Purpose
Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have shown promise in treating Duchenne muscular dystrophy (DMD). We evaluated a semi-mechanistic pharmacokinetic (PK) and ...pharmacodynamic (PD) model to capture the relationship between plasma and muscle tissue exposure/response in
mdx
mice treated by mouse surrogate PPMO.
Methods
A single or repeated (every 4 weeks for 20 weeks) intravenous PPMO dose was administered to
mdx
mice (n = 6/timepoint). A PK/PD model was built to characterize data via sequential modeling. A 2-compartment model was used to describe plasma PK. A simultaneous tissue PK/PD model was subsequently developed: 2-compartment model to describe muscle PK; linked to an indirect response model describing stimulation of synthesis of skipped transcript, which was in turn linked to stimulation of synthesis of dystrophin protein expression.
Results
Model performance assessment via goodness-of-fit plots, visual predictive checks, and accurate parameter estimation indicated robust fits of plasma PK and muscle PK/PD data. The model estimated a PPMO tissue half-life of 5 days—a useful parameter in determining the longevity of PPMOs in tissue and their limited accumulation after multiple doses. Additionally, the model successfully described dystrophin expression after single dosing and associated protein accumulation after multiple dosing (increasing ~ twofold accumulation from the first to last dose).
Conclusions
This first PK/PD model of a PPMO in a DMD disease model will help characterize and predict the time course of PK/PD biomarkers in
mdx
mice. Furthermore, the model framework can be used to develop clinical PK/PD models and can be extended to other exon-skipping therapies and species.
The age-related effects of GDF11 have been a subject of controversy. Here, we find that elevated GDF11 causes signs of cachexia in mice: reduced food intake, body weight, and muscle mass. GDF11 also ...elicited a significant elevation in plasma Activin A, previously shown to contribute to the loss of skeletal muscle. The effects of GDF11 on skeletal muscle could be reversed by administration of antibodies to the Activin type II receptors. In addition to the effects on muscle, GDF11 increased plasma GDF15, an anorectic agent. The anorexia, but not the muscle loss, could be reversed with a GDF15-neutralizing antibody. GDF15 upregulation is due to GDF11-induced recruitment of SMAD2/3 to the GDF15 promoter. Inhibition of GDF15 can restore appetite but cannot restore the GDF11-induced loss of muscle mass, which requires blockade of ActRII signaling. These findings are relevant for treatment of cachexia.
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•Increased levels of GDF11 cause increased circulating Activin A and GDF15 in mice•Supraphysiologic levels of GDF11 induce cachexia, anorexia, and muscle loss•Blockade of GDF15 spares anorexia, but not muscle loss•Blockade of the GDF11 receptor ActRII spares muscle loss and decreases anorexia
Jones et al. find that high levels of GDF11 in mice induce symptoms of cachexia: skeletal muscle loss and anorexia. The anorexia is due to GDF11-dependent upregulation of GDF15. Downregulation of GDF11 in settings in which it exists at high levels is predicted to be beneficial.
Inflammation in the central nervous system (CNS) is observed in many neurological disorders. Nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling plays an ...essential role in modulating neuroinflammation. CYR119 is a CNS-penetrant sGC stimulator that amplifies endogenous NO-sGC-cGMP signaling. We evaluated target engagement and the effects of CYR119 on markers of neuroinflammation in vitro in mouse microglial cells and in vivo in quinolinic acid (QA)-induced and high-fat diet-induced rodent neuroinflammation models.
Target engagement was verified in human embryonic kidney (HEK) cells, rat primary neurons, mouse SIM-A9 cells, and in rats by measuring changes in cGMP and downstream targets of sGC signaling phosphorylated vasodilator-stimulated phosphoprotein (pVASP), phosphorylated cAMP-response element binding (pCREB). In SIM-A9 cells stimulated with lipopolysaccharides (LPS), markers of inflammation were measured when cells were treated with or without CYR119. In rats, microinjections of QA and vehicle were administered into the right and left hemispheres of striatum, respectively, and then rats were dosed daily with either CYR119 (10 mg/kg) or vehicle for 7 days. The activation of microglia ionized calcium binding adaptor molecule 1 (Iba1) and astrocytes glial fibrillary acidic protein (GFAP) was measured by immunohistochemistry. Diet-induced obese (DIO) mice were treated daily with CYR119 (10 mg/kg) for 6 weeks, after which inflammatory genetic markers were analyzed in the prefrontal cortex.
In vitro, CYR119 synergized with exogenous NO to increase the production of cGMP in HEK cells and in primary rat neuronal cell cultures. In primary neurons, CYR119 stimulated sGC, resulting in accumulation of cGMP and phosphorylation of CREB, likely through the activation of protein kinase G (PKG). CYR119 attenuated LPS-induced elevation of interleukin 6 (IL-6) and tumor necrosis factor (TNF) in mouse microglial cells. Following oral dosing in rats, CYR119 crossed the blood-brain barrier (BBB) and stimulated an increase in cGMP levels in the cerebral spinal fluid (CSF). In addition, levels of proinflammatory markers associated with QA administration or high-fat diet feeding were lower in rodents treated with CYR119 than in those treated with vehicle.
These data suggest that sGC stimulation could provide neuroprotective effects by attenuating inflammatory responses in nonclinical models of neuroinflammation.
Effective treatments for neurodegenerative diseases remain elusive and are critically needed since the burden of these diseases increases across an aging global population. Nitric oxide (NO) is a ...gasotransmitter that binds to soluble guanylate cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Impairment of this pathway has been demonstrated in neurodegenerative diseases. Normalizing deficient NO-cGMP signaling could address multiple pathophysiological features of neurodegenerative diseases. sGC stimulators are small molecules that synergize with NO, activate sGC, and increase cGMP production. Many systemic sGC stimulators have been characterized and advanced into clinical development for a variety of non-central nervous system (CNS) pathologies. Here, we disclose the discovery of CY6463, the first brain-penetrant sGC stimulator in clinical development for the treatment of neurodegenerative diseases, and demonstrate its ability to improve neuronal activity, mediate neuroprotection, and increase cognitive performance in preclinical models. In several cellular assays, CY6463 was demonstrated to be a potent stimulator of sGC. In agreement with the known effects of sGC stimulation in the vasculature, CY6463 elicits decreases in blood pressure in both rats and mice. Relative to a non-CNS penetrant sGC stimulator, rodents treated with CY6463 had higher cGMP levels in cerebrospinal fluid (CSF), functional-magnetic-resonance-imaging-blood-oxygen-level-dependent (fMRI-BOLD) signals, and cortical electroencephalographic (EEG) gamma-band oscillatory power. Additionally, CY6463 improved cognitive performance in a model of cognitive disruption induced by the administration of a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. In models of neurodegeneration, CY6463 treatment increased long-term potentiation (LTP) in hippocampal slices from a Huntington’s disease mouse model and decreased the loss of dendritic spines in aged and Alzheimer’s disease mouse models. In a model of diet-induced obesity, CY6463 reduced markers of inflammation in the plasma. Furthermore, CY6463 elicited an additive increase in cortical gamma-band oscillatory power when co-administered with donepezil: the standard of care in Alzheimer’s disease. Together, these data support the clinical development of CY6463 as a novel treatment for neurodegenerative disorders.
Praliciguat is a soluble guanylate cyclase stimulator that elicits hemodynamic, anti-inflammatory, and antifibrotic effects in preclinical models of metabolic dysfunction. We assessed the metabolic ...effects of praliciguat in a mouse diet-induced obesity (DIO) model housed at thermoneutrality. At 6 weeks old, male C57BL/6N mice were either maintained on low-fat diet (LFD, lean mice) or placed on 60% high-fat diet (HFD, DIO mice). At 14 weeks old, the DIO mice were either maintained on HFD or switched to HFD with praliciguat (6-mg/kg). Day 28 samples were collected for biomarker analysis. In a second study under the same paradigm, indirect calorimetry was performed on days 8, 9, 20, 21, 32, and 33 and an oral lipid tolerance test (LTT) on day 38. Mice treated 28 days with praliciguat had lower levels of fasting plasma insulin, C-peptide, triglycerides, and HOMA-IR (homeostatic model assessment for insulin resistance) than DIO controls. In addition, energy expenditure was higher in praliciguat-treated than in DIO control mice on days 9, 20, 32, and 33; and day-38 triglycerides were lower. HFD-induced increases in gene expression of liver TNF-ɑ, lipoprotein lipase (
), and patatin-like phospholipase domain-containing protein 3 (
) in control DIO mice were attenuated in praliciguat-treated DIO mice. The positive metabolic effects observed in praliciguat-treated mice were associated with the restoration of liver PI3K (pAKT-Thr308) signaling, but not MAPK (pERK). In conclusion, praliciguat-treated DIO mice had increased energy utilization, improved insulin sensitivity, and lower plasma triglycerides. These results illustrate metabolic effects associated with praliciguat treatment in DIO mice.
The purpose of this work is to develop a mathematical model of energy balance and body weight regulation that can predict species-specific response to common pre-clinical interventions. To this end, ...we evaluate the ability of a previously published mathematical model of mouse metabolism to describe changes in body weight and body composition in rats in response to two short-term interventions. First, we adapt the model to describe body weight and composition changes in Sprague-Dawley rats by fitting to data previously collected from a 26-day caloric restriction study. The calibrated model is subsequently used to describe changes in rat body weight and composition in a 23-day cannabinoid receptor 1 antagonist (CB1Ra) study. While the model describes body weight data well, it fails to replicate body composition changes with CB1Ra treatment. Evaluation of a key model assumption about deposition of fat and fat-free masses shows a limitation of the model in short-term studies due to the constraint placed on the relative change in body composition components. We demonstrate that the model can be modified to overcome this limitation, and propose additional measurements to further test the proposed model predictions. These findings illustrate how mathematical models can be used to support drug discovery and development by identifying key knowledge gaps and aiding in the design of additional experiments to further our understanding of disease-relevant and species-specific physiology.
Drugs acting at cannabinoid type 1 receptors (CB1) have modulatory effects on glutamate and GABA neurotransmission in basal ganglia; thus, they potentially affect motor behavior in the parkinsonian ...setting. Preclinical trials with diverse cannabinoid agents have shown varied results, and the precise effects of blocking cannabinoid CB1 receptors remain uncertain. We tested behavioral effects of the selective antagonist 1-7-(2-chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo1,5-a-1,3,5triazin-4-yl-3-ethylaminoazetidine-3-carboxylic acid amide benzenesulfonate (CE) as monotherapy and in combination with l-DOPA in treatment-naive and L-DOPA-primed 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated rhesus monkeys with moderate and severe parkinsonism. Motor disability and L-DOPA-induced dyskinesias were scored with a standardized scale after subcutaneous drug administration, and plasma levels of L-DOPA were determined by high-performance liquid chromatography/electrochemical detection. CE doses ranged from 0.03 to 1 mg/kg, and L-DOPA methyl ester doses were selected as optimal and suboptimal doses (maximal and 50% of maximal responses, respectively). CE had no intrinsic effects on motor behavior regardless of the degree of parkinsonism (moderate or severe groups) or previous drug exposure ("de novo" or after L-DOPA priming). Initial CE administration did not affect development of L-DOPA antiparkinsonian responses. In coadministration trials, CE, in a dose-dependent manner, increased responses to L-DOPA (suboptimal doses). These effects were seen in both moderate and severely parkinsonian monkeys as a 30% increase of, predominantly, response duration with no effects on L-DOPA pharmacokinetics. CE did not modify levodopa-induced dyskinesias. These results suggest that selective cannabinoid CB1 antagonists may enhance the antiparkinsonian action of dopaminomimetics and possibly facilitate the use of lower doses, thereby reducing side effects.
Background Interventions for T2DM have in part aimed to mimic exercise. Here, we have compared the independent and combined effects of a PPARδ agonist and endurance training mimetic (GW501516) and a ...myostatin antibody and resistance training mimetic (PF-879) on metabolic and performance outcomes in obese insulin resistant mice. Methodology/Principal Findings Male ob/ob mice were treated for 6 weeks with vehicle, GW501516, PF-879, or GW501516 in combination with PF-879. The effects of the interventions on body composition, glucose homeostasis, glucose tolerance, energy expenditure, exercise capacity and metabolic gene expression were compared at the end of study. GW501516 attenuated body weight and fat mass accumulation and increased the expression of genes of oxidative metabolism. In contrast, PF-879 increased body weight by driving muscle growth and altered the expression of genes involved in insulin signaling and glucose metabolism. Despite their differences, both interventions alone improved glucose homeostasis. Moreover, GW501516 more effectively improved serum lipids, and PF-879 uniquely increased energy expenditure, exercise capacity and adiponectin levels. When combined the robust effects of GW501516 and/or PF-879 on body weight, adiposity, muscle mass, glycemia, serum lipids, energy expenditure and exercise capacity were highly conserved. Conclusions/Significance The data, for the first time, demonstrate postnatal inhibition of myostatin not only promotes gains in muscle mass similar to resistance training,but improves metabolic homeostasis. In several instances, these effects were either distinct from or complimentary to those of GW501516. The data further suggest that strategies to increase muscle mass, and not necessarily oxidative capacity, may effectively counter insulin resistance and T2DM.
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