Long-Term Study on Therapeutic Strategy for Treatment of Eisenmenger Syndrome Patients: A Case Series Study.

Eisenmenger syndrome (ES) refers to congenital heart diseases (CHD) with reversal flow associated with increased pulmonary pressure and irreversible pulmonary vascular remodeling. Previous reports showed limited therapeutic strategies in ES. In this study, 5 ES patients (2 males and 3 females), who had been followed regularly at our institution from 2010 to 2019, were retrospectively reviewed. We adopted an add-on combination of sildenafil, bosentan, and iloprost and collected the clinical characteristics and outcomes as well as findings of echocardiography, computed tomography, pulmonary perfusion-ventilation scans, positron emission tomography, and biomarkers. The age of diagnosis in these ES patients ranged from 23 to 54 years (38.2 ± 11.1 years; mean ± standard deviation), and they were followed for 7 to 17 years. Their mean pulmonary arterial pressure and pulmonary vascular resistance index were 56.4 ± 11.3 mmHg and 24.7 ± 8.5 WU.m2, respectively. Intrapulmonary arterial thrombosis was found in 4 patients, ischemic stroke was noted in 2 patients, and increased glucose uptake of the right ventricle was observed in 4 patients. No patient mortality was seen within 5 years of follow-up. Subsequently, 2 patients died of right ventricular failure, 1 died of sepsis related to brain abscess, and another died of sudden death. The life span of these patients was 44-62 years. Although these patients showed longer survival, the beneficial data on specific-target pharmacologic interventions in ES is still preliminary. Thus, larger trials are warranted, and the study of cardiac remodeling in ES from various CHD should be explored.

ENERGI-F703 gel, as a new topical treatment for diabetic foot and leg ulcers: A multicenter, randomized, double-blind, phase II trial.

Background: Diabetic foot and leg ulcers are a major cause of disability among patients with diabetes mellitus. A topical gel called ENERGI-F703, applied twice daily and with adenine as its active pharmaceutical ingredient, accelerated wound healing in diabetic mice. The current study evaluated the safety and efficacy of ENERGI-F703 for patients with diabetic foot and leg ulcers. Methods: This randomized, double-blind, multicenter, phase II trial recruited patients from eight medical centers in Taiwan. Patients with intractable diabetic foot and leg ulcers (Wagner Grade 1-3 without active osteomyelitis) were randomly assigned (2:1) to receive topical ENERGI-F703 gel or vehicle gel twice daily for 12 weeks or until complete ulcer closure. The investigator, enrolled patients and site personnel were masked to treatment allocation. Intention to treat (ITT) population and safety population were patient to primary analyses and safety analyses, respectively. Primary outcome was complete ulcer closure rate at the end of treatment. This trial is registered with ClinicalTrials.gov, number NCT02672436. Findings: Starting from March 15th, 2017 to December 26th, 2019, 141 patients were enrolled as safety population and randomized into ENERGI-F703 gel (n = 95) group or vehicle gel (n = 46) group. In ITT population, ENERGI-F703 (n = 90) and vehicle group showed ulcer closure rates of 36.7% (95% CI = 26.75% - 47.49%) and 26.2% (95% CI = 13.86% - 42.04%) with difference of 9.74 % (95 % CI = -6.74% - 26.23%) and 25% quartiles of the time to complete ulcer closure of 69 days and 84 days, respectively. There were 25 (26.3%) patients in ENERGI-F703 group and 11 (23.9%) patients in vehicle group experiencing serious adverse events and five deaths occurred during the study period, none of them related to the treatment. Interpretation: Our study suggests that ENERGI-F703 gel is a safe and well-tolerated treatment for chronic diabetic foot and leg ulcers. Further studies are needed to corroborate our findings in light of limitations. Funding: Energenesis Biomedical Co., Ltd.

Modulation of adenine phosphoribosyltransferase-mediated salvage pathway to accelerate diabetic wound healing.

Adenine phosphoribosyltransferase (APRT) is the key enzyme involved in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotides. To evaluate the role of APRT in the repair processes of cutaneous wounds in healthy skin and in diabetic patients, a diabetic mouse model (db/db) and age-matched wild-type mice were used. Moreover, the topical application of adenine was assessed. In vitro studies, analytical, histological, and immunohistochemical methods were used. Diabetic mice treated with adenine exhibited elevated ATP levels in organismic skin and accelerated wound healing. In vitro studies showed that APRT utilized adenine to rescue cellular ATP levels and proliferation from hydrogen peroxide-induced oxidative damage. HPLC-ESI-MS/MS-based analysis of total adenylate nucleotides in NIH-3T3 fibroblasts demonstrated that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided additional AMP for the further generation of ATP. These data indicate an upregulation of APRT in skin wounds, highlighting its role during the healing of diabetic wounds through regulation of the nucleotide pool after injury. Furthermore, topical adenine supplementation resulted in an enlargement of the adenylate pool needed for the generation of ATP, an important molecule for wound repair.

Post-infectious Bronchiolitis Obliterans: HRCT, DECT, Pulmonary Scintigraphy Images, and Clinical Follow-up in Eight Children.

Background: Bronchiolitis obliterans (BO), first mentioned in 1901, is a severe and rare chronic lung disease in children. BO has various etiologies and the most common in children is post-infectious BO (PIBO). High resolution CT (HRCT) is an often-used image tool for the diagnosis of BO, and pulmonary scintigraphy is an alternative tool that can functionally evaluate BO. Recently, dual-energy computed tomography (DECT) have also been applied to BO for its accuracy and safety. Here we described the characteristics of HRCT, pulmonary scintigraphy, DECT, and the clinical profiles of patients with PIBO. Methods: This is a retrospective and descriptive study. Data were collected from patients diagnosed with PIBO from 2014 to 2019 in the Pediatric Cardiopulmonary Outpatient Clinics of Kaohsiung Medical University Hospital. The diagnosis was based on clinical, chest X-ray, and HRCT findings. Clinical profile, radiological characteristics, and images of pulmonary scintigraphy were documented. Results: Eight children (4 boys and 4 girls) were diagnosed with PIBO at a mean age of 25.8 months (range 15 to 41 months). Two of our patients developed pulmonary hypertension. The most common HRCT finding is mosaic pattern, where match ventilation/perfusion (V/Q) defects is a general feature in pulmonary scintigraphy. DECT pulmonary blood vasculature images revealed various degrees of decreased perfusion and is compatible with the decreased perfusion on pulmonary scintigraphy. Conclusion: The therapeutic strategy of PIBO is still lacking of standardization. HRCT and V/Q scans are important image tools in diagnosis and follow-up of BO. DECT may be used in BO patients as it has no additional radiation exposure and add value on functional information of HRCT.

Adenine causes cell cycle arrest and autophagy of chronic myelogenous leukemia K562 cells via AMP-activated protein kinase signaling.

AMP-activated protein kinase (AMPK) is known as a pivotal regulator of cellular metabolism. Mounting evidences have demonstrated that AMPK activation exerts tumor suppressive activity on leukemia cells. The present study reported that adenine, an AMPK activator, triggers cell cycle arrest and autophagy of human chronic myelogenous leukemia K562 cells consequently suppressing cell viability. The present findings revealed that adenine treatment (4.0-8.0 mM) significantly inhibited the viability of K562 cells to 69.3±2.5% (24 h) and 53.4±2.1% (48 h) of the control. Flow cytometric analysis revealed that there was a significant accumulation in G2/M phase, but not sub-G1 phase K562 cells following exposure to adenine. Additional investigation demonstrated that adenine treatments significantly increased the number of acidic vesicular organelles and the level of autophagosomal microtubule associated protein 1 light chain 3 α (LC3) marker. By contrast, cleavage of caspase-9, caspase-3 and poly-ADP-ribose polymerase was insignificantly affected in K562 cells following adenine treatment. In K562 cells, adenine was able to markedly promote the phosphorylation of AMPKα and suppress the phosphorylation of mammalian target of rapamycin (mTOR), a downstream target of AMPK. In addition, inhibiting AMPK phosphorylation using dorsomorphin restored mTOR phosphorylation, inhibited the accumulation of LC3 and significantly recovered the suppressed cell viability in response to adenine. Taken together, the present results demonstrated that adenine induced G2/M phase arrest and autophagic cell death, consequently suppressing the viability of K562 cells, which may attribute to the AMPK activation triggered by adenine. These findings provide evidence that adenine may be beneficial to chronic myelogenous leukemia therapy by suppressing excessive cell proliferation.

Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

The AMP-activated protein kinase (AMPK) signaling system plays a key role in cellular stress by repressing the inflammatory responses induced by the nuclear factor-kappa B (NF-κB) system. Previous studies suggest that the anti-inflammatory role of AMPK involves activation by adenine, but the mechanism that allows adenine to produce these effects has not yet been elucidated. In human umbilical vein endothelial cells (HUVECs), adenine was observed to induce the phosphorylation of AMPK in both a time- and dose-dependent manner as well as its downstream target acetyl Co-A carboxylase (ACC). Adenine also attenuated NF-κB targeting of gene expression in a dose-dependent manner and decreased monocyte adhesion to HUVECs following tumor necrosis factor (TNF-α) treatment. The short hairpin RNA (shRNA) against AMPK α1 in HUVECs attenuated the adenine-induced inhibition of NF-κB activation in response to TNF-α, thereby suggesting that the anti-inflammatory role of adenine is mediated by AMPK. Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC. Similarly, the expression of a shRNA against APRT nullified the anti-inflammatory effects of adenine in HUVECs. These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK.

Purine analogue ENERGI-F706 induces apoptosis of 786-O renal carcinoma cells via 5'-adenosine monophosphate-activated protein kinase activation.

Purine compounds are known to activate 5'-adenosine monophosphate-activated protein kinase (AMPK), which has important roles in treatments for renal cell carcinoma. The present study was aimed to investigate the effects of the purine analogue ENERGI­F706 on the human renal carcinoma cell line 786­O and the underlying mechanisms. The results revealed that ENERGI­F706 (0.2­0.6 mg/ml) significantly decreased the cell viability to up to 36.4±2.4% of that of the control. Compared to 786­O cells, ENERGI­F706 exerted less suppressive effects on the viability of the human non­tumorigenic renal cell line HK­2. Flow cytometric analysis showed that ENERGI­F706 contributed to cell cycle arrest at S­phase and triggered apoptosis of 786­O cells. Immunoblot analysis revealed that anti­apoptotic B­cell lymphoma 2 (Bcl­2) levels were reduced and pro­apoptotic Bcl­2­associated X protein levels were diminished. In addition, activation of caspase­9, caspase­3 and poly(adenosine diphosphate ribose) polymerase (PARP) was promoted in 786­O cells in response to ENERGI­F706. Effects of ENERGI­F706 on AMPK cascades were investigated and the results showed that ENERGI­F706 enhanced phosphorylation of AMPKα (T172) and p53 (S15), a downstream target of AMPK. In addition, the AMPK activation, p53 (S15) phosphorylation, reduction of Bcl­2, cleavage of caspase­3 and PARP as well as suppressed cell viability induced by ENERGI­F706 were reversed in the presence of AMPK inhibitor compound C (dorsomorphin). In conclusion, the findings of the present study revealed that ENERGI­F706 significantly suppressed the viability of 786­O cells via induction of cell cycle arrest and apoptosis, attributing to AMPK and p53 activation and subsequent cell cycle regulatory and apoptotic signaling. It was therefore indicated that ENERGI­F706 may be suitable for the treatment of renal cell carcinoma.

Identification of adenine modulating AMPK activation in NIH/3T3 cells by proteomic approach.

AMP-activated protein kinase (AMPK) is a metabolic master switch maintaining the energy homeostasis in cells and thought to modulate cellular response to stresses. Adenine as well as a pharmacological AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), induced the phosphorylation of AMPK and acetyl-CoA carboxylase in NIH/3T3 cells. Administration of adenine or AICAR increased the expression and translocation of glucose transporter 4, enhanced the cellular glucose uptake, and elevated the intracellular ATP level. To better understand the proteomic changes in response to exogenous adenine treatment, we performed two-dimensional difference gel electrophoresis (2DE-DIGE) and grouped protein spots with similar intensities prior to MS analysis. These process allowed us to exclude these constant expressed proteins, reduce the coverage from abundant signals and increase the identification of middle/lower expressed proteins. Bioinformatics analysis on the proteomic alterations suggested that both of adenine and AICAR could induce up-regulation of a panel of proteins associated with glucose metabolism. We also found that adenine upregulated expression of the glycolytic enzyme, hexokinase 2, indicating a link between adenine and AMPK-mediated glycolysis. Taken together, by demonstrating the adenine-mediated proteome changes in NIH/3T3 cells, our study provides useful information for the characteristics of adenine-induced AMPK activation and development of efficient AMPK activator. BIOLOGICAL SIGNIFICANCE: AMPK is a fuel sensing enzyme that responds to a central role of energy homeostasis and contributes to the acceleration of insulin signaling. Recently, we have shown that exogenous adenine exerted anti-inflammatory effects through activation of AMPK, suggesting the treatment is a potent therapeutic strategy against hyperglycemia. Adenine had similar effects with 5-amino-4-imidazole-carboxamide riboside (AICAR, an AMPK activator) in modulating glucose uptake via AMPK-mediated signaling. In this study, we performed a 2DE-DIGE/MS-based approach to investigate the mechanism of exogenous adenine in NIH/3T3 cells. Our results provide evidence of a novel role for adenine in AMPK-mediated signaling and glucose metabolism and suggest potential therapeutic perspectives in insulin resistance and metabolic dysfunctions.

AMPK activation inhibits expression of proinflammatory mediators through downregulation of PI3K/p38 MAPK and NF-κB signaling in murine macrophages.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a central role in energy homeostasis and regulation of inflammatory responses. The present study is aimed to investigate the anti-inflammatory effects of ENERGI-F704, a nucleobase analogue isolated from bamboo leaves, on expression of proinflammatory mediators in murine macrophage RAW264.7 in response to lipopolysaccharide (LPS). ENERGI-F704 enhanced phosphorylation of AMPK(T172) but insignificantly affected the viability of RAW264.7 cells. Further investigation showed that ENERGI-F704 decreased mRNA expression of interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS) induced by LPS, as well as suppressed the production of prostaglandin E2 (PGE2) and nitric oxide (NO). Additionally, the inhibitory effects of ENERGI-F704 on the LPS-induced proinflammatory mediators were diminished by pretreatment of AMPK inhibitor Compound C. ENERGI-F704 also inhibited LPS-triggered activation of nuclear factor kappa B (NF-κB), phosphatidylinositol 3-kinase (PI3K), and p38 mitogen-activated protein kinase (p38), whereas extracellular signal-regulated kinase (Erk)1/2 and c-Jun N-terminal kinase (JNK) were insignificantly influenced. Our findings indicate that ENERGI-F704 may exert anti-inflammatory activity on RAW264.7 cells in response to LPS through the activation of AMPK and suppression of PI3K/P38/NF-κB signaling and the consequent decreased expression of proinflammatory mediators, suggesting that ENERGI-F704 is beneficial to the amelioration of inflammatory disorders.

Amelioration of LPS-induced inflammation response in microglia by AMPK activation.

Adenosine 5'-monophosphate-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis via modulating metabolism of glucose, lipid, and protein. In addition to energy modulation, AMPK has been demonstrated to associate with several important cellular events including inflammation. The results showed that ENERGI-F704 identified from bamboo shoot extract was nontoxic in concentrations up to 80 µM and dose-dependently induced phosphorylation of AMPK (Thr-172) in microglia BV2 cells. Our findings also showed that the treatment of BV2 with ENERGI-F704 ameliorated the LPS-induced elevation of IL-6 and TNF-α production. In addition, ENERGI-F704 reduced increased production of nitric oxide (NO) and prostaglandin E2 (PGE2) via downregulating the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), respectively. Moreover, ENERGI-F704 decreased activated nuclear translocation and protein level of NF-κB. Inhibition of AMPK with compound C restored decreased NF-κB translocation by ENERGI-F704. In conclusion, ENERGI-F704 exerts inhibitory activity on LPS-induced inflammation through manipulating AMPK signaling and exhibits a potential therapeutic agent for neuroinflammatory disease.

Prediction of H3 receptor occupancy diurnal fluctuations using population modeling and simulation with focus on guiding dose selection in a Phase IIa study.

PURPOSE: 4-[(1S,2S)-2-(4-cyclobutylpiperazine-1-carbonyl)cyclopropyl]benzamide ("AZ1") is a histamine 3 (H3) autoreceptor in vivo antagonist. Sleep disturbance is a well-known class-effect for H3 antagonists and associated with high H3 receptor occupancy (RO) at night. The objective of the present work was to investigate if it was possible to obtain large diurnal fluctuations in RO for AZ1 and to suggest suitable doses for a Phase IIa study. METHODS: Four Phase I studies were pooled and used to build a population pharmacokinetic model in NONMEM. Based on simulations of the PK model and the reported Ki-value for H3 RO from a human PET-study, RO vs. time profiles were simulated. RESULTS: The model well described the AZ1 pharmacokinetics. Simulations predicting plasma concentration and RO vs. time profiles for several doses were explored and doses with a wide range of fluctuation in RO over the dosing interval could be identified. CONCLUSIONS: By using population modeling and simulations of PK data and the Ki-value from a human PET study, predictions of RO vs. time for unstudied doses of AZ1 was made. Using this methodology it was possible to suggest doses with expected large diurnal fluctuations in RO.

A new nucleic acid-based agent inhibits cytotoxic T lymphocyte-mediated immune disorders.

BACKGROUND: Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and graft-versus-host disease (GVHD) are distinct immune reactions elicited by drugs or allogeneic antigens; however, they share a pathomechanism with the activation of cytotoxic T lymphocytes (CTLs). CTLs produce cytotoxic proteins, cytokines, chemokines, or immune alarmins, such as granulysin (GNLY), leading to the extensive tissue damage and systemic inflammation seen in patients with SJS/TEN or GVHD. Currently, there is no effective therapeutic agent specific for CTL-mediated immune disorders. OBJECTIVES: By targeting GNLY(+) CTLs, we aimed to develop a nucleic acid-based agent consisting of an anti-CD8 aptamer with GNLY small interfering RNA (siRNA). METHODS: We performed systematic evolution of ligands using exponential enrichment to select and identify effective anti-CD8 aptamers. We developed an aptamer-siRNA chimera using a "sticky bridge" method by conjugating the aptamer with siRNA. We analyzed the inhibitory effects of the aptamer-siRNA chimera on CTL responses in patients with SJS/TEN or GVHD. RESULTS: We identified a novel DNA aptamer (CD8AP17s) targeting CTLs. This aptamer could be specifically internalized into human CTLs. We generated the CD8AP17s aptamer-GNLY siRNA chimera, which showed a greater than 79% inhibitory effect on the production of GNLY by drug/alloantigen-activated T cells. The CD8AP17s aptamer-GNLY siRNA chimera decreased cytotoxicity in in vitro models of both SJS/TEN (elicited by drug-specific antigen) and GVHD (elicited by allogeneic antigens). CONCLUSIONS: Our results identified a new nucleic acid-based agent (CD8 aptamer-GNLY siRNA chimera) that can significantly inhibit CTL-mediated drug hypersensitivity, such as that seen in patients with SJS/TEN, as well as the alloreactivity seen in patients with GVHD. This study provides a novel therapeutic strategy for CTL-mediated immune disorders.

SCUBE3 regulation of early lung cancer angiogenesis and metastatic progression.

Signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3) is strongly expressed in extremely invasive lung carcinoma. We showed in our previous study that SCUBE3 triggers the transforming growth factor-ß pathway and subsequently promotes tumor angiogenesis and the epithelial-mesenchymal transition (EMT). However, the role of SCUBE3 in early tumor expansion hasn't been fully demonstrated in vivo. The present study used dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to temporally assess tumor angiogenesis in SCUBE3-knockdown and control non-small-cell lung carcinoma (NSCLC) cancer cells in the early tumor stage (weeks 1-3). We further evaluated the metastatic potential of the SCUBE3-knockdown and control tumor cells using a circulating tumor cell (CTC) assay. The differences in gene expression profile between these cell lines were determined using microarray analysis. The results show that SCUBE3 knockdown was associated with lower vascular permeability in the tumor and effectively inhibited the metastatic potential of NSCLC, as evidenced by the decreased CTCs in the mice bearing SCUBE3-knockdown tumors. Microarray analysis revealed that several genes involved in angiogenesis and EMT were down-regulated in SCUBE3-knockdown tumors, including matrix metalloproteinases (MMPs) 2, 9, and 14, (MMP-2, MMP-9, and MMP-14, respectively), fibronectin (FN-1), lysyl oxidase (LOX), hairy/enhancer-of-split related with YRPW motif protein 1 (HEY1), early growth response protein 1 (EGR1), and interleukin 8 (IL-8). Together these data suggest that SCUBE3 is a potential target for pharmacological intervention. The findings of the present study also show that differences in vascular permeability precede the CTCs detection, indicating that DCE-MRI may be a sensitive biomarker for assessing tumor invasiveness.

In vitro metabolism of α7 neuronal nicotinic receptor agonist AZD0328 and enzyme identification for its N-oxide metabolite.

1. AZD0328 was pharmacologically characterized as a α7 neuronal nicotinic receptor agonist intended for treatment of Alzheimer's disease. In vitro AZD0328 cross species metabolite profile and enzyme identification for its N-oxide metabolite were evaluated in this study. 2. AZD0328 was very stable in the human hepatocyte incubation, whereas extensively metabolized in rat, dog and guinea pig hepatocyte incubations. The N-oxidation metabolite (M6) was the only metabolite detected in human hepatocyte incubations, and it also appeared to be the major in vitro metabolic pathway in a number of preclinical species. In addition, N-glucuronide metabolite of AZD0328 was observed in human liver microsomes. 3. Other metabolic pathways in the preclinical species include hydroxylation in azabicyclo octane or furopyridine part of the molecule. Pyridine N-methylation of AZD0328 (M2) was identified as a dog specific metabolite, not observed in human or other preclinical species. 4. Multiple enzymes including CYP2D6, CYP3A4/5, FMO1 and FMO3 catalyzed AZD0328 metabolism. The potential for AZD0328 to be inhibited clinically by co-administered drugs or genetic polymorphism is relative low.

Pharmacokinetics of 8-hour intravenous infusion of NXY-059: a phase I, randomized, double-blind (within dose panels), placebo-controlled study in healthy Chinese volunteers.

BACKGROUND: NXY-059 is a free radical-trapping neuroprotectant that has been reported to reduce infarct size and preserve brain function in experimental models of acute ischemic stroke. NXY-059 administered as an 8- or 72-hour IV infusion has been reported to be well tolerated in healthy young (age, 20-45 years) and older (55-75 years) white volunteers. NXY-059 is no longer in development following a lack of efficacy found in a Phase III trial in patients with acute ischemic stroke. OBJECTIVES: The primary objectives of this study were to determine the pharmacokinetic (PK) properties of an 8-hour IV infusion of NXY-059 in healthy Chinese volunteers and to compare those data with those previously reported in the white population, therefore exploring any differences in PK properties between the 2 ethnic groups. Secondary objectives were to evaluate PK linearity and tolerability. METHODS: This Phase I, randomized, double-blind (within dose panels), placebo-controlled study was conducted at Peking Union Medical College Hospital, Beijing, China. Healthy male and female Chinese volunteers aged 20 to 45 years were recruited. NXY-059 was administered as a continuous 8-hour IV infusion, starting with a 1-hour loading dose (dosing rate, 3 x maintenance infusion rate) followed by a 7-hour maintenance dose infusion. Subjects were randomly assigned, in a 3:1 ratio, to receive doses calculated (based on creatinine clearance in individual subjects) to achieve 1 of 3 concentration targets, or inactive vehicle (sodium chloride; placebo). The target unbound plasma NXY-059 concentrations during constant rate infusion (steady state) (Cu(ss)) in the 3 dose panels were 100,200, and 300 micromol/L. An explorative bridging analysis was used to compare PK data from this study with those previously reported in the white population. Linearity of NXY-059 PK properties was assessed. Tolerability was assessed using adverse events (spontaneous reporting, study staff observation, and open questioning), physical examination, including vital sign measurement; and electrocardiography and laboratory analysis. RESULTS: Thirty-six subjects were randomized (mean age, 32 years [range, 20-41 years]; mean body mass index, 22.6 kg/m(2) [range, 20-26 kg/m(2)]). The target exposures of NXY-059 were achieved (mean [SD] Cu(ss) values, 98.3 [8.9], 202.1 [18.3], and 287.9 [25.4] micromol/L, respectively). Steady-state concentrations appeared to have been reached after 4 hours. From the bridging analysis, comparison of PK properties in the 27 Chinese volunteers versus those in 28 white volunteers found similar total plasma clearance of NXY-059 (estimated Chinese:white clearance ratio, 1.077 [95% CI, 1.009-1.150]). There were no apparent differences in other PK parameters between the 2 ethnic groups. The PK properties of NXY-059 in Chinese volunteers were suggestive of linearity. A total of 7 adverse events were reported, all of mild intensity, in the NXY-059 and placebo groups (thirst and polyuria [each in 2 subjects who received NXY-059 and 1 subject who received placebo]; urinary tract infection [1 subject who received NXY-059]). CONCLUSIONS: The results from the present study suggest that the PK properties of NXY-059 were similar in the Chinese and historical white healthy volunteer populations.

A phase 1, placebo-controlled, randomised, double-blind (within dose panels) study evaluating the safety, tolerability and pharmacokinetics of intravenous NXY-059 in Japanese subjects.

OBJECTIVE: NXY-059 has a proposed mechanism of action of free-radical trapping and has been studied in clinical trials based on positive effects seen in experimental models of acute ischaemic stroke. This study evaluated the safety, tolerability and pharmacokinetics of NXY-059 in healthy Japanese male subjects compared with previous data from healthy Caucasian subjects. RESEARCH DESIGN AND METHODS: The primary objective of this phase 1, double-blind, randomised, placebo-controlled, dose-escalating study was to evaluate the safety and tolerability of an 8- or 24-h intravenous infusion of NXY-059 targeting an unbound plasma concentration of 25-300 micromol/L in healthy Japanese male subjects (20-45 years). NXY-059 pharmacokinetics were also assessed, and any differences in pharmacokinetics between Japanese and previously studied Caucasian subjects (20-45 years) were explored. RESULTS: NXY-059 was generally well tolerated and no safety concerns were identified. There was a similar incidence of adverse events between subjects receiving NXY-059 or placebo, and no obvious trend towards an increased incidence of adverse events with increasing doses of NXY-059 was observed. In addition, there was no evidence of any vasoirritative effects or changes in renal function. The tolerability profile was similar in Caucasian subjects. The pharmacokinetic results indicate proportional exposure of 8-h and 24-h infusions of NXY-059 resulting in mean unbound steady state plasma concentrations up to 417 micromol/L and 379 micromol/L, respectively. Plasma clearance values for NXY-059 were similar in both Japanese and Caucasian subjects. CONCLUSIONS: This study suggests that the tolerability and pharmacokinetics of NXY-059 in healthy Japanese male subjects and Caucasians are similar.

Effect of NXY-059, a novel neuroprotectant, on the pharmacokinetics of a single dose of digoxin in healthy subjects.

OBJECTIVE: NXY-059 is a novel free-radical trapping neuroprotectant. Digoxin treatment is common in acute ischaemic stroke, the intended patient population for NXY-059. Since both digoxin and NXY-059 are eliminated primarily renally, with a substantial contribution by active renal secretion, and because digoxin has a narrow therapeutic window, this open, randomised, crossover, two-period study investigated whether NXY-059 affects the pharmacokinetics (PK) of digoxin. RESEARCH DESIGN AND METHODS: Twenty-two healthy subjects received 0.5 mg oral digoxin 2 h after the start of 60-h intravenous infusions of NXY-059 and placebo separated by a 14-day washout. Blood and urine were collected for 60 h. Digoxin concentrations were measured by a novel liquid chromatography-mass spectrometry assay. MAIN OUTCOME MEASURES: The ratio of the geometric mean (90% confidence interval) between NXY-059 and placebo for the digoxin area under the concentration-versus-time curve was 0.91 (0.83-0.99) and was within the predefined range for no interaction (0.80-1.25). No safety concerns were raised in the study. No serious adverse events were recorded. The most common adverse event was headache with similar frequencies in the two treatments. CONCLUSIONS: NXY-059 had no clinically significant effect on the PK of digoxin.