A Novel TGFß Receptor Inhibitor, IPW-5371, Prevents Diet-induced Hepatic Steatosis and Insulin Resistance in Irradiated Mice.

As the number of cancer survivors increases and the risk of accidental radiation exposure rises, there is a pressing need to characterize the delayed effects of radiation exposure and develop medical countermeasures. Radiation has been shown to damage adipose progenitor cells and increase liver fibrosis, such that it predisposes patients to developing metabolic-associated fatty liver disease (MAFLD) and insulin resistance. The risk of developing these conditions is compounded by the global rise of diets rich in carbohydrates and fats. Radiation persistently increases the signaling cascade of transforming growth factor ß (TGFß), leading to heightened fibrosis as characteristic of the delayed effects of radiation exposure. We investigate here a potential radiation medical countermeasure, IPW-5371, a small molecule inhibitor of TGFßRI kinase (ALK5). We found that mice exposed to sub-lethal whole-body irradiation and chronic Western diet consumption but treated with IPW-5371 had a similar body weight, food consumption, and fat mass compared to control mice exposed to radiation. The IPW-5371 treated mice maintained lower fibrosis and fat accumulation in the liver, were more responsive to insulin and had lower circulating triglycerides and better muscle endurance. Future studies are needed to verify the improvement by IPW-5371 on the structure and function of other metabolically active tissues such as adipose and skeletal muscle, but these data demonstrate that IPW-5371 protects liver and whole-body health in rodents exposed to radiation and a Western diet, and there may be promise in using IPW-5371 to prevent the development of MAFLD.

Effects of Simulated 5-Ion Galactic Cosmic Radiation on Function and Structure of the Mouse Heart.

Missions into deep space will expose astronauts to the harsh space environment, and the degenerative tissue effects of space radiation are largely unknown. To assess the risks, in this study, male BALB/c mice were exposed to 500 mGy 5-ion simulated GCR (GCRsim) at the NASA Space Radiation Laboratory. In addition, male and female CD1 mice were exposed to GCRsim and administered a diet containing Transforming Growth Factor-beta (TGF-ß)RI kinase (ALK5) inhibitor IPW-5371 as a potential countermeasure. An ultrasound was performed to investigate cardiac function. Cardiac tissue was collected to determine collagen deposition, the density of the capillary network, and the expression of the immune mediator toll-like receptor 4 (TLR4) and immune cell markers CD2, CD4, and CD45. In male BALB/c mice, the only significant effects of GCRsim were an increase in the CD2 and TLR4 markers. In male CD1 mice, GCRsim caused a significant increase in total collagens and a decrease in the expression of TLR4, both of which were mitigated by the TGF-ß inhibitor diet. In female CD1 mice, GCRsim caused an increase in the number of capillaries per tissue area in the ventricles, which may be explained by the decrease in the left ventricular mass. However, this increase was not mitigated by TGF-ß inhibition. In both male and female CD1 mice, the combination of GCRsim and TGF-ß inhibition caused changes in left ventricular immune cell markers that were not seen with GCRsim alone. These data suggest that GCRsim results in minor changes to cardiac tissue in both an inbred and outbred mouse strain. While there were few GCRsim effects to be mitigated, results from the combination of GCRsim and the TGF-ß inhibitor do point to a role for TGF-ß in maintaining markers of immune cells in the heart after exposure to GCR.

IPW-5371 mitigates the delayed effects of acute radiation exposure in WAG/RijCmcr rats when started 15 days after PBI with bone marrow sparing.

PURPOSE: To test IPW-5371 for the mitigation of the delayed effects of acute radiation exposure (DEARE). Survivors of acute radiation exposure are at risk for developing delayed multi-organ toxicities; however, there are no FDA-approved medical countermeasures (MCM) to mitigate DEARE. METHODS: WAG/RijCmcr female rat model of partial-body irradiation (PBI), by shielding part of one hind leg, was used to test IPW-5371 (7 and 20 mg kg-1 d-1) for mitigation of lung and kidney DEARE when started 15 d after PBI. Rats were fed known amounts of IPW-5371 using a syringe, instead of delivery by daily oral gavage, sparing exacerbation of esophageal injury by radiation. The primary endpoint, all-cause morbidity was assessed over 215 d. Secondary endpoints: body weight, breathing rate and blood urea nitrogen were also assessed. RESULTS: IPW-5371 enhanced survival (primary endpoint) as well as attenuated secondary endpoints of lung and kidney injuries by radiation. CONCLUSION: To provide a window for dosimetry and triage, as well as avoid oral delivery during the acute radiation syndrome (ARS), the drug regimen was started at 15 d after 13.5 Gy PBI. The experimental design to test mitigation of DEARE was customized for translation in humans, using an animal model of radiation that was designed to simulate a radiologic attack or accident. The results support advanced development of IPW-5371 to mitigate lethal lung and kidney injuries after irradiation of multiple organs.

Rat Models of Partial-body Irradiation with Bone Marrow-sparing (Leg-out PBI) Designed for FDA Approval of Countermeasures for Mitigation of Acute and Delayed Injuries by Radiation.

ABSTRACT: The goal of this study was to develop rat models of partial body irradiation with bone-marrow sparing (leg-out PBI) to test medical countermeasures (MCM) of both acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE) under the FDA animal rule. The leg-out PBI models were developed in female and male WAG/RijCmcr rats at doses of 12.5-14.5 Gy. Rats received supportive care consisting of fluids and antibiotics. Gastrointestinal ARS (GI-ARS) was assessed by lethality to d 7 and diarrhea scoring to d 10. Differential blood counts were analyzed between d 1-42 for the natural history of hematopoietic ARS (H-ARS). Lethality and breathing intervals (BI) were measured between d 28-110 to assess delayed injury to the lung (L-DEARE). Kidney injury (K-DEARE) was evaluated by measuring elevation of blood urea nitrogen (BUN) between d 90-180. The LD50/30, including both lethality from GI-ARS and H-ARS, for female and male rats are 14.0 Gy and 13.5 Gy, respectively, while the LD50/7 for only GI-ARS are 14.3 Gy and 13.6 Gy, respectively. The all-cause mortalities, including ARS and L-DEARE, through 120 d (LD50/120) are 13.5 Gy and 12.9 Gy, respectively. Secondary end points confirmed occurrence of four distinct sequelae representing GI, hematopoietic, lung, and kidney toxicities after leg-out PBI. Adult rat models of leg-out PBI showed the acute and long-term sequelae of radiation damage that has been reported in human radiation exposure case studies. Sex-specific differences were observed in the DRR between females and males. These rat models are among the most useful for the development and approval of countermeasures for mitigation of radiation injuries under the FDA animal rule.

Blood-brain barrier dysfunction in aging induces hyperactivation of TGFß signaling and chronic yet reversible neural dysfunction.

Aging involves a decline in neural function that contributes to cognitive impairment and disease. However, the mechanisms underlying the transition from a young-and-healthy to aged-and-dysfunctional brain are not well understood. Here, we report breakdown of the vascular blood-brain barrier (BBB) in aging humans and rodents, which begins as early as middle age and progresses to the end of the life span. Gain-of-function and loss-of-function manipulations show that this BBB dysfunction triggers hyperactivation of transforming growth factor-ß (TGFß) signaling in astrocytes, which is necessary and sufficient to cause neural dysfunction and age-related pathology in rodents. Specifically, infusion of the serum protein albumin into the young rodent brain (mimicking BBB leakiness) induced astrocytic TGFß signaling and an aged brain phenotype including aberrant electrocorticographic activity, vulnerability to seizures, and cognitive impairment. Furthermore, conditional genetic knockdown of astrocytic TGFß receptors or pharmacological inhibition of TGFß signaling reversed these symptomatic outcomes in aged mice. Last, we found that this same signaling pathway is activated in aging human subjects with BBB dysfunction. Our study identifies dysfunction in the neurovascular unit as one of the earliest triggers of neurological aging and demonstrates that the aging brain may retain considerable latent capacity, which can be revitalized by therapeutic inhibition of TGFß signaling.

Macrocyclic MEK1/2 inhibitor with efficacy in a mouse model of cardiomyopathy caused by lamin A/C gene mutation.

Signaling mediated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) is involved in numerous cellular processes. Mitogen-activated protein kinase kinases (MEK1/2) catalyze the phosphorylation of ERK1/2, converting it into an active kinase that regulates the expression of numerous genes and cellular processes. Inhibitors of MEK1/2 have demonstrated preclinical and clinical efficacy in certain cancers and types of cardiomyopathy. We report the synthesis of a novel, allosteric, macrocyclic MEK1/2 inhibitor that potently inhibits ERK1/2 activity in cultured cells and tissues of mice after systemic administration. Mice with dilated cardiomyopathy caused by a lamin A/C gene mutation have abnormally increased cardiac ERK1/2 activity. In these mice, this novel MEK1/2 inhibitor is well tolerated, improves left ventricular systolic function, decreases left ventricular fibrosis, has beneficial effects on skeletal muscle structure and pathology and prolongs survival. The novel MEK1/2 inhibitor described herein may therefore find clinical utility in the treatment of this rare cardiomyopathy, other types of cardiomyopathy and cancers in humans.

IPW-5371 Proves Effective as a Radiation Countermeasure by Mitigating Radiation-Induced Late Effects.

There is an ongoing and significant need for radiation countermeasures to reduce morbidities and mortalities associated with exposure of the heart and lungs from a radiological or nuclear incidents. Radiation-induced late effects occur months to years after exposure, stemming from significant tissue damage and remodeling, resulting in fibrosis and loss of function. TGF-ß is reported to play a role in both pulmonary and cardiac fibrosis. We investigated the ability of a small molecule TGF-ß receptor 1 inhibitor, IPW-5371, to mitigate the effects of thoracic irradiation in C57L/J mice, a murine model that most closely resembles that observed in humans in the induction of fibrosis and dose response. To simulate a radiological event, radiation was administered in two doses: 5 Gy total-body irradiation (eliciting a whole-body response) and immediately after that, a thoracic "top-up" of 6.5 Gy irradiation, for a total dose of 11.5 Gy to the thorax. IPW-5371 was administered once daily, orally, starting 24 h postirradiation for 6 or 20 weeks at a dose of 10 mg/kg or 30 mg/kg. Animals were monitored for a period of 180 days for survival, and cardiopulmonary injury was assessed by echocardiography, breathing rate and arterial oxygen saturation. Exposure of the thorax (11.5 Gy) induced both pulmonary and cardiac injury, resulting in a reduced life span with median survival of 135 days. IPW-5371 treatment for 6 weeks, at both 10 mg/kg and 30 mg/kg, delayed disease onset and mortality, with median survival of 165 days. Twenty weeks of IPW-5371 treatment at 30 mg/kg preserved arterial O2 saturation and cardiac contractile reserve and resulted in significant decreases in breathing frequency and cardiac and pulmonary fibrosis. This led to dramatic improvement in survival compared to the irradiated, vehicle-treated group (P < 0.001), and was statistically insignificant from the nonirradiated group. We observed that IPW-5371 treatment resulted in decreased pSmad3 tissue levels, confirming the effect of IPW-5371 on TGF-ß signaling. These results demonstrate that IPW-5371 represents a potentially promising radiation countermeasure for the treatment of radiation-induced late effects.

3-heterocyclyl quinolone inhibitors of the HCV NS5B polymerase.

The discovery and optimization of a novel class of quinolone small-molecules that inhibit NS5B polymerase, a key enzyme of the HCV viral life-cycle, is described. Our research led to the replacement of a hydrolytically labile ester functionality with bio-isosteric heterocycles. An X-ray crystal structure of a key analog bound to NS5B facilitated the optimization of this series of compounds to afford increased activity against the target enzyme and in the cell-based replicon assay system.

Diamino-1,2,4-triazole derivatives are selective inhibitors of TYK2 and JAK1 over JAK2 and JAK3.

Tyrosine kinase 2 (TYK2) is required for signaling of interleukin-23 (IL-23), which plays a key role in rheumatoid arthritis. Presented is the design and synthesis of 1,2,4-triazoles, and the evaluation of their inhibitory activity against the Janus associated kinases TYK2 and JAKs 1-3.

Assessment of the water quality and ecosystem health of the Great Barrier Reef (Australia): conceptual models.

Run-off containing increased concentrations of sediment, nutrients, and pesticides from land-based anthropogenic activities is a significant influence on water quality and the ecologic conditions of nearshore areas of the Great Barrier Reef World Heritage Area, Australia. The potential and actual impacts of increased pollutant concentrations range from bioaccumulation of contaminants and decreased photosynthetic capacity to major shifts in community structure and health of mangrove, coral reef, and seagrass ecosystems. A detailed conceptual model underpins and illustrates the links between the main anthropogenic pressures or threats (dry-land cattle grazing and intensive sugar cane cropping) and the production of key contaminants or stressors of Great Barrier Reef water quality. The conceptual model also includes longer-term threats to Great Barrier Reef water quality and ecosystem health, such as global climate change, that will potentially confound direct model interrelationships. The model recognises that system-specific attributes, such as monsoonal wind direction, rainfall intensity, and flood plume residence times, will act as system filters to modify the effects of any water-quality system stressor. The model also summarises key ecosystem responses in ecosystem health that can be monitored through indicators at catchment, riverine, and marine scales. Selected indicators include riverine and marine water quality, inshore coral reef and seagrass status, and biota pollutant burdens. These indicators have been adopted as components of a long-term monitoring program to enable assessment of the effectiveness of change in catchment-management practices in improving Great Barrier Reef (and adjacent catchment) water quality under the Queensland and Australian Governments' Reef Water Quality Protection Plan.

Inhibiting TGF-beta signaling restores immune surveillance in the SMA-560 glioma model.

Transforming growth factor-beta (TGF-beta) is a proinvasive and immunosuppressive cytokine that plays a major role in the malignant phenotype of gliomas. One novel strategy of disabling TGF-beta activity in gliomas is to disrupt the signaling cascade at the level of the TGF-beta receptor I (TGF-betaRI) kinase, thus abrogating TGF-beta-mediated invasiveness and immune suppression. SX-007, an orally active, small-molecule TGF-betaRI kinase inhibitor, was evaluated for its therapeutic potential in cell culture and in an in vivo glioma model. The syngeneic, orthotopic glioma model SMA-560 was used to evaluate the efficacy of SX-007. Cells were implanted into the striatum of VM/Dk mice. Dosing began three days after implantation and continued until the end of the study. Efficacy was established by assessing survival benefit. SX-007 dosed at 20 mg/kg p.o. once daily (q.d.) modulated TGF-beta signaling in the tumor and improved the median survival. Strikingly, approximately 25% of the treated animals were disease-free at the end of the study. Increasing the dose to 40 mg/kg q.d. or 20 mg/kg twice daily did not further improve efficacy. The data suggest that SX-007 can exert a therapeutic effect by reducing TGF-beta-mediated invasion and reversing immune suppression. SX-007 modulates the TGF-beta signaling pathway and is associated with improved survival in this glioma model. Survival benefit is due to reduced tumor invasion and reversal of TGF-beta-mediated immune suppression, allowing for rejection of the tumor. Together, these results suggest that treatment with a TGF-betaRI inhibitor may be useful in the treatment of glioblastoma.

Small-scale temporal variation and the effect of urbanisation on extracellular enzyme activity in streams.

The activity of six extracellular enzymes involved in the degradation of dissolved organic carbon compounds was measured in two highly urbanised and two minimally impacted streams east of Melbourne, Australia, using 4-methylumbelliferyl-substrates. Small-scale temporal variation in enzyme activity was determined by repeatedly sampling the same point in the water column, while the effect of flow was determined by sampling in regions of higher and lower flow in both stream types. Replicate samples showed that enzyme activity was not significantly different over small (minutes) time scales. On five of six sampling occasions the enzyme activity was unaffected by flow. On one sampling occasion in a minimally disturbed stream, the difference between the high- and low-flow regions was statistically significant (ANOSIM, Global R= 0.78, P= 0.03). Enzyme activity profiles (activities of the suite of enzymes) of the streams in urbanised catchments were different to those in minimally disturbed catchments. The measurements made in four different streams showed high reproducibility over short time periods (minutes) which lends greater credibility to analogous spatial studies. Although these results determined that small-scale temporal variability was not significant, and that the effects of flow were generally minimal, it is recommended that spatial and temporal variability in the stream be at least considered before any studies measuring extracellular enzyme activity in stream waters are carried out. Such an approach will lead to conclusions from measurements that are not likely to be confounded by variables such as flow rate or time.

Assessing long-term pH change in an Australian river catchment using monitoring and palaeolimnological data.

Reviews of stream monitoring data suggest that there has been significant acidification (>1.0 pH unit at some sites) of Victorian streamwaters over the past 3 decades. To assess whether these declines are within the range of natural variability, we developed a diatom model for inferring past pH and applied it to a ca. 3500-yr diatom record from a flood plain lake, Callemondah 1 Billabong, on the Goulburn River, which has among the most substantial observed pH declines. The model has a jackkniffed r2 between diatom inferred and measured pH of 0.77 and a root mean square error of prediction of 0.35 pH units. In the pre-European period, pH was stable (range 6.5-6.7) for approximately 3000 yr. Since European settlement around 160 yr ago, diatom-inferred billabong pH has increased significantly by >0.5 units. We hypothesize that this increase in pH is related to processes associated with land clearance (e.g., increased base cation load and decreased organic acid load). There is no evidence of the recent monitored declines in the Callemondah record, which may indicate that that flood plain lakes and the main stream are experiencing divergent pH trends or that the temporal resolution in the billabong sediment record is insufficient to register recent declines.

Asymmetric synthesis of the carbapenam core from serine.

The stereospecific synthesis of the functionalized carbapenam core 16 from the serine-derived trisubstituted pyrrolidine 9 is reported. The synthetic strategy relies on synthesizing an appropriately functionalized pyrrolidine, followed by an intramolecular azetidone formation utilizing a modified Mukiyama reagent. The efficient one-pot conversion of the benzenesulfonamide-protected pyrrolidine 9 to the Cbz-protected pyrrolidine 10 is also reported.

Nutrient budget for Saguling Reservoir, West Java, Indonesia.

A preliminary nutrient budget for Saguling Reservoir is reported as a first attempt to quantify the behaviour of nutrients entering this reservoir. This work is part of a larger Indonesia-Australia collaborative research and training project, involving Padjadjaran University and Monash University, established to study nutrient dynamics in Saguling Reservoir. Saguling Reservoir, the first of a chain of three large reservoirs (Saguling, Cirata and Jatilahur), built on the Citarum River in central Java, was completed in 1985. It has already become highly polluted, particularly with domestic and industrial effluent (organic matter, nutrients, heavy metals) from the urban areas of Bandung (population 2 million). The reservoir experiences major water quality problems, including excessive growths of floating plants, toxic cyanobacterial blooms and regular fish-kills. The work reported in this paper shows that Saguling receives a very large nutrient load from the city of Bandung and because of this, is highly eutrophic. It is unlikely that the water quality of Saguling will improve until a substantial part of Bandung is sewered and adequate discharge controls are placed on the many industries in the region upstream of the reservoir.

Luxury uptake of phosphorus by sediment bacteria.

This note reports the results of experiments aimed at confirming the luxury uptake of phosphorus (P) by sediment bacteria as polyphosphate (Poly-P). Aerobic suspensions of sediments from two different sites were spiked with 1 mg P/L as orthophosphate and augmented with acetate (a fermentation product) or glucose. The orthophosphate was rapidly taken up over a period of a few hours. When these aerobic uptake experiments were made anaerobic and additional organic carbon added, only the acetate-amended sediment released a significant amount of the added phosphorus. It was hypothesised that during the aerobic stage, and with the addition of acetate, some of the phosphorus was accumulated as Poly-P by sediment microorganisms, which was released during the subsequent anaerobic stage (provided acetate was still present). Two lines of evidence--transmission electron microscope analysis of sediment bacteria and 31P-NMR analysis of sediment extracts--are presented to support the hypothesis that a portion of the phosphorus taken up during the aerobic experiments was stored as Poly-P.

Conformationally Constrained 7-Azabicyclo[2.2.1]heptane Amino Acids. Synthesis of a Glutamic Acid Analogue.

We report the synthesis of 2-substituted 7-azabicyclo[2.2.1]heptane glutamic acid analogue 27 from L-serine. Hemiaminal intermediate 2 can be converted to the 2S,3S,5S-trisubstituted pyrrolidine 3 by a tandem Wittig/Michael reaction or to the 2S,3S,5R-trisubstituted pyrrolidine 4 via an iodosulfonamidation reaction. The key transannular alkylation step to form the [2.2.1] ring system involves a beta-elimination of a silyl ether followed by cyclization to afford tert-butyl 7-benzyloxycarbonyl-7-azabicyclo[2.2.1]-2-heptene-1-carboxylate (20). Selective functionalization at C-2 was accomplished by the direct reduction with SmI(2) of 2-keto-3-silyl ether 23 to the C-2 ketone 24, which was converted to alpha,beta-unsaturated ester 25. Stereospecific reduction of the double bond from the exo face leads to a single protected glutamate analogue, tert-butyl (1S,2R,4R)-7-benzyloxycarbonyl-2-(methoxycarbonylmethyl)-7-azabicyclo[2.2.1]heptane-1-carboxylate (27).

Bacterial colonisation and infection in spina bifida babies.

A Series of bacteriological investigations was carried out on 17 babies with spina bifida. Results showed that despite colonisation of their eyes, nose, throat, skin, umbilicus, wounds, urine and faeces with potentially pathogenic microorganisms, infections other than those of the urinary tract and brain were relatively uncommon.