy and FCD after surgery were candidates for the analysis. The three-dimensional T1-weighted MRI scans of the patients were analyzed as test cases using the MAP. RESULTS: The MRI scans of 87 patients were included in the analysis. The radiologist detected abnormal findings correlated with FCD (RAD positive [RAD(+)]) in 34 cases (39.1%), while the MAP could detect FCD in 25.3% of cases. A combination of the MAP (MAP[+] cases) with interpretations by the radiologist increased the detection to 42.5% (37 cases). The lesion detection rate was not different according to the type of reference scanners except in one case. MAP(+)/RAD(-) presented in three cases, all of which had FCD type IIa. The detection rate was slightly higher using the same kind of scanner as a reference, but not significantly (35.0% vs. 22.4% p=0.26). CONCLUSIONS: The results of postprocessing in the MAP for detecting FCD did not depend on the type of reference scanner, and the MAP was the strongest in detecting FCD IIa. We suggested that the MAP could be widely utilized without developing institutional standards and could become an effective tool for detecting FCD lesions.

KRIBB11 inhibits HSP70 synthesis through inhibition of heat shock factor 1 function by impairing the recruitment of positive transcription elongation factor b to the hsp70 promoter.

Heat shock factor 1 (HSF1) is the master switch for heat shock protein (HSP) expression in eukaryotes. A synthetic chemical library was screened to identify inhibitors of HSF1 using a luciferase reporter under the control of a heat shock element. A compound named KRIBB11 (N(2)-(1H-indazole-5-yl)-N(6)-methyl-3-nitropyridine-2,6-diamine) was identified for its activity in abolishing the heat shock-induced luciferase activity with an IC(50) of 1.2 µmol/liter. When the cells were exposed to heat shock in the presence of KRIBB11, the induction of HSF1 downstream target proteins such as HSP27 and HSP70 was blocked. In addition, treatment of HCT-116 cells with KRIBB11 induced growth arrest and apoptosis. Markers of apoptosis, such as cleaved poly(ADP-ribose) polymerase, were detected after KRIBB11 treatment. Biotinyl-KRIBB11 was synthesized as an affinity probe for the identification of KRIBB11 target proteins. Using affinity chromatography and competition assays, KRIBB11 was shown to associate with HSF1 in vitro. Chromatin immunoprecipitation analysis showed that KRIBB11 inhibited HSF1-dependent recruitment of p-TEFb (positive transcription elongation factor b) to the hsp70 promoter. Finally, intraperitoneal treatment of nude mice with KRIBB11 at 50 mg/kg resulted in a 47.4% (p < 0.05) inhibition of tumor growth without body weight loss. Immunoblotting assays showed that the expression of HSP70 was lower in KRIBB11-treated tumor tissue than in control tissues. Because HSPs are expressed at high levels in a wide range of tumors, these results strengthen the rationale for targeting HSF1 in cancer therapy.

Phosphatase of regenerating liver-3 promotes migration and invasion by upregulating matrix metalloproteinases-7 in human colorectal cancer cells.

Phosphatase of regenerating liver (PRL)-3, a member of a subgroup of protein tyrosine phosphatases that can stimulate the degradation of the extracellular matrix, is over-expressed in metastatic colorectal cancer (CRC) relative to primary tumors. To determine whether PRL-3-induced enhancement of migration and invasion is dependent on the expression of matrix metalloproteinases (MMPs), PRL-3 was expressed in DLD-1 human CRC cells. The motility, migration and invasion characteristics of the cells were examined, and metastasis to the lung was confirmed in a nude mouse using PRL-3-overexpressing DLD-1 cells [DLD-1 (PRL-3)]. Migration and invasion of the cells were inhibited by phosphatase and farnesyltransferase inhibitors. Expression of MMPs was enhanced 3- to 10-fold in comparison to control cells, and migration and invasion were partially inhibited by small interfering RNA (siRNA) knockdown of MMP-2, -13 or -14. Importantly, siRNA knockdown of MMP-7 completely inhibited the migration and invasion of DLD-1 (PRL-3) cells, whereas overexpression of MMP-7 increased migration. The expression of MMP-7 was also downregulated by phosphatase and farnesyltransferase inhibitors. It was found that PRL-3 induced MMP-7 through oncogenic pathways including PI3K/AKT and ERK and that there is a relationship between the expression of PRL-3 and MMP-7 in human tumor cell lines. The expression of MMP-13 and -14 was very sensitive to the inhibition of farnesyltransferase; however, the migration and invasion of DLD-1 (PRL-3) cells did not strongly depend on the expression of MMP-13 or -14. These results suggest that the migration and invasion of PRL-3-expressing CRC cells depends primarily on the expression of MMP-7.

Novel Thioxothiazolo[3,4-a]quinazolin-5(4H)-one Derivatives as BKCa Channel Activators for Urinary Incontinence.

Overactive bladder (OAB) is a syndrome causing a sudden and unstoppable need to urinate with significant global prevalence. Several drugs are used to treat OAB; however, they have various side effects. Therefore, new treatment options for OAB are required. A series of novel 5-oxo-N-phenyl-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline-3-carboxamide derivatives were synthesized and evaluated for their large-conductance voltage- and Ca2+-activated K+ channel activation through a cell-based fluorescence assay and electrophysiological recordings. Several compounds, including a 7-bromo substituent on the heterocyclic system, showed increased channel currents. Among the derivatives, compound 12h exhibited potent in vitro activity with a half-maximal effective concentration (EC50) of 2.89 µM, good oral pharmacokinetic properties (area under the curve and half-life), and in vivo efficacy in a spontaneously hypertensive rat model.

2-Hydroxycurcuminoid induces apoptosis of human tumor cells through the reactive oxygen species-mitochondria pathway.

2-Hydroxycinnamaldehyde (HCA) and curcumin have been reported to have antitumor effects against various human tumor cells in vitro and in vivo by generation of ROS. Aldehyde-free HCA analogs were synthesized based on the structure of curcumin, which we have called 2-hydroxycurcuminoids. The hydroxyl group of curcuminoids enhances the ability to generate ROS. 2-Hydroxycurcuminoid (HCC-7) strongly inhibited the growth of SW620 colon tumor cells with a GI(50) value of 7µM, while the parent compounds, HCA and curcumin, displayed GI(50) values of 12 and 30µM, respectively. HCC-7 was found to induce apoptosis through the reactive oxygen species-mitochondria pathway and cell cycle arrest at G2/M phase.

Cosmomycin C inhibits signal transducer and activator of transcription 3 (STAT3) pathways in MDA-MB-468 breast cancer cell.

The signal transducer and activator of transcription 3 (STAT3) is constitutively activated in cancer cells. Therefore, blocking the aberrant activity of STAT3 in tumor cells is a validated therapeutic strategy. To discover novel inhibitors of STAT3 activity, we screened against microbial natural products using a dual-luciferase assay. Using the microbial metabolome library, we identified cosmomycin C (CosC), which was isolated from the mycelium extract of Streptomyces sp. KCTC19769, as a STAT3 pathway inhibitor. CosC inhibited STAT3 (Tyr705) phosphorylation and subsequent nuclear translocation in MDA-MB-468 breast cancer cells. CosC-mediated inhibition of STAT3 signaling pathway was confirmed by suppressed expression of STAT3 downstream target proteins including cyclin D1, Bcl-xL, survivin, Mcl-1, and VEGF in CosC-treated MDA-MB-468 cells. Flow cytometry showed that CosC caused accumulation in the G(0)-G(1) phase of the cell cycle and induced apoptosis via PARP cleavage and caspase-3 activation. Based on these findings, CosC may be a potential candidate for modulation of STAT3 pathway.

Interplay among Conformation, Intramolecular Hydrogen Bonds, and Chameleonicity in the Membrane Permeability and Cyclophilin A Binding of Macrocyclic Peptide Cyclosporin O Derivatives.

A macrocyclic peptide scaffold with well-established structure-property relationship is desirable for tackling undruggable targets. Here, we adopted a natural macrocycle, cyclosporin O (CsO) and its derivatives (CP1-3), and evaluated the impact of conformation on membrane permeability, cyclophilin A (CypA) binding, and the pharmacokinetic (PK) profile. In nonpolar media, CsO showed a similar conformation to cyclosporin A (CsA), a well-known chameleonic macrocycle, but less chameleonic behavior in a polar environment. The weak chameleonicity of CsO resulted in decreased membrane permeability; however, the more rigid conformation of CsO was not detrimental to its PK profile. CsO exhibited a higher plasma concentration than CsA, which resulted from minimal CypA binding and lower accumulation in red blood cells and moderate oral bioavailability (F = 12%). Our study aids understanding of CsO, a macrocyclic peptide that is less explored than CsA but with greater potential for diversity generation and rational design.