Toxicological and pharmacokinetic properties of QPI-1007, a chemically modified synthetic siRNA targeting caspase 2 mRNA, following intravitreal injection.

We report the toxicological and pharmacokinetic properties of the synthetic, small interfering RNA (siRNA), QPI-1007, following intravitreal administration. QPI-1007 is a chemically modified siRNA designed to act via the RNA interference (RNAi) pathway to temporarily inhibit expression of the caspase 2 protein and is being developed as a neuroprotectant for the treatment of nonarteritic anterior ischemic optic neuropathy and other optic neuropathies such as glaucoma that result in the death of retinal ganglion cells. The half-life of QPI-1007 in the vitreous and retina/choroid in the Dutch Belted rabbit was about 2 days, and there was no sign of accumulation after repeated administrations at either 2- or 4-week dosing intervals in the rabbit. QPI-1007 was well tolerated in Dutch Belted rabbits following single or repeated intravitreal administrations of up to 11 doses over 9 months. Test-article-related effects were limited to the eyes, with minimal to mild vitreal cellular infiltration being the major finding, which was reversible. In repeated-dose studies, a modest reduction in B-wave amplitude obtained by electroretinography was observed in animals treated with the highest dose level tested (3 mg, which is equivalent to a 12 mg/eye human dose) that was not considered to be clinically meaningful. Administration in the rat of either a single bolus intravenous (i.v.) injection of 100 mg/kg or daily bolus i.v. injections of 75 mg/kg/day for 28 days failed to elicit any macroscopic or microscopic changes, suggesting a low risk for systemic toxicity. QPI-1007 was negative in three genetic toxicity studies. Overall, the nonclinical studies support the further development of QPI-1007.

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.

Toxicological and pharmacokinetic properties of chemically modified siRNAs targeting p53 RNA following intravenous administration.

We report the toxicological and pharmacokinetic properties of the synthetic, small interfering RNA I5NP following intravenous administration in rodents and nonhuman primates. I5NP is designed to act via the RNA interference (RNAi) pathway to temporarily inhibit expression of the pro-apoptotic protein p53 and is being developed to protect cells from acute ischemia/reperfusion injuries such as acute kidney injury that can occur during major cardiac surgery and delayed graft function that can occur following renal transplantation. Following intravenous administration, I5NP was very rapidly cleared from plasma was distributed predominantly to the kidney, with very low levels in liver and other tissues. Doses of 800 mg/kg I5NP in rodents, and 1,000 mg/kg I5NP in nonhuman primates, were required to elicit adverse effects, which in the monkey were isolated to direct effects on the blood that included a sub-clinical activation of complement and slightly increased clotting times. In the rat, no additional adverse effects were observed with a rat analogue of I5NP, indicating that the effects likely represent class effects of synthetic RNA duplexes rather than toxicity related to the intended pharmacologic activity of I5NP. Taken together, these data support clinical testing of intravenous administration of I5NP for the preservation of renal function following acute ischemia/reperfusion injury.

First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin disorder.

The rare skin disorder pachyonychia congenita (PC) is an autosomal dominant syndrome that includes a disabling plantar keratoderma for which no satisfactory treatment is currently available. We have completed a phase Ib clinical trial for treatment of PC utilizing the first short-interfering RNA (siRNA)-based therapeutic for skin. This siRNA, called TD101, specifically and potently targets the keratin 6a (K6a) N171K mutant mRNA without affecting wild-type K6a mRNA. The safety and efficacy of TD101 was tested in a single-patient 17-week, prospective, double-blind, split-body, vehicle-controlled, dose-escalation trial. Randomly assigned solutions of TD101 or vehicle control were injected in symmetric plantar calluses on opposite feet. No adverse events occurred during the trial or in the 3-month washout period. Subjective patient assessment and physician clinical efficacy measures revealed regression of callus on the siRNA-treated, but not on the vehicle-treated foot. This trial represents the first time that siRNA has been used in a clinical setting to target a mutant gene or a genetic disorder, and the first use of siRNA in human skin. The callus regression seen on the patient's siRNA-treated foot appears sufficiently promising to warrant additional studies of siRNA in this and other dominant-negative skin diseases.

Ocular biodistribution of bevasiranib following a single intravitreal injection to rabbit eyes.

PURPOSE: The primary objective of these investigations was to determine the ocular biodistribution of bevasiranib, a small interfering RNA (siRNA) targeting vascular endothelial growth factor A (VEGF-A), following a single intravitreal injection to rabbit eyes. METHODS: A tissue distribution and pharmacokinetic study was conducted with (3)H-bevasiranib prepared in balanced-salt solution (BSS). Single doses of either 0.5 mg/eye or 2.0 mg/eye of (3)H-bevasiranib were given by intravitreal injection to Dutch-Belted rabbits (both eyes were treated). Subgroups of rabbits were serially-sacrificed at various times up to 7 days following dosing for collection of tissue samples. The right eye of each rabbit was collected whole, and the left eye was dissected to isolate five ocular tissues. All samples were analyzed by liquid scintillation counting to determine the concentrations of bevasiranib equivalents. An ocular disposition study was also performed with non-radiolabeled bevasiranib, which was administered to Dutch-Belted rabbit eyes via intravitreal injection at a dose of 2.0 mg/eye. Twenty-four hours post-dose, the eyes were enucleated and dissected into eight individual ocular structures that were analyzed for intact bevasiranib using a locked nuleic acid (LNA) noncompetitive hybridization-ligation enzyme-linked immunosorbent assay. RESULTS: Following intravitreal injection of 0.5 mg or 2.0 mg radiolabeled bevasiranib to Dutch-Belted rabbits, bevasiranib was detected in the vitreous, iris, retina, retinal pigment epithelium (RPE), and sclera (+choroid). As expected, the highest concentrations were found in the vitreous, and vitreous levels steadily decreased over time, while concentrations of radioactivity in the other ocular tissues increased to maximum values between 24 h and 72 h after dosing. Of these tissues, the highest concentration of radioactivity was detected in the retina. The LNA assay further confirmed the presence of intact bevasiranib in these tissues 24 h following intravitreal injection of non-radiolabeled bevasiranib (2 mg/eye). CONCLUSIONS: These studies demonstrate distribution of bevasiranib throughout the eye following intravitreal injection, including extensive uptake into the retina.

Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA.

The results of administering escalating, i.v. doses of targeted nanoparticles containing a siRNA targeting the M2 subunit of ribonucleotide reductase to non-human primates are reported. The nanoparticles consist of a synthetic delivery system that uses a linear, cyclodextrin-containing polycation, transferrin (Tf) protein targeting ligand, and siRNA. When administered to cynomolgus monkeys at doses of 3 and 9 mg siRNA/kg, the nanoparticles are well tolerated. At 27 mg siRNA/kg, elevated levels of blood urea nitrogen and creatinine are observed that are indicative of kidney toxicity. Mild elevations in alanine amino transferase and aspartate transaminase at this dose level indicate that the liver is also affected to some extent. Analysis of complement factors does not reveal any changes that are clearly attributable to dosing with the nanoparticle formulation. Detection of increased IL-6 levels in all animals at 27 mg siRNA/kg and increased IFN-gamma in one animal indicate that this high dose level produces a mild immune response. Overall, no clinical signs of toxicity clearly attributable to treatment are observed. The multiple administrations spanning a period of 17-18 days enable assessment of antibody formation against the human Tf component of the formulation. Low titers of anti-Tf antibodies are detected, but this response is not associated with any manifestations of a hypersensitivity reaction upon readministration of the targeted nanoparticle. Taken together, the data presented show that multiple, systemic doses of targeted nanoparticles containing nonchemically modified siRNA can safely be administered to non-human primates.

Complement activation is responsible for acute toxicities in rhesus monkeys treated with a phosphorothioate oligodeoxynucleotide.

The objective of this study was to define the role of complement activation in the acute and transient toxicities associated with administration of phosphorothioate oligonucleotides in monkeys. In the absence of complement inhibitor, complement activation blocker-2 (CAB-2), i.v. infusion of 20 mg/kg ISIS 2302 produced increases in the concentrations of the complement split products Bb and C5a (100- and 7-fold, respectively). Monkeys also experienced marked changes in bloodpressure (hypertension and hypotension), clinical signs of toxicity (lethargy and periorbital edema), fluctuations in circulating neutrophil counts, and elevations in serum cytokine levels (45-, 12-, and 4-fold increases in IL-6, MCP-1, and IL-12, respectively). Changes occurred at or near the end of infusion and returned to normal over time. One of the three animals died approximately 4 h following infusion of 20 mg/kg ISIS 2302 alone. In contrast, prior treatment with CAB-2 effectively blocked complement activation, as well as the ISIS 2302-induced hemodynamic and clinical responses. Importantly, plasma concentration of ISIS 2302 were unaffected by CAB-2 pretreatment. Thus, the protection afforded by CAB-2 was due to its inhibition of complement activation rather than to any impact on the disposition of ISIS 2302. These results clearly demonstrate the causal relationship between activation of the alternative complement pathway and the hemodynamic and clinical responses associated with rapid infusion of phosphorothioate oligonucleotides. Demonstration of this relationship underscores the importance of avoiding complement activation in patients to ensure the continued safe use of phosphorothioate oligodeoxynucleotides.

Effect of silica and volcanic ash on the content of lung alveolar and tissue phospholipids

Silica or volcanic ash (VA) was administered to rats via intratracheal instillation and the changes in extracelular (i.e. lavage fluid) and tissue phospholipids, as well as various biochemical parameters, were monitored over a 6 month period. VA produced relatively minor (up tp 2.8-fold) increases in lung tissue or lavage fluid phospholipids that were maximal al 1 month postinstillation. These increases were quantitatively similar to the increases in protein and DNA content of lung tissue and lavage fluid induced by VA and, thus, may be attributable to hypercellularity and accumulation of cellular breakdown products in the alveolar lumen. Instillation of silica produced a much greater (up to 11 fold) increase than VA in total phospholipid over time, primarily due to a 14-fold increase in phosphatidylcholine (PC). The accumulation of PC was more pronounced in the lavage fluid during the first month following silica instillation, but thereafter progresses more rapidly in the lung tissue. The relatively small increased (1.3- to 3.5-fold) in other phospholipids induced by silica appeared to be nonspecific, since they did not differ greatly from the increases in lung weight, DNA and protein. Collectively, these results indicate that intratracheal instillation of silica induces selective accumulation of lung PC, implying enhanced synthesis and secretion of pulmonary surfactant from alveolar epithelial Type II cells into the lumen(AU)