Phosphorothioate Antisense Oligonucleotides Research Articles

Functional Rescue of Dystrophin-deficient mdx Mice by a Chimeric Peptide-PMO

Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy.

B08 Reduction of prolonged cag repeat containing alleles in Huntington's disease using antisense oligonucleotides

BackgroundHuntington's disease (HD) is a devastating disease for which currently no therapy is available. It is a progressive autosomal dominant neurodegenerative disorder that is caused by a CAG repeat expansion...

Post-natal stress-induced endocrine and metabolic alterations in mice at adulthood involve different pro-opiomelanocortin-derived peptides

In previous investigations we added a physical stress (mild pain) to the “classical” post-natal psychological stress in male mice, and we found that this combination produced a series of dysmetabolic signs very similar to mild human type-2 diabetes. Here, for the first time we demonstrate that within this diabetes model at least two groups of signs depend on the unbalance of two different endogenous systems. Newborn male mice were daily exposed to stressful procedures for 21 days (brief mother separation plus sham injection). Other groups underwent the same procedure, and also received naloxone (Na) to block μ–δ endogenous receptors, or a phosphorothioate antisense oligonucleotide (AS) directed against pro-opiomelanocortin (POMC)-mRNA [to block adrenocorticotropin (ACTH)- and POMC-derived opioid peptides]. Adult mice which received only post-natal stress increased body weight (+7.5%), abdominal overweight (+74%), fasting glycemia (+43%), plasma corticosterone (+110%), plasma (+169%) and pituitary (+153%) ACTH levels. Conversely, hypothalamic ACTH and corticotropin-releasing hormone (CRH) were reduced (−70% and −75%, respectively). Neonatal AS administration reverted all parameters to control values. Neonatal naloxone had little or no influence on glucose, corticosterone, ACTH, CRH levels, whereas it prevented body overweight and abdominal overweight. We conclude that, within this type-2 diabetes model in male mice at least two endocrino-neurohumoral systems are damaged, one concerning the opioid system, and the other concerning HPA hormones. The use of the two drugs was of primary importance to demonstrate this statement, and to demonstrate that these two groups of signs could be defined as “separate entities” following our complex post-natal stress model.

In vitro and in vivo characterisation of a novel c-FLIP-targeted antisense phosphorothioate oligonucleotide

Previous studies have suggested that the caspase 8 inhibitor FLIP is a promising anti-cancer therapeutic target. In this study, we characterised a novel FLIP-targeted antisense phosphorothioate oligonucleotide (AS PTO). FLIP AS and control PTOs were assessed in vitro in transient transfection experiments and in vivo using xenograft models in Balb/c nude mice. FLIP expression was assessed by QPCR and Western. Apoptosis induction was determined by flow cytometry and Western. Of 5 sequences generated, one potently down-regulated FLIP. AS PTO-mediated down-regulation of FLIP resulted in caspase 8 activation and apoptosis induction in non-small cell lung (NSCLC) cells but not in normal lung cells. Similar results were observed in colorectal and prostate cancer cells. Furthermore, the FLIP AS PTO sensitized cancer cells but not normal lung cells to apoptosis induced by rTRAIL. Moreover, the FLIP AS PTO enhanced chemotherapy-induced apoptosis in NSCLC cells. Importantly, compared to a control non-targeted PTO, intra-peritoneal delivery of FLIP AS PTO inhibited the growth of NSCLC xenografts and enhanced the in vivo antitumour effects of cisplatin. We have identified a novel FLIP-targeted AS PTO that has in vitro and in vivo activity and which therefore has potential for further pre-clinical development.

Off‐Target Effects Related to the Phosphorothioate Modification of Nucleic Acids

Phosphorothioate antisense oligonucleotides have been widely used in clinical studies for rational sequence-specific gene silencing. However, several sequence-unspecific off-target effects have been recently described for this compound class. In contrast to siRNA-mediated knockdown of the same gene, the bcl-2-targeted oblimersen (Genasense, G3139) downregulates a number of proteins involved in apoptotic resistance and several glycolytic enzymes in 607B human melanoma cells. Regardless of their target, phosphorothioate-modified antisense and siRNA compounds, but not oligonucleotides with a phosphodiester backbone, resulted in a similar impact on the proteome. Unspecifically downregulated proteins include cancer markers involved in apoptotic resistance and endoplasmatic reticulum (ER) stress such as the 78 kDa glucose regulated protein (GRP 78), protein disulfide isomerase A3 (PDIA3, GRP 58), calumenin, and galectin-1, as well as the glycolytic enzymes triose phosphate isomerase, glyceraldehyde phosphodehydrogenase, and phosphoglycerate mutase. The depletion of the glycolytic enzymes is reflected by a decrease in L-lactate production, indicating a partial reversal of the Warburg effect. Compared with other phosphorothioate oligonucleotides, oblimersen generally led to a more pronounced effect both in terms of the number of influenced proteins and the extent of downregulation, suggesting a synergistic effect of Bcl-2 downregulation.

Abstract 3716: Targeted therapy of high-grade gliomas using TGF-beta2 inhibitor trabedersen (AP 12009): Results of the Phase IIb study as basis for the Phase III SAPPHIRE Study

Abstract Background: High-grade gliomas (HGG) strongly overexpress TGF-beta 2. Trabedersen, a phosphorothioate antisense oligonucleotide is a TGF-beta 2 inhibitor administrated intratumorally using convection-enhanced delivery (CED). So far, clinical studies in HGG have included three Phase I/II studies and one randomized, active-controlled, open-label, multinational dose-finding Phase IIb study. Methods: Main objectives of the Phase IIb study were to compare response rate, survival, and safety of 2 doses of trabedersen (10 µM or 80 µM) vs. standard chemotherapy (TMZ or PCV). 145 patients with recurrent or refractory HGG (AA WHO grade III or GBM WHO grade IV) were randomized. Trabedersen was given intratumorally by convection-enhanced delivery via a single multi-hole catheter with up to 11 treatment cycles (7-d-on, 7-d-off / cycle). Results: As in the previous Phase I/II studies, treatment with trabedersen led to long-lasting remissions in patients with recurrent or refractory AA or GBM. Trabedersen showed a good tolerability and safety profile. Overall, 10 µM was superior to 80 µM trabedersen in both efficacy and safety. Overall, the highest efficacy was observed in AA patients treated with the lower dose of trabedersen. The 10 µM trabedersen group showed a consistently higher survival rate compared to 80 µM trabedersen and standard chemotherapy (at 24 months: 83.3%, 53.3% and 41.7%, respectively). Duration of response was about 3 times longer in the 10 µM trabedersen group (29.1 months) compared to the standard chemotherapy group (8.0 months). The median overall survival was higher in both trabedersen groups than in the chemotherapy control group, with a remarkable survival benefit of 10 µM trabedersen over chemotherapy of 17.4 months. In GBM patients, trabedersen was as efficacious as standard chemotherapy. Furthermore, in a prespecified subgroup (age ≤55 years, KPS >80%), trabedersen patients had a markedly higher 2-year survival rate compared to standard chemotherapy (40.0 vs. 13.3%). Conclusion and Outlook: Trabedersen treatment is safe and has a clear clinical benefit in HGG. A randomized, multinational and active-controlled Phase III study (SAPPHIRE) with trabedersen as monotherapy in patients with recurrent or refractory AA (WHO grade III) has started and patient enrollment is ongoing. In this pivotal study, patients are randomized either to treatment with 10 µM trabedersen or to standard chemotherapy (TMZ or BCNU). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3716.

Nanoparticle formulation enhances the delivery and activity of a vascular endothelial growth factor antisense oligonucleotide in human retinal pigment epithelial cells.

The objective of this study was to investigate the delivery and activity of a vascular endothelial growth factor (VEGF) antisense oligonucleotide in a human retinal pigment epithelial cell line (ARPE-19) using a biodegradable nanoparticulate delivery system. A 19-mer antisense phosphorothioate oligonucleotide (PS-ODN) complementary to bases 6-24 relative to the translational start site of the VEGF mRNA, a sense PS-ODN and a mismatch PS-ODN were examined for the inhibition of secretion and mRNA expression of VEGF using an enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. Nanoparticles of the antisense oligonucleotides were formulated using a poly(lactide-co-glycolide) (50:50) copolymer using a double emulsion solvent evaporation method. After preparing nanoparticles, drug loading, encapsulation efficiency and particle size were determined. The cells were exposed to either plain solution of oligonucleotide or nanoparticles of oligonucleotide from Day 3 through Day 6. Alternatively, the cells were incubated with PS-ODNs and lipofectin for 4 h on Day 4. In all studies, VEGF secretion and mRNA expression were determined on Day 6. The particle size, drug loading and encapsulation efficiency were 252 nm, 5.5% and 16.5%, respectively. The antisense PS-ODN inhibited VEGF mRNA and protein secretion when delivered using nanoparticles or lipofectin but not in its free form. This was consistent with the ability of nanoparticles and lipofectin to elevate the cellular uptake of the oligonucleotide by 4-fold and 13-fold, respectively. Neither mismatch nor sense oligonucleotides inhibited VEGF secretion. In conclusion, biodegradable nanoparticles enhance cellular delivery of a VEGF antisense oligonucleotide and inhibit VEGF secretion and mRNA expression in a human retinal pigment epithelial cell line.

Abstract C12: Preclinical characterization of a novel FLIP-targeted antisense phosphorothioate oligonucleotide

Abstract FLIP (FLICE-inhibitory protein) is a major inhibitor of death receptor-mediated activation of caspase 8 (FLICE, Fas-associated death domain-like interleukin-1 -converting enzyme). We have previously found that FLIP is not only a key regulator of death receptor-mediated apoptosis, but also inhibits chemotherapy-induced apoptosis in several cancers. Furthermore, we found that silencing FLIP expression using siRNA approaches was in itself sufficient to induce apoptosis in several different cancer models. These results suggest that FLIP is a promising therapeutic target in several cancers. In this study, we describe the pre-clinical characterisation of a novel FLIP-targeted antisense phosphorothioate oligonucleotide (AS PTO). Of 5 potential sequences identified, one AS PTO potently down-regulated FLIP. Down-regulation of FLIP by this AS PTO resulted in caspase 8 activation and apoptosis induction in non-small cell lung (NSCLC), colorectal and prostate cancer cell lines. Furthermore, the FLIP AS PTO sensitized cancer cells to apoptosis induced by the clinically relevant death ligand TRAIL (TNF-related apoptosis-inducing ligand). Moreover, the FLIP AS PTO also sensitized NSCLC cells to apoptosis induced by the chemotherapeutic agents cisplatin and taxol. Importantly, compared to a control non-targeted PTO, the FLIP-targeted AS PTO demonstrated in vivo antitumor activity against NSCLC xenografts and enhanced the antitumor effects of cisplatin. In summary, we have identified a novel FLIP-targeted AS PTO that has in vitro and in vivo activity and which may therefore have potential for clinical development. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C12.

Nasal delivery of antisense oligonucleotides: in vitro evaluation of a thiomer/glutathione microparticulate delivery system

Purpose: The aim of this study was to develop a nasal mucoadhesive microparticulate delivery system for phosphorothioate antisense oligonucleotides (PTO-ODNs) utilizing the thiomer technology.Methods: PTO-ODN microparticles, coated with either the mucoadhesive polymer polycarbophil-cysteine (PCP-Cys) or unmodified PCP and reduced glutathione (GSH) were prepared by the emulsification solvent evaporation technique. Particle size, drug load, decrease in thiol groups on microparticles, swelling properties, release of incorporated PTO-ODN, and mucoadhesive properties were examined. Permeation enhancing effect of the deployed thiomer conjugate was investigated on excised porcine respiratory mucosa of the nasal cavity.Results: Results demonstrated that microparticles were almost of spherical structure displaying particle diameter up to 30 μm. In addition, a controlled drug release of the incorporated PTO-ODN was achieved from these particles. Mucoadhesion studies revealed that thiolated PCP-Cys microparticles display 3-fold higher mucoadhesive properties than the corresponding unthiolated polycarbophil microparticles. The uptake of PTO-ODN, incubated in thiolated polycarbophil and glutathione microparticles, from the nasal mucosa was 2.2-fold improved.Conclusions: According to these results, the thiolated polycarbophil/reduced GSH microparticles might be a promising formulation for systemic delivery of PTO-ODNs via the nasal route.

A proteomic study reveals unspecific apoptosis induction and reduction of glycolytic enzymes by the phosphorothioate antisense oligonucleotide oblimersen in human melanoma cells

The question of specificity and the elucidation of the exact molecular mechanism of action of post-transcriptional gene silencing agents are two major challenges for their establishment as therapeutics. A proteomic off-target effect study (2-DE with MS) in combination with DIGE comparing the phosphorothioate antisense oligonucleotide oblimersen (Genasense, G3139) to a Bcl-2-targeting siRNA-sequence on human melanoma cells showed that additional off-target effects contribute to the apoptotic effect of oblimersen. When both oligonucleotides were transfected with lipofectamine 2000, only oblimersen increased apoptosis as determined by annexin staining and caspase activity measurement. In contrast to the highly specific siRNA, the expression level of a number of proteins was found to be altered after oblimersen treatment. Several proteins linked to apoptosis and stress response, among those galectin-1, cofilin-1, GRP78, HSP60, nucleophosmin, and peroxiredoxins, were identified and found to be down-regulated after oblimersen treatment. A down-regulation of enolase-1 and three other glycolytic enzymes indicates a reversion of the cancer-related Warburg effect. The observed effects may be caused by a phosphorothioate mediated blockage of the mitochondrial voltage dependent anion channel (VDAC).

Antisense oligonucleotide pharmacokinetics and metabolism

Background: The use of oligonucleotides as therapeutic agents has elicited a great deal of interest. Basic understanding and evaluation of the pharmacokinetic properties of oligonucleotides is foundational to their appropriate design and application. Objective: To review the primary pharmacokinetic properties that drive successful use and delivery of oligonucleotides. Methods: The primary data set available in the published literature for summarizing the pharmacokinetic properties of oligonucleotides exists for single strand phosphorothioate antisense oligonucleotides and their chimeric chemical modifications (second generation). Where possible, data from other classes of compounds are contrasted with this base class. Results/conclusion: Although there are several different classes of oligonucleotides being developed as therapeutic agents, their pharmacokinetic properties by class are primarily a function of their backbone chemistry and the resulting chemical relationship to biological stability and plasma protein binding properties.

Lack of Pharmacokinetic Interaction of Mipomersen Sodium (ISIS 301012), a 2′-O-Methoxyethyl Modified Antisense Oligonucleotide Targeting Apolipoprotein B-100 Messenger RNA, with Simvastatin and Ezetimibe

Mipomersen sodium (ISIS 301012) is a 20-mer phosphorothioate antisense oligonucleotide that is complementary to human apolipoprotein B-100 (apoB-100) messenger RNA and subsequently reduces translation of ApoB-100 protein, the major apolipoprotein of very low-density lipoprotein, intermediate-density lipoprotein and low-density lipoprotein (LDL). Mipomersen sodium is currently being studied in phase II/III clinical studies to determine its clinical utility as add-on therapy to HMG-CoA reductase inhibitors or other lipid-lowering agents in subjects with hypercholesterolaemia. The aim of this study was to characterize the pharmacokinetic interactions of mipomersen sodium with simvastatin and ezetimibe. Another aim was to evaluate the ability of mipomersen sodium to inhibit major cytochrome P450 (CYP) isoenzymes in vitro. In a phase I clinical study, ten healthy subjects per cohort received a single oral dose of simvastatin 40 mg or ezetimibe 10 mg followed by four 2-hour intravenous doses of mipomersen sodium 200 mg over an 8-day period, with simvastatin 40 mg or ezetimibe 10 mg being administered again with the last dose of mipomersen sodium. Mipomersen sodium pharmacokinetic profiles were assessed following the first dose (mipomersen sodium alone) and the last dose (mipomersen sodium in combination with simvastatin or ezetimibe). Plasma samples for measurement of simvastatin, simvastatin acid, and free and total ezetimibe concentrations were collected at various timepoints following their first and last oral dosing. A comparative pharmacokinetic analysis was performed to determine if there were any effects resulting from coadministration of mipomersen sodium with these lipid-lowering drugs. In addition to the clinical pharmacokinetic analysis, the ability of mipomersen sodium to inhibit the major CYP isoform enzymes (namely CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) was evaluated in cryo-preserved human hepatocytes in vitro. The area under the plasma concentration-time curve (AUC) from 0 to 24 hours (AUC(24)), maximum plasma concentration and apparent elimination half-life values of mipomersen sodium were similar when administered alone and in combination with oral simvastatin or oral ezetimibe. The 90% confidence intervals of the geometric least squares means ratios (%Reference) of the mipomersen sodium AUC(24) values were 93.6, 107 when administered together with simvastatin, and 92.4, 111 when administered with ezetimibe. Therefore, there were no large deviations outside the default no-effect boundaries (80-125%) for total exposure (the AUC) of mipomersen sodium in combination with either simvastatin or ezetimibe. Similarly, large deviations outside the default no-effect boundaries were not observed for simvastatin, simvastatin acid, or free and total ezetimibe exposure in combination with mipomersen sodium. In cryo-preserved human hepatocytes, mipomersen sodium exhibited no cytotoxicity. Significant cell uptake was demonstrated by analysing cell-associated concentrations of mipomersen sodium. All evaluated enzyme activities had <10% inhibition at tested concentrations up to 800 microg/mL (approximately 100 micromol/L) of mipomersen sodium, and dose-dependent inhibition was not observed. Therefore, mipomersen sodium is not considered an inhibitor of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 enzyme activities. These data provide evidence that mipomersen sodium exhibits no clinically relevant pharmacokinetic interactions with the disposition and clearance of simvastatin or ezetimibe, and vice versa. Moreover, mipomersen sodium does not inhibit any of the major CYP enzymes that were evaluated. Taken together, the results from this study support the use of mipomersen sodium in combination with oral lipid-lowering agents.

Syndecan-2 downregulation impairs angiogenesis in human microvascular endothelial cells

The formation of new blood vessels, or angiogenesis, is a necessary process during development but also for tumour growth and other pathologies. It is promoted by different growth factors that stimulate endothelial cells to proliferate, migrate, and generate new tubular structures. Syndecans, transmembrane heparan sulphate proteoglycans, bind such growth factors through their glycosaminoglycan chains and could transduce the signal to the cytoskeleton, thus regulating cell behaviour. We demonstrated that syndecan-2, the major syndecan expressed by human microvascular endothelial cells, is regulated by growth factors and extracellular matrix proteins, in both bidimensional and tridimensional culture conditions. The role of syndecan-2 in “ in vitro” tumour angiogenesis was also examined by inhibiting its core protein expression with antisense phosphorothioate oligonucleotides. Downregulation of syndecan-2 reduces spreading and adhesion of endothelial cells, enhances their migration, but also impairs the formation of capillary-like structures. These results suggest that syndecan-2 has an important function in some of the necessary steps that make up the angiogenic process. We therefore propose a pivotal role of this heparan sulphate proteoglycan in the formation of new blood vessels.

Custirsen (OGX-011): a second-generation antisense inhibitor of clusterin for the treatment of cancer

Background: Clusterin is a stress-induced cytoprotective chaperone protein, regulated by HSF1, and functions similarly to a small heat-shock protein. Clusterin is expressed in a variety of cancers and associated with broad-spectrum treatment resistance. Custirsen (OGX-011) is a 2′-methoxyethyl modified phosphorothioate antisense oligonucleotide that is complementary to clusterin mRNA; it is currently in clinical trials for patients with cancer. Objective/methods: To review the literature on the role of clusterin in cancer progression and treatment resistance, and to summarize completed and ongoing clinical trials with custirsen. Results/conclusions: Custirsen is well tolerated in humans and biologically active in inhibiting expression of clusterin in patients with cancer. Randomized trials of custirsen in combination with chemotherapy are planned in patients with castration-resistant prostate cancer.

Identification of Mg2+-dependent Neutral Sphingomyelinase 1 as a Mediator of Heat Stress-induced Ceramide Generation and Apoptosis

Neutral sphingomyelinases (SMases) are involved in the induction of ceramide-mediated proapoptotic signaling under heat stress conditions. Although ceramide is an important mediator of apoptosis, the neutral SMase that is activated under heat stress has not been identified. In this study, we cloned an Mg(2+)-dependent neutral SMase from a zebrafish embryonic cell cDNA library using an Escherichia coli expression-cloning vector. Screening of the clones using an SMase activity assay with C(6)-7-nitro-2-1,3-benzoxadiazol-4-yl-sphingomyelin as the substrate resulted in the isolation of one neutral SMase cDNA clone. This cDNA encoded a polypeptide of 420 amino acids (putative molecular weight: 46,900) containing two predicted transmembrane domains in its C-terminal region. The cloned neutral SMase 1 acted as a mediator of stress-induced apoptosis. Bacterially expressed recombinant neutral SMase 1 hydrolyzed [choline-methyl-(14)C]sphingomyelin optimally at pH 7.5 in the presence of an Mg(2+) ion. In zebrafish embryonic cells, the endogenous SMase enzyme was localized in the microsomal fraction. In FLAG-tagged SMase-overexpressing cells, neutral SMase 1 colocalized with a Golgi marker in a cytochemical analysis. Inactivation of the enzyme by an antisense phosphorothioate oligonucleotide repressed the induction of ceramide generation, caspase-3 activation, and apoptotic cell death by heat stress. Thus, neutral SMase 1 participates in an inducible ceramide-mediating, proapoptotic signaling pathway that operates in heat-induced apoptosis in zebrafish embryonic cells.

C-myc Antisense Oligonucleotides Sensitize Human Colorectal Cancer Cells to Chemotherapeutic Drugs

Background/Aims: Overexpression of the c-myc oncogene frequently occurs in both colon tumors and colon carcinoma cell lines. We examined the sensitization of human colorectal cancer cells to chemotherapeutic drugs using c-myc antisense (AS) phosphorothioate oligonucleotides ([S]ODNs). Methods: Cancer cells were treated with c-myc [S]ODNs, taxol, 5-fluorouracil (5-FU), doxorubicin and vinblastine individually and in combination. The antiproliferative effects, type of interaction between c-myc [S]ODNs and cytotoxic drugs, cell cycle, apoptosis and expression of cell-cycle- and apoptosis-regulatory genes were evaluated. Results: After treatment withc-myc AS[S]ODNs, the growth of cancer cells was markedly inhibited in a dose- and time-dependent manner and the levels of c-myc mRNA and protein were greatly decreased (p < 0.0001). The combinations of c-myc AS[S]ODNs and cytotoxic drugs produced greater growth inhibition of human colorectal cancer cells compared to single treatment with either c-myc AS[S]ODNs (p < 0.006) or cytotoxic drugs (p < 0.0001). These combinations exhibited time- and dose-dependent additive and/or synergistic antiproliferative effects. Cancer cells treated with cytotoxic drugs were growth arrested in the S phase. In contrast, cells treated with either c-myc AS[S]ODNs or by the combination of c-myc AS[S]ODNs and cytotoxic drugs were growth arrested in the G₂/M and S phases. The combination treatments also exhibited a marked apoptotic effect compared to single treatments. c-myc AS[S]ODN treatment reduced the mRNA levels of Bcl2, BclxL, cdk2, cyclin E1, cdk1 and cyclin B1, while increasing the mRNA levels of p21, p27, bax and caspase-3. Conclusion: This two-hit approach may be important in the quest to overcome drug resistance in cancer patients whose tumors carry an overexpressed c-myc gene.

Anti‐apoptotic role of EGF in HaCaT keratinocytes via a PPARβ‐dependent mechanism

Epidermal growth factor (EGF) plays an important role in epithelial cell proliferation and apoptosis. Our recent studies found that EGF-attenuated tumor necrosis factor-alpha induced HaCaT keratinocyte apoptosis, and this effect was accompanied by up-regulation of the expression of peroxisome proliferator-activated receptor beta (PPARbeta). However, little is known about whether PPARbeta is functionally involved in the inhibition of keratinocyte apoptosis by EGF. Here, we showed that EGF up-regulated the DNA-binding and transcriptional regulation activities of PPARbeta. Antisense phosphorothioate oligonucleotides against PPARbeta markedly inhibited de novo synthesis of PPARbeta and attenuated the protective effect of EGF on tumor necrosis factor-alpha-induced apoptosis. L165041, a specific PPARbeta ligand, significantly enhanced the transcriptional regulation activity of PPARbeta and increased the protective effect of EGF. These results suggest a molecular mechanism by which EGF protects HaCaT keratinocytes against apoptosis in a PPARbeta-dependent manner.

Analysis of the related substances in phosphorothioate antisense oligonucleotide FT19

A reversed-phase high performance liquid chromatographic (RP-HPLC) method was developed for the simultaneous determination of the contents of adapalene and the preservatives (2-phenoxyethanol and methyl-4-hydroxybenzoate) in adapalene gels. The chromatographic analysis was carried out on a Tigerkin C18 column (150 mm x 4.6 mm, 5 microm). The mobile phase was 0.02 mol/L ammonium acetate buffer (pH 3.0) and tetrahydrofuran-acetonitrile with gradient elution, and the detection wavelength was set at 270 nm. The calibration curves were linear over the ranges of 10-100 mg/L (r = 0.9999), 4-40 mg/L (r = 0.9999) and 4-40 mg/L (r = 0. 9999) for 2-phenoxyethanol, methyl-4-hydroxybenzoate and adapalene, respectively. The average recoveries of the three substances were within 98.0%-98.6%. The method is simple, reliable and suitable for the simultaneous determination of adapalene, 2-phenoxyethanol and methyl-4-hydroxybenzoate in adapalene gels.

Advantageous toxicity profile of inhaled antisense oligonucleotides following chronic dosing in non-human primates

TPI ASM8 and TPI 1100 are two products containing modified phosphorothioate antisense oligonucleotides (AONs), which are undergoing development for the treatment of asthma and chronic obstructive pulmonary disease (COPD), respectively. TPI ASM8 is comprised of two AONs, one targeting the human chemokine receptor 3 (CCR3) and the other targeting the common beta-chain of the IL-3/IL-5/GM-CSF receptors. TPI 1100 is also a dual-AON compound targeting the phosphodiesterase (PDE) 4 and 7 isotypes. For both products, the AONs are present in a 1:1 ratio by weight. Both products will be administered by inhalation to patients, and TPI ASM8 is currently undergoing Phase 2 clinical trials. As part of the safety assessment of both products, the toxicity and disposition (i.e., pharmacokinetics of the AON components in plasma and tissues) were investigated in 14-day inhalation studies in monkeys at doses ranging from 0.05 to 2.5 mg/kg/day. Results indicated that both products were safe and well tolerated at all dose levels. Reversible treatment-related alterations were only observed at the high dose levels tested and were limited to changes in the respiratory tract which were characterized primarily by the presence of alveolar macrophages in the absence of a generalized inflammatory response. Plasma pharmacokinetic profiles showed very low plasma concentrations, and no plasma accumulation was observed after repeated doses. While significant amounts of the AONs of both TPI ASM8 and TPI 1100 were measured in trachea and lung, only limited amounts of the AONs could be measured in kidney and liver, which, in combination with the low plasma level data, is indicative of very low systemic exposure. Taken together, these results demonstrate that these two new AON-based products are safe and that delivery via the inhaled route achieves localized deposition in the pulmonary tract with very limited systemic exposure and reduced toxicity compared to other routes of AON administration.

The role of peroxisome proliferator-activated receptor-β/δ in epidermal growth factor-induced HaCaT cell proliferation

Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) expression and activation is involved in the cell proliferation. However, little is known about the role of PPARβ/δ in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPARβ/δ mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, an EGF receptor (EGFR) special inhibitor, caused attenuation of PPARβ/δ protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPARβ/δ binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPARβ/δ caused selectively inhibition of PPARβ/δ protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPARβ/δ, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPARβ/δ up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPARβ/δ promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPARβ/δ expression in a c-Jun-dependent manner and PPARβ/δ plays a vital role in EGF-stimulated proliferation of HaCaT cells.