Targeted tissue delivery of RNA therapeutics using antibody-oligonucleotide conjugates (AOCs).

Although targeting TfR1 to deliver oligonucleotides to skeletal muscle has been demonstrated in rodents, effectiveness and pharmacokinetic/pharmacodynamic (PKPD) properties remained unknown in higher species. We developed antibody-oligonucleotide conjugates (AOCs) towards mice or monkeys utilizing anti-TfR1 monoclonal antibodies (αTfR1) conjugated to various classes of oligonucleotides (siRNA, ASOs and PMOs). αTfR1 AOCs delivered oligonucleotides to muscle tissue in both species. In mice, αTfR1 AOCs achieved a > 15-fold higher concentration to muscle tissue than unconjugated siRNA. A single dose of an αTfR1 conjugated to an siRNA against Ssb mRNA produced > 75% Ssb mRNA reduction in mice and monkeys, and mRNA silencing was greatest in skeletal and cardiac (striated) muscle with minimal to no activity in other major organs. In mice the EC50 for Ssb mRNA reduction in skeletal muscle was >75-fold less than in systemic tissues. Oligonucleotides conjugated to control antibodies or cholesterol produced no mRNA reduction or were 10-fold less potent, respectively. Tissue PKPD of AOCs demonstrated mRNA silencing activity primarily driven by receptor-mediated delivery in striated muscle for siRNA oligonucleotides. In mice, we show that AOC-mediated delivery is operable across various oligonucleotide modalities. AOC PKPD properties translated to higher species, providing promise for a new class of oligonucleotide therapeutics.

Safety Assessment of Formulation Vehicles Following Intravitreal Administration in Rabbits.

PURPOSE: Evaluate 21 formulation vehicles administered to rabbits after intravitreal injection for tolerability and safety. METHODS: Forty-two Dutch Belted rabbits were anesthetized, and the eyes received a single intravitreal injection of the excipient formulation. Clinical signs and ocular irritation responses were recorded twice daily for 7 days and microscopic evaluation of the eyes, optic nerve, and eyelids was completed at 1-week post treatment. RESULTS: Saline (≥ 300 mOsm and ≤ 592 mOsm at pH 7.0 or 300 mOsm at pH 8.0) and 10 formulation excipients; (10% w/v PEG 3350 at pH 7.4, 1% polysorbate 21 at pH 7.4, PVA at pH 7.0, 0.2% polysorbate 80 at pH 7.2, 0.2% Pluronic F108® at pH 7.3, 2%, 100 mM sodium sulfate at pH 3.2, 2 mM sodium glycocholate at pH 7.4, and 275 mM D-mannitol pH 7.0 in sterile water, and 100 mM sodium phosphate in combination with 0.9% NaCl 300 mOsm and 0.01% or 0.05% polysorbate 80 at pH 7.4) considered as formulation vehicles for intravitreal injectables, were well-tolerated in rabbits. Clinical signs were transient and microscopic changes were not observed. CONCLUSIONS: Of the 21 formulation vehicles evaluated, 10 formulation vehicles were well-tolerated in rabbits and feasible candidates for future investigations.

Integrated Safety Assessment of 2'-O-Methoxyethyl Chimeric Antisense Oligonucleotides in NonHuman Primates and Healthy Human Volunteers.

The common chemical and biological properties of antisense oligonucleotides provide the opportunity to identify and characterize chemical class effects across species. The chemical class that has proven to be the most versatile and best characterized is the 2'-O-methoxyethyl chimeric antisense oligonucleotides. In this report we present an integrated safety assessment of data obtained from controlled dose-ranging studies in nonhuman primates (macaques) and healthy human volunteers for 12 unique 2'-O-methoxyethyl chimeric antisense oligonucleotides. Safety was assessed by the incidence of safety signals in standardized laboratory tests for kidney and liver function, hematology, and complement activation; as well as by the mean test results as a function of dose level over time. At high doses a number of toxicities were observed in nonhuman primates. However, no class safety effects were identified in healthy human volunteers from this integrated data analysis. Effects on complement in nonhuman primates were not observed in humans. Nonhuman primates predicted safe doses in humans, but over predicted risk of complement activation and effects on platelets. Although limited to a single chemical class, comparisons from this analysis are considered valid and accurate based on the carefully controlled setting for the specified study populations and within the total exposures studied.

Identification and characterization of modified antisense oligonucleotides targeting DMPK in mice and nonhuman primates for the treatment of myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults. DM1 is caused by an expanded CTG repeat in the 3'-untranslated region of DMPK, the gene encoding dystrophia myotonica protein kinase (DMPK). Antisense oligonucleotides (ASOs) containing 2',4'-constrained ethyl-modified (cEt) residues exhibit a significantly increased RNA binding affinity and in vivo potency relative to those modified with other 2'-chemistries, which we speculated could translate to enhanced activity in extrahepatic tissues, such as muscle. Here, we describe the design and characterization of a cEt gapmer DMPK ASO (ISIS 486178), with potent activity in vitro and in vivo against mouse, monkey, and human DMPK. Systemic delivery of unformulated ISIS 486718 to wild-type mice decreased DMPK mRNA levels by up to 90% in liver and skeletal muscle. Similarly, treatment of either human DMPK transgenic mice or cynomolgus monkeys with ISIS 486178 led to up to 70% inhibition of DMPK in multiple skeletal muscles and ∼50% in cardiac muscle in both species. Importantly, inhibition of DMPK was well tolerated and was not associated with any skeletal muscle or cardiac toxicity. Also interesting was the demonstration that the inhibition of DMPK mRNA levels in muscle was maintained for up to 16 and 13 weeks post-treatment in mice and monkeys, respectively. These results demonstrate that cEt-modified ASOs show potent activity in skeletal muscle, and that this attractive therapeutic approach warrants further clinical investigation to inhibit the gain-of-function toxic RNA underlying the pathogenesis of DM1.

Antisense inhibition of coagulation factor XI prolongs APTT without increased bleeding risk in cynomolgus monkeys.

A strategy to produce sufficient anticoagulant properties with reduced risk of bleeding may be possible through inhibition of factor XI (FXI), a component of the intrinsic coagulation cascade. The objective of this work was to determine the safety profile of ISIS 416858, a 2'-methoxyethoxy (2'-MOE) antisense oligonucleotide inhibitor of FXI, with focus on assessment of bleeding risk. Cynomolgus monkeys administered ISIS 416858 (4, 8, 12, and 40 mg/kg/wk, subcutaneous) for up to 13 weeks produced a dose-dependent reduction in FXI (mRNA in liver and plasma activity) and a concomitant increase in activated partial thromboplastin time (APTT). ISIS 416858 (20 or 40 mg/kg/wk) reduced plasma FXI activity by 80% at 4 weeks of treatment that resulted in a 33% increase in APTT by 13 weeks with no effects on PT, platelets, or increased bleeding following partial tail amputation or gum and skin laceration. The dose-dependent presence of basophilic granules in multiple tissues in ISIS 416858-treated animals was an expected histologic change for a 2'-MOE antisense oligonucleotide, and no toxicity was attributed to hepatic FXI reduction. Basophilic granules reflect cellular drug uptake and subsequent visualization on hematoxylin staining. These results suggest that ISIS 416858 has an acceptable preclinical safety profile and is a promising clinical candidate to treat thrombotic disease.

Inhibition of Complement Factor 3 in Geographic Atrophy with NGM621: Phase 1 Dose-Escalation Study Results.

PURPOSE: To evaluate the safety and tolerability of single and multiple intravitreal injections of NGM621 in patients with geographic atrophy (GA) and to characterize the pharmacokinetics and immunogenic potential. DESIGN: Multicenter, open-label, single- and multiple-dose phase 1 study. METHODS: Fifteen patients enrolled at 4 sites in the United States. Participants had GA secondary to age-related macular degeneration, lesion size ≥2.5 mm2, best-corrected visual acuity of 4 to 54 letters (20/80 to 20/800 Snellen equivalent) in the study eye, and no history of choroidal neovascularization in either eye. Patients who met eligibility criteria were treated in a single ascending-dose phase (2 mg, 7.5 mg, and 15 mg) or received 2 doses of NGM621 (15 mg) 4 weeks apart in the multidose phase and were monitored for 12 weeks (85 days). Assessments included adverse events, best-corrected visual acuity, low-luminance visual acuity, vital signs, clinical laboratory evaluations, GA lesion area as measured by fundus autofluorescence, spectral domain optical coherence tomography, and pharmacokinetic, immunogenicity, and pharmacodynamic assessments. RESULTS: All 15 participants completed the 12-week study. There were no serious adverse events, no drug-related adverse events, and no choroidal neovascularization developed in either eye. Mean visual acuity and GA lesion area appeared stable through week 12 for all cohorts. Pharmacokinetic analyses indicated that NGM621 serum exposures appeared to be dose proportional, and no antidrug antibodies were identified at any of the evaluated time points. CONCLUSIONS: In this small, open-labeled, 12-week phase 1 study, NGM621 was safe and tolerable when administered intravitreally up to 15 mg..

Sulindac metabolism and synergy with tumor necrosis factor-alpha 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-alpha (TNF-alpha) in plasma was significantly increased by LPS at 1 h, and SLD enhanced this response. Etanercept, a soluble TNF-alpha receptor, reduced SLD/LPS-induced liver injury, suggesting a role for TNF-alpha. 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-alpha augmented the cytotoxicity of SLD sulfide in primary hepatocytes and HepG2 cells. These results suggest that TNF-alpha can enhance SLD sulfide-induced hepatotoxicity, thereby contributing to liver injury in SLD/LPS-cotreated rats.

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. METHODS: Buffer capacity was evaluated using 7.5, 13.3, and 44.2 mm l-carnosine in a pH range of 5.5-7.5. Accelerated chemical stability was determined by HPLC at l-carnosine concentrations of 5-100 mm. Combinations of 7.5 mm l-carnosine with 0.06-0.6% (w/v) gellan gum were characterised rheologically. l-Carnosine-buffered solutions of gellan gum were tested for acute topical ocular tolerance in vivo in pigmented rabbits. A unique formulation combining timolol (which lowers intraocular pressure) in l-carnosine-buffered gellan gum was compared with Timoptic-XE in normotensive dogs. KEY FINDINGS: l-Carnosine exhibited optimal pharmaceutical characteristics for use as a buffer in chronically administered topical ocular formulations. Enhancement trends were observed in solution-to-gel transition of l-carnosine-buffered vehicles containing gellan gum vs comparators. Topical tolerability of l-carnosine-buffered gellan gum formulations and lowering of intraocular pressure were equivalent with timolol and Timoptic-XE. CONCLUSIONS: Functional synergy between excipients in gellan gum formulations buffered with l-carnosine has potential for topical ocular dosage forms with sustained precorneal residence.

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.

Assessment of the Effects of 2'-Methoxyethyl Antisense Oligonucleotides on Platelet Count in Cynomolgus Nonhuman Primates.

Decreases in platelet (PLT) counts observed in nonhuman primates (NHPs) given 2'-O-methoxyethyl modified antisense inhibitors (2'-MOE ASOs) have been reported, but the incidence and severity of the change vary considerably between sequences, studies, and animals. This article will broadly illustrate the spectrum of effects on PLT count in NHPs. From queries of an NHP safety database representing over 102 independent 2'-MOE ASOs, from 61 studies and >2200 NHPs, two patterns of PLT changes emerged. The first is a consistent and reproducible decrease in group mean values, observed with about 30% of the compounds, in which PLT count typically remains ≥150K cells/µL. The second is a sporadic decrease in PLTs to <50K cells/µL (2%-4% incidence at doses >5 mg/kg) that is often not reproducible. In both cases, the reduction in PLT count is dose dependent and reversible. The human relevance of PLT change observed in NHPs was investigated using ISIS 404173. In a chronic NHP study (20 mg/kg/wk for 26 weeks), a gradual decrease in group mean PLT count was observed at ≥10 mg/kg/wk, which plateaued by 13 weeks generally within the normal range and was maintained through 26 weeks of treatment. However, PLT counts <50K cells/µL occurred in 1 of 16 NHP at 10 mg/kg/wk and 3 of 16 NHP at 20 mg/kg/wk. In a 26-week double-blind, placebo-controlled Phase 2 trial, 62 patients were treated with 200 mg/wk ISIS 404173 (∼3.3 mg/kg/wk) there was an increased incidence of PLT count >30% decreased compared to baseline but no incidence of PLT <75K cells/µL. Based on these data, the consistent, self-limiting PLT reduction seen in NHP may translate to humans, but these changes appear to be of limited clinical significance. However, NHPs appear to overpredict the incidence of sporadic PLT <50K cells/µL compared to humans.

The role of hepatocellular oxidative stress in Kupffer cell activation during 1,2-dichlorobenzene-induced hepatotoxicity.

1,2-dichlorobenzene (1,2-DCB), an industrial solvent, is a known hepatotoxicant. Two oxidative events in the liver contribute to 1,2-DCB-induced liver injury: an initial hepatocellular oxidative stress, followed by oxidant stress associated with an inflammatory response. We hypothesize that the initial hepatocellular oxidative event triggers molecular and cellular processes within hepatocytes that lead to the production of factors that contribute to Kupffer cell (KC) activation and upregulation of the inflammatory cascade. To investigate the molecular effects of 1,2-DCB, primary cultures of Fischer-344 (F-344) and Sprague-Dawley (SD) rat hepatocytes were incubated with 1,2-DCB (3.6-12.4 mumol) and examined for enhanced DNA-binding activity of the oxidant-sensitive transcription factors activator protein-1 (AP-1), nuclear factor-kappa B (NF-kappaB), and electrophile responsive element (EpRE), and production and release of the chemokine cytokine-induced neutrophil chemoattractant (CINC). In F-344 rat hepatocytes, the activities of AP-1 and NF-kappaB were increased by as much as 3-fold by 6 h of 1,2-DCB treatment, when compared to control. Nuclear translocation of EpRE was also enhanced by 3-fold and occurred 2 h following 1,2-DCB treatment. These events were greater in F-344 than in SD rat hepatocytes incubated with 1,2-DCB. Moreover, F-344 rat hepatocytes produced and released CINC following incubation with 1,2-DCB, but SD rat hepatocytes did not. Lastly, conditioned media from 1,2-DCB-treated F-344 rat hepatocytes stimulated KC activity as determined by enhanced NF-kappaB-binding activity and increased nitric oxide production. Collectively, these data suggest that the mechanisms of 1,2-DCB-induced hepatotoxicity involve intercellular communication whereby compromised hepatocytes may signal KC activation via the production and release of oxidant-sensitive chemokines and cytokines.

Utilization of causal reasoning of hepatic gene expression in rats to identify molecular pathways of idiosyncratic drug-induced liver injury.

Drug-induced liver injury (DILI) represents a leading cause of acute liver failure. Although DILI can be discovered in preclinical animal toxicology studies and/or early clinical trials, some human DILI reactions, termed idiosyncratic DILI (IDILI), are less predictable, occur in a small number of individuals, and do not follow a clear dose-response relationship. The emergence of IDILI poses a critical health challenge for patients and a financial challenge for the pharmaceutical industry. Understanding the cellular and molecular mechanisms underlying IDILI is key to the development of models that can assess potential IDILI risk. This study used Reverse Causal Reasoning (RCR), a method to assess activation of molecular signaling pathways, on gene expression data from rats treated with IDILI or pharmacologic/chemical comparators (NON-DILI) at the maximum tolerated dose to identify mechanistic pathways underlying IDILI. Detailed molecular networks involved in mitochondrial injury, inflammation, and endoplasmic reticulum (ER) stress were found in response to IDILI drugs but not negative controls (NON-DILI). In vitro assays assessing mitochondrial or ER function confirmed the effect of IDILI compounds on these systems. Together our work suggests that using gene expression data can aid in understanding mechanisms underlying IDILI and can guide in vitro screening for IDILI. Specifically, RCR should be considered for compounds that do not show evidence of DILI in preclinical animal studies positive for mitochondrial dysfunction and ER stress assays, especially when the therapeutic index toward projected human maximum drug plasma concentration is low.

Recommendations for safety pharmacology evaluations of oligonucleotide-based therapeutics.

This document was prepared by the Safety Pharmacology Subcommittee of the Oligonucleotide Safety Working Group (OSWG), a group of industry and regulatory scientists involved in the development and regulation of therapeutic oligonucleotides. The mission of the Subcommittee was to develop scientific recommendations for the industry regarding the appropriate scope and strategies for safety pharmacology evaluations of oligonucleotides (ONs). These recommendations are the consensus opinion of the Subcommittee and do not necessarily reflect the current expectations of regulatory authorities. 1) Safety pharmacology testing, as described in the International Conference on Harmonisation (ICH) S7 guidance, is as applicable to ONs as it is to small molecule drugs and biotherapeutics. 2) Study design considerations for ONs are similar to those for other classes of drugs. In general, as with other therapeutics, studies should evaluate the drug product administered via the clinical route. Species selection should ideally consider relevance of the model with regard to the endpoints of interest, pharmacological responsiveness, and continuity with the nonclinical development program. 3) Evaluation of potential effects in the core battery (cardiovascular, central nervous, and respiratory systems) is recommended. In general: a. In vitro human ether-a-go-go-related gene (hERG) testing does not provide any specific value and is not warranted. b. Emphasis should be placed on in vivo evaluation of cardiovascular function, typically in nonhuman primates (NHPs). c. Due to the low level of concern, neurologic and respiratory function can be assessed concurrently with cardiovascular safety pharmacology evaluation in NHPs, within repeat-dose toxicity studies, or as stand-alone studies. In the latter case, rodents are most commonly used. 4) Other dedicated safety pharmacology studies, beyond the core battery, may have limited value for ONs. Although ONs can accumulate in the kidney and liver, evaluation of functional changes in these organs, as well as gastrointestinal (GI) and unintended "pro-inflammatory" effects, may be best evaluated during repeat-dose toxicity studies. Broad receptor- or ligand-binding profiling has not historically been informative for most ON subclasses, but may have value for investigative purposes.

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.

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.

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.

Inhibiting matrix metalloproteinases with prinomastat produces abnormalities in fetal growth and development in rats.

BACKGROUND: Matrix metalloproteinases (MMPs) play key roles in remodeling of the extracellular matrix during embryogenesis and fetal development. The objective of this study was to determine the effects of prinomastat, a potent selective MMP inhibitor, on fetal growth and development. METHODS: Prinomastat (25, 100, 250 mg/kg/day, p.o.) was administered to pregnant female Sprague-Dawley rats on gestational days (GD) 6-17. A Cesarian section was carried out on GD 20 and the fetuses were evaluated for viability and skeletal and soft tissue abnormalities. RESULTS: Prinomastat treatment at the 250 mg/kg/day dose produced a decrease in body weight and food consumption in the dams. A dose-dependent increase in post-implantation loss was observed in the 100 and 250 mg/kg/day-dose groups, resulting in only 22% of the dams having viable litters for evaluation at the 250 mg/kg/day dose. Fetal skeletal tissue variations and malformations were present in all prinomastat treated groups and their frequency increased with dose. Variations and malformation in fetal soft tissue were also increased at the 100 and 250 mg/kg/day doses. Prinomastat also interfered with fetal growth of rat embryo cultures in vitro. CONCLUSIONS: These data confirm that MMP inhibition has a profound effect on fetal growth and development in vivo and in vitro.

CpG and Non-CpG oligodeoxynucleotides induce differential proinflammatory gene expression profiles in liver and peripheral blood leukocytes in mice.

Proinflammatory effects caused by oligodeoxynucleotides (ODN) include cytokine production, splenomegaly and infiltration of mononuclear cells into tissues. Presence of one or more CpG motifs in an ODN sequence confers potency for proinflammatory properties. The objective of this research was to characterize the proinflammatory effects produced by CpG containing ODN as compared to non-CpG ODN using gene array analysis. Female CD-1 mice were administered equipotent dose regimens of a CpG ODN (ISIS 12449, 4 mg/kg sc, single or repeat dose for 7 d) or a non-CpG ODN (ISIS 2302, 50 mg/kg sc, q2d for 1 or 3 weeks) and tissues (liver and peripheral blood leukocytes) were harvested for immunohistochemical analysis or gene array analysis. Splenomegaly, a marker of ODN-induced inflammation, was greatest (3-fold above control) with ISIS 12449 when given at multiple doses. Immunohistochemical staining identified mainly monocytes/macrophages as the immune cell infiltrates in the liver following ISIS 12449 or ISIS 2302 treatment. Gene analysis of liver tissue indicated enhanced expression of chemokines (MIG, MIP-2beta, MCP-1, IL-1beta, CCR3), cell surface markers (CD14, CD18, CD86, CD11c, P-selectin), intracellular markers (NF-kappaBp65, MyD88, Survivin) and markers of cell proliferation (PCNA, Ki-67, CD71) was produced with ISIS 12449 or ISIS 2302. Although CpG and non-CpG containing ODN produced similar gene expression profiles, notable differences were observed to suggest that their mechanisms of immune modulation are not completely overlapping. MIG and MIP 1beta were identified as potential biomarker for immune stimulation that may be used to further study the species specificity, sequence/structure dependence and time course of proinflammatory ODN and antisense inhibitors used as therapeutics.

Biliary excretion of [(GS)(2)AsSe](-) after intravenous injection of rabbits with arsenite and selenate.

It has been shown that the seleno-bis (S-glutathionyl) arsinium ion, [(GS)(2)AsSe](-), is the major arsenic and selenium excretory product in bile of rabbits treated with arsenite and selenite [Gailer, J., Madden, S., Buttigieg, G. A., Denton, M. B., and Younis, H. S. (2002) Appl. Organomet. Chem. 16, 72-75]. To investigate the in vivo interaction between the other environmentally common oxy-anions of arsenic and selenium in mammals, we have intravenously injected rabbits with different combinations of the arsenic and selenium oxo-anions (arsenite + selenate, arsenate + selenite, and arsenate + selenate) and analyzed the collected bile and whole blood samples by X-ray absorption spectroscopy. Only the injection of arsenite and selenate led to the biliary excretion of [(GS)(2)AsSe](-) within 25 min. Whole blood collected from these animals (25 min postinjection) contained predominantly unchanged selenate, which suggests the presence of a mammalian selenate reductase in the liver. The lack of any significant biliary excretion of [(GS)(2)AsSe](-) in the other treatment groups implies that arsenate was not reduced in the liver on the time scale of our experiments. The relevance of these results for the human toxicology of arsenic and selenium is discussed.