Non-steroidal mineralocorticoid antagonists and hyperkalemia monitoring in chronic kidney disease patients associated with type II diabetes: a narrative review.

Chronic kidney disease (CKD) is a prevalent complication of Type II diabetes (T2D). The coexistence of CKD with T2D is comparable to cardiovascular disease (CVD) when the estimated glomerular filtration rate declines below 60 ml/min/1.73 m2. Screening and early detection of people with high risk for CKD would be beneficial in managing CKD progress and the associated complications such as CV complications. Renin-angiotensin-aldosterone system inhibitors (RAASi) have demonstrated beneficial effects in delaying CKD progression, but they carry the risk of hyperkalemia. Nonsteroidal mineralocorticoid antagonists (nsMRA), such as finerenone, exhibit considerable efficacy in their anti-inflammatory, antifibrotic, and renal protective effects with demonstrable reductions in CV complications. In addition, nsMRAs do not cause significant changes in serum potassium levels compared to traditional steroidal MRA. Ongoing research explores the capacity of the sodium-glucose transport protein 2 inhibitors (SGLT-2i), combined with nsMRA, to produce synergistic renal protective effects and reduce the risk of hyperkalemia. Also, a dedicated renal outcomes study (FLOW study) involving a once-weekly injectable Glucagon-like peptide-1 receptor agonist, semaglutide, was halted early by the data monitoring committee due to having achieved the predefined efficacy endpoint and considerations related to renal disease. In CKD patients with T2D on nsMRA, hyperkalemia management requires a comprehensive approach involving lifestyle adjustments, dietary modifications, regular serum potassium level monitoring, and potassium binders, if necessary. Withholding or down-titration of nsMRAs with close monitoring of serum potassium levels may be required in patients with concerning potassium levels. In light of the current state of knowledge, this review article explores the perspectives and approaches that HCPs may consider when monitoring and managing hyperkalemia in CKD patients with T2D.

Chronic Kidney Disease (CKD) is a common and serious problem among people with Type II Diabetes (T2D). People who have CKD with T2D are at a higher risk for heart disease after normal kidney function declines below certain levels. Renin-angiotensin-aldosterone system inhibitors are a group of medications that can help delay CKD progression but may cause a rise in circulating potassium levels. Nonsteroidal mineralocorticoid antagonist (nsMRA), such as finerenone, can reduce kidney inflammation and damage, with noted cardiovascular benefits, and with less effect on serum potassium levels as compared to their steroid-based counterparts. Researchers are studying whether combining blood sugar medications such as sodium-glucose transport protein-2 inhibitors (SGLT-2i) and finerenone can help protect the kidneys and heart. They also want to see if this combination can prevent high potassium levels. This article talks about ways to check and monitor potassium levels in CKD patients with T2D who may be taking nsMRA. To manage high potassium levels in people with CKD and T2D, doctors may suggest lifestyle changes, dietary adjustments, potassium-lowering medication, or adjustment of other medications with close monitoring of potassium levels.

Persistent effects of the orexin-1 receptor antagonist SB-334867 on naloxone precipitated morphine withdrawal symptoms and nociceptive behaviors in morphine dependent rats.

AIM OF THE STUDY: In this study, we investigated the effect of long-term administration of orexin receptor 1 (OXR1) antagonist on naloxone-precipitated morphine withdrawal symptoms and nociceptive behaviors in morphine-dependent rats. MATERIALS AND METHODS: Wistar rats received subcutaneous (s.c.) injections of morphine (6, 16, 26, 36, 46, 56, and 66 mg/kg, 2 ml/kg) at an interval of 24 h for 7 days. In chronic groups, the OXR1 antagonist, SB-334867 (20 mg/kg, i.p.), or its vehicle, was injected repetitively from postnatal day 1 (PND1)-PND23 and then for the following seven days before each morphine injection. Meanwhile, in acute groups, SB-334867, or its vehicle, was administered before each morphine injection. In groups of rats that were designated for withdrawal experiments, naloxone (2.5 mg/kg, i.p.) was administered after the last injection of morphine. In the formalin-induced pain, the effect of OXR1 inhibition on the antinociceptive effects of morphine was measured by injecting formalin after the final morphine injection. RESULTS: Animals that received long-term SB-334867 administration before morphine injection demonstrated a significant reduction in chewing, defecation, diarrhea, grooming, teeth chattering, wet-dog shake, and writhing. Inhibiting OXR1 for a long time increased formalin-induced nociceptive behaviors in interphase and phase II of the formalin-induced pain. CONCLUSIONS: Our results indicated that the inhibition of OXR1 significantly reduces the development of morphine dependence and behavioral signs elicited by the administration of naloxone in morphine-dependent rats. Furthermore, the prolonged blockade of OXR1 might be involved in formalin-induced nociceptive behaviors.

Orexin receptors in the CA1 region of hippocampus modulate the stress-induced antinociceptive responses in an animal model of persistent inflammatory pain.

Stress activates multiple neural pathways and neurotransmitters that often suppress pain perception, the phenomenon called stress-induced analgesia (SIA). Orexin neurons from the lateral hypothalamus project to entire brain structures such as the hippocampus. The present study examined this hypothesis that orexinergic receptors in the CA1 region of the hippocampus may play a modulatory role in the development of SIA in formalin test as an animal model of persistent inflammatory pain. One hundred-two adult male Wistar rats were administered with intra-CA1 orexin-1 receptor (OX1r) antagonist, SB334867, at the doses of 3, 10, 30, and 100 nmol or TCS OX2 29 as orexin-2 receptor (OX2r) antagonist at the doses of 1, 3, 10, and 30 nmol. Five min later, rats were exposed to forced swim stress (FSS) for a 6-min period. Then, pain-related behaviors induced by formalin injection were measured at the 5-min blocks during a 60-min period of formalin test. The current study indicated that solely stress exposure elicits antinociception in the early and late phases of the formalin test. The FSS-induced analgesia was prevented by intra-CA1 administration of SB334867 or TCS OX2 29 during either phase of the formalin test. Moreover, the contribution of the OX2r in the mediation of analgesic effect of stress was more prominent than that of the OX1r during both phases of the formalin test. It is suggested that OX1r and OX2r in the CA1 region of the hippocampus are involved in stress-induced analgesia in the animal model of persistent inflammatory pain.

Casein Kinase II exacerbates rheumatoid arthritis via promoting Th1 and Th17 cell inflammatory responses.

OBJECTIVES: Studies have demonstrated that CK2 is engaged in CD4+ T cell proliferation and activation. We investigated the potential involvement of CK2 in the pathogenesis of rheumatoid arthritis (RA). METHODS: Peripheral blood and synovial fluid mononuclear cells (PBMC and SFMC) of RA patients, as well as splenocytes of collagen-induced arthritis (CIA) mice were treated with different doses of CK2 inhibitor CX4945 in vitro. Then, the Th1, Th2, Th17, and Treg cell responses were analyzed. In addition, CIA mice were administrated with CX4945 via oral gavage. Accordingly, the arthritis scores, bone destruction, tissue damage, and the CD4+ T cell subsets were assessed. RESULTS: The expression of CK2 was upregulated in CD4+ T cells under RA circumstance. In vitro CX4945 treatment significantly inhibited the Th1 and Th17 cell responses, while promoted the Th2 cell responses in RA patient PBMC, SFMC and CIA mouse splenocytes, dampening IFN-γ and IL-17A production. Moreover, administration of CX4945 ameliorated the severity of arthritis in CIA mice, along with decreased Th1 and Th17 cells. However, CX4945 seemed to have minimal effect on RA Treg cells. CONCLUSION: CK2 serves as an important regulator of the Th1 and Th17 cell axes in RA, thus contributing to the disease aggravation.

Establishment of a Rapid Detection System for ISG20-Dependent SARS-CoV-2 Subreplicon RNA Degradation Induced by Interferon-α.

Inhaled nebulized interferon (IFN)-α and IFN-ß have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 µM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.

[18F]F-ET-OTSSP167 Targets Maternal Embryo Leucine Zipper Kinase for PET Imaging of Triple-Negative Breast Cancer.

Maternal embryo leucine zipper kinase (MELK) is a serine/threonine kinase and is highly expressed in triple-negative breast cancer (TNBC). This study aimed to develop a 18F-radiolabeled tracer based on the structure of a small-molecule MELK inhibitor OTSSP167 and evaluate its application for PET imaging of MELK expression in TNBC. OTSSP167 was modified with ethylene glycol to adjust its pharmacokinetics and was then radiolabeled with 18F to obtain [18F]F-ET-OTSSP167 at a labeling yield of 7.14 ± 2.19% and a molar activity of 16.23 ± 1.13 MBq/nmol. In vitro binding assays showed differentiated binding affinities of [18F]F-ET-OTSSP167 in different breast cancer cell lines, with high uptake in MDA-MB-231 (mild MELK expression) and low uptake in MCF-7 (negative MELK expression). PET imaging revealed that MDA-MB-231 tumors could be clearly delineated in vivo, while low tracer uptake was observed in MCF-7 tumors. These findings were confirmed by ex vivo biodistribution studies and were consistent with the immunohistochemistry and tissue staining results. Tracer accumulation in MDA-MB-231 tumors was significantly inhibited by excess amounts of OTSSP167, indicating high specificity of the tracer. In summary, [18F]F-ET-OTSSP167, an easily-prepared probe, can be used to visualize MELK positive tumors, demonstrating its promising clinical potential in selecting patients for MELK inhibitor therapy.

Effects of Finerenone Combined with Empagliflozin in a Model of Hypertension-Induced End-Organ Damage.

INTRODUCTION: The nonsteroidal mineralocorticoid receptor (MR) antagonist finerenone and sodium-glucose cotransporter-2 (SGLT2) inhibitors have demonstrated clinical benefits in CKD patients with type 2 diabetes. Clinical data analyzing the potential value of a combination therapy are currently limited. We therefore investigated cardiorenal protection of respective mono- and combination therapy in a preclinical model of hypertension-induced end-organ damage. METHODS: Cardiovascular (CV) morbidity and mortality were studied in hypertensive, N(ω)-nitro-L-arginine methyl ester-treated, renin-transgenic (mRen2)27 rats. Rats (10- to 11-week-old females, n = 13-17/group) were treated once daily orally for up to 7 weeks with placebo, finerenone (1 and 3 mg/kg), empagliflozin (3 and 10 mg/kg), or a combination of the respective low doses. Key outcome parameters included mortality, proteinuria, plasma creatinine and uric acid, blood pressure, and cardiac and renal histology. RESULTS: Placebo-treated rats demonstrated a 50% survival rate over the course of 7 weeks. Drug treatment resulted in variable degrees of survival benefit, most prominently in the low-dose combination group with a survival benefit of 93%. Monotherapies of finerenone or empagliflozin dose-dependently reduced proteinuria, while low-dose combination revealed an early, sustained, and over-additive reduction in proteinuria. Empagliflozin induced a strong and dose-dependent increase in urinary glucose excretion which was not influenced by finerenone coadministration in the combination arm. Low-dose combination but not respective low-dose monotherapies significantly reduced plasma creatinine and plasma uric acid after 6 weeks. Treatment with finerenone and the low-dose combination significantly decreased systolic blood pressure after 5 weeks. There was a dose-dependent protection from cardiac and kidney fibrosis and vasculopathy with both agents, while low-dose combination therapy was more efficient than the respective monotherapy dosages on most cardiorenal histology parameters. DISCUSSION/CONCLUSIONS: Nonsteroidal MR antagonism by finerenone and SGLT2 inhibition by empagliflozin confer CV protection in preclinical hypertension-induced cardiorenal disease. Combination of these 2 independent modes of action at low dosages revealed efficacious reduction in important functional parameters such as proteinuria and blood pressure, plasma markers including creatinine and uric acid, cardiac and renal lesions as determined by histopathology, and mortality indicating a strong potential for combined clinical use in cardiorenal patient populations.

Central blockade of orexin type 1 receptors reduces naloxone induced activation of locus coeruleus neurons in morphine dependent rats.

Orexin neuropeptides are implicated in the expression of morphine dependence. Locus coeruleus (LC) nucleus is an important brain area involving in the development of withdrawal signs of morphine and contains high expression of orexin type 1 receptors (OX1Rs). Despite extensive considerations, effects of immediate inhibition of OX1Rs by a single dose administration of SB-334867 prior to the naloxone-induced activation of LC neurons remains unknown. Therefore, we examined the direct effects of OX1Rs acute blockade on the neuronal activity of the morphine-dependent rats which underwent naloxone administration. Adult male rats underwent subcutaneous administration of 10 mg/kg morphine (two times/day) for a ten-day period. On the last day of experiment, intra-cerebroventricular administration of 10 µg/µl antagonist of OX1Rs, SB-334867, was performed just before intra-peritoneal injection of 2 mg/kg naloxone. Thereafter, in vivo extracellular single unit recording was employed to evaluate the electrical activity of LC neuronal cells. The outcomes demonstrated that morphine tolerance developed following ten-day of injection. Then, naloxone administration causes hyperactivity of LC neuronal cells, whereas a single dose administration of SB-334867 prior to naloxone prevented the enhanced activity of neurons upon morphine withdrawal. Our findings indicate that increased response of LC neuronal cells to applied naloxone could be prevented by the acute inhibition of the OX1Rs just before the naloxone treatment.

Successful Partnerships: Exploring the Potential of Immunogenic Signals Triggered by TMZ, CX-4945, and Combined Treatment in GL261 Glioblastoma Cells.

BACKGROUND: The relevance of the cancer immune cycle in therapy response implies that successful treatment may trigger the exposure or the release of immunogenic signals. Previous results with the preclinical GL261 glioblastoma (GB) showed that combination treatment of temozolomide (TMZ) + CX-4945 (protein kinase CK2 inhibitor) outperformed single treatments, provided an immune-friendly schedule was followed. Our purpose was to study possible immunogenic signals released in vitro by GB cells. METHODS: GL261 GB cells were treated with TMZ and CX-4945 at different concentrations (25 µM-4 mM) and time frames (12-72 h). Cell viability was measured with Trypan Blue and propidium iodide. Calreticulin exposure was assessed with immunofluorescence, and ATP release was measured with bioluminescence. RESULTS: TMZ showed cytostatic rather than cytotoxic effects, while CX-4945 showed remarkable cytotoxic effects already at low concentrations. Calreticulin exposure after 24 h was detected with TMZ treatment, as well as TMZ/CX-4945 low concentration combined treatment. ATP release was significantly higher with CX-4945, especially at high concentrations, as well as with TMZ/CX-4945. CONCLUSIONS: combined treatment may produce the simultaneous release of two potent immunogenic signals, which can explain the outperformance over single treatments in vivo. A word of caution may be raised since in vitro conditions are not able to mimic pharmacokinetics observed in vivo fully.

Determination of finerenone - a novel, selective, nonsteroidal mineralocorticoid receptor antagonist - in human plasma by high-performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study in venous and capillary human plasma.

A straightforward and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay allowing the sensitive and selective quantitation of finerenone (BAY 94-8862) in lithium heparin human plasma is described. Finerenone is a novel, selective, nonsteroidal mineralocorticoid receptor antagonist that is in phase III clinical trials for the treatment of chronic kidney disease. Finerenone quantitation is performed after addition of its stable isotope-labelled internal standard (ISTD) by protein precipitation with acidified acetonitrile followed by HPLC-MS/MS separation and detection. The determination of finerenone concentrations was validated for a plasma volume of 0.100 mL and subsequently also for a lower plasma volume of 0.010 mL, collected e.g. in paediatric studies. The analytical range was from 0.100 µg/L (lower limit of quantification) to 200 µg/L (upper limit of quantification). Inter-day accuracy was 99.7-105.0% for the plasma volume of 0.100 mL and 101.1-104.5% for the plasma volume of 0.010 mL. Inter-day precision was ≤ 7.0%, independent of the extracted plasma volume. A moderate, concentration-independent matrix effect on ionisation was observed for both finerenone and its ISTD of 0.535-0.617, which is fully compensated by the ISTD (ISTD-normalised matrix factors were 0.98-1.03). The assay was successfully applied with both validated plasma volumes to a clinical phase I study in which the pharmacokinetics of 20 mg finerenone were compared in capillary plasma (0.010 mL) and venous plasma (0.100 mL) in a concentration range from the lower limit of quantification to 310 µg/L (capillary plasma) and 252 µg/L (venous plasma). The area under the plasma concentration versus time curve was similar in both matrices, while maximum concentrations were 37% higher in capillary plasma. In conclusion, capillary sampling should not bias pharmacokinetic exposure estimates compared with venous plasma values, if limited to sampling times in the distribution and elimination phases of finerenone.

A Case of Pediatric Subcutaneous Abscess Caused by Mycobacterium mageritense Infection.

Soft tissue infections with Mycobacterium mageritense are uncommon. We report the case of a 5-year-old girl who developed a subcutaneous abscess in her right ankle caused by M. mageritense. She had a history of acute encephalopathy and adrenal insufficiency and was hospitalized for acute pancreatitis. During hospitalization, the patient developed fever and tachycardia. Blood culture was positive for gram-positive bacilli. Although initial testing with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) reported a different organism, a repeat test identified M. mageritense. One month after the positive blood culture, she developed redness and swelling in the right ankle. The pus from the subcutaneous abscess after drainage grew M. mageritense, which was further confirmed by the sequencing of housekeeping genes. Based on sensitivity testing, the patient was treated with tosufloxacin and linezolid. The local inflammatory signs gradually improved on starting the treatment. The antibiotics were administered for 6 months, and she experienced no relapse during the 8 months of follow-up after the completion of therapy. This is the first case report of a pediatric M. mageritense infection, which also highlights an important potential pitfall of MALDI-TOF MS. Further, we observe that the choice of antimicrobials for the treatment of M. mageritense is more limited in children than in adults.

Blockade of orexin receptors in the ventral tegmental area reduced the extinction period of the lateral hypothalamic-induced conditioned place preference in rats.

The orexinergic connection between the lateral hypothalamus (LH) and the ventral tegmental area (VTA) is involved in modulating the reward circuit. The conditioned place preference (CPP) can be induced by microinjection of carbachol, a cholinergic agonist, into the LH. The current research was conducted to understand whether intra-VTA orexin receptors (OXRs) could influence the duration of the extinction period or maintenance of the intra-LH carbachol-induced CPP. To this end, the rats unilaterally received intra-LH carbachol (250 nM) within a 3-day conditioning period. Animals that have already passed the conditioning test were unilaterally administered by intra-VTA microinjection of SB334867, an OX1R antagonist, or TCS OX2 29, an OX2R antagonist during the extinction phase of the LH stimulation-induced CPP. For the first time, our data indicated that daily intra-VTA administration of either SB334867 (30 nM) or TCS OX2 29 (10 and 30 nM) during the extinction period decreased the maintenance of intra-LH carbachol-induced CPP. In conclusion, OXRs in the VTA play crucial roles in the maintenance of reward processes.

Comparing mTOR inhibitor Rapamycin with Torin-2 within the RIST molecular-targeted regimen in neuroblastoma cells.

The prognosis for patients with relapsed or refractory high-risk neuroblastoma remains dismal and novel therapeutic options are urgently needed. The RIST treatment protocol has a multimodal metronomic therapy design combining molecular-targeted drugs (Rapamycin and Dasatinib) with chemotherapy backbone (Irinotecan and Temozolomide), which is currently verified in a phase II clinical trial (NCT01467986). With the availability of novel and more potent ATP competitive mTOR inhibitors, we expect to improve the RIST combination therapy. By comparing the IC50 values of Torin-1, Torin-2, AZD3147 and PP242 we established that only Torin-2 inhibited cell viability of all three MycN-amplified neuroblastoma cell lines tested at nanomolar concentration. Single treatment of both mTOR inhibitors induced a significant G1 cell cycle arrest and combination treatment with Dasatinib reduced the expression of cell cycle regulator cyclin D1 or increased the expression of cell cycle inhibitor p21. The combinatorial index depicted for both mTOR inhibitors a synergistic effect with Dasatinib. Interestingly, compared to Rapamycin, the combination treatment with Torin-2 resulted in a broader mTOR pathway inhibition as indicated by reduced phosphorylation of AKT (Thr308, Ser473), 4E-BP (Ser65), and S6K (Thr389). Furthermore, substituting Rapamycin in the modified multimodal RIST protocol with Torin-2 reduced cell viability and induced apoptosis despite a significant lower Torin-2 drug concentration applied. The efficacy of nanomolar concentrations may significantly reduce unwanted immunosuppression associated with Rapamycin. However, at this point we cannot rule out that Torin-2 has increased toxicity due to its potency in more complex systems. Nonetheless, our results suggest that including Torin-2 as a substitute for Rapamycin in the RIST protocol may represent a valid option to be evaluated in prospective clinical trials for relapsed or treatment-refractory high-risk neuroblastoma.

Poly (ADP-Ribose) Polymerase Inhibitor Treatment as a Novel Therapy Attenuating Renal Ischemia-Reperfusion Injury.

Intrarenal robust inflammatory response following ischemia-reperfusion injury (IRI) is a major factor in the pathogenesis of renal injury in ischemic acute kidney injury (AKI). Although numerous studies have investigated various agents of immune modulation or suppression for ischemic AKI, few showed reproducible effects. We hypothesized that poly (ADP-ribose) polymerase (PARP) inhibitor may favorably change post-ischemic intrarenal immunologic micromilieu by reducing damage-associated molecular pattern (DAMP) signals and improve renal outcome in ischemic AKI. The effects of JPI-289 (a PARP inhibitor) on early renal injury in a murine IRI model and hypoxic HK-2 cell model were investigated. Bilateral IRI surgery was performed in three groups of 9-week-old male C57BL/6 mice (control, JPI-289 50 mg/kg, and JPI-289 100 mg/kg; n = 9-10 in each group). Saline or JPI-289 was intraperitoneally injected. Renal function deterioration was significantly attenuated in the JPI-289 treatment groups in a dose-dependent manner. Inflammatory cell infiltration and proinflammatory cytokine/chemokine expressions in the post-ischemic kidneys were also attenuated by JPI-289 treatment. JPI-289 treatment at 0.5 and 0.75 µg/ml facilitated the proliferation of hypoxic HK-2 cells. PARP inhibition with JPI-289 treatment showed favorable effects in ischemic AKI by attenuating intrarenal inflammatory cascade in a murine model and facilitating proliferation of hypoxic HK-2 cells.

Penetration, distribution, and elimination of remofuscin/soraprazan in Stargardt mouse eyes following a single intravitreal injection using pharmacokinetics and transmission electron microscopic autoradiography: Implication for the local treatment of Stargardt's disease and dry age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in older people in the developed world while Stargardt's disease (SD) is a juvenile macular degeneration and an orphan disease. Both diseases are untreatable and are marked by accumulation of lipofuscin advancing to progressive deterioration of the retinal pigment epithelium (RPE) and retina and subsequent vision loss till blindness. We discovered that a small molecule belonging to the tetrahydropyridoether class of compounds, soraprazan renamed remofuscin, is able to remove existing lipofuscin from the RPE. This study investigated the drug penetration, distribution, and elimination into the eyes of a mouse model for increased lipofuscinogenesis, following a single intravitreal injection. We measured the time course of concentrations of remofuscin in different eye tissues using high-performance liquid chromatography combined with mass spectroscopy (HPLC-MS). We also visualized the penetration and distribution of 3 H-remofuscin in eye sections up to 20 weeks post-injection using transmission electron microscopic (TEM) autoradiography. The distribution of silver grains revealed that remofuscin accumulated specifically in the RPE by binding to the RPE pigments (melanin, lipofuscin and melanolipofuscin) and that it was still detected after 20 weeks. Importantly, the melanosomes in choroidal melanocytes only rarely bind remofuscin emphasizing its potential to serve as an active ingredient in the RPE for the treatment of SD and dry AMD. In addition, our study highlights the importance of electron microscopic autoradiography as it is the only method able to show drug binding with a high intracellular resolution.

Synthesis and preliminary studies of 11C-labeled tetrahydro-1,7-naphthyridine-2-carboxamides for PET imaging of metabotropic glutamate receptor 2.

Selective modulation of metabotropic glutamate receptor 2 (mGlu2) represents a novel therapeutic approach for treating brain disorders, including schizophrenia, depression, Parkinson's disease (PD), Alzheimer's disease (AD), drug abuse and addiction. Imaging mGlu2 using positron emission tomography (PET) would allow for in vivo quantification under physiological and pathological conditions and facilitate drug discovery by enabling target engagement studies. In this paper, we aimed to develop a novel specific radioligand derived from negative allosteric modulators (NAMs) for PET imaging of mGlu2. Methods. A focused small molecule library of mGlu2 NAMs with tetrahydro naphthyridine scaffold was synthesized for pharmacology and physicochemical evaluation. GIRK dose-response assays and CNS panel binding selectivity assays were performed to study the affinity and selectivity of mGlu2 NAMs, among which compounds 14a and 14b were selected as PET ligand candidates. Autoradiography in SD rat brain sections was used to confirm the in vitro binding specificity and selectivity of [11C]14a and [11C]14b towards mGlu2. In vivo binding specificity was then studied by PET imaging. Whole body biodistribution study and radiometabolite analysis were conducted to demonstrate the pharmacokinetic properties of [11C]14b as most promising PET mGlu2 PET ligand. Results. mGlu2 NAMs 14a-14g were synthesized in 14%-20% yields in five steps. NAMs 14a and 14b were selected to be the most promising ligands due to their high affinity in GIRK dose-response assays. [11C]14a and [11C]14b displayed similar heterogeneous distribution by autoradiography, consistent with mGlu2 expression in the brain. While PET imaging study showed good brain permeability for both tracers, compound [11C]14b demonstrated superior binding specificity compared to [11C]14a. Further radiometabolite analysis of [11C]14b showed excellent stability in the brain. Conclusions. Compound 14b exhibited high affinity and excellent subtype selectivity, which was then evaluated by in vitro autoradiography and in vivo PET imaging study after labeling with carbon-11. Ligand [11C]14b, which we named [11C]MG2-1904, demonstrated high brain uptake and excellent in vitro/in vivo specific binding towards mGlu2 with high metabolic stability in the brain. As proof-of-concept, our preliminary work demonstrated a successful example of visualizing mGlu2in vivo derived from NAMs, which represents a promising chemotype for further development and optimization aimed for clinical translation.

Aaptamine attenuates the proliferation and progression of non-small cell lung carcinoma.

CONTEXT: Aaptamine is a potent ocean-derived non-traditional drug candidate against human cancers. However, the underlying molecular mechanisms governing aaptamine-mediated repression of lung cancer cells remain largely undefined. OBJECTIVE: To examine the inhibitory effect of aaptamine on proliferation and progression of non-small cell lung carcinoma (NSCLC) and dissect the potential mechanisms involved in its anticancer functions. MATERIALS AND METHODS: In vitro assays of cell proliferation, cell cycle analysis, clonal formation, apoptosis and migration were performed to examine the inhibitory effects of aaptamine (8, 16 and 32 µg/mL) on NSCLC cells. The expression levels of proteins were analysed using western blotting analysis when cells were treated with a single drug or a combination treatment for 48 h. RESULTS: Aaptamine significantly inhibited A549 and H1299 cells proliferation with IC50 values of 13.91 and 10.47 µg/mL. At the concentrations of 16 and 32 µg/mL, aaptamine significantly reduced capacities in clonogenicity, enhanced cellular apoptosis and decreased the motile and invasive cellular phenotype. In addition, aaptamine arrested cell cycle at G1 phase via selectively abating cell cycle regulation drivers (CDK2/4 and Cyclin D1/E). Western blotting results showed that aaptamine attenuated the protein expression of MMP-7, MMP-9 and upregulated the expression of cleaved-PARP and cleaved-caspase 3. Moreover, aaptamine inhibited PI3K/AKT/GSK3ß signalling cascades through specifically degrading the phosphorylated AKT and GSK3ß. DISCUSSION AND CONCLUSIONS: Aaptamine retarded the proliferation and invasion of NSCLC cells by selectively targeting the pathway PI3K/AKT/GSK3ß suggesting it as a potential chemotherapeutic agent for repressing tumorigenesis and progression of NSCLC in humans.

Translational pharmacology of an inhaled small molecule αvß6 integrin inhibitor for idiopathic pulmonary fibrosis.

The αvß6 integrin plays a key role in the activation of transforming growth factor-ß (TGFß), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvß6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvß6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFß signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvß6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvß6, induces prolonged inhibition of TGFß signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.

Activation orexin 1 receptors in the ventrolateral periaqueductal gray matter attenuate nitroglycerin-induced migraine attacks and calcitonin gene related peptide up-regulation in trigeminal nucleus caudalis of rats.

This study aims to explore whether orexin 1 receptors (Orx1R) in the ventrolateral periaqueductal gray matter (vlPAG) play a role in the modulation of migraine headaches in adult male Wistar rats. To model chronic migraine-associated pain, nitroglycerin (NTG) (5 mg/kg/IP) was administered to test subjects every second day for 9 days. After the last NTG injection, rats were randomly separated into the following groups (n = 6): orexin-A (OrxA) groups that received intra-vlPAG OrxA (25, 50, and 100 pM), an Orx1R antagonist group, a SB-334867 (20 µM) group; and a SB-334867 (20 µM) + OrxA (100 pM) group. After 10 min, migraine-associated behavioral symptoms were recorded in all animals for up to 90 min. Light-dark chamber and hot plate tests were used for assessing light aversion and thermal hyperalgesia, respectively. Calcitonin gene-related peptide (CGRP)-positive cells were detected in the trigeminal nucleus caudalis (Vc) by immunofluorescence microscopy. NTG caused significant freezing behavior, which was prevented by all OrxA doses. Moreover, OrxA (100 pM) could obstruct NTG-induced increases in facial rubbing and decreases in climbing and body grooming. Furthermore, NTG-induced light aversion and thermal hyperalgesia were attenuated by OrxA at doses of 50 and 100 pM. The effects of OrxA were significantly blocked by SB-334867 (20 µM). Besides, OrxA (100 pM) decreased NTG-induced CGRP upregulation. The data revealed that the activation of Orx1Rs in the vlPAG is effective in relieving NTG-induced migraine symptoms mainly by the downregulation of CGRP in the Vc of rats.

First-in-Human Evaluation of the Safety, Tolerability, and Pharmacokinetics of a Neuroprotective Poly (ADP-ribose) Polymerase-1 Inhibitor, JPI-289, in Healthy Volunteers.

BACKGROUND: Poly (ADP-ribose) polymerase-1 (PARP-1) inhibitor has therapeutic potential for acute ischemic stroke by suppressing microglial activation and facilitating neuroprotection. In this first-in-human study, we investigate the safety, tolerability and pharmacokinetics (PK) of JPI-289 in healthy male volunteers. SUBJECTS AND METHODS: In single ascending dose (SAD) study, 35, 75, 150, 300, 600 mg JPI-289 or placebo was infused intravenously over 30 minutes to 40 subjects. In multiple ascending dose (MAD) study, 150, 300, 450 mg JPI-289 or placebo was infused over 1 hour every 12 hours to each of 24 subjects for 3.5 days (7 times). The plasma and urine concentrations of JPI-289 and its metabolites were determined. RESULTS: In the SAD study, AUClast and Cmax tended to increase supra-proportionally especially at higher doses in SAD study. However, Cmax showed dose-proportionality in the range of 75-600mg. JPI-289 reached a mean Tmax within 0.50 hour after dosing and a mean elimination half-life (t1/2) was 2.18 to 3.21 hours. In the MAD study, observed accumulation index ranged from 1.52 to 1.76. The effective half-life of JPI-289 was 1.88 to 3.05 hours, indicating that the plasma JPI-289 concentration rapidly reaches steady state. % recovered of JPI-289 measured in urine was 1.59-9.05%. In both studies, concentration of metabolites was less than 10% of JPI-289. Adverse events reported in the study were all mild in intensity and resolved without any sequelae. CONCLUSION: The tolerable dose ranges and pharmacokinetic characteristics of JPI-289 evaluated in these studies will be useful in further clinical development of JPI-289.