Safety, Pharmacokinetic, and Pharmacodynamic Evaluation of a 2'-(2-Methoxyethyl)-D-ribose Antisense Oligonucleotide-Triantenarry N-Acetyl-galactosamine Conjugate that Targets the Human Transmembrane Protease Serine 6.

Cellular uptake of antisense oligonucleotides (ASOs) is one of the main determinants of in vivo activity and potency. A significant advancement in improving uptake into cells has come through the conjugation of ASOs to triantenarry N-acetyl-galactosamine (GalNAc3), a ligand for the asialoglycoprotein receptor on hepatocytes. The impact for antisense oligonucleotides, which are already taken up into hepatocytes, is a 10-fold improvement in potency in mice and up to a 30-fold potency improvement in humans, resulting in overall lower effective dose and exposure levels. 2'-Methoxyethyl-modified antisense oligonucleotide conjugated to GalNAc3 (ISIS 702843) is specific for human transmembrane protease serine 6 and is currently in clinical trials for the treatment of ß-thalassemia. This report summarizes a chronic toxicity study of ISIS 702843 in nonhuman primates (NHPs), including pharmacokinetic and pharmacology assessments. Suprapharmacologic doses of ISIS 702843 were well tolerated in NHPs after chronic dosing, and the data indicate that the overall safety profile is very similar to that of the unconjugated 2'-(2-methoxyethyl)-D-ribose (2'-MOE) ASOs. Notably, the GalNAc3 moiety did not cause any new toxicities nor exacerbate the known nonspecific class effects of the 2'-MOE ASOs. This observation was confirmed with multiple GalNAc3-MOE conjugates by querying a data base of monkey studies containing both GalNAc3-conjugated and unconjugated 2'-MOE ASOs. SIGNIFICANCE STATEMENT: This report documents the potency, pharmacology, and overall tolerability profile of a triantenarry N-acetyl-galactosamine (GalNAc3)-conjugated 2'-(2-methoxyethyl)-D-ribose (2'-MOE) antisense oligonucleotide (ASO) specific to transmembrane protease serine 6 after chronic treatment in the cynomolgus monkey. Collective analysis of 15 independent GalNAc3-conjugated and unconjugated 2'-MOE ASOs shows the consistency in the dose response and character of hepatic and platelet tolerability across sequences that will result in much larger safety margins for the GalNAc3-conjugated 2'-MOE ASOs when compared with the unconjugated 2'-MOE ASOs given the increased potency.

IONIS-PKKRx a Novel Antisense Inhibitor of Prekallikrein and Bradykinin Production.

Kallikrein is the key contact system mediator responsible for the conversion of high-molecular-weight kininogen into the inflammatory vasodilator peptide bradykinin, a process regulated by C1-esterase inhibitor (C1-INH). In hereditary angioedema (HAE), genetic mutations result in deficient or dysfunctional C1-INH and dysregulation of the contact system leading to recurrent, sometimes fatal, angioedema attacks. IONIS-PKKRx is a second-generation 2'-O-(2-methoxyethyl)-modified chimeric antisense oligonucleotide, designed to bind and selectively reduce prekallikrein (PKK) mRNA in the liver. IONIS-PKKRx demonstrated dose-dependent reduction of human prekallikrein hepatic mRNA and plasma protein in transgenic mice and dose- and time-dependent reductions of plasma PKK in Cynomolgus monkeys. Similar dose-dependent reductions of plasma PKK levels were observed in healthy human volunteers accompanied by decreases in bradykinin generation capacity with an acceptable safety and tolerability profile. These results highlight a novel and specific approach to target PKK for the treatment of HAE and other diseases involving contact system activation and overproduction of bradykinin.

Chronic Toxicity Assessment of 2'-O-Methoxyethyl Antisense Oligonucleotides in Mice.

Advances in antisense oligonucleotide (ASO) chemistry and screening have enabled the design and selection of molecules that are optimized for a particular therapeutic application in terms of both potency and tolerability. The most-well studied of the chemically modified ASOs are single-stranded antisense inhibitors with phosphorothioate backbones and 2'-O-methoxyethyl modifications (2'-MOE ASO). The 2'-MOE chemical modification in the design of the ASO has conferred increased hybridization affinity, increased stability, and/or enhanced tissue residence time, resulting in better potency and pharmacokinetics. Compound screening and selection are also important in optimizing the tolerability of intended therapeutic antisense inhibitors. In this study, we report the chronic toxicity of multiple 2'-MOE ASOs in mice for several representative compounds that have progressed to later phases of clinical development. The results show that these 2'-MOE ASOs selected for development have consistent behavior between sequences, have tolerability profiles suitable for chronic administration, and exhibit a relative lack of progression of findings observed in subchronic studies in mice.

Disposition and Pharmacokinetics of a GalNAc3-Conjugated Antisense Oligonucleotide Targeting Human Lipoprotein (a) in Monkeys.

Triantennary N-acetyl galactosamine (GalNAc3)-conjugated antisense oligonucleotides (ASOs) have greatly improved potency due to receptor-mediated uptake into hepatocyte. The disposition and pharmacokinetics of ISIS 681257, a GalNAc3-conjugated ASO, were studied in monkeys. Following subcutaneous (SC) injection, ISIS 681257 was rapidly absorbed into the systemic circulation, with peak plasma levels observed within hours after dosing. After reaching Cmax, plasma concentrations rapidly declined in a multiexponential manner and were characterized by a dominant initial rapid distribution phase in which drug transferred to tissues from circulation, followed by a much slower terminal elimination phase (half-life of 4 weeks). Intact ISIS 681257 is the major full-length oligonucleotide species in plasma (≥70%). In tissues, the conjugated-GalNAc sugar moiety was rapidly metabolized, leaving the fully unconjugated form as the only full-length oligonucleotide detected at 48 h after dosing. Unconjugated ISIS 681257 cleared slowly from tissues with a half-life of 4 weeks. ISIS 681257 was highly bound to plasma proteins (>97% bound), which limited its urinary excretion. Disposition of ISIS 681257 in plasma and liver appeared nonlinear over the 1-40 mg/kg dose range studied. The plasma and liver tissue concentration data were well described by a population based mixed-effects modeling approach with Michaelis-Menten uptake from plasma to liver. Safety data from the study and the good exposure, as well as the extended half-life of the unconjugated ASO in the liver, support further development and less frequent dosing in Phase I clinical study.

Disposition and Pharmacology of a GalNAc3-conjugated ASO Targeting Human Lipoprotein (a) in Mice.

Triantennary N-acetyl galactosamine (GalNAc3)-conjugated antisense oligonucleotides (ASOs) have greatly improved potency via receptor-mediated uptake. In the present study, the in vivo pharmacology of a 2'-O-(2-methoxyethyl)-modified ASO conjugated with GalNAc3 (ISIS 681257) together with its unmodified congener (ISIS 494372) targeting human apolipoprotein (a) (apo(a)), were studied in human LPA transgenic mice. Further, the disposition kinetics of ISIS 681257 was studied in CD-1 mice. ISIS 681257 demonstrated over 20-fold improvement in potency over ISIS 494372 as measured by liver apo(a) mRNA and plasma apo(a) protein levels. Following subcutaneous (SC) dosing, ISIS 681257 cleared rapidly from plasma and distributed to tissues. Intact ISIS 681257 was the major full-length oligonucleotide species in plasma. In tissues, however, GalNAc sugar moiety was rapidly metabolized and unconjugated ISIS 681257 accounted > 97% of the total exposure, which was then cleared slowly from tissues with a half-life of 7-8 days, similar to the half-life in plasma. ISIS 681257 is highly bound to plasma proteins (> 94% bound), which limited its urinary excretion. This study confirmed dose-dependent exposure to the parent drug ISIS 681257 in plasma and rapid conversion to unconjugated ASO in tissues. Safety data and the extended half-life support its further development and weekly dosing in phase 1 clinical studies.

Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys.

Triantennary N-acetyl galactosamine (GalNAc3) is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc3 via a trishexylamino (THA)-C6 cluster significantly enhances antisense oligonucleotide (ASO) potency. Herein, the biotransformation, disposition, and elimination of the THA cluster of ION-681257, a GalNAc3-conjugated ASO currently in clinical development, are investigated in rats and monkey. Rats were administered a single subcutaneous dose of (3)H-radiolabeled ((3)H placed in THA) or nonradiolabeled ION-681257. Mass balance included radiometric profiling and metabolite fractionation with characterization by mass spectrometry. GalNAc3-conjugated ASOs were extensively distributed into liver. The THA-C6 triantenerrary GalNAc3 conjugate at the 5'-end of the ASO was rapidly metabolized and excreted with 25.67 ± 1.635% and 71.66 ± 4.17% of radioactivity recovered in urine and feces within 48 hours postdose. Unchanged drug, short-mer ASOs, and linker metabolites were detected in urine. Collectively, 14 novel linker associated metabolites were discovered including oxidation at each branching arm, initially by monooxidation at the ß-position followed by dioxidation at the α-arm, and lastly, tri and tetra oxidations on the two remaining ß-arms. Metabolites in bile and feces were identical to urine except for oxidized linear and cyclic linker metabolites. Enzymatic reaction phenotyping confirmed involvement of N-acetyl-ß-glucosaminidase, deoxyribonuclease II, alkaline phosphatase, and alcohol + aldehyde dehydrogenases on the complex metabolism pathway for THA supplementing in vivo findings. Lastly, excreta from monkeys treated with ION-681257 revealed the identical series as observed in rat. In summary, our findings provide an improved understanding of GalNAc3-conjugated-ASO metabolism pathways which facilitate similar development programs.

Inhibiting C-reactive protein for the treatment of cardiovascular disease: promising evidence from rodent models.

Raised blood C-reactive protein (CRP) level is a predictor of cardiovascular events, but whether blood CRP is causal in the disease process is unknown. The latter would best be defined by pharmacological inhibition of the protein in the context of a randomized case-control study. However, no CRP specific drug is currently available so such a prospective study cannot be performed. Blood CRP is synthesized primarily in the liver and the liver is an organ where antisense oligonucleotide (ASO) drugs accumulate. Taking advantage of this we evaluated the efficacy of CRP specific ASOs in rodents with experimentally induced cardiovascular damage. Treating rats for 4 weeks with a rat CRP-specific ASO achieved >60% reduction of blood CRP. Notably, this effect was associated with improved heart function and pathology following myocardial infarction (induced by ligation of the left anterior descending artery). Likewise in human CRP transgenic mice treated for 2 weeks with a human CRP-specific ASO, blood human CRP was reduced by >70% and carotid artery patency was improved (2 weeks after surgical ligation). CRP specific ASOs might pave the way towards a placebo-controlled trial that could clarify the role of CRP in cardiovascular disease.

Pharmacodynamics and subchronic toxicity in mice and monkeys of ISIS 388626, a second-generation antisense oligonucleotide that targets human sodium glucose cotransporter 2.

ISIS 388626, a 2'-methoxyethyl (MOE)-modified antisense oligonucleotide (ASO) that targets human sodium glucose cotransporter 2 (SGLT2) mRNA, is in clinical trials for the management of diabetes. SGLT2 plays a pivotal role in renal glucose reabsorption, and inhibition of SGLT2 is anticipated to reduce hyperglycemia in diabetic subjects by increasing urinary glucose elimination. To selectively inhibit SGLT2 in the kidney, ISIS 388626 was designed as a "shortmer" ASO, consisting of only 12 nucleotides with two 2'-MOE-modified nucleotides at the termini. Mice and monkeys received up to 30 mg/kg/week ISIS 388626 via subcutaneous injection for 6 or 13 weeks. Dose-dependent decreases in renal SGLT2 mRNA expression were observed, which correlated with dose-related increases in glucosuria without concomitant hypoglycemia. There were no histologic changes in the kidney attributed to SGLT2 inhibition after 6 or 13 weeks of treatment. The remaining changes observed in these studies were typical of those produced in these species by the administration of oligonucleotides, correlated with high doses of ISIS 388626, and were unrelated to the inhibition of SGLT2 expression. The kidney contained the highest concentration of ISIS 388626, and dose-dependent basophilic granule accumulation in tubular epithelial cells of the kidney, which is evidence of oligonucleotide accumulation in these cells, was the only histologic change identified. No changes in kidney function were observed. These results revealed only readily reversible changes after the administration of ISIS 388626 and support the continued investigation of the safety and efficacy of ISIS 388626 in human trials.

Renal uptake and tolerability of a 2'-O-methoxyethyl modified antisense oligonucleotide (ISIS 113715) in monkey.

The primary target organ for uptake of systemically administered phosphorothioate oligonucleotides is the kidney cortex and the proximal tubular epithelium in particular. To determine the effect of oligonucleotide uptake on renal function, a detailed renal physiology study was performed in cynomolgus monkeys treated with 10-40 mg/kg/week ISIS 113715 for 4 weeks. The concentrations of oligonucleotide in the kidney cortex ranged from 1400 to 2600 µg/g. These concentrations were associated with histologic changes in proximal tubular epithelial cells that ranged from the appearance of cytoplasmic basophilic granules to atrophic and degenerative changes at higher concentrations. However, there were no renal functional abnormalities as determined by the typical measurements of blood urea nitrogen, serum creatinine, creatinine clearance, or urine specific gravity. Nor were there changes in glomerular filtration rate, or renal blood flow. Specific urinary markers of tubular epithelial cell damage, such as N-acetyl-glucosaminidase, and α-glutathione-s-transferase were not affected. Tubular function was further evaluated by monitoring the urinary excretion of amino acids, ß(2)-microglobulin, or glucose. Renal function was challenged by administering a glucose load and by examining concentrating ability after a 4-h water deprivation. Neither challenge produced any evidence of change in renal function. The only change observed was a low incidence of increased urine protein/creatinine ratio in monkeys treated with ≥40 mg/kg/week which was rapidly reversible. Collectively, these data indicate that ISIS 113715-uptake by the proximal tubular epithelium has little or no effect on renal function at concentrations of 2600 µg/g.

Chronic administration of belimumab, a BLyS antagonist, decreases tissue and peripheral blood B-lymphocyte populations in cynomolgus monkeys: pharmacokinetic, pharmacodynamic, and toxicologic effects.

The tolerability, pharmacodynamic effects, and pharmacokinetics of belimumab (LymphoStat-B) were evaluated in cynomolgus monkeys. Belimumab is a fully human IgG1lambda antibody directed against B-lymphocyte stimulator (BLyS) protein. BLyS is a TNF family member that supports B-lymphocyte maturation and survival and has been implicated in the pathogenesis of autoimmune diseases and B-lymphocyte malignancies. Belimumab was developed to antagonize BLyS activity in autoimmune diseases and B-lymphocyte malignancies, where undesirable effects of B-lymphocyte activity may cause or contribute to disease. Pharmacodynamic effects of belimumab were monitored by immunophenotyping of peripheral blood. Pathology end points, including tissue immunophenotyping, are described after 13 and 26 weeks of treatment and after a 34-week treatment-free (recovery) period. Belimumab was safe and well tolerated in repeat-dose toxicology studies at 5-50 mg/kg for up to 26 weeks. Monkeys exposed to belimumab had significant decreases in peripheral blood B lymphocytes by 13 weeks of exposure, continuing into the recovery period, despite total lymphocyte counts similar to the controls. There were concomitant decreases in spleen and lymph node B-lymphocyte representation after 13 or 26 weeks of treatment with belimumab. Microscopically, monkeys treated with belimumab for 13 or 26 weeks had decreases in the number and size of lymphoid follicles in the white pulp of the spleen. All findings were generally reversible within a 34-week recovery period. These data confirm the specific pharmacologic activity of belimumab in reducing B lymphocytes in the cynomolgus monkey. The favorable safety profile and lack of treatment-related infections also support continued clinical development of belimumab.

Functional analysis of a carboxyl-terminal phosphorylation mutant of the bovine papillomavirus E1 protein.

The papillomavirus E1 protein is essential for viral DNA replication, and phosphorylation of E1 appears to regulate protein function and DNA replication. Serine 584 of bovine papillomavirus E1 is in a conserved motif resembling a CK2 consensus site, and is phosphorylated by CK2 in vitro. Mutation of serine 584 to alanine eliminates replication of the viral genome in transient replication assays. Wild-type and mutant E1 proteins were expressed from recombinant baculoviruses and used to assess biochemical functions of the amino acid 584 substitution. Helicase enzyme activity, E1 binding to the viral E2 protein and to cellular DNA polymerase alpha-primase were all unaffected in the mutant protein. Binding of E1 to viral replication origin DNA sequences was reduced in the mutant, but not eliminated. The carboxyl-terminal region of the protein appears to play a role in regulating E1 function, and adds to a complex picture emerging for papillomavirus DNA replication control.