LRG1-Targeted Nintedanib Delivery for Enhanced Renal Fibrosis Mitigation.

Renal fibrosis lacks effective nephroprotective drugs in clinical settings due to poor accumulation of therapeutic agents in damaged kidneys, underscoring the urgent need for advanced renal-targeted delivery systems. Herein, we exploited the significantly increased expression of the leucine-rich α-2 glycoprotein 1 (LRG1) protein during renal fibrosis to develop a novel drug delivery system. Our engineered nanocarrier, DENNM, preferentially targets fibrotic kidneys via the decorated ET peptide's high affinity for LRG1. Once internalized by damaged renal cells, DENNM releases its encapsulated nintedanib, triggered by the active caspase-3 protease, disrupting the nanomedicine's structural integrity. The released nintedanib effectively reduces the level of expression of the extracellular matrix and impedes the progression of renal fibrosis by inhibiting the transforming growth factor-ß (TGF-ß)-Smad2/3 pathway. Our comprehensive in vitro and in vivo studies validate DENNM's antifibrotic efficacy, emphasizing LRG1's potential in renal targeted drug delivery and introducing an innovative approach to nanomedicine for treating renal fibrosis.

VSMC-specific EP4 deletion exacerbates angiotensin II-induced aortic dissection by increasing vascular inflammation and blood pressure.

Prostaglandin E2 (PGE2) plays an important role in vascular homeostasis. Its receptor, E-prostanoid receptor 4 (EP4) is essential for physiological remodeling of the ductus arteriosus (DA). However, the role of EP4 in pathological vascular remodeling remains largely unknown. We found that chronic angiotensin II (AngII) infusion of mice with vascular smooth muscle cell (VSMC)-specific EP4 gene knockout (VSMC-EP4-/-) frequently developed aortic dissection (AD) with severe elastic fiber degradation and VSMC dedifferentiation. AngII-infused VSMC-EP4-/- mice also displayed more profound vascular inflammation with increased monocyte chemoattractant protein-1 (MCP-1) expression, macrophage infiltration, matrix metalloproteinase-2 and -9 (MMP2/9) levels, NADPH oxidase 1 (NOX1) activity, and reactive oxygen species production. In addition, VSMC-EP4-/- mice exhibited higher blood pressure under basal and AngII-infused conditions. Ex vivo and in vitro studies further revealed that VSMC-specific EP4 gene deficiency significantly increased AngII-elicited vasoconstriction of the mesenteric artery, likely by stimulating intracellular calcium release in VSMCs. Furthermore, EP4 gene ablation and EP4 blockade in cultured VSMCs were associated with a significant increase in MCP-1 and NOX1 expression and a marked reduction in α-SM actin (α-SMA), SM22α, and SM differentiation marker genes myosin heavy chain (SMMHC) levels and serum response factor (SRF) transcriptional activity. To summarize, the present study demonstrates that VSMC EP4 is critical for vascular homeostasis, and its dysfunction exacerbates AngII-induced pathological vascular remodeling. EP4 may therefore represent a potential therapeutic target for the treatment of AD.

Disruption of prostaglandin E2 receptor EP4 impairs urinary concentration via decreasing aquaporin 2 in renal collecting ducts.

The antidiuretic hormone arginine vasopressin is a systemic effector in urinary concentration. However, increasing evidence suggests that other locally produced factors may also play an important role in the regulation of water reabsorption in renal collecting ducts. Recently, prostaglandin E2 (PGE2) receptor EP4 has emerged as a potential therapeutic target for the treatment of nephrogenic diabetes insipidus, but the underlying mechanism is unknown. To evaluate the role of EP4 in regulating water homeostasis, mice with renal tubule-specific knockout of EP4 (Ksp-EP4(-/-)) and collecting duct-specific knockout of EP4 (AQP2-EP4(-/-)) were generated using the Cre-loxP recombination system. Urine concentrating defect was observed in both Ksp-EP4(-/-) and AQP2-EP4(-/-) mice. Decreased aquaporin 2 (AQP2) abundance and apical membrane targeting in renal collecting ducts were evident in Ksp-EP4(-/-) mice. In vitro studies demonstrated that AQP2 mRNA and protein levels were significantly up-regulated in mouse primary inner medullary collecting duct (IMCD) cells after pharmacological activation or adenovirus-mediated overexpression of EP4 in a cAMP/cAMP-response element binding protein-dependent manner. In addition, EP4 activation or overexpression also increased AQP2 membrane accumulation in a mouse IMCD cell line (IMCD3) stably transfected with the AQP2 gene, mainly through the cAMP/protein kinase A and extracellular signal-regulated kinase pathways. In summary, the EP4 receptor in renal collecting ducts plays an important role in regulating urinary concentration under physiological conditions. The ability of EP4 to promote AQP2 membrane targeting and increase AQP2 abundance makes it a potential therapeutic target for the treatment of clinical disorders including acquired and congenital diabetes insipidus.

Fabrication and Characterization of Carbon Fiber-Reinforced Nano-Hydroxyapatite/Polyamide46 Biocomposite for Bone Substitute.

BACKGROUND Ideal bone repair material should be of good biocompatibility and high bioactivity. Besides, their mechanical properties should be equivalent to those of natural bone. The objective of this study was to fabricate a novel biocomposite suitable for load-bearing bone defect repair. MATERIAL AND METHODS A novel biocomposite composed of carbon fiber, hydroxyapatite and polyamide46 (CF/HA/PA46) was fabricated, and its mechanical performances and preliminary cell responses were evaluated to explore its feasibility for load-bearing bone defect repair. RESULTS The resultant CF/HA/PA46 biocomposite showed a bending strength of 159-223 MPa, a tensile strength of 127-199 MPa and a tensile modulus of 7.7-10.8 GPa, when the CF content was 5-20% (mass fraction) in biocomposite. The MG63 cells, showing an osteogenic phenotype, were well adhered and spread on the surface of the CF/HA/PA46 biocomposite. Moreover, the cells vitality and differentiation on the CF/HA/PA46 biocomposite surface were obviously increased during the culture time and there was no significant difference between the CF/HA/PA46 biocomposite and HA/PA (as control) at all the experimental time (P>0.05). CONCLUSIONS The addition of CF into HA/PA46 composite manifest improved the mechanical performances and showed favorable effects on biocompatibility of MG63 cells. The obtained biocomposite has high potential for bone repair in load-bearing sites.

Farnesoid X receptor (FXR) gene deficiency impairs urine concentration in mice.

The farnesoid X receptor (FXR) is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. FXR is mainly expressed in liver and small intestine, where it plays an important role in bile acid, lipid, and glucose metabolism. The kidney also has a high FXR expression level, with its physiological function unknown. Here we demonstrate that FXR is ubiquitously distributed in renal tubules. FXR agonist treatment significantly lowered urine volume and increased urine osmolality, whereas FXR knockout mice exhibited an impaired urine concentrating ability, which led to a polyuria phenotype. We further found that treatment of C57BL/6 mice with chenodeoxycholic acid, an FXR endogenous ligand, significantly up-regulated renal aquaporin 2 (AQP2) expression, whereas FXR gene deficiency markedly reduced AQP2 expression levels in the kidney. In vitro studies showed that the AQP2 gene promoter contained a putative FXR response element site, which can be bound and activated by FXR, resulting in a significant increase of AQP2 transcription in cultured primary inner medullary collecting duct cells. In conclusion, the present study demonstrates that FXR plays a critical role in the regulation of urine volume, and its activation increases urinary concentrating capacity mainly via up-regulating its target gene AQP2 expression in the collecting ducts.

FAM3A activates PI3K p110α/Akt signaling to ameliorate hepatic gluconeogenesis and lipogenesis.

UNLABELLED: FAM3A belongs to a novel cytokine-like gene family, and its physiological role remains largely unknown. In our study, we found a marked reduction of FAM3A expression in the livers of db/db and high-fat diet (HFD)-induced diabetic mice. Hepatic overexpression of FAM3A markedly attenuated hyperglycemia, insulin resistance, and fatty liver with increased Akt (pAkt) signaling and repressed gluconeogenesis and lipogenesis in the livers of those mice. In contrast, small interfering RNA (siRNA)-mediated knockdown of hepatic FAM3A resulted in hyperglycemia with reduced pAkt levels and increased gluconeogenesis and lipogenesis in the livers of C57BL/6 mice. In vitro study revealed that FAM3A was mainly localized in the mitochondria, where it increases adenosine triphosphate (ATP) production and secretion in cultured hepatocytes. FAM3A activated Akt through the p110α catalytic subunit of PI3K in an insulin-independent manner. Blockade of P2 ATP receptors or downstream phospholipase C (PLC) and IP3R and removal of medium calcium all significantly reduced FAM3A-induced increase in cytosolic free Ca(2+) levels and attenuated FAM3A-mediated PI3K/Akt activation. Moreover, FAM3A-induced Akt activation was completely abolished by the inhibition of calmodulin (CaM). CONCLUSION: FAM3A plays crucial roles in the regulation of glucose and lipid metabolism in the liver, where it activates the PI3K-Akt signaling pathway by way of a Ca(2+) /CaM-dependent mechanism. Up-regulating hepatic FAM3A expression may represent an attractive means for the treatment of insulin resistance, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD).

Integrating Full Bayesian Inference and Student's t-Distribution Method for Enhanced Outlier Handling in Caffeine Population Pharmacokinetics: Assessing Drug-Drug Interactions with Enasidenib in Relapsed or Refractory AML and MDS Patients.

As the first-in-class, selective, and potent inhibitor of the isocitrate dehydrogenase-2 (IDH2) mutant protein, enasidenib was approved by the US Food and Drug Administration (FDA) in 2017 for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with an IDH2 mutation. Known for its interactions with various cytochrome P450 (CYP) enzymes and transporters in vitro, a clinical pharmacokinetics (PK) trial was initiated to assess the impact of multiple doses of enasidenib on the single-dose PK of sensitive probe substrates of several cytochrome P450 enzymes and transporters. In this study, a population pharmacokinetic analysis approach was employed to address challenges posed by high, nonzero baseline caffeine concentrations. Moreover, we integrated full Bayesian inference into this approach innovatively for a more detailed understanding of parameter uncertainty and greater modeling flexibility, alongside Student's t-distribution for robust error modeling in handling the abnormal outlier caffeine concentration data observed in this trial. Our analyses demonstrated that multiple doses of enasidenib altered caffeine clearance to a clinically meaningful extent, as evidenced by an approximate 8-fold decrease. This finding led to a specific recommendation in the package insert to avoid the concurrent use of certain CYP1A2 substrates with enasidenib, unless directed otherwise in the prescribing information. Furthermore, this research underlines the technical benefits of integrating full Bayesian inference and incorporating Student's t-distribution for residual error modeling in the PK field.

Pediatric model-based dose optimization using a pooled exposure-response safety analysis for nivolumab and nivolumab plus ipilimumab combination in melanoma.

An exposure-response (E-R) safety analysis was conducted across adult and pediatric (<18 years) studies to evaluate the potential impact of higher nivolumab and/or ipilimumab exposures in adolescents (≥12 to <18 years) versus adults with melanoma using the approved adult dosing regimens for nivolumab alone or in combination with ipilimumab. Data from 3507 patients across 15 studies were used to examine the relationship between nivolumab-ipilimumab daily average exposure and time to grade 2+ immune-mediated adverse events (gr2+ IMAEs). Results from the E-R safety model showed ipilimumab, but not nivolumab, exposure to be a statistically significant predictor of gr2+ IMAEs. Significant covariates included sex (41% higher risk for women than men), line of therapy (19% higher for first-line than later-line), and treatment setting (26% lower for adjuvant than advanced melanoma). Younger age and lower body weight (BW) were each associated with a lower risk of gr2+ IMAEs (hazard ratio [HR]: 0.830 for 15-year-olds versus 60-year-olds and 0.84 for BW 52 kg versus 75 kg). For adolescents with melanoma treated with nivolumab in the advanced or adjuvant settings, these results are supportive of nivolumab flat dosing regimens for adolescents greater than or equal to 40 kg and BW-based dosing for adolescents less than 40 kg. These results also support adult weight-based dosing regimens for nivolumab plus ipilimumab in adolescents with advanced melanoma. This analysis suggests that although higher exposures are predicted in adolescents with lower weight compared with adults, there is no predicted immune-mediated safety risk when treated with the approved adult dosing of nivolumab with/without ipilimumab.

Nivolumab and ipilimumab population pharmacokinetics in support of pediatric dose recommendations-Going beyond the body-size effect.

Body size has historically been considered the primary source of difference in the pharmacokinetics (PKs) of monoclonal antibodies (mAbs) between children aged greater than or equal to 2 years and adults. The contribution of age-associated differences (e.g., ontogeny) beyond body-size differences in the pediatric PKs of mAbs has not been comprehensively evaluated. In this study, the population PK of two mAbs (nivolumab and ipilimumab) in pediatric oncology patients were characterized. The effects of age-related covariates on nivolumab or ipilimumab PKs were assessed using data from 13 and 10 clinical studies, respectively, across multiple tumor types, including melanoma, lymphoma, central nervous system tumors (CNSTs), and other solid tumors. Clearance was lower in pediatric patients (aged 1-17 years) with solid tumors or CNST than in adults after adjusting for other covariates, including the effect of body size. In contrast, clearance was similar in pediatric patients with lymphoma to that in adults with lymphoma. The pediatric effects characterized have increased the accuracy of the predictions of the model, facilitating its use in subsequent exposure comparisons between pediatric and adult patients, as well as for exposure-response analyses to inform pediatric dosing. This study approach may be applicable to the optimization of pediatric dosing of other mAbs and possibly other biologics.

Significance of the great protection of the Yangtze River: Riverine input contributes primarily to the presence of PAHs and HMs in its estuary and the adjacent sea.

The Yangtze River protection strategies are expected to improve the water quality and ecological function of the Yangtze River Estuary (YRE). The concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) and 6 heavy metals (HMs) in the YRE were measured and the riverine fluxes were calculated subsequently. In particular, the concentrations of low molecular weight PAHs (LMW-PAHs), arsenic (As) and mercury (Hg) in seawater decreased over time, while those of other studied pollutants did not change a lot. In sediments, the concentration changes for all the pollutants were insignificant. For the present pollutants, the river input is the dominant source, and the flux decreased after the protection. The contribution of the discharge from wastewater treatment plants (WWTPs) was quantified. Its influence cannot be ignored. The seafood quality remained stable and the risk via diet was insignificant. Long-term monitoring is necessary, and the positive impact of the Protection Strategy is gradually emerging.

Model-Based Approach to Selecting Pegfilgrastim Dose for Pharmacokinetic and Pharmacodynamic Similarity Studies in Biosimilar Development.

This study applied modeling and simulation (M&S) approaches to evaluate the sensitivity of pegfilgrastim pharmacokinetics (PKs) and pharmacodynamics (PDs) to changes in dose amount, and linear or nonlinear clearance (CL) over pegfilgrastim subcutaneous dose of 2-6 mg. A previously published model was adapted to better describe pegfilgrastim PK and PD data in healthy subjects and used in simulation. Nonlinear CL accounts for 98% and 77%, respectively, of the total CL of pegfilgrastim at 2 and 6 mg. The sensitivity analyses showed: (i) PK of 2 and 6 mg doses are similarly sensitive to detect differences for a 5% change in dose; (ii) PK of 2 mg dose is more sensitive to changes in receptor binding affinity, a model parameter for nonlinear CL, and a product quality attribute characterized with orthogonal methods as part of demonstrating analytical similarity between products; (iii) PK of approved 6 mg dose is more sensitive to changes in linear CL, which has not been associated with any specific product quality attributes, and (iv) the PDs are not sensitive to changes in linear or nonlinear CL. Taken together, our analyses support that the approved pegfilgrastim dose of 6 mg is appropriate for detecting differences between a biosimilar and the reference products in pegfilgrastim PK and PD similarity studies. The described M&S approaches can be adopted to support dose selection for biosimilars with nonlinear PK and complex PK-PD interplay.

Clinical characterization and proteomic profiling of lean nonalcoholic fatty liver disease.

Introduction: Obesity has been historically associated with nonalcoholic fatty liver disease (NAFLD), but it can also occur in lean individuals. However, limited data is available on this special group. To investigate the clinical and proteomic characteristics of lean subjects with NAFLD, and to identify potential clinical variables and plasma proteins for diagnosing NAFLD in lean individuals, we collected clinical data from a large cohort of 2,236 subjects. Methods: Diagnosis of NAFLD relied on detecting pronounced hepatic steatosis through abdominal ultrasonography. Participants were categorized into four groups based on body mass index: overweight NAFLD, overweight control, lean NAFLD, and lean control. Plasma proteomic profiling was performed on samples from 20 subjects in each group. The lean NAFLD group was compared to both lean healthy and obese NAFLD groups across all data. Results and discussion: The results indicated that the lean NAFLD group exhibited intermediate metabolic profiles, falling between those of the lean healthy and overweight NAFLD groups. Proteomic profiling of plasma in lean subjects with or without NAFLD revealed 45 statistically significant changes in proteins, of which 37 showed high diagnostic value (AUC > 0.7) for lean NAFLD. These potential biomarkers primarily involved lipid metabolism, the immune and complement systems, and platelet degranulation. Furthermore, AFM, GSN, CFH, HGFAC, MMP2, and MMP9 have been previously associated with NAFLD or NAFLD-related factors such as liver damage, insulin resistance, metabolic syndromes, and extracellular homeostasis. Overall, lean individuals with NAFLD exhibit distinct clinical profiles compared to overweight individuals with NAFLD. Despite having worse metabolic profiles than their healthy counterparts, lean NAFLD patients generally experience milder systemic metabolic disturbances compared to obese NAFLD patients. Additionally, the plasma proteomic profile is significantly altered in lean NAFLD, highlighting the potential of differentially expressed proteins as valuable biomarkers or therapeutic targets for diagnosing and treating NAFLD in this population.

Cord-Blood Engraftment Using an Enhanced Dual-Conditioning Regimen for Malignant Hematologic Diseases.

To explore a more effective conditioning regimen for umbilical cord blood transplantation (UCBT) to treat hematologic malignancies, we conducted a cohort study of a fludarabine/busulfan/cytarabine plus cyclophosphamide 200 mg/kg regimen. Forty-two consecutive patients with leukemia, myelodysplastic syndrome, or lymphoma received the regimen. The median number of infused total nucleated cells per kilogram was 5.5 × 107 (1.81-20.6), the median number of infused CD34+ cells per kilogram was 1.58 × 105 (0.58-6.6), and the median follow-up for surviving patients was 37 months (4.0-79.5 months). The cumulative incidence of neutrophil engraftment at 31 days was 100% [95% confidence interval (CI): 0.9159-1.0], and the median time to neutrophil engraftment was 19 days. The cumulative incidence of nonrelapse mortality was 12.76% (95% CI: 0.0455-0.2356) at 180 days and 3 years. The 3-year overall survival (OS) and disease-free survival (DFS) rates were 71.6% and 59.6%, respectively. Especially in patients who received transplants in the early and intermediate stages, the 3-year OS and DFS rates were 90.3% (95% CI: 0.805-1.0) and 76.2% (95% CI: 0.608-0.956), respectively. The regimen significantly improved engraftment and survival, indicating that the high graft failure of UCBT was caused by rejection.

Serum Metabolomics in Patients with Coexisting NAFLD and T2DM Using Liquid Chromatography-Mass Spectrometry.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM) frequently coexist and can act synergistically to drive adverse outcomes of one another. This study aimed to unravel the metabolomic changes in patients with NAFLD and T2DM, to identify potential noninvasive biomarkers, and to provide insights for understanding the link between NAFLD and T2DM. METHODS: Three hundred participants aged 35 to 70 years who were diagnosed with NAFLD (n = 100), T2DM (n = 100), or a comorbidity of NAFLD and T2DM (n = 100) were included in this study. Anthropometrics and routine blood chemistry were assessed after overnight fast. The global serum metabolomic analysis was performed by ultra-performance liquid chromatography-Orbitrap mass spectrometry. Multivariate data analysis methods were utilized to identify the potential biomarkers. RESULTS: A set of serum biomarkers that could effectively separate NAFLD from NAFLD + T2DM and T2DM from NAFLD + T2DM were identified. We found that patients with coexisting NAFLD and T2DM had significantly higher levels of total protein (TP), triglycerides (TG), glucose in urine, and gamma-hydroxybutyric acid than those with NAFLD and had significant increased levels of TP, albumin, alanine aminotransferase, aspartate aminotransferase, total cholesterol, cholinesterase, TG, low-density lipoprotein, and apolipoprotein A when compared to patients with T2DM. CONCLUSION: The metabolomics results provide evidence that the comorbidity of NAFLD and T2DM considerably altered patients' metabolomics patterns compared to those of patients with only NAFLD or T2DM.

Preclinical Characterization of the Selective JAK1 Inhibitor LW402 for Treatment of Rheumatoid Arthritis.

BACKGROUND: Research on JAK family members as therapeutic targets for autoimmune diseases has brought tofacitinib and baricitinib into clinical for the treatment of rheumatoid arthritis and other autoimmune diseases. Despite the potent efficacy of these first-generation JAK inhibitors, their broad-spectrum JAK inhibition and adverse events warrant development of a JAK1-specific inhibitor to improve their safety profile. METHODS: In this study, we characterized a JAK1-specific inhibitor, LW402, on biochemical and human whole-blood assays. We further evaluated the therapeutic efficacy of LW402 in a rat adjuvant-induced arthritis (rAIA) model and a mouse collagen-induced arthritis (mCIA) model. The safety of LW402 was evaluated in both SpragueDawley rats and cynomolgus monkeys. RESULTS: LW402 exhibited potent nanomolar activity against JAK1 and showed a 45-fold selectivity for inhibition of JAK1- over JAK2-dependent signaling induced by either IL6 or GM-CSF in human whole-blood assays. In the rAIA model, oral dosing of LW402 resulted in a dose-dependent improvement in disease symptoms, including reduction in paw swelling, marked reduction in the inflammatory-cell infiltration to synovial tissue, and protection of articular cartilage and bone from damage. The therapeutic efficacy of LW402 correlated well with the plasma exposure of LW402 and the extent of pSTAT3 inhibition in white blood cells. LW402 also effectively eased disease symptoms in the mCIA model. Toxicity studies in the Sprague Dawley rats and cynomolgus monkeys established a ≥5x therapeutic window for LW402 as drug exposures of toxicity study NOAEL dose and pharmacology study ED50 dose were compared. CONCLUSION: We developed a novel JAK1-specific inhibitor LW402 with potent efficacy in rAIA and mCIA models. We established a good safety profile for LW402 in toxicity studies, and the overall superiority of LW402 should translated well to the clinical setting for the treatment of RA and other autoimmune diseases.

Sweritranslactone D, a hepatoprotective novel secoiridoid dimer with tetracyclic lactone skeleton from heat-transformed swertiamarin.

Swertia mileensis, known as Qing-Ye-Dan (QYD), has been documented in Chinese Pharmacopoeia to cure hepatitis. Interestingly, its announced main active component, swertiamarin, could not be detected in the decoction, which indicated that the efficacy of QYD might be attributed to heat-transformed products of swertiamarin (HTPS). Further investigation on HTPS led to the isolation of sweritranslactone D (1), a novel secoiridoid dimer possessing a tetracyclic lactone skeleton, with better hepatoprotective activity than N-acetyl-L-cysteine in vitro.

Endothelial cell prostaglandin E2 receptor EP4 is essential for blood pressure homeostasis.

Prostaglandin E2 and its cognate EP1-4 receptors play important roles in blood pressure (BP) regulation. Herein, we show that endothelial cell-specific (EC-specific) EP4 gene-knockout mice (EC-EP4-/-) exhibited elevated, while EC-specific EP4-overexpression mice (EC-hEP4OE) displayed reduced, BP levels compared with the control mice under both basal and high-salt diet-fed conditions. The altered BP was completely abolished by treatment with l-NG-nitro-l-arginine methyl ester (l-NAME), a competitive inhibitor of endothelial nitric oxide synthase (eNOS). The mesenteric arteries of the EC-EP4-/- mice showed increased vasoconstrictive response to angiotensin II and reduced vasorelaxant response to acetylcholine, both of which were eliminated by l-NAME. Furthermore, EP4 activation significantly reduced BP levels in hypertensive rats. Mechanistically, EP4 deletion markedly decreased NO contents in blood vessels via reducing eNOS phosphorylation at Ser1177. EP4 enhanced NO production mainly through the AMPK pathway in cultured ECs. Collectively, our findings demonstrate that endothelial EP4 is essential for BP homeostasis.

6-MOMIPP, a novel brain-penetrant anti-mitotic indolyl-chalcone, inhibits glioblastoma growth and viability.

PURPOSE: 3-(6-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (6-MOMIPP) is a novel indole-based chalcone that disrupts microtubules. The present study aims to define the mechanism through which 6-MOMIPP induces cell death and to evaluate the efficacy of the compound in penetrating the blood-brain barrier and inhibiting growth of glioblastoma xenografts. METHODS: The effects of 6-MOMIPP were evaluated in cultured U251 glioblastoma cells, using viability, flow cytometry, and tubulin polymerization assays. Scintillation proximity and tubulin crosslinking methods were used to identify the binding site of 6-MOMIPP on tubulin, and western blots were performed to define the signaling pathways that contribute to cell death. LC/MS assays were used to study the pharmacokinetic behavior of 6-MOMIPP in mice. Subcutaneous and intracerebral xenograft models were utilized to assess the effects of 6-MOMIPP on growth of U251 glioblastoma in vivo. RESULTS: The findings indicate that 6-MOMIPP targets the colchicine site on ß-tubulin. At concentrations ≥ 250 nm, 6-MOMIPP induces mitotic arrest, caspase activation and loss of cell viability. Cells are protected by caspase inhibitors, pointing to an apoptotic mechanism of cell death. Loss of cell viability is preceded by activation of Cdk1(Cdc2) and phosphorylation of Bcl-2 and Bcl-xL. Inhibition of both events with a Cdk1 inhibitor prevents cell death. 6-MOMIPP has broad activity against the viability of multiple glioblastoma, melanoma and lung carcinoma cell lines. Viability of normal cells, including differentiated neurons, is not significantly affected at a drug concentration (1 µM) that reduces viability in most cancer lines. Pharmacokinetic studies in mice show that concentrations of 6-MOMIPP in the brain mirror those in the plasma, indicating that 6-MOMIPP readily penetrates the blood-brain barrier. Studies with mice bearing human U251 glioblastoma xenografts demonstrate that 6-MOMIPP is effective in suppressing growth of subcutaneous and intracerebral tumors without causing general toxicity. CONCLUSIONS: The results indicate that 6-MOMIPP is a novel microtubule disruptor that targets the colchicine binding site on ß-tubulin to induce mitotic arrest and cell death. The ability of 6-MOMIPP to penetrate the blood-brain barrier and inhibit growth of glioblastoma xenografts suggests that it warrants further preclinical evaluation as potential small-molecule therapeutic that may have advantages in treating primary and metastatic brain tumors.