Biodistribution of ⁶⁸Ga-labelled phosphodiester, phosphorothioate, and 2′-O-methyl phosphodiester oligonucleotides in normal rats

Abstract

Antisense oligonucleotides may hybridise with high selectivity to mRNA sequences allowing monitoring of gene expression or inhibition of the manifestation of altered genes inducing diseases. As part of the development of positron emission tomography methods, 17-mer antisense phosphodiester (PO), phosphorothioate (PS) and 2′-O-methyl phosphodiester (OMe) oligonucleotides specific for point mutationally activated human K-ras oncogene were labelled with ⁶⁸Ga radionuclide via a chelator coupled to the probe. Hybridisation in solution and non-denaturing polyacrylamide gel electrophoresis (PAGE) with a subsequent exposure of the gels was performed to verify the hybridisation ability after labelling. The biodistribution was studied in male Sprague–Dawley rats by injecting 2 MBq of ⁶⁸Ga-oligonucleotides via the tail vein and measuring the organ radioactivity concentration after 20, 60 and 120 min or using whole-body autoradiography with 10 MBq ⁶⁸Ga-oligonucleotide and 20 min incubation time. Control experiments were performed with ⁶⁸GaCl₃ and ⁶⁸Ga–chelator complex. The results revealed that ⁶⁸Ga-labelling did not change the hybridisation abilities of the oligonucleotides. The biodistribution pattern depended on the nature of the oligonucleotide backbone. Bone marrow, kidney, liver, spleen and urinary bladder were the five organs of highest uptake with each oligonucleotide. The PO accumulated highly in the liver, whereas high kidney uptake dominated the PS and OMe patterns. Intact PS and OMe were detected in plasma samples taken 20 and 60 min after injection. This study supplies a base for the further development of ⁶⁸Ga-labelled oligonucleotides as pharmacokinetic tools and a potential future use for in vivo imaging of gene expression.

Introduction

In today's molecular medicine, the hypothesis is that many diseases result from altered patterns of gene expression, which converts cells into the “sick” phenotypes. For example, alterations in the cellular genes, which directly or indirectly control cell growth and differentiation, are generally considered the main cause of cancer. Members of the ras gene family, H-ras, K-ras and N-ras, are thought to be involved in normal cell growth and maturation. However, a point mutation induced by carcinogens or environmental factors causing an amino acid alteration at one of the three critical positions in the protein results in conversion to a form that is involved in the formation of tumours. Approximately 10–20% of all human tumours have a mutation in one of the ras oncogenes; however over 90% of pancreatic adenocarcinomas, about 50% of adenocarcinomas of colon, lung adenocarcinomas, thyroid carcinomas, and a large fraction of haematological malignancies have been found to be associated with point mutationally activated ras oncogenes (Bos, 1989).

The growing number of identified genes behind various human diseases has promoted the idea of using this information for treatment by “knocking down” their expressions. Antisense oligonucleotide may be such a molecule that could reach this goal on the basis of arresting the mRNA in the cell and thereby preventing it from being translated into a protein. These short, synthetic nucleic acids manifest the inhibition effect on the gene expression by hybridising with their complementary “sense” sequences in mRNA through Watson–Crick base-pairing. The therapeutic utility of antisense nucleic acids was already suggested by Stephenson following the first encouraging experiments (Zamecnik and Stephenson, 1978), which entailed the testing of several antisense oligonucleotides in different in vitro model systems. However, the most intensive research concerning the in vivo applications has been conducted in the past decade. Successful “knock-down” of gene expression has been demonstrated in cell cultures (Monia et al., 1992, Kita et al., 1999, Shi and Siemann, 2002, Yoo et al., 2004), and in small animal experiments (Lai et al., 1996, Lopes de Menezes et al., 2000, Fluiter et al., 2002). A number of human trials have started with the aim of inhibiting disease-related genes in viral infections (Amado et al., 1999, Detrick et al., 2001), cardiovascular diseases (Phillips et al., 2000), and cancer (Nesterova and Cho-Chung, 2000, Yang et al., 2001, Abaza et al., 2003). There is at least one antisense drug available, Vitravene (Marwick, 1998), and others are in clinical trials (Opalinska and Gewirtz, 2002, Dean and Bennett, 2003, Crooke, 2004).

For effective inhibition, the antisense oligonucleotides should be specific and accessible to the target, stable in vivo, and possess minimal non-specific interactions. The fact that natural oligonucleotides with their phosphodiester backbone proved unstable to serum and cellular nucleases (Sands et al., 1994) led to the search for analogues with improved stability. The phosphorothioate oligonucleotide, which is resistant to metabolism (Agrawal and Iyer, 1997, Henry et al., 1999), is the most studied analogue. Methylphosphonate, phosphoramidate, 2′-O-methyl RNA, morpholino oligonucleotide, mixed-backbone oligonucleotide, peptide nucleic acid (PNA) and locked nucleic acid (LNA) are other modified alternatives, which have been tested (Wahlestedt et al., 2000, Younes et al., 2002) to find good candidates. Obviously, any chemical modification of the oligonucleotide could be expected to have effect on the specific hybridisation, sensitivity to nucleases, elimination, membrane passage, protein binding etc., and thereby may cause major alterations in the pharmacokinetics, essential for its biological activity. A method allowing pharmacokinetic measurements of oligonucleotides in vivo would be of value to characterise this important aspect of antisense oligonucleotides having a therapeutic potential (Tavitian et al., 1998).

Positron emission tomography (PET) is a most advanced technology allowing to image biological processes (e.g. molecular interactions) in vivo and to obtain knowledge about the fate of drugs and substances pharmacokinetics, pharmacodynamics, and metabolism (Långström and Bergström, 1995, Jones, 1996, Långström et al., 1999, Paans and Vaalburg, 2000, Eckelman, 2002). Therefore, it is logical to suggest this technology as a tool in the development of antisense therapy with respect to selective accumulation in target organs or for the assessment of drug concentration in organs where side effects may be induced. Additionally, PET might be able to offer a non-invasive diagnostic tool to analyse gene expression, converting in vitro (in situ) hybridisation to in vivo hybridisation.

To be able to record antisense oligonucleotides in vivo with PET, methods were developed by which different oligonucleotides and other analogues may be labelled using ¹⁸F, ⁷⁶Br, ¹²⁵I and ⁶⁸Ga as radionuclides (Yngve et al., 1999, Kühnast et al., 2000, Velikyan et al., 2004a). In our previous publication, we examined ⁷⁶Br-labelled antisense oligonucleotides of different lengths (Wu et al., 2000). Following up and extending the used preclinical methods, we have in the present study in vitro and ex vivo investigated ⁶⁸Ga-labelled 17-mer phosphodiester (PO), phosphorothioate (PS) and 2′-O-methyl phosphodiester (OMe) antisense oligonucleotides specific for point mutationally activated K-ras oncogene to assess the effect of labelling on the hybridisation abilities and to obtain information about biodistribution and metabolism in rats. The present publication summarises the experiments performed in Uppsala, and in this manner, it is a co-paper of the publication by Roivainen et al. reporting the Turku part of the K-ras antisense oligonucleotide study including in vitro, ex vivo and also in vivo experiments (Roivainen et al., 2004). We chose the K-ras oncogene as a model gene towards in vivo imaging because of its significance in cancer biology and oncology.