Bombesin receptor antagonists may be preferable to agonists for tumor targeting

Two bombesin analogs, Demobesin 4 and Demobesin 1, were characterized in vitro as gastrin-releasing peptide (GRP) receptor agonist and antagonist, respectively, and were compared as (99m)Tc-labeled ligands for their in vitro and in vivo tumor-targeting properties. METHODS: N(4)-[Pro(1),Tyr(4),Nle(14)]Bombesin (Demobesin 4) and N(4)-[d-Phe(6),Leu-NHEt(13),des-Met(14)]bombesin(6-14) (Demobesin 1) were characterized in vitro for their binding properties with GRP receptor autoradiography using GRP receptor-transfected HEK293 cells, PC3 cells, and human prostate cancer specimens. Their ability to modulate calcium mobilization in PC3 and transfected HEK293 cells was analyzed as well as their ability to trigger internalization of the GRP receptor in transfected HEK293 cells, as determined qualitatively by immunofluorescence microscopy and quantitatively by enzyme-linked immunosorbent assay (ELISA). Further, their internalization properties as (99m)Tc-labeled radioligands were tested in vitro in both cell lines. Finally, their biodistribution was analyzed in PC3 tumor-bearing mice. RESULTS: A comparable binding affinity with the 50% inhibitory concentration (IC(50)) in the nanomolar range was measured for Demobesin 4 and Demobesin 1 in all tested tissues. Demobesin 4 behaved as an agonist by strongly stimulating calcium mobilization and by triggering GRP receptor internalization. Demobesin 1 was ineffective in stimulating calcium mobilization and in triggering GRP receptor internalization. However, in these assays, it behaved as a competitive antagonist as it reversed completely the agonist-induced effects in both systems. (99m)Tc-Labeled Demobesin 1 was only weakly taken up by PC3 cells or GRP receptor-transfected HEK293 cells (10% and 5%, respectively, of total added radioactivity) compared with (99m)Tc-labeled Demobesin 4 (45% of total added radioactivity in both cell lines). Remarkably, the biodistribution study revealed a much more pronounced uptake at 1, 4, and 24 h after injection of (99m)Tc-labeled Demobesin 1 in vivo into PC3 tumors than (99m)Tc-labeled Demobesin 4. In vivo competition experiments demonstrated a specific uptake in PC3 tumors and in physiologic GRP receptor-expressing tissues. The tumor-to-kidney ratios were 0.7 for Demobesin 4 and 5.2 for Demobesin 1 at 4 h. CONCLUSION: This comparative in vitro/in vivo study with Demobesin 1 and Demobesin 4 indicates that GRP receptor antagonists may be superior targeting agents to GRP receptor agonists, suggesting a change of paradigm in the field of bombesin radiopharmaceuticals.

Regulation of Id1 expression by SRC: implications for targeting of the bone morphogenetic protein pathway in cancer

Deregulated activation of the Src tyrosine kinase and heightened Id1 expression are independent mediators of aggressive tumor biology. The present report implicates Src signaling as a critical regulator of Id1 gene expression. Microarray analyses showed that Id family genes were among the most highly down-regulated by incubation of A549 lung carcinoma cells with the small-molecule Src inhibitor AZD0530. Id1 transcript and protein levels were potently reduced in a dose-dependent manner concomitantly with the reduction of activated Src levels. These effects were conserved across a panel of lung, breast, prostate, and colon cancer cell lines and confirmed by the ability of PP2, Src siRNA, and Src-blocking peptides to suppress Id1 expression. PP2, AZD0530, and dominant-negative Src abrogated Id1 promoter activity, which was induced by constitutively active Src. The Src-responsive region of the Id1 promoter was mapped to a region 1,199 to 1,360 bps upstream of the translation start site and contained a Smad-binding element. Src was also required for bone morphogenetic protein-2 (BMP-2)-induced Id1 expression and promoter activity, was moderately activated by BMP-2, and complexed with Smad1/5. Conversely, Src inhibitors blocked Smad1/5 nuclear translocation and binding to the Src-responsive region of the Id1 promoter. Consistent with a role for Src and Id1 in cancer cell invasion, Src inhibitors and Id1 siRNA decreased cancer cell invasion, which was increased by Id1 overexpression. Taken together, these results reveal that Src positively interacts with the BMP-Smad-Id pathway and provide new ways for targeted inhibition of Id1.

Drug targeting using OX7-immunoliposomes: correlation between Thy1.1 antigen expression and tissue distribution in the rat

OX7 monoclonal antibody F((ab')2) fragments directed against Thy1.1 antigen can be used for drug targeting by coupling to the surface of drug-loaded liposomes. Such OX7-conjugated immunoliposomes (OX7-IL) were used recently for drug delivery to rat glomerular mesangial cells, which are characterized by a high level of Thy1.1 antigen expression. In the present study, the relationship between OX7-IL tissue distribution and target Thy1.1 antigen localization in different organs in rat was investigated. Western blot and immunohistofluorescence analysis revealed a very high Thy1.1 expression in brain cortex and striatum, thymus and renal glomeruli. Moderate Thy1.1 levels were observed in the collecting ducts of kidney, lung tissue and spleen. Thy1.1 was not detected in liver and heart. There was a poor correlation between Thy1.1 expression levels and organ distribution of fluorescence- or (14)C-labeled OX7-IL. The highest overall organ density of OX7-IL was observed in the spleen, followed by lung, liver and kidney. Heart and brain remained negative. With respect to intra-organ distribution, a localized and distinct signal was observed in renal glomerular mesangial cells only. As a consequence, acute pharmacological (i.e. toxic) effects of doxorubicin-loaded OX7-IL were limited to renal glomeruli. The competition with unbound OX7 monoclonal antibody F((ab')2) fragments demonstrated that the observed tissue distribution and acute pharmacological effects of OX7-IL were mediated specifically by the conjugated OX7 antibody. It is concluded that both the high target antigen density and the absence of endothelial barriers are needed to allow for tissue-specific accumulation and pharmacological effects of OX7-IL. The liposomal drug delivery strategy used is therefore specific toward renal glomeruli and can be expected to reduce the risk of unwanted side effects in other tissues.

Natalizumab: targeting alpha4-integrins in multiple sclerosis

In 1992, it was shown that monoclonal antibodies blocking alpha(4)-integrins prevent the development of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis (MS). As alpha(4)beta(1)-integrin was demonstrated to mediate the attachment of immune-competent cells to inflamed brain endothelium in experimental autoimmune encephalomyelitis, the therapeutic effect was attributed to the inhibition of immune cell extravasation and inflammation in the central nervous system. This novel therapeutic approach was rapidly and successfully translated into the clinic. The humanized anti-alpha(4)-integrin antibody natalizumab demonstrated an unequivocal therapeutic effect in preventing relapses and slowing down the pace of neurological deterioration in patients with relapsing-remitting MS in phase II and phase III clinical trials. The occurrence of 3 cases of progressive multifocal leukoencephalopathy in patients treated with natalizumab led to the voluntary withdrawal of the drug from the market. After a thorough safety evaluation of all patients receiving this drug in past and ongoing studies for MS and Crohn's disease, natalizumab again obtained approval in the US and the European Community. A treatment targeting leukocyte trafficking in MS has now re-entered the clinic. Further thorough evaluation is necessary for a better understanding of the risk-benefit balance of this new treatment option for relapsing MS. In this review, we discuss the basic mechanism of action, key clinical results of clinical trials and the emerging indication of natalizumab in MS.

Selective pharmacological targeting of a DEAD box RNA helicase

RNA helicases represent a large family of proteins implicated in many biological processes including ribosome biogenesis, splicing, translation and mRNA degradation. However, these proteins have little substrate specificity, making inhibition of selected helicases a challenging problem. The prototypical DEAD box RNA helicase, eIF4A, works in conjunction with other translation factors to prepare mRNA templates for ribosome recruitment during translation initiation. Herein, we provide insight into the selectivity of a small molecule inhibitor of eIF4A, hippuristanol. This coral-derived natural product binds to amino acids adjacent to, and overlapping with, two conserved motifs present in the carboxy-terminal domain of eIF4A. Mutagenesis of amino acids within this region allowed us to alter the hippuristanol-sensitivity of eIF4A and undertake structure/function studies. Our results provide an understanding into how selective targeting of RNA helicases for pharmacological intervention can be achieved.

Targeting vessels to treat hepatocellular carcinoma

The process of blood vessel proliferation, known as angiogenesis, is essential during embryonic development and organogenesis. In adult life, it participates in normal tissue repair, wound healing, and cyclical growth of the corpus luteum and the endometrium. Crucial as it is, angiogenesis can become pathological, and abnormal angiogenesis contributes to the pathogenesis of inflammatory and neoplasic diseases. The present review highlights the evidence for the role of angiogenesis in HCC (hepatocellular carcinoma) and discusses the increasing importance of inhibitors of angiogenesis in HCC therapy.