Husam S. Younis

Underlying mitochondrial dysfunction triggers flutamide-induced oxidative liver injury in a mouse model of idiosyncratic drug toxicity.

Flutamide, a widely used nonsteroidal anti-androgen, but not its bioisostere bicalutamide, has been associated with idiosyncratic drug-induced liver injury. Although the susceptibility factors are unknown, mitochondrial injury has emerged as a putative hazard of flutamide. To explore the role of mitochondrial sensitization in flutamide hepatotoxicity, we determined the effects of superimposed drug stress in a murine model of underlying mitochondrial abnormalities. Male wild-type or heterozygous Sod2(+/-) mice were injected intraperitoneously with flutamide (0, 30 or 100 mg/kg/day) for 28 days. A kinetic pilot study revealed that flutamide (100 mg/kg/day) caused approximately 10-fold greater exposure than the reported therapeutic mean plasma levels. Mutant (5/10), but not wild-type, mice in the high-dose group exhibited small foci of hepatocellular necrosis and an increased number of apoptotic hepatocytes. Hepatic GSSG/GSH, protein carbonyl levels, and serum lactate levels were significantly increased, suggesting oxidant stress and mitochondrial dysfunction. Measurement of mitochondrial superoxide in cultured hepatocytes demonstrated that mitochondria were a significant source of flutamide-enhanced oxidant stress. Indeed, mitochondria isolated from flutamide-treated Sod2(+/-) mice exhibited decreased aconitase activity as compared to vehicle controls. A transcriptomics analysis using MitoChips revealed that flutamide-treated Sod2(+/-) mice exhibited a selective decrease in the expression of all complexes I and III subunits encoded by mitochondrial DNA. In contrast, Sod2(+/-) mice receiving bicalutamide (50 mg/kg/day) did not reveal any hepatic changes. These results are compatible with our concept that flutamide targets hepatic mitochondria and exerts oxidant stress that can lead to overt hepatic injury in the presence of an underlying mitochondrial abnormality.

PD0325901, a Mitogen-Activated Protein Kinase Kinase Inhibitor, Produces Ocular Toxicity in a Rabbit Animal Model of Retinal Vein Occlusion

OBJECTIVE PD0325901, a selective inhibitor of mitogen-activated protein kinase kinase (MEK), was associated with the occurrence of ocular retinal vein occlusion (RVO) during clinical trials in patients with solid tumors. As previous animal safety studies in rats and dogs did not identify the eye as a target organ of toxicity, this work was conducted to develop a rabbit model of ocular toxicity with PD0325901. METHODS Dutch-Belted rabbits were administered a single intravitreal injection of PD0325901 (0.5 or 1 mg/eye) or saline control, and ophthalmic examinations and retinal angiography were conducted over a 2-week period post-dose. In addition, mechanism of ocular toxicity was further explored in rat with microarray analysis. RESULTS PD0325901 treatment produced RVO with retinal vasculature leakage and hemorrhage within 48-h postinjection in Dutch-Belted rabbits. Subsequent retinal detachment and degeneration were also detected on day 8 postinjection. To evaluate the potential mechanism(s) of PD0325901-mediated RVO, male Brown Norway rats were orally administered PD0325901 (45 mg/kg/day) up to 5 days and retinal tissue was collected for gene array analysis. Although PD0325901 did not produce clinical evidence of RVO in rats, retinal gene expression suggested an increased oxidative stress and inflammatory response, endothelium and blood-retinal barrier damage, and prothrombotic effects. Moreover, soluble endothelial protein C receptor (sEPCR), a biomarker for RVO, was elevated in human umbilical vascular endothelial cells (HUVECs) cultured with PD0325901. CONCLUSIONS This work has developed a rabbit model of PD0325901-induced RVO that may be used to characterize the cellular and molecular mechanisms of this effect in humans.

Hepatotoxic Interaction of Sulindac with Lipopolysaccharide: Role of the Hemostatic System

Sulindac (SLD) is a nonsteroidal anti-inflammatory drug (NSAID) that has been associated with a greater incidence of idiosyncratic hepatotoxicity in human patients than other NSAIDs. One hypothesis regarding idiosyncratic adverse drug reactions is that interaction of a drug with a modest inflammatory episode precipitates liver injury. In this study, we tested the hypothesis that lipopolysaccharide (LPS) interacts with SLD to cause liver injury in rats. SLD (50 mg/kg) or its vehicle was administered to rats by gavage 15.5 h before LPS (8.3 x 10(5) endotoxin unit/kg) or its saline vehicle (i.v.). Thirty minutes after LPS treatment, SLD or vehicle administration was repeated. Rats were killed at various times after treatment, and serum, plasma, and liver samples were taken. Neither SLD nor LPS alone caused liver injury. Cotreatment with SLD/LPS led to increases in serum biomarkers of both hepatocellular injury and cholestasis. Histological evidence of liver damage was found only after SLD/LPS cotreatment. As a result of activation of hemostasis induced by SLD/LPS cotreatment, fibrin and hypoxia were present in liver tissue before the onset of hepatotoxicity. Heparin treatment reduced hepatic fibrin deposition and hypoxia and protected against liver injury induced by SLD/LPS cotreatment. These results indicate that cotreatment with nontoxic doses of LPS and SLD causes liver injury in rats, and this could serve as a model of human idiosyncratic liver injury. The hemostatic system is activated by SLD/LPS cotreatment and plays an important role in the development of SLD/LPS-induced liver injury.

Sulindac Metabolism and Synergy with Tumor Necrosis Factor-α in a Drug-Inflammation Interaction Model of Idiosyncratic Liver Injury

Sulindac (SLD) is a nonsteroidal anti-inflammatory drug (NSAID) that has been associated with a greater incidence of idiosyncratic hepatotoxicity in human patients than other NSAIDs. In previous studies, cotreatment of rats with SLD and a modestly inflammatory dose of lipopolysaccharide (LPS) led to liver injury, whereas neither SLD nor LPS alone caused liver damage. In studies presented here, further investigation of this animal model revealed that the concentration of tumor necrosis factor-α (TNF-α) in plasma was significantly increased by LPS at 1 h, and SLD enhanced this response. Etanercept, a soluble TNF-α receptor, reduced SLD/LPS-induced liver injury, suggesting a role for TNF-α. SLD metabolites in plasma and liver were determined by LC/MS/MS. Cotreatment with LPS did not increase the concentrations of SLD or its metabolites, excluding the possibility that LPS contributed to liver injury through enhanced exposure to SLD or its metabolites. The cytotoxicities of SLD and its sulfide and sulfone metabolites were compared in primary rat hepatocytes and HepG2 cells; SLD sulfide was more toxic in both types of cells than SLD or SLD sulfone. TNF-α augmented the cytotoxicity of SLD sulfide in primary hepatocytes and HepG2 cells. These results suggest that TNF-α can enhance SLD sulfide-induced hepatotoxicity, thereby contributing to liver injury in SLD/LPS-cotreated rats.

Sulindac metabolism and synergy with TNF in a drug-inflammation interaction model of idiosyncratic liver injury

Sulindac (SLD) is a nonsteroidal anti-inflammatory drug (NSAID) that has been associated with a greater incidence of idiosyncratic hepatotoxicity in human patients than other NSAIDs. In previous studies, cotreatment of rats with SLD and a modestly inflammatory dose of lipopolysaccharide (LPS) led to liver injury, whereas neither SLD nor LPS alone caused liver damage. In studies presented here, further investigation of this animal model revealed that the concentration of tumor necrosis factor-α (TNF-α) in plasma was significantly increased by LPS at 1 h, and SLD enhanced this response. Etanercept, a soluble TNF-α receptor, reduced SLD/LPS-induced liver injury, suggesting a role for TNF-α. SLD metabolites in plasma and liver were determined by LC/MS/MS. Cotreatment with LPS did not increase the concentrations of SLD or its metabolites, excluding the possibility that LPS contributed to liver injury through enhanced exposure to SLD or its metabolites. The cytotoxicities of SLD and its sulfide and sulfone metabolites were compared in primary rat hepatocytes and HepG2 cells; SLD sulfide was more toxic in both types of cells than SLD or SLD sulfone. TNF-α augmented the cytotoxicity of SLD sulfide in primary hepatocytes and HepG2 cells. These results suggest that TNF-α can enhance SLD sulfide-induced hepatotoxicity, thereby contributing to liver injury in SLD/LPS-cotreated rats.

Trovafloxacin, a fluoroquinolone antibiotic with hepatotoxic potential, causes mitochondrial peroxynitrite stress in a mouse model of underlying mitochondrial dysfunction

Trovafloxacin (TVX) is a fluoroquinolone antibiotic whose therapeutic use was severely restricted due to an unacceptable risk of idiosyncratic liver injury. Oxidative stress and mitochondrial injury have been implicated in fluoroquinolone toxicity, but the mechanisms underlying liver injury are poorly understood. Because TVX-induced hepatotoxicity cannot be modeled in normal healthy rodents, we asked whether an underlying genetic defect (heterozygous deficiency in mitochondrial superoxide dismutase, Sod2) might aggravate TVX-induced mitochondrial adverse effects. Wild-type and Sod2(+/-) mice were treated with vehicle or alatrofloxacin (the prodrug of TVX, 33mg/kg/day, ip) for 28 days. We found that hepatic protein carbonyls were increased by 2.5-fold and hepatic mitochondrial aconitase activity was decreased by 20% in mutant, but not wild-type mice. Because aconitase is a major target of peroxynitrite, we determined the extent of nitrotyrosine residues in hepatic mitochondrial proteins. Trovafloxacin significantly increased nitrotyrosine in Sod2(+/-) mice only. Using the NO-selective probe DAF-2, we found that TVX increased the production of mitochondrial NO in immortalized human hepatocytes. Similarly, mitochondrial Ca(2+) was increased by TVX, suggesting Ca(2+)-dependent activation of mitochondrial NOS activity. Furthermore, the transcript levels of the mtDNA-encoded gene Cox2/mtCo2 were decreased in Sod2(+/-) mice only, while the expression of nDNA-encoded mitochondrial genes was not significantly altered in both genotypes, suggesting selective effects on mtDNA expression. The amount of mtDNA (copy number) was, however, unchanged. These data indicate that TVX enhances hepatic mitochondrial peroxynitrite stress in mice with underlying increased basal levels of superoxide, leading to the disruption of critical mitochondrial enzymes and gene regulation.

Oxidative stress is important in the pathogenesis of liver injury induced by sulindac and lipopolysaccharide cotreatment

Among currently prescribed nonsteroidal anti-inflammatory drugs, sulindac (SLD) is associated with the greatest incidence of idiosyncratic hepatotoxicity in humans. Previously, an animal model of SLD-induced idiosyncratic hepatotoxicity was developed by cotreating rats with a nonhepatotoxic dose of LPS. Tumor necrosis factor-alpha (TNF) was found to be critically important to the pathogenesis. In this study, the mechanism of liver injury induced by SLD/LPS cotreatment was further explored. Protein carbonyls, products of oxidative stress, were elevated in liver mitochondria of SLD/LPS-cotreated rats. The results of analyzing gene expression in livers of rats before the onset of liver injury indicated that genes associated with oxidative stress were selectively regulated by SLD/LPS cotreatment. Antioxidant treatment with either ebselen or dimethyl sulfoxide attenuated SLD/LPS-induced liver injury. The role of oxidative stress was further investigated in vitro. SLD sulfide, the toxic metabolite of SLD, enhanced TNF-induced cytotoxicity and caspase 3/7 activity in HepG2 cells. SLD sulfide also increased dichlorofluorescein fluorescence, suggesting generation of reactive oxygen species (ROS). Hydrogen peroxide and TNF cotreatment of HepG2 cells caused greater cytotoxicity than either treatment alone. Either antioxidant tempol or a pancaspase inhibitor Z-VAD-FMK decreased cell death as well as caspase 3/7 activity induced by SLD sulfide/TNF coexposure. These results indicate that SLD/LPS treatment causes oxidative stress in livers of rats and suggest that ROS are important in SLD/LPS-induced liver injury in vivo. Furthermore, ROS contribute to the cytotoxic interaction of SLD and TNF by activating caspase 3/7.

Neutrophil-cytokine interactions in a rat model of sulindac-induced idiosyncratic liver injury.

Previous studies indicated that lipopolysaccharide (LPS) interacts with the nonsteroidal anti-inflammatory drug sulindac (SLD) to produce liver injury in rats. In the present study, the mechanism of SLD/LPS-induced liver injury was further investigated. Accumulation of polymorphonuclear neutrophils (PMNs) in the liver was greater in SLD/LPS-cotreated rats compared to those treated with SLD or LPS alone. In addition, PMN activation occurred specifically in livers of rats cotreated with SLD/LPS. The hypothesis that PMNs and proteases released from them play critical roles in the hepatotoxicity was tested. SLD/LPS-induced liver injury was attenuated by prior depletion of PMNs or by treatment with the PMN protease inhibitor, eglin C. Previous studies suggested that tumor necrosis factor-α (TNF) and the hemostatic system play critical roles in the pathogenesis of liver injury induced by SLD/LPS. TNF and plasminogen activator inhibitor-1 (PAI-1) can contribute to hepatotoxicity by affecting PMN activation and fibrin deposition. Therefore, the role of TNF and PAI-1 in PMN activation and fibrin deposition in the SLD/LPS-induced liver injury model was tested. Neutralization of TNF or inhibition of PAI-1 attenuated PMN activation. TNF had no effect on PAI-1 production or fibrin deposition. In contrast, PAI-1 contributed to fibrin deposition in livers of rats treated with SLD/LPS. In summary, PMNs, TNF and PAI-1 contribute to the liver injury induced by SLD/LPS cotreatment. TNF and PAI-1 independently contributed to PMN activation, which is critical to the pathogenesis of liver injury. Moreover, PAI-1 contributed to liver injury by promoting fibrin deposition.

l-Carnosine: multifunctional dipeptide buffer for sustained-duration topical ophthalmic formulations

Objectives The use of L‐carnosine as an excipient in topical ophthalmic formulations containing gellan gum, a carbohydrate polymer with in‐situ gelling properties upon mixing with mammalian tear fluid, was developed as a novel platform to extend precorneal duration. Specific utilisation of L‐carnosine as a buffer in gellan gum carrying vehicles was characterised.

An Assessment of the Ocular Safety of Inactive Excipients Following Sub-Tenon Injection in Rabbits

PURPOSE This work characterized the safety and toleration of inactive excipients following sub-Tenon (ST) administration. METHODS Rabbits were anesthetized and eyes received an ST injection of the following test excipients: carboxy methylcellulose (CMC; low [90 kDa], mid [250 kDa], and high [700 kDa] molecular weight [MW], 0.25%-1.0% w/v), polysorbate 80 (0.02 and 0.2% w/v), polyethylene glycol 3350 (PEG; 0.2 and 1.0% w/v), poloxamer 188 (0.01 and 0.25% w/v), poloxamer 182 (2% w/v), benzyl alcohol (BA; 4% w/v), benzalkonium chloride (BAC; 0.02%, 0.04%, and 0.05% w/v), and methylcellulose (MC; 0.25% w/v). After a 1-week observation period for clinical signs of ocular tolerability, the animals were euthanized and eyes were collected for histologic examination. RESULTS The ocular tolerability of the tested excipients were ranked as follows from the innocuous to most deleterious: saline approximately PEG (1% w/v) approximately polysorbate 80 (0.2% w/v) > CMC (0.25% w/v, 90 kDa) > MC (0.25% w/v) approximately poloxomer 188 (0.25% w/v) approximately sodium citrate (pH 9) BAC (0.05% w/v) > CMC (0.5% w/v, 700 kDa) > poloxomer 182 (2% w/v) > BA (4% w/v). Clinical signs of ocular irritation were limited to redness and chemosis observed with most test excipients. The BA excipient also produced corneal opacity. Microscopic findings included histiocytic infiltration (BAC, BA, CMC, MC, and poloxamer 188), heterophilic inflammation (BA, CMC, and poloxamer 182), and edema (BAC, BA, CMC, and poloxamer 182) in episcleral tissue. The severity of the clinical and hisopathologic effects increased with the concentration of the test excipients administered. CONCLUSIONS This research has evaluated the safety profile of inactive excipients that may be used to formulate new chemical entities for the treatment of ocular disease following a ST injection.