The antisense oligonucleotide trabedersen (AP 12009) for the targeted inhibition of TGF-β2.

Despite remarkable advances in cancer research, patients with malignant tumors such as high-grade glioma or advanced pancreatic carcinoma still face a poor prognosis. Because of the severe morbidity and mortality of such malignant tumor types, the identification of suitable molecular drug targets for causal treatment approaches is an important area of current research. Transforming growth factor-beta 2 (TGF-β2) is an attractive target because it regulates key mechanisms of carcinogenesis, in particular immunosuppression and metastasis, and is frequently overexpressed in malignant tumors.

Transforming growth factor-beta 2 gene silencing with trabedersen (AP 12009) in pancreatic cancer.

Pancreatic cancer is one of the most aggressive human cancers with a 5-year survival rate of <5%. Overexpression of transforming growth factor-beta 2 (TGF-β2) in pancreatic malignancies is suggested to be a pivotal factor for malignant progression by inducing immunosuppression, metastasis, angiogenesis and proliferation. Trabedersen (AP 12009) is a phosphorothioate antisense oligodeoxynucleotide specific for human TGF-β2 mRNA and was successfully tested in a randomized, active-controlled phase IIb clinical study in patients with high-grade glioma.

Targeted therapy for high-grade glioma with the TGF-β2 inhibitor trabedersen: results of a randomized and controlled phase IIb study.

This randomized, open-label, active-controlled, dose-finding phase IIb study evaluated the efficacy and safety of trabedersen (AP 12009) administered intratumorally by convection-enhanced delivery compared with standard chemotherapy in patients with recurrent/refractory high-grade glioma. One hundred and forty-five patients with central reference histopathology of recurrent/refractory glioblastoma multiforme (GBM) or anaplastic astrocytoma (AA) were randomly assigned to receive trabedersen at doses of 10 or 80 µM or standard chemotherapy (temozolomide or procarbazine/lomustine/vincristine). Primary endpoint was 6-month tumor control rate, and secondary endpoints included response at further timepoints, survival, and safety.

Antisense therapeutics for tumor treatment: the TGF-beta2 inhibitor AP 12009 in clinical development against malignant tumors.

Overexpression of the cytokine transforming growth factor-beta 2 (TGF-beta2) is a hallmark of various malignant tumors including pancreatic carcinoma, malignant glioma, metastasizing melanoma, and metastatic colorectal carcinoma. This is due to the pivotal role of TGF-beta2 as it regulates key mechanisms of tumor development, namely immunosuppression, metastasis, angiogenesis, and proliferation. The antisense technology is an innovative technique offering a targeted approach for the treatment of different highly aggressive tumors and other diseases.

Inhibition of TGF-beta2 with AP 12009 in recurrent malignant gliomas: from preclinical to phase I/II studies.

Transforming growth factor-beta2 (TGF-beta2) is known to suppress the immune response to cancer cells and plays a pivotal role in tumor progression by regulating key mechanisms including proliferation, metastasis, and angiogenesis. For targeted protein suppression the TGF-beta2-specific antisense oligodeoxynucleotide AP 12009 was developed. In vitro experiments have been performed to prove specificity and efficacy of the TGF-beta2 inhibitor AP 12009 employing patient-derived malignant glioma cells as well as peripheral blood mononuclear cells (PBMCs) from patients.

Targeted tumor therapy with the TGF-beta 2 antisense compound AP 12009.

TGF-beta overexpression is a hallmark of various malignant tumors. This is due to the pivotal role of TGF-beta as it regulates key mechanisms of tumor development, namely immunosuppression, metastasis, angiogenesis, and proliferation. We have developed a new immunotherapeutic approach for the treatment of malignant tumors based on the specific inhibition of TGF-beta2 by the antisense oligodeoxynucleotide AP 12009.

Intracerebral and intrathecal infusion of the TGF-beta 2-specific antisense phosphorothioate oligonucleotide AP 12009 in rabbits and primates: toxicology and safety.

Here, we provide first evidence that long-term continuous infusion of highly purified antisense phosphorothioate oligodeoxynucleotides (S-ODN) into brain parenchyma is well tolerated and thus highly suitable for in vivo application. AP 12009 is an S-ODN for the therapy of malignant glioma. It is directed against human transforming growth factor-beta (TGF-beta2) mRNA.

Inhibition of immunosuppressive effects of melanoma-inhibiting activity (MIA) by antisense techniques.

Melanoma inhibitory activity (MIA) is an 11 kD protein secreted by malignant melanomas. Recent studies revealed an interaction of MIA with epitopes of extracellular matrix proteins including fibronectin. Structural homology of MIA with the binding sites of alpha4beta1 integrin results in complex interactions of MIA with molecules binding to alpha4beta1 integrin.

Antisense oligomers for selective suppression of MCP-1 synthesis in human pulmonary endothelial cells.

Endothelial synthesis of the C-C chemokine monocyte chemotactic protein-1 (MCP-1) has been implicated in the regulation of monocyte recruitment for extravascular pools under both physiologic and inflammatory conditions. We designed and characterized five antisense phosphorothioate oligodeoxynucleotides (PS-ODN) targeting MCP-1 secretion by human pulmonary artery endothelial cells (HPAEC) and pulmonary microvascular endothelial cells (HMVEC-L). The most effective PS-ODN (MCP-1 AS 2) dose-dependently suppressed the secretion of MCP-1 but not the secretion of the C-X-C chemokine interleukin-8 (IL-8) in both HPAEC and HMVEC-L in the nanomolar concentration range.

Clearance kinetics, biodistribution, and organ saturability of phosphorothioate oligodeoxynucleotides in mice.

We examined the dynamics of removal from circulation, tissue distribution, and persistence of phosphorothioate oligodeoxynucleotides (S-ODN) anti-tumor-necrosis-factor and a control of random sequence (randomer) in mice. After intravenous injection, the majority (96%) of S-ODN cleared rapidly from the circulation in the first two phases. In the first phase, 37.

Changes in the expression of synapsin I and II messenger RNA during postnatal rat brain development.

Synapsin Ia, Ib, IIa, and IIb are neuronal phosphoproteins, which are supposed to play a role in the short-term regulation of neurotransmitter release. Besides a high degree of homology among the four synapsin subtypes, there are structural differences in the 3'end of their coding region. Here we present the first extensive study of the expression of their gene transcripts by using in situ hybridization and northern blot analysis.

Transforming growth factor-beta-mediated autocrine growth regulation of gliomas as detected with phosphorothioate antisense oligonucleotides.

Transforming growth factors-beta 1 and -beta 2 (TGF-beta 1 and -beta 2) are important growth-regulatory proteins for astroglial neoplasms. We analyzed their role in tumor-cell proliferation in 12 glioma cell lines, employing phosphorothioate antisense oligodeoxynucleotides (S-ODNs, 14 mer), specifically targeted against the coding sequences of TGF-beta 1-mRNA and TGF-beta 2-mRNA. TGF-beta 1-S-ODNs inhibited cell proliferation in 5 of 12 gliomas, whereas TGF-beta 2-S-ODNs reduced the cell proliferation in all glioma cell lines, compared to nonsense-S-ODN-treated and S-ODN-untreated cells as controls.

Antisense-mediated inhibition of protein synthesis : rational drug design, pharmacokinetics, intracerebral application, and organ uptake of phosphorothioate oligodeoxynucleotides.

There is hardly any class of drugs for which the term "ratronal drug design" is more appropriate than for the currently developing antisense therapeutics. The specrficlty of the hybridization reaction and the surprisingly efficient uptake of synthetrc oligonucleotide derivatives provide a new class of selective protein synthesis inhibitors. Concurrently with the development of the antisense technology, elucidation of the pathogenetic role of mdrvrdual proteins for certain diseases is rapidly progressing, most notably in the fields of cancer research and virology.

Transforming growth factor-beta-mediated regulation of human peripheral blood mononuclear cell proliferation as detected with phosphorothioate antisense oligodeoxynucleotides.

Transforming growth factor-beta (TGF-beta) is a potent cytokine that has influence upon immunosuppressive as well as proinflammatory processes. In this context we have investigated the role of endogenous TGF-beta in human peripheral blood mononuclear cells (PBMCs) by TGF-beta 1 phosphorothioate antisense oligodeoxynucleotides (TGF-beta 1-S-ODNs). In short-term cultures (up to 3 days), TGF-beta 1-S-ODNs-treated, interleukin 2-activated PBMCs displayed a growth advantage (up to 148%) compared to nonsense or untreated controls as measured by cell counting and [3H]thymidine incorporation.

Inhibition of memory consolidation after active avoidance conditioning by antisense intervention with ependymin gene expression.

A rapid increase in ependymin mRNA expression demonstrated by semiquantitative in situ hybridization after avoidance conditioning on goldfish suggested a molecular demand for newly synthesized ependymin translation product. To inhibit de novo synthesis of ependymin molecules without interference with preexisting ones, 18 mer anti-ependymin mRNA-phosphorothioate oligodeoxynucleotides (S-ODNs) were injected into the perimeningeal brain fluid before active avoidance training. S-ODN-injected animals learned the avoidance response; however, they were amnesic in the test.

Learning-induced expression of meningeal ependymin mRNA and demonstration of ependymin in neurons and glial cells.

The turnover of a CNS-specific cell adhesion glycoprotein, ependymin, has earlier been found to increase during periods of neuronal plasticity. Here, ependymin mRNA expression was analyzed by semiquantitative in situ hybridization in goldfish. Learning of an active avoidance response resulted in a significant increase in ependymin mRNA expression 20 min to 4 h after acquisition of the task.

Effects of transforming growth factor-beta 1 on collagen synthesis, integrin expression, adhesion and invasion of glioma cells.

Transforming growth factor-beta 1 (TGF-beta 1) as a potent modulator of cell-extracellular matrix (ECM) interactions may be related to poorly understood ECM-associated features of glioblastomas, such as diffuse brain invasion, rarity of extracranial metastasis and marked ECM production in vitro. We therefore studied TGF-beta 1 expression in glioblastoma biopsy specimens and cell lines by using reverse transcription-polymerase chain reaction (RT-PCR). The cell lines were also examined by Western blotting and immunocytochemistry.

Block of c-Fos and JunB expression by antisense oligonucleotides inhibits light-induced phase shifts of the mammalian circadian clock.

Light-induced phase shifts of circadian rhythmic locomotor activity are associated with the expression of c-Jun, JunB, c-Fos and FosB transcription factors in the rat suprachiasmatic nucleus, as shown in the present study. In order to explore the importance of c-Fos and JunB, the predominantly expressed AP-1 proteins for the phase-shifting effects of light, we blocked the expression of c-Fos and JunB in the suprachiasmatic nucleus of male rats, housed under constant darkness, by intracerebroventricular application of 2 microliters of 1 mM antisense phosphorothioate oligodeoxynucleotides (ASO) specifically directed against c-fos and junB mRNA. A light pulse (300 lux for 1 h) at circadian time 15 induced a significant phase shift (by 125 +/- 15 min) of the circadian locomotor activity rhythm, whereas application of ASO 6 h before the light pulse completely prevented this phase shift.

Reversal of multiple drug resistance in vitro by phosphorothioate oligonucleotides and ribozymes.

In the present study we investigated the effectiveness of 14, 15 and 18 nucleotide antisense phosphorothioate oligonucleotides (S-ODNs) directed to four different regions of the published mdr-1 gene sequence to reduce the level of mdr-1 gene product (p170, P-glycoprotein) and its function in the over-expressing cell lines Lo-VoDxR, S180DxR and KBChR8-5. The highest efficiency in reduction of multiple drug resistance was obtained at a concentration of 2 microM. In proliferation assays a growth reduction of 50% was observed after exposure of doxorubicin-resistant cells to S-ODN3.

Differential expression of c-jun, junB and junD in rat hippocampal slices.

The temporal expression pattern of jun genes was studied in the hippocampal slice preparation. Slices were kept either in a physiological Ringer solution or a modified medium, substituting calcium (Ca2+) by magnesium (Mg2+). All three jun genes were expressed in a circumscribed, independent fashion with respect to distribution, intensity and time course.

Design and application of antisense oligonucleotides in cell culture, in vivo, and as therapeutic agents.

1. Synthetic oligonucleotides can inhibit the expression of a gene in a sequence specific manner on the transcriptional and translational level. These molecules are usually referred to as antisense oligonucleotides.

The role of Jun transcription factor expression and phosphorylation in neuronal differentiation, neuronal cell death, and plastic adaptations in vivo.

1. To investigate the role of the Jun transcription factors in neuronal differentiation, programmed neuronal cell death, and neuronal plasticity, we used phosphorothioate oligodeoxynucleotides (S-ODN) to inhibit selectively the expression of c-Jun, JunB, and JunD. 2.

Sequence-specific impairment of learning by c-jun antisense oligonucleotides.

Hybridization studies revealed a differential accumulation of c-jun and jun B mRNA in the hippocampus and cerebral cortex of rats trained on a foot-shock-motivated brightness discrimination. Supposing that early gene expression is functionally significant for plastic changes in the brain, we used antisense phosphorothioate oligodeoxynucleotides (S-ODN) in vivo to study effects of specific inhibition of c-jun and jun B gene expression on learning and memory formation in rat brain. Discrimination performance of rats was impaired after intrahippocampal injection of anti-c-jun S-ODN but not of anti-jun B S-ODN.

Inhibition of p185c-erbB-2 proto-oncogene expression by antisense oligodeoxynucleotides down-regulates p185-associated tyrosine-kinase activity and strongly inhibits mammary tumor-cell proliferation.

The c-erbB-2 proto-oncogene codes for a 185-kd putative growth factor receptor that is highly homologous to but distinct from the epidermal growth factor (EGF) receptor. Amplification and overexpression of c-erbB-2 occurs in a number of human tumors, in some of which it is a negative prognostic factor. This study investigates the possibility of inhibiting tumor-cell proliferation by blocking c-erbB-2 expression in the human mammary carcinoma cell line SK-Br-3 using chemically modified antisense oligodeoxynucleotides.