Effect of renal function and dialysis modality on daprodustat and predominant metabolite exposure.

BACKGROUND: Current therapies for anemia of chronic kidney disease (CKD) include administration of supplemental iron (intravenous and/or oral), blood transfusions and replacement of erythropoietin through the administration of recombinant human erythropoietin (rhEPO) and rhEPO analogs, each with limitations. Daprodustat is an orally active, small molecule hypoxia-inducible factor-prolyl hydroxylase inhibitor that is currently in Phase 3 clinical studies. As it is well appreciated that the kidney represents a major route of elimination of many drugs, and daprodustat will be administered to patients with advanced CKD as well as patients with end-stage kidney disease, it is important to characterize the pharmacokinetic profile in these patient populations to safely dose this potential new medicine. METHODS: The primary objective of these studies, conducted under two separate protocols and with identical assessments and procedures, was to characterize the steady-state pharmacokinetics of daprodustat and the six predominant metabolites (i.e. metabolites present in the highest concentration in circulation) in subjects with normal renal function, anemic non-dialysis (ND)-dependent CKD subjects (CKD Stage 3/4) and anemic subjects on either hemodialysis (HD) or peritoneal dialysis (PD). All enrolled subjects were administered daprodustat 5 mg once daily for 14 days (all except HD subjects) or 15 days (for HD subjects). Blood, urine and peritoneal dialysate were collected at various times for measurement of daprodustat, predominant metabolite, erythropoietin and hepcidin levels. RESULTS: The pharmacokinetic properties of steady-state daprodustat peak plasma concentration (Cmax), area under the plasma daprodustat concentration-time curve (AUC) and the time of Cmax (tmax) were comparable between all cohorts in this study. In addition, there was no clinically relevant difference in these properties in the HD subjects between a dialysis and ND day. For CKD Stage 3/4, HD (dialysis day) and PD subjects, the AUC of all daprodustat metabolites assessed was higher, while the C max was slightly higher than that in subjects with normal renal function. Over the course of the 14 or 15 days of daprodustat administration, hemoglobin levels were seen to be relatively stable in the subjects with normal renal function, CKD Stage 3/4 and PD subjects, while HD subjects had a decrease of 1.9 gm/dL. All renally impaired subjects appeared to have similar erythropoietin responses to daprodustat, with approximately a 3-fold increase in these levels. In subjects with minimal to no change in hemoglobin levels, hepcidin levels remained relatively stable. Daprodustat, administered 5 mg once daily for 14-15 days, was generally well tolerated with a safety profile consistent with this patient population. CONCLUSION: These studies demonstrated no clinically meaningful change in the pharmacokinetic properties of daprodustat when administered to subjects with various degrees of renal impairment, while for CKD Stage 3/4, HD (dialysis day) and PD subjects, the C max and AUC of all daprodustat metabolites assessed were higher than in subjects with normal renal function. Administration of daprodustat in this study appeared to be generally safe and well tolerated.

Bioequivalence Studies of a Reformulated Dutasteride and Tamsulosin Hydrochloride Combination Capsule and a Commercially Available Formulation.

A dutasteride 0.5 mg and tamsulosin hydrochloride 0.4 mg combination (DTC) capsule (Duodart® ) was reformulated to reduce the capsule size and enhance product stability. Bioequivalence of the reformulated DTC capsule with the commercial formulation was evaluated in 2 single-dose, open-label, randomized, 2-way crossover studies in healthy adult male volunteers. Subjects in a fasted or fed state received a single oral dose of either the reformulated DTC or the commercial formulation followed by a 28-day washout period between treatments. Blood samples were taken predose and up to 72 hours postdose for pharmacokinetic (PK) analysis of dutasteride and tamsulosin serum concentrations. From the serum concentration-vs-time data, a noncompartmental method was used to calculate the maximum observed serum concentration (Cmax ) and area under the serum concentration-time curve (AUC0-t ) for dutasteride and tamsulosin, and AUC0-∞ for tamsulosin. The 90% confidence intervals for the ratios of the Cmax and AUC0-t (for dutasteride and tamsulosin) and for AUC0-∞ (for tamsulosin) were all completely contained within the range of 80% to 125%; therefore, the reformulated DTC capsule is bioequivalent to the commercial formulation under both fed and fasted states.

Temporal profiles of cytoskeletal protein loss following traumatic axonal injury in mice.

To examine the time course and relative extent of proteolysis of neurofilament and tubulin proteins after traumatic axonal injury (TAI), anesthetized mice were subjected to optic nerve stretch injury. Immunohistochemistry confirmed neurofilament accumulation within axonal swellings at 4, 24, and 72 h postinjury (n = 4 injured and 2 sham per time point). Immunoblotting of optic nerve homogenates (n = 5 injured and 1 sham at 0.5, 4, 24 or 72 h) revealed calpain-mediated spectrin proteolytic fragments after injury. Protein levels for NF68 progressively decreased from 0.5 h to 24 h postinjury, while NF200 and alpha-tubulin levels decreased acutely (0.5-4 h), with a secondary decline at 72 h postinjury. These data demonstrate that diffusely distributed TAI is associated not only with a localized accumulation of neurofilament proteins, but also significant decreases in total cytoskeletal protein levels which may be mediated, in part, by calpains. Protection of the axonal cytoskeleton represents a potential therapeutic target for axonal damage associated with injury or neurodegenerative diseases.

Mechanisms of cell death and neuroprotection by poloxamer 188 after mechanical trauma.

The mechanisms of cell death and the progressive degeneration of neural tissue following traumatic brain injury (TBI) have come under intense investigation. However, the complex interactions among the evolving pathologies in multiple cell types obscure the causal relationships between the initial effects of the mechanical trauma at the cellular level and the long-term dysfunction and neuronal death. We used an in vitro model of neuronal injury to study the mechanisms of cell death in response to a well-defined mechanical insult and found that the majority of dead cells were apoptotic. We have previously reported that promotion of membrane repair acutely with the non-ionic surfactant poloxamer 188 (P188) restored cell viability to control values at 24 h postinjury. Here, we showed that P188 significantly inhibits apoptosis and prevents necrosis. We also examined the role of mitogen-activated protein kinases (MAPKs) in cell death. There was a rapid, transient activation of extracellular signal-regulated kinases, c-Jun N-terminal kinase, and p38s after mechanical insult. Of these, activation of the proapoptotic p38 was the greatest. Treatment with P188 inhibited p38 activation; however, direct inhibition of p38 by SB203580, which selectively inhibits the activity of the p38 MAPK, provided only partial inhibition of apoptosis and had no effect on necrosis. These data suggest that multiple signaling pathways may be involved in the long-term response of neurons to mechanical injury. Furthermore, that the membrane resealing action of P188 provides such significant protection from both necrosis and apoptosis suggests that acute membrane damage due to trauma is a critical precipitating event that is upstream of the many signaling cascades contributing to the subsequent pathology.

The effect of poloxamer-188 on neuronal cell recovery from mechanical injury.

Neuronal injury resulting from mechanical deformation is poorly characterized at the cellular level. The immediate structural consequences of the mechanical loading lead to a variety of inter- and intra-cellular signaling events that interact on multiple time and length scales. Thus, it is often difficult to establish cause-and-effect relationships such that appropriate treatment strategies can be devised. In this report, we showed that treating mechanically injured neuronal cells with an agent that promotes the resealing of disrupted plasma membranes rescues them from death at 24 h post-injury. A new in vitro model was developed to allow uniform mechanical loading conditions with precisely controlled magnitude and onset rate of loading. Injury severity increased monotonically with increasing peak shear stress and was strongly dependent on the rate of loading as assessed with the MTT cell viability assay, 24 h post-injury. Mechanical injury produced an immediate disruption of membrane integrity as indicated by a rapid and transient release of LDH. For the most severe injury, cell viability decreased approximately 40% with mechanical trauma compared to sham controls. Treatment of cells with Poloxamer 188 at 15 min post-injury restored long-term viability to control values. These data establish membrane integrity as a novel therapeutic target in the treatment of neuronal injury.