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Purpose The aim of this study to evaluate the effect of mipomersen on QT intervals in a phase I dose escalation, placebo-controlled study, and a thorough QT (tQT) study in healthy subjects. Methods In the initial phase I study, 29 healthy subjects received either single or multiple (for 4 weeks) ascending doses of mipomersen (50–400 mg) administered subcutaneously (SC) or via a 2-h intravenous (IV) infusion, and 7 subjects received placebo. In the confirmative tQT study, 58 healthy subjects received placebo, 400 mg IV moxifloxacin, 200 mg SC, or 200 mg IV of mipomersen in a double-blind, 4-way crossover design with a minimum 5-day washout between treatments. ECG measurements were performed at baseline and selected time points (including T max ). The correlation between QTcF intervals corrected for baseline and time-matched placebo when available with PK plasma exposure was evaluated by linear regression analysis. Results In the phase I study, no positive correlation between the PK exposure and ∆QTcF or ∆∆QTcF was observed within the wide dose or exposure range tested. Similar results were observed in the tQT study, where the predicted ΔΔQTcF and its upper bound of the 90 % CI at C max of therapeutic and supratherapeutic dose were approximately −1.7 and 2.9 ms, respectively. Conclusions Mipomersen showed no effect on QT intervals in both the phase I dose escalation study and the tQT study. These results support the proposal that QT assessment can be made in a phase I dose escalation study, and no tQT study may be necessary if the phase I dose escalation study showed a negative QT effect.

Background and aims: To evaluate the safety and efficacy of the intercellular adhesion molecule 1 (ICAM-1) antisense phosphorothioate oligonucleotide alicaforsen (ISIS 2302) in Crohn's disease. Methods: Active (Crohn's disease activity index (CDAI) 200–350), steroid dependent (prednisone 10–40 mg) Crohn's patients were randomised into three treatment groups: placebo versus ISIS 2302 (2 mg/kg intravenously three times a week) for two or four weeks. Patients were treated in months 1 and 3, with steroid withdrawal attempted by week 10. The primary end point (steroid free remission) was a CDAI <150 off steroids at the end of week 14. Results: A total of 299 patients were enrolled, with a mean baseline CDAI of 276 and steroid dose of 23 mg/day. Rates of steroid free remission were equivalent for the two and four week ISIS 2302 groups (20.2% and 21.2%) and the placebo group (18.8%). At week 14, steroid withdrawal was successful in more ISIS 2302 patients compared with placebo treated patients (78% v 64%; p=0.032). Steroid free remission was highly correlated with exposure (p=0.0064). Other clinical responses were correlated with exposure, with significant results versus placebo being observed in the highest area under the curve subgroup. CDAI scores decreased by 136 (112) at week 14 versus 52 (107) for placebo (p=0.027) and inflammatory bowel disease score questionnaire improved by 43 (31) versus 15 (36) for placebo (p=0.027). Conclusions: Although the primary outcomes failed to demonstrate efficacy, pharmacodynamic modelling suggests that alicaforsen (ISIS 2302) may be an effective therapy for steroid dependent Crohn's disease.

Mipomersen (Kynamro ® ), a second-generation 2′- O -methoxyethyl chimeric antisense oligonucleotide (ASO), inhibits the synthesis of apolipoprotein B (apoB) and is indicated in the US as an adjunct therapy for homozygous familial hypercholesterolemia (HoFH) at a dose of 200 mg subcutaneously (SC) once weekly. The pharmacokinetic (PK) properties of mipomersen are generally consistent across all species studied, including mouse, rat, monkey, and humans. After SC administration, mipomersen is rapidly and extensively absorbed. It has an apparent plasma and tissue terminal elimination half-life of approximately 30 days. Mipomersen achieves steady-state tissue concentrations within approximately 4–6 months of once-weekly dosing. It does not exhibit PK-based drug–drug interactions with other concomitant medications, either involving competition for plasma protein binding or alterations in disposition of any evaluated drugs. Furthermore, mipomersen does not prolong the corrected QT (QTc) interval. There have been no ethnic- or gender-related differences in PK observed. In clinical trials, both as a single agent and in the presence of maximal lipid-lowering therapy, mipomersen has demonstrated significant dose-dependent reductions in all measured apoB-containing atherogenic lipoproteins. Overall, mipomersen has well-characterized PK and pharmacodynamic properties in both animals and humans, and is an efficacious adjunct treatment for patients with HoFH.

Mipomersen sodium (Kynamro™) (henceforth mipomersen) is a second-generation antisense oligonucleotide inhibitor of apolipoprotein B-100, which is the main structural component of atherogenic lipid particles. Mipomersen is administered via subcutaneous injection and is indicated as adjunctive treatment for homozygous familial hypercholesterolaemia (HoFH). The drug was developed by Isis Pharmaceuticals, which now collaborates with Genzyme Corporation for on-going development and product marketing. Multinational phase III trials of mipomersen as adjunctive therapy were completed in patients with HoFH, severe FH, heterozygous FH (HeFH) with coronary artery disease (CAD), and in those with hypercholesterolaemia at high risk of CAD. Mipomersen 200 mg once weekly has been approved in the USA as an adjunct to lipid-lowering medications and diet in HoFH patients and is undergoing regulatory review in the EU for the same indication. Genzyme is also conducting a multinational phase III, open-label extension study to evaluate long-term treatment in HoFH and HeFH patients, as well as a multinational trial to evaluate a three-times-per-week mipomersen regimen in patients with severe FH. This article summarises the milestones in the development of once-weekly, subcutaneous mipomersen leading to this first approval.

The species sensitivity and mechanism of complement pathway activation by a phosphorothioate oligonucleotide were investigated in monkey and human serum. Increasing concentrations of a phosphorothioate oligonucleotide, ISIS 2302, were incubated in either monkey or human serum. Complement activation in monkey serum was selective for the alternative pathway and occurred at concentrations ≥50 μg/mL ISIS 2302. By comparison, complement activation in human serum was absent. A similar difference in sensitivity for activation was also observed for a representative 2′-methoxyethyl (MOE)–modified oligonucleotide. The absence of oligonucleotide-induced complement activation was also observed in dogs. Protein binding with ISIS 2302 and enzyme competition studies suggested that factor H was important in oligonucleotide-mediated complement activation process, and addition of factor H to serum effectively prevented the activation in monkey serum. Furthermore, based on the immunoassay for factor H, there was an apparent decrease in factor H concentration as the ISIS 2302 concentration increased. This result suggests that ISIS 2302 binds to factor H and interferes with the factor H antibody from the immunoassay. Factor H is a regulatory protein that limits alternative pathway activation. Disruption of factor H interaction with C3 convertase by oligonucleotide could promote activation in this pathway.

Insufficient efficacy and/or specificity of antisense oligonucleotides limit their in vivo usefulness. We demonstrate here that a high-affinity DNA analog, locked nucleic acid (LNA), confers several desired properties to antisense agents. Unlike DNA, LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. However, like DNA, the LNA/DNA copolymers were capable of activating RNase H, an important antisense mechanism of action. In contrast to phosphorothioate-containing oligonucleotides, isosequential LNA analogs did not cause detectable toxic reactions in rat brain. LNA/DNA copolymers exhibited potent antisense activity on assay systems as disparate as a G-protein-coupled receptor in living rat brain and an Escherichia coli reporter gene. LNA-containing oligonucleotides will likely be useful for many antisense applications.

1. Introduction 345 2. Peptide nucleic acid (PNA) 346 3. ‘Cell penetrating peptides’ (CPPs) 346 4. Endosomal escape 347 5. Cellular delivery of PNA 347 6. In vivo bioavailability of PNA 349 7. References 350 Recent results on the cellular delivery of antisense peptide nucleic acids (PNA) via peptide conjugation is briefly discussed, in particular in the context of endosomal entrapment and escape.

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibited target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipulation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

Antisense technology may play a major role in cancer chemotherapy. It is clearly a tool of exceptional value in the functionalization of genes and their validation as potential targets for cancer chemotherapy. Additionally, there is now substantial evidence that antisense drugs are safe, and a growing body of data showing activity in animal models of human disease including cancer, and suggesting efficacy in patients with cancer. In this article, I review the progress in the technology, the anticancer antisense drugs in development and potential roles that antisense technology might play.