Safety evaluation of 2'-deoxy-2'-fluoro nucleotides in GalNAc-siRNA conjugates.

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

Incidental Ultrastructural Findings in the Sural Nerve and Dorsal Root Ganglion of Aged Control Sprague Dawley Rats in a Nonclinical Carcinogenicity Study.

Xenobiotic-induced peripheral nerve damage is a growing concern. Identifying relative risks that a new drug may cause peripheral nerve injury over long periods of administration is gathering importance in the evaluation of animal models. Separating out age-related changes in peripheral nerves of rats caused by compression injury from drug-induced effects has been difficult. Biopsy of the sural nerve is utilized in humans for investigations of peripheral neuropathy, because it is largely removed from the effects of nerve compression. This study used transmission electron microscopy to identify incidental findings in the sural nerves and dorsal root ganglia of aged control rats over time. The goal was to establish a baseline understanding of the range of possible changes that could be noted in controls compared to rats treated with any new investigative drug. In this evaluation, most sural nerve fibers from aged control rats had few ultrastructural abnormalities of pathologic significance. However, glycogenosomes, polyglucosan bodies, swollen mitochondria, autolysosomes, split myelin, Schwann cell processes, and endoneural macrophages with phagocytosed debris (considered an indication of ongoing degenerative changes) were occasionally noted.

Nonclinical Safety Profile of Revusiran, a 1st-Generation GalNAc-siRNA Conjugate for Treatment of Hereditary Transthyretin-Mediated Amyloidosis.

Revusiran is a 1st-generation short interfering RNA targeting transthyretin conjugated to an N-acetylgalactosamine ligand to facilitate delivery to hepatocytes via uptake by the asialoglycoprotein receptors. Revusiran, in development for the treatment of hereditary transthyretin-mediated amyloidosis, was discontinued after an imbalance in deaths in the "ENDEAVOUR" phase 3 clinical trial. Nonclinical safety assessments included safety pharmacology, acute and repeat-dose toxicity, genotoxicity, and carcinogenicity. There were no effects on cardiovascular or respiratory function in monkeys after single doses of up to 100 mg/kg. No neurological effects were noted in monkeys in repeat-dose studies up to 300 mg/kg. Revusiran was well tolerated in repeat-dose mouse (weekly doses) and rat and monkey (five daily doses followed by weekly doses) toxicity studies. The no observed adverse effect level (NOAEL) in rats was 30 mg/kg based on reversible microscopic changes in liver that were accompanied by correlating elevations in clinical chemistry at higher doses. Dose-limiting toxicity was absent in monkeys, and the NOAEL was 200 mg/kg. There was no evidence of genotoxicity in vitro or in vivo at limit doses or carcinogenicity in a 2-year study in rats at doses up to 100 mg/kg. Overall, these results demonstrate that revusiran had a favorable nonclinical safety profile.

Different effects of angiotensin receptor blockade on end-organ damage in salt-dependent and salt-independent hypertension.

BACKGROUND: Although angiotensin II type 1 receptor blockers have emerged as effective antihypertensive agents, it is not known how efficacious these agents are in treating hypertension-associated target organ damage. METHODS AND RESULTS: The present study was undertaken to compare the effect of angiotensin type 1 receptor inhibition on the progression of the organ damage observed in 2 models of hypertension, namely, salt-sensitive and nitric oxide synthase inhibition-mediated hypertension. Effective (16.4 micromol/kg) and ineffective (0.8 to 4.9 micromol/kg) antihypertensive doses of candesartan cilexetil were initiated after hypertension was established. Both low- and high-dose candesartan cilexetil significantly reduced cardiac and renal damage in the nitric oxide synthase inhibitor model of hypertension (P < 0.05 versus untreated); however, high-dose candesartan caused a significant increase in renal damage in the Dahl salt-sensitive model of hypertension (P < 0.05 versus untreated). Interestingly, the beneficial end-organ effects of candesartan in the nitric oxide synthase inhibition model were independent of sustained antihypertensive actions of candesartan, whereas the exacerbation of renal injury with candesartan in the Dahl salt-sensitive model was inversely related to its blood pressure-lowering effect. CONCLUSIONS: These data show that angiotensin type 1 blockade reduces injury in the l-nitroarginine methyl ester model but increases tissue injury in the salt-sensitive model. These data suggest that angiotensin II via angiotensin type 1 receptor activation contributes to organ damage in nitric oxide-deficient salt-independent hypertension but is protective in salt-induced hypertension. These data further suggest that (1) renal injury may evolve independently of blood pressure and (2) the effectiveness of an antihypertensive agent in ameliorating renal injury may depend on the etiology of the hypertension.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in hamster aortic atherosclerosis: correlation with in-situ zymography.

Atherogenesis requires extracellular matrix (ECM) alterations, a process possibly mediated by matrix-degrading metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs). The objective of this study was to examine the immunohistochemical expression patterns of MMPs-1, -2, -3 and -9 and their tissue inhibitors, TIMPs-1, -2, -3 and -4 during the three major stages of atherosclerotic lesion development in hypercholesterolemic Syrian Golden hamsters. Aortic atherosclerotic lesions (fatty streak, fibro-fatty and advanced) were histologically characterized in treated hamsters at 12, 24, and 49 weeks. The immunochemistry expression of these MMPs and TIMPs were examined in treated aortic sections with lesions and control aortic sections without lesions. MMP activity in control aortas and atherosclerotic lesions was characterized by in-situ zymography. Positive immunoreactivity for MMPs-2, -3, -9 and TIMPs-1, -2,-3, and -4 was observed in both control and atherosclerotic aortic arch segments, while MMP-1 was only observed in atherosclerotic lesions. Using in-situ zymography, we identified casein and gelatin degradation in fatty streak, fibro-fatty and advanced lesions. The immunohistochemical expression of these MMPs and TIMPs were examined in treated aortic sections with lesions and control aortic sections without lesions. In all lesion stages, substrate degradation was inhibited with 1,10-phenanthroline. Degradation of these substrates was not observed in control aortas. In addition, substrate degradation was inhibited with 1,10-phenanthroline. These findings suggested that in control segments, the net proteolytic balance was shifted in favor of MMP inhibition. Alternatively, despite the colocalization of MMPs and TIMPs in the treated segments, net proteolytic balance favored the catalytic MMPs.

Besnoitiosis in a miniature donkey.

A 1-year-old male miniature donkey (Equus asinus) from a herd of eight was presented with a 9-month history of pruritic dermatitis, lethargy and anorexia. Physical examination revealed diffuse lichenification and scales involving the skin of the face, head and dorsum from the neck to the pelvis. The main histological alteration within the superficial and deep dermis was the presence of multiple large, spherical, thick walled, protozoal Besnoitia cysts. In addition, the inflammatory response consisted of a moderate, superficial and deep perivascular, mixed mononuclear cell infiltrate, with epidermal hyperplasia and compact orthokeratosis. Based upon the large size of the protozoal cysts and the ultrastructural features of the bradyzoites contained therein (conoid, polar ring, rhoptries, micronemes and microtubules), a diagnosis of cutaneous besnoitiosis was established. Treatment with trimethoprim-sulphamethoxazole resulted in significant clinical improvement. To our knowledge, this is the first reported case of besnoitiosis in a miniature donkey in North America.