Antisense oligonucleotides (ASOs) have potential as anti-cancer agents by specifically modulating genes involved in tumorigenesis. However, little is known about ASO biodistribution and tissue pharmacokinetics (PKs) in humans, including whether sufficient delivery to target tumor tissue may be achieved. In this preliminary study in human subjects, we used combined positron emission and computed tomography (PET-CT) imaging and subsequent modeling analysis of acquired dynamic data, to examine the in vivo biodistribution and PK properties of LY2181308 - a second generation ASO which targets the apoptosis inhibitor protein survivin. Following radiolabeling of LY2181308 with methylated carbon-11 ([(11)C]methylated-LY2181308), micro-doses (<1mg) were administered to three patients with solid tumors enrolled in a phase I trial. Moderate uptake of [(11)C]methylated-LY2181308 was observed in tumors (mean=32.5ng*h /mL, per mg administered intravenously). Highest uptake was seen in kidney and liver and lowest uptake was seen in lung and muscle. One patient underwent repeat analysis on day 15 of multiple dose therapy, during administration of LY2181308 (750mg), when altered tissue PKs and a favorable change in biodistribution was seen. [(11)C]methylated-LY2181308 exposure increased in tumor, lung and muscle, whereas renal and hepatic exposure decreased. This suggests that biological barriers to ASO tumor uptake seen at micro-doses were overcome by therapeutic dosing. In addition, (18)F-labeled fluorodeoxyglucose (FDG) scans carried out in the same patient before and after treatment showed up to 40% decreased tumor metabolism. For the development of anti-cancer ASOs, the results provide evidence of LY2181308 tumor tissue delivery and add valuable in vivo pharmacological information. For the development of novel therapeutic agents in general, the study exemplifies the merits of applying PET imaging methodology early in clinical investigations.
Transforming growth factor beta (TGFbeta) plays an important role in cancer, but accurate measurement of circulating TGFbeta is complicated by the high TGFbeta content of platelets which can release TGFbeta ex vivo. We evaluated the use of citrate-theophylline-adenosine-dipyridamole (CTAD) tubes to reduce preanalytical variation in TGFbeta measurements caused by ex vivo platelet activation. CTAD substantially reduced ex vivo platelet activation relative to traditional plasma collections in normal donors, which correlated with a decrease in measured TGFbeta levels. We show that TGFbeta levels are elevated in the majority of cancer patients with skeletal metastases, and that within-patient variability of these levels is relatively low over several weeks. Patients with elevated TGFbeta could be subdivided into groups with or without evidence of platelet contribution to measured TGFbeta levels. The use of CTAD tubes allows a better determination of a patient's TGFbeta status, and may improve classification of patients with oncologic disease.
Even though myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, the molecular alterations that lead to marrow failure have not been well elucidated. We have previously shown that the myelosuppressive TGF-β pathway is constitutively activated in MDS progenitors. Because there is conflicting data about upregulation of extracellular TGF-β levels in MDS, we wanted to determine the molecular basis of TGF-β pathway overactivation and consequent hematopoietic suppression in this disease. We observed that SMAD7, a negative regulator of TGF-β receptor I (TBRI) kinase, is markedly decreased in a large meta-analysis of gene expression studies from MDS marrow-derived CD34(+) cells. SMAD7 protein was also found to be significantly decreased in MDS marrow progenitors when examined immunohistochemically in a bone marrow tissue microarray. Reduced expression of SMAD7 in hematopoietic cells led to increased TGF-β-mediated gene transcription and enhanced sensitivity to TGF-β-mediated suppressive effects. The increased TGF-β signaling due to SMAD7 reduction could be effectively inhibited by a novel clinically relevant TBRI (ALK5 kinase) inhibitor, LY-2157299. LY-2157299 could inhibit TGF-β-mediated SMAD2 activation and hematopoietic suppression in primary hematopoietic stem cells. Furthermore, in vivo administration of LY-2157299 ameliorated anemia in a TGF-β overexpressing transgenic mouse model of bone marrow failure. Most importantly, treatment with LY-2157199 stimulated hematopoiesis from primary MDS bone marrow specimens. These studies demonstrate that reduction in SMAD7 is a novel molecular alteration in MDS that leads to ineffective hematopoiesis by activating of TGF-β signaling in hematopoietic cells. These studies also illustrate the therapeutic potential of TBRI inhibitors in MDS.
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