Safety evaluation of the interchangeable use of robenacoxib in commercially-available tablets and solution for injection in cats.

BACKGROUND: Robenacoxib (Onsior™) is a non-steroidal anti-inflammatory drug developed for canine and feline use for the control of pain and inflammation. It is available as both tablets and solution for injection. The objective of this study was to evaluate the safety of the interchangeable use of commercially available robenacoxib formulations when administered to cats orally using 6 mg tablets and subcutaneously using a solution for injection containing 20 mg/mL. Thirty-four naïve healthy 4-month old cats were enrolled in this 37-day study and were randomized to four groups (three robenacoxib and one control). One robenacoxib group received the maximum recommended dose (MRD) rate of each formulation, while the other two received two and three times this dose rate. The cats underwent three 10-day treatment cycles comprised of seven days of once daily oral administration followed by three days of subcutaneous administration. The third cycle was followed by an additional seven days of oral treatment. The control group received oral empty gelatin capsules or subcutaneous saline injections. Assessment of safety was based on general health observations, clinical observations, physical, ophthalmic, electrocardiographic and neurological examinations, clinical pathology evaluations, food consumption, body weight, and macroscopic and microscopic examinations. Blood samples were collected for toxicokinetic evaluation. RESULTS: Blood concentrations of robenacoxib confirmed systemic exposure of all treated cats. All cats were in good health through study termination and there were no serious adverse events during the study. There were no changes in body weight, food consumption, ophthalmic, physical or neurological examinations during the study. Treatment-related abnormalities were of low occurrence at all doses and included injection site changes (transient edema with minimal or mild, subacute/chronic inflammation histologically) and prolongation of the QT interval. These findings were consistent with previously observed findings in studies with robenacoxib administered separately orally or subcutaneously in cats. Thus, there were no adverse effects that could be attributed specifically to the interchangeable use of oral and injectable robenacoxib. CONCLUSIONS: This 37-day laboratory study supports the safety of interchanging robenacoxib injection at a daily dose of 2 mg/kg with robenacoxib tablets at a daily dose of 1 mg/kg, or vice versa.

Urinary mitochondrial DNA is a biomarker of mitochondrial disruption and renal dysfunction in acute kidney injury.

Recent studies show the importance of mitochondrial dysfunction in the initiation and progression of acute kidney injury (AKI). However, no biomarkers exist linking renal injury to mitochondrial function and integrity. To this end, we evaluated urinary mitochondrial DNA (UmtDNA) as a biomarker of renal injury and function in humans with AKI following cardiac surgery. mtDNA was isolated from the urine of patients following cardiac surgery and quantified by quantitative PCR. Patients were stratified into no AKI, stable AKI, and progressive AKI groups based on Acute Kidney Injury Network (AKIN) staging. UmtDNA was elevated in progressive AKI patients and was associated with progression of patients with AKI at collection to higher AKIN stages. To evaluate the relationship of UmtDNA to measures of renal mitochondrial integrity in AKI, mice were subjected to sham surgery or varying degrees of ischemia followed by 24 h of reperfusion. UmtDNA increased in mice after 10-15 min of ischemia and positively correlated with ischemia time. Furthermore, UmtDNA was predictive of AKI in the mouse model. Finally, UmtDNA levels were negatively correlated with renal cortical mtDNA and mitochondrial gene expression. These translational studies demonstrate that UmtDNA is associated with recovery from AKI following cardiac surgery by serving as an indicator of mitochondrial integrity. Thus UmtDNA may serve as valuable biomarker for the development of mitochondrial-targeted therapies in AKI.

Suppression of mitochondrial biogenesis through toll-like receptor 4-dependent mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling in endotoxin-induced acute kidney injury.

Although disruption of mitochondrial homeostasis and biogenesis (MB) is a widely accepted pathophysiologic feature of sepsis-induced acute kidney injury (AKI), the molecular mechanisms responsible for this phenomenon are unknown. In this study, we examined the signaling pathways responsible for the suppression of MB in a mouse model of lipopolysaccharide (LPS)-induced AKI. Downregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of MB, was noted at the mRNA level at 3 hours and protein level at 18 hours in the renal cortex, and was associated with loss of renal function after LPS treatment. LPS-mediated suppression of PGC-1α led to reduced expression of downstream regulators of MB and electron transport chain proteins along with a reduction in renal cortical mitochondrial DNA content. Mechanistically, Toll-like receptor 4 (TLR4) knockout mice were protected from renal injury and disruption of MB after LPS exposure. Immunoblot analysis revealed activation of tumor progression locus 2/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (TPL-2/MEK/ERK) signaling in the renal cortex by LPS. Pharmacologic inhibition of MEK/ERK signaling attenuated renal dysfunction and loss of PGC-1α, and was associated with a reduction in proinflammatory cytokine (e.g., tumor necrosis factor-α [TNF-α], interleukin-1ß) expression at 3 hours after LPS exposure. Neutralization of TNF-α also blocked PGC-1α suppression, but not renal dysfunction, after LPS-induced AKI. Finally, systemic administration of recombinant tumor necrosis factor-α alone was sufficient to produce AKI and disrupt mitochondrial homeostasis. These findings indicate an important role for the TLR4/MEK/ERK pathway in both LPS-induced renal dysfunction and suppression of MB. TLR4/MEK/ERK/TNF-α signaling may represent a novel therapeutic target to prevent mitochondrial dysfunction and AKI produced by sepsis.

Renal cortical hexokinase and pentose phosphate pathway activation through the EGFR/Akt signaling pathway in endotoxin-induced acute kidney injury.

While disruption of energy production is an important contributor to renal injury, metabolic alterations in sepsis-induced AKI remain understudied. We assessed changes in renal cortical glycolytic metabolism in a mouse model of sepsis-induced AKI. A specific and rapid increase in hexokinase (HK) activity (∼2-fold) was observed 3 h after LPS exposure and maintained up to 18 h, in association with a decline in renal function as measured by blood urea nitrogen (BUN). LPS-induced HK activation occurred independently of HK isoform expression or mitochondrial localization. No other changes in glycolytic enzymes were observed. LPS-mediated HK activation was not sufficient to increase glycolytic flux as indicated by reduced or unchanged pyruvate and lactate levels in the renal cortex. LPS-induced HK activation was associated with increased glucose-6-phosphate dehydrogenase activity but not glycogen production. Mechanistically, LPS-induced HK activation was attenuated by pharmacological inhibitors of the EGF receptor (EGFR) and Akt, indicating that EGFR/phosphatidylinositol 3-kinase/Akt signaling is responsible. Our findings reveal LPS rapidly increases renal cortical HK activity in an EGFR- and Akt-dependent manner and that HK activation is linked to increased pentose phosphate pathway activity.

Formoterol restores mitochondrial and renal function after ischemia-reperfusion injury.

Mitochondrial biogenesis may be an adaptive response necessary for meeting the increased metabolic and energy demands during organ recovery after acute injury, and renal mitochondrial dysfunction has been implicated in the pathogenesis of AKI. We proposed that stimulation of mitochondrial biogenesis 24 hours after ischemia/reperfusion (I/R)-induced AKI, when renal dysfunction is maximal, would accelerate recovery of mitochondrial and renal function in mice. We recently showed that formoterol, a potent, highly specific, and long-acting ß2-adrenergic agonist, induces renal mitochondrial biogenesis in naive mice. Animals were subjected to sham or I/R-induced AKI, followed by once-daily intraperitoneal injection with vehicle or formoterol beginning 24 hours after surgery and continuing through 144 hours after surgery. Treatment with formoterol restored renal function, rescued renal tubules from injury, and diminished necrosis after I/R-induced AKI. Concomitantly, formoterol stimulated mitochondrial biogenesis and restored the expression and function of mitochondrial proteins. Taken together, these results provide proof of principle that a novel drug therapy to treat AKI, and potentially other acute organ failures, works by restoring mitochondrial function and accelerating the recovery of renal function after injury has occurred.

Suppressed mitochondrial biogenesis in folic acid-induced acute kidney injury and early fibrosis.

Acute kidney injury (AKI) is a disease with mitochondrial dysfunction and a newly established risk factor for the development of chronic kidney disease (CKD) and fibrosis. We examined mitochondrial homeostasis in the folic acid (FA)-induced AKI model that develops early fibrosis over a rapid time course. Mice given a single dose of FA had elevated serum creatinine (3-fold) and urine glucose (2.2-fold) 1 and 2 d after injection that resolved by 4d. In contrast, peroxisome proliferator gamma coactivator 1α (PGC-1α) and mitochondrial transcription factor A (TFAM), critical transcriptional regulators of mitochondrial biogenesis (MB), were down-regulated ∼80% 1d after FA injection and remained depressed through 14 d. Multiple electron transport chain and ATP synthesis genes were also down-regulated from 1 to 14 d after FA, including NADH dehydrogenase (ubiquinone) 1 beta subcomplex 8 (NDUFß8), ATP synthase subunit ß (ATPS-ß), and cytochrome C oxidase subunit I (COXI). Mitochondrial DNA copy number was reduced ∼50% from 2 to 14 d after FA injection. Protein levels of early fibrosis markers α-smooth muscle actin and transforming growth factor ß1 were elevated at 6 and 14 d after FA. Picrosirius red staining and collagen 1A2 (COL1A2) IHC revealed staining for mature collagen deposition at 14 d. We propose that mitochondrial dysfunction induced by AKI is a persistent cellular injury that promotes progression to fibrosis and CKD, and that this model can be used to test mitochondrial therapeutics that limit progression to fibrosis and CKD.

cGMP-selective phosphodiesterase inhibitors stimulate mitochondrial biogenesis and promote recovery from acute kidney injury.

Recent studies demonstrate that mitochondrial dysfunction is a mediator of acute kidney injury (AKI). Consequently, restoration of mitochondrial function after AKI may be key to the recovery of renal function. Mitochondrial function can be restored through the generation of new, functional mitochondria in a process called mitochondrial biogenesis (MB). Despite its potential therapeutic significance, very few pharmacological agents have been identified to induce MB. To examine the efficacy of phosphodiesterase (PDE) inhibitors (PDE3: cAMP and cGMP activity; and PDE4: cAMP activity) in stimulating MB, primary cultures of renal proximal tubular cells (RPTCs) were treated with a panel of inhibitors for 24 hours. PDE3, but not PDE4, inhibitors increased the FCCP-uncoupled oxygen consumption rate (OCR), a marker of MB. Exposure of RPTCs to the PDE3 inhibitors, cilostamide and trequinsin, for 24 hours increased peroxisome proliferator-activated receptor γ coactivator-1α, and multiple mitochondrial electron transport chain genes. Cilostamide and trequinsin also increased mRNA expression of mitochondrial genes and mitochondrial DNA copy number in mice renal cortex. Consistent with these experiments, 8-Br-cGMP increased FCCP-uncoupled OCR and mitochondrial gene expression, whereas 8-Br-cAMP had no effect. The cGMP-specific PDE5 inhibitor sildenafil also induced MB in RPTCs and in vivo in mouse renal cortex. Treatment of mice with sildenafil after folic acid-induced AKI promoted restoration of MB and renal recovery. These data provide strong evidence that specific PDE inhibitors that increase cGMP are inducers of MB in vitro and in vivo, and suggest their potential efficacy in AKI and other diseases characterized by mitochondrial dysfunction and suppressed MB.

Mitochondrial Homeostasis in Acute Organ Failure.

The kidneys compose approximately 0.5% of the body mass but consume about 10% of the oxygen in cellular respiration. This discordance is due to the high energy demands on the kidney for reabsorption of filtered blood components and makes the kidney sensitive to mitochondrial stress, the primary source of cellular ATP. Regardless of the etiology, acute kidney injury (AKI) almost always involves aspects of mitochondrial dysfunction. Recent evidence from experimental models suggests that preserving mitochondrial function or promoting mitochondrial repair rescues renal function during AKI. In this review we discuss the effect of AKI on disruption of mitochondrial homeostasis, and how the dynamic processes of mitochondrial biogenesis, fission/fusion, and mitophagy influence renal injury and recovery.

DNA polymerase eta participates in the mutagenic bypass of adducts induced by benzo[a]pyrene diol epoxide in mammalian cells.

Y-family DNA-polymerases have larger active sites that can accommodate bulky DNA adducts allowing them to bypass these lesions during replication. One member, polymerase eta (pol eta), is specialized for the bypass of UV-induced thymidine-thymidine dimers, correctly inserting two adenines. Loss of pol eta function is the molecular basis for xeroderma pigmentosum (XP) variant where the accumulation of mutations results in a dramatic increase in UV-induced skin cancers. Less is known about the role of pol eta in the bypass of other DNA adducts. A commonly encountered DNA adduct is that caused by benzo[a]pyrene diol epoxide (BPDE), the ultimate carcinogenic metabolite of the environmental chemical benzo[a]pyrene. Here, treatment of pol eta-deficient fibroblasts from humans and mice with BPDE resulted in a significant decrease in Hprt gene mutations. These studies in mammalian cells support a number of in vitro reports that purified pol eta has error-prone activity on plasmids with site-directed BPDE adducts. Sequencing the Hprt gene from this work shows that the majority of mutations are G>T transversions. These data suggest that pol eta has error-prone activity when bypassing BPDE-adducts. Understanding the basis of environmental carcinogen-derived mutations may enable prevention strategies to reduce such mutations with the intent to reduce the number of environmentally relevant cancers.

Translesion synthesis polymerases in the prevention and promotion of carcinogenesis.

A critical step in the transformation of cells to the malignant state of cancer is the induction of mutations in the DNA of cells damaged by genotoxic agents. Translesion DNA synthesis (TLS) is the process by which cells copy DNA containing unrepaired damage that blocks progression of the replication fork. The DNA polymerases that catalyze TLS in mammals have been the topic of intense investigation over the last decade. DNA polymerase η (Pol η) is best understood and is active in error-free bypass of UV-induced DNA damage. The other TLS polymerases (Pol ι, Pol κ, REV1, and Pol ζ) have been studied extensively in vitro, but their in vivo role is only now being investigated using knockout mouse models of carcinogenesis. This paper will focus on the studies of mice and humans with altered expression of TLS polymerases and the effects on cancer induced by environmental agents.

Micro- and nanotechnology approaches for capturing circulating tumor cells.

Circulating tumor cells (CTC) are cells that have detached from primary tumors and circulate in the bloodstream where they are carried to other organs, leading to seeding of new tumors and metastases. CTC have been known to exist in the bloodstream for more than a century. With recent progress in the area of micro- and nanotechnology, it has been possible to adopt new approaches in CTC research. Microscale and nanoscale studies can throw some light on the time course of CTC appearance in blood and CTC overexpression profiles for cancer-related markers and galvanize the development of drugs to block metastases. CTC counts could serve as endpoint biomarkers and as prognostic markers for patients with a metastatic disease. This paper reviews some of the recent researches on using micro- and nanotechnology to capture and profile CTC.