Evidence of Antisense Tumor Targeting in Mice - Bioconjugate

Datta E. Ponde , ZiFen Su , Alan Berezov , Hongtao Zhang , Abbas Alavi , Mark I. Greene , Ramachandran Murali. Bioorganic & Medicinal Chemistry Letter...
1 downloads 0 Views 519KB Size
Download PDF
Bioconjugate Chem. 2004, 15, 1475−1480

1475

TECHNICAL NOTES Evidence of Antisense Tumor Targeting in Mice K. Nakamura,† C. Fan,‡,§ G. Liu,‡ S. Gupta,‡ J. He,‡ S. Dou,‡ A. Kubo,† M. Rusckowski,‡ and D. J. Hnatowich*,‡ Department of Radiology, Keio University School of Medicine, Tokyo, Japan, Department of Radiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, and Department of Nuclear Medicine, West China Hospital, Sichuan University, Chengdu, China. Received April 13, 2004; Revised Manuscript Received May 27, 2004

Even though increased accumulations of radiolabeled antisense DNAs compared to control DNAs are becoming a routine observation in cultured tumor cells, trustworthy evidence of tumor targeting in vivo by an antisense mechanism remains elusive. The goal of this study was to obtain convincing evidence of antisense tumor targeting in nude mice by using two different tumors and both intratumoral (i.t.) and intravenous (i.v.) administration of radiolabeled antisense and control sense DNAs. Both the MDR++ cell line KB-G2 and its parent MDR+ cell line KB-31 were used in this study. The antisense (AS) DNA was directed against the AUG start codon of the MDR1 mRNA and, along with the sense (S) control DNA, was a uniform phosphorothioate administered naked. In previous cell culture studies from our laboratories, the accumulation of this AS DNA was strikingly high in KB-G2 cells and only average in KB-31 cells, a fact we attribute to the 1000-fold higher expression by RT-PCR of MDR1 mRNA in the former cell line. In this study, both DNAs were radiolabeled with 99mTc via MAG3 and administered i.t. or i.v. at 1 µg (100 µCi) per animal 24 h prior to sacrifice and dissection in mice bearing thigh tumors of about 1 g. Following i.t. administration, no statistically significant differences (Student’s t test, p < 0.05, N ) 4) between the AS and S DNA biodistributions in normal tissues were observed except in the KB-G2 mice in which muscle levels were lower for the S control. In contrast, tumor levels in the KB-G2 animals were significantly higher for the AS DNA vs S DNA (14.7 vs 8.5% ID/g) while this difference (8.6 vs 4.3% ID/g) was insignificant in the KB-31 animals. The whole body images obtained just prior to sacrifice clearly show improved targeting of AS DNA vs S DNA in the KB-G2 but not the KB-31 animals. Calculations based on these results show that about 60 000 AS DNAs accumulated specifically (i.e. AS DNA - S DNA) per KB-G2 tumor cell following i.t. administration. When administered i.v. rather than i.t., higher tumor levels in KB-G2 animals compared to KB-31 were not observed, most likely because of the lower dosage reaching the tumors. When the KB-G2 and KB-31 results are combined, no statistically significant differences between the AS and S DNA biodistributions in normal tissues were observed except in blood in which S DNA levels were higher and in spleen in which they were lower. In contrast, tumor levels were significantly higher for the AS DNA vs S DNA (0.100 vs 0.063% ID/g). Calculations based on these results show that about 400 AS DNAs accumulated specifically per tumor cell following i.v. administration. Therefore evidence for tumor targeting in vivo by an antisense mechanism has been obtained in that statistically higher tumor accumulations of the 99mTc-AS DNA were observed compared to the control 99mTc-S DNA both following i.t. and i.v. administrations. The successful localization of AS DNA in tumor demonstrates that in vivo AS targeting of tumor is feasible although improvements in tumor delivery and normal tissue clearance are needed for practical antisense imaging.

INTRODUCTION

Before radiolabeled antisense (AS) oligomers merit serious consideration as targeting modalities for tumor and other tissues, it must first be demonstrated to the * Corresponding author: D. J. Hnatowich, Ph.D., Professor of Radiology, Division of Nuclear Medicine, Department of Radiology, H2-579, University of Massachusetts Medical School 55 Lake Ave. North, Worcester, MA 01655. Tel: (508) 856-4256. Fax: (508) 856-4572. E-mail: [email protected]. † Keio University School of Medicine. ‡ University of Massachusetts Medical School. § Sichuan University

greatest possible extent that localization follows an antisense mechanism. To prove an antisense mechanism may be extremely difficult especially in the case of phosphorothioate DNAs that show high binding affinities for proteins. One obvious concern is the possibility of binding by an aptameric mechanism in which the AS oligomer, but not its control oligomer, assumes a particular configuration that encourages binding to cellular proteins by a sequence-specific but non-antisense mechanism (1). If increased accumulations compared to control DNAs are observed for a variety of DNAs, radiolabeled by different methods, with antisense base sequence against different messenger RNA (mRNA) targets and

10.1021/bc0499073 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/05/2004

1476 Bioconjugate Chem., Vol. 15, No. 6, 2004

in different cell types with irrelevant base sequences (e.g. sense, random, scrambled), then an aptameric mechanism for the increased accumulations may be largely excluded. Since oligomers with different base sequences will show differences in behavior, a large number of such studies with positive accumulations compared to controls will be necessary to exclude aptameric effects as a plausible mechanism. In general, to help establish an antisense mechanism by a different means, at a minimum, four criteria should be met: (1) higher accumulations must be demonstrated for the AS oligomer in comparison to a control oligomer designed to be as identical as possible to that of the study DNA but with restricted or nonexistent hybridization affinities for the mRNA target; (2) higher accumulations compared to controls should be demonstrated for the AS oligomer in a variety of cell types, hopefully with accumulations in proportion to the estimated mRNA target concentrations of each cell type; (3) the higher accumulations should be demonstrated for different AS oligomers directed against different mRNA targets as evidence against aptameric effects; and (4) the higher accumulations of the AS oligomer should be shown to decrease with increasing AS DNA concentrations as evidence of specific binding. While a direct demonstration of an antisense mechanism remains elusive even in cell culture, indirect evidence of this mechanism is now common. We have observed statistically significant increased accumulation of radioactivity in LS174T colon cancer cells and ACHN kidney cancer cells with endogenously labeled 35S as well as 99mTc-radiolabeled uniform phosphorothioate MAG3DNA against the regulatory subunit I alpha (RIR) mRNA of protein kinase A (PKA) compared to sense and random DNA controls but not in HC2 cells with irrelevant murine RIR mRNA (2). An increased accumulation of radioactivity in MDA-MB-231 breast cancer cells with 99mTc-labeled Hynic-DNA antisense to the c-myc oncogene compared to the S DNA control has also been observed (3). Recently a statistically significant increased accumulation was also observed in targeting of multidrug resistance mRNA in epidermal carcinoma cells KB-G2 and KB-31 with 99mTc-labeled AS MAG3-DNA compared to the S DNA control (4). Should an AS oligomer assume a particular configuration that encourages increased cellular accumulation by an aptameric effect, this would be extremely unlikely to be the case for each of the large variety of base sequences, labeling methods, mRNA targets and cell types described in the above studies. Another test of specific mechanism of localization is to demonstrate that accumulation decreases with increasing dosage as binding becomes saturated. When ACHN cells were incubated with 99mTc-labeled AS DNA with increasing concentrations of unlabeled AS DNA in the range 7 to 100 nM and compared to the accumulations of 99mTc-labeled S DNA with increasing concentrations of unlabeled S DNA in the same range, the difference in AS compared to S DNA accumulations decreased and became statistically insignificant as the concentration increased (2). More definitive results have recently been observed for 99mTc-labeled AS and control S DNAs in the KB-G2 and KB-31 cells as well as TCO-1 cells, another multidrug resistance positive cell type (4). These studies in cell culture therefore generally satisfy the above four conditions necessary to claim evidence of an antisense mechanism. The more important remaining challenge is therefore to demonstrate evidence of an antisense mechanism in vivo. Herein we report on the first results from these laboratories of in vivo antisense

Technical Notes

targeting of tumor in mice. The MDR++ cell line KB-G2 and its parent MDR+ cell line KB-31 were again used along with the same AS and control S DNAs used previously with these cell types. The expression of MDR1 has been reported to be 1000-fold higher by RT-PCR for KB-G2 cells compared to KB-31 cells (4). In that investigation, the increased expression of MDR in the KB-G2 cells compared to KB-31 cells and the specificity of the AS DNA for its target were both confirmed in cell culture using 99mTc-sestamibi, a substrate of MDR. Because delivery is an important issue in antisense targeting, especially when oligomers are to be administered naked as in this case, intratumor (i.t.) as well as intravenous (i.v.) administrations were performed. MATERIALS AND METHODS

Uniform phosphorothioate DNAs were obtained for this investigation with a primary amine on the 5′-end via a six-carbon alkyl linker (Qiagen, Alameda, CA). All DNAs were 20-mer and all were HPLC purified by the supplier. The base sequence of the AS DNA was 5′-CCA-TCCCGA-CCT-CGC-GCT-CC while that of the S control was 5 ‘-GGA-GCG-CGA-GGT-CGG-GAT-GG (5). S-Acetyl NHSMAG3 was synthesized in house (6) and its structure confirmed by elemental analysis, proton nuclear magnetic resonance, and mass spectroscopy. The P4 medium for separation was purchased (Bio-Rad Laboratories Inc., Hercules, CA). The 99mTc-pertechnetate was eluted from a 99Mo-99mTc generator (Bristol-Myers Squibb Medical Imaging, Inc., North Billerica, MA, or Daiichi Radioisotopes, Tokyo, Japan). The two cell lines were a generous gift from Dr. I. Sugawara, Research Institute of Tuberculosis, Tokyo, Japan. Other chemicals were obtained from various suppliers and used without purification. DNA Conjugation and Radiolabeling. Both DNAs were conjugated with S-acetyl NHS-MAG3 via the 5′derivatized amine as previously described (7). A solution of 1 mg (about 0.15 µmol) of DNA in 200 µL of 0.2 M pH 8.0 HEPES buffer was added to a vial containing 1.72.0 mg (about 4.5 µmol) S-acetyl NHS-MAG3. The vial was vortexed immediately and incubated for 1 h at room temperature. The DNA was then purified on a 0.7 × 20 cm P4 column with either 0.25 M pH 5.2 NH4OAc buffer or 0.05 M PBS pH 7.2 buffer as eluant. The peak fractions were pooled and the DNA concentration quantitated by UV absorbance at 265 nm. Radiolabeling of the DNAs was achieved by introducing about 1 mCi (20-40 µL) of 99mTc-pertechnetate generator eluate into a combined solution consisting of 50 µL (about 10 µg) MAG3-conjugated DNA in either buffer, 25 µL of 50 µg/µL Na2tartrate‚2H2O in a pH 9.2 buffer, and 5 µL of fresh 4 µg/µL SnCl2‚2H2O in 10 mM HCl. The final pH was about 7.8. After vortexing and then heating for 20 min in boiling water, the labeling was confirmed by C18 SepPak (Waters, Milford, MA) in which the first elution with PBS removes radiolabeled pertechnetate and tartrate, the second elution with 40% acetonitrile removes radiolabeled DNA and the SepPak retained radiolabeled colloids. In most cases, the radiochemical purity exceeded 90% without purification, and the specific radioactivity was about 100 µCi/µg. Biodistribution Studies. All animal studies at UMMS were performed with the approval of the UMMS Institutional Animal Care and Use Committee while all animal studies at Keio University School of Medicine were performed with the approval of The Laboratory Animal Care and use Committee. For the i.t. studies, the cells were grown in D-MEM supplemented with 10% fetal bovine serum (FBS), 100

Bioconjugate Chem., Vol. 15, No. 6, 2004 1477

Technical Notes Table 1. Biodistribution of 99mTc-Labeled Antisense and Sense DNA Obtained 24 h Postintratumoral Administration to Nude Mice Bearing KB-G2 Tumors (% ID/g (SD) N ) 4)

Table 2. Biodistribution of 99mTc-Labeled Antisense and Sense DNA Obtained 24 h Postintratumoral Administration to Nude Mice Bearing KB-31 Tumors (% ID/g (SD) N ) 4)

tissue

antisense

sense

P value

tissue

antisense

sense

P value

blood heart lung liver spleen small intestines large intestines kidney muscle tumor

0.018 (0.003) 0.016 (0.002) 0.023 (0.005) 0.215 (0.080) 0.052 (0.024) 0.247 (0.070) 3.01 (0.43) 0.243 (0.018) 0.027 (0.007) 14.74 (3.24)

0.021(0.005) 0.018 (0.003) 0.030 (0.013) 0.301 (0.200) 0.083 (0.056) 0.493 (0.389) 5.27 (2.76) 0.323 (0.083) 0.015 (0.005) 8.52 (3.45)

0.377 0.415 0.319 0.455 0.354 0.260 0.158 0.107 0.027a 0.039a

blood heart lung liver spleen small intestines large intestines kidney muscle tumor

0.019 (0.005) 0.018 (0.006) 0.025 (0.006) 0.230 (0.120) 0.057 (0.016) 0.189 (0.081) 2.86 (0.89) 0.200 (0.009) 0.014 (0.006) 8.65 (5.00)

0.023 (0.011) 0.020 (0.015) 0.053 (0.057) 0.291 (0.245) 0.079 (0.059) 0.156 (0.121) 3.70 (3.07) 0.276 (0.164) 0.063 (0.039) 4.30 (3.38)

0.546 0.847 0.369 0.695 0.497 0.663 0.619 0.389 0.050 0.199

a

Difference statistically significant (Student’s t test).

units/mL penicillin, and 100 µg/mL streptomycin (GibcoInvitrogen, Carlsbad, CA). Cells were maintained as monolayers in a humidified 5% carbon dioxide atmosphere, normally in T75 flasks (Falcon, Becton Dickinson, Lincoln Park, NJ). The cells were trypsinated in the T75 flasks at 80-90% confluence using 0.05% trypsin/0.02% EDTA and were then suspended in MEM with 10% FBS. For the i.v. studies, the cells were grown in RPMI medium (RPMI 1640, Gibco BRL Products, Gaithersburg, MD) with 2.0 mg/L sodium bicarbonate, supplemented with 10% FBS. The cells were trypsinated in the T75 flasks at 80-90% confluence using 0.5% trypsin and were then suspended in PBS. Nude mice (i.t. studies: NIH Swiss, Taconic Farms, Germantown, NY, 30-40 g; i.v. studies: BALB/c AnNCrjnu male, Oriental, Tokyo, 25-30 g) were each injected subcutaneously in the left thigh with a 0.1 mL suspension containing 107 KB-G2 or KB-31 tumor cells with greater than 95% viability. Animals were used 10 days (KB-31) or14 days (KB-G2) later when the tumors were about 1.0 cm in any dimension. Each tumored nude mouse was injected with 1.0 µg of DNA (100 µCi) in 100 µL i.v. via a tail vein or with 50 µL i.t. Animals were sacrificed under halothane anesthesia at 24 h, blood and other organs were removed, weighed, and counted in a NaI(Tl) well counter along with a standard of the injectate. Statistical significance between tissue radioactivity levels in animals receiving AS vs S DNA was established by the Student’s t test two-tailed distribution and paired. In the case of animals receiving radiolabeled DNA by i.t. administration, prior to sacrifice, the animals were imaged simultaneously in groups of four while resting on the collimator of an Elscint APEX 409 M large view gamma camera (Hackensack, NJ). RESULTS

The biodistribution results obtained 24 h post i.t. administration of radiolabeled AS and S DNAs to nude mice bearing KB-G2 and KB-31 tumors are presented in Tables 1 and 2, respectively. The only normal tissues showing statistically significant differences in radioactivity accumulation of AS DNA compared to S DNA are in the KB-G2 animals in which muscle levels were lower for the S DNA. Possibly because of differences in pharmacokinetic properties of the DNAs resulting from the different base sequence, tissue levels tend to be higher in general following administration of the S DNA when administered i.t. That this difference was not seen following i.v. administration may be due to pharmacokinetic differences following delivery into the vasculature compared to the lymphatics. While the tumor accumulations were higher in both animal models following administration of the AS DNA

Table 3. Biodistribution of 99mTc-Labeled Antisense and Sense DNA Obtained 24 h Postintravenous Administration to Tumor Bearing Nude Mice. Results for KB-31 and KB-G2 Combined (% ID/g (SD) N ) 5-6) tissue

antisense

sense

P value

blood heart lung liver spleen small intestines large intestines kidney muscle tumor

0.022 (0.003) 0.055 (0.012) 0.106 (0.021) 2.58 (0.18) 0.688 (0.060) 0.151 (0.083) 0.519 (0.212) 0.778 (0.086) 0.016 (0.004) 0.100 (0.011)

0.017 (0.002) 0.049 (0.012) 0.081 (0.024) 2.46 (0.19) 1.00 (0.12) 0.098 (0.024) 0.376 (0.217) 0.699 (0.145) 0.018 (0.006) 0.063 (0.009)

0.018a 0.433 0.108 0.155 0.003a 0.189 0.302 0.324 0.719 4.8 × 10-8 a

a

Difference statistically significant (Student’s t test).

compared to S DNA, this difference was significant only in the KB-G2 case. The absence of significance in the KB31 case may be related to the large standard deviations of the tumor values resulting from the i.t. administrations. That the accumulations of AS DNA in KB-G2 tumor compared to KB-31 were significantly higher (P ) 0.047) may be related to the higher expression levels of the target MDR1 mRNA in the former tumor type. The higher accumulation of S DNAs in KB-G2 tumors compared to KB-31 tumors failed to reach significance (P ) 0.13). These biodistribution results are faithfully reflected in the whole body radioactivity images obtained at 22 h postadministration as shown in Figures 1 and 2. Each figure is a composite in which scintigraphic whole body images have been superimposed on a photographic image taken during imaging to provide accurate registration. In each image, two animals receiving the AS DNA have been imaged simultaneously with two animals receiving the S DNA, as indicated in the figures. The higher radioactivity levels in tumor (left thighs) in animals receiving the AS DNA compared to animals receiving the S DNA is readily apparent in Figure 1 for KB-G2 tumored mice. The higher levels are less evident in Figure 2 for KB-31 animals. From the specific radioactivity of the labeled DNAs, the dosage administered, the percent accumulating in each gram of tumor and under the assumption of 108 tumored cells per gram, a simple calculation will show that about 66 000 AS DNAs accumulated specifically (i.e. AS DNA accumulation - S DNA accumulation) per KBG2 cell while for KB-31, this value was 53 000 per cell. Table 3 presents biodistribution results obtained in the identical fashion to that of Tables 1 and 2 except that administration was intravenous rather than intratumoral. This study was performed separately for both KBG2 and KB-31 animals. However, unlike the i.t. study, no differences were apparent between the biodistributions obtained in KB-G2 and KB-31 animals following

1478 Bioconjugate Chem., Vol. 15, No. 6, 2004

Technical Notes

Figure 1. A composite obtained at 22 h postadministration in KB-G2 tumored mice. The scintigraphic whole body image has been superimposed on a photographic image taken during imaging to provide accurate registration. Two animals receiving the AS DNA are on the right while two identical animals receiving the control S DNA are on the left. In all cases, the tumor is in the left thigh.

Figure 2. A composite obtained at 22 h postadministration in KB-31 tumored mice. The scintigraphic whole body image has been superimposed on a photographic image taken during imaging to provide accurate registration. Two animals receiving the AS DNA are on the right while two identical animals receiving the control S DNA are on the left. In all cases, the tumor is in the left thigh.

i.v. administration (data not presented). Whereas tumor levels for both DNAs were higher in KB-G2 compared to KB-31 animals following i.t. administration, tumor levels were identical following i.v. administration. This may be due to the much smaller dosage of DNA reaching the tumor following i.v. administration compared to i.t. such that the difference in the number of mRNA targets between the two tumors may be immaterial under the i.v. conditions. The KB-G2 and KB-31 results have therefore been combined in Table 3 to improve the estimate of statistical significance. Among the normal tissues considered, only blood and spleen showed statistically significant accumulations between AS and S DNAs with the former showing lower levels and the latter higher levels for the S DNA. The accumulation of AS DNA was higher in most normal organs as shown in Table 3 although these differences are not significant. Since the mouse MDR1 gene is reported to be expressed in most mouse organs such as liver, kidney, lung, and heart and since there is reported to be homology in the mouse and human gene (8), some

of the observed increased accumulations of AS DNA in normal tissues may be specific. Possibly because the variability in tumor accumulations was much smaller following i.v. vs i.t. administration, the higher accumulation of the AS DNA in tumor compared to S DNA following i.v. administration is considerably more statistically significant compared to that of the KB-G2 tumors following i.t. administration. The identical calculation applied to animals administered i.v. as that described above for animals receiving i.t. injections show that about 400 AS DNAs accumulated specifically in each tumor cell. DISCUSSION

An evaluation of studies in cell culture with AS oligomers permits the conclusion that, thus far, results have been very positive (2). Accumulations have been observed by what appears to be an antisense mechanism, and the magnitude of these accumulations have been much greater than that predicted on the basis of estimated steady-state mRNA levels (9). The more difficult

Technical Notes

challenge that remains is now to demonstrate that AS oligomers will accumulate in target tissues in vivo by an antisense mechanism. Among the factors that will make this challenge difficult is insufficient delivery of AS DNA into tumor cells when administered i.v. While in culture, cellular accumulations are at levels that would certainly provide adequate nuclear medicine images; the barriers to in vivo cellular accumulations will reduce these levels considerably. Another factor contributing to the difficulties ahead may be nonspecific accumulations of radiolabeled DNAs in nontarget tissues leading to high background radioactivity levels. An impression that antisense imaging may not be achievable has been reinforced by the general absence of positive results thus far in the limited number of published studies of in vivo antisense targeting (10, 11). Recently positive images were reported in mouse xenografts in which the target mRNA could be upregulated by epidermal growth factor (12). Superior images were obtained following 111 In-labeled DNA administration in mice bearing the tumor following upregulation. However, since no significant differences were observed between the AS DNA and its random control DNA, the improvement in imaging with upregulation may not be due to an antisense mechanism. The possibility that the intratumor administration of epidermal growth factor has altered tumor phenotype to explain the increased accumulations cannot be excluded. Recently, several other in vitro and/or in vivo studies have been reported using control tumors (13-15). While useful in other ways, the results of these investigations shed little light on whether accumulations were by an antisense mechanism since control oligomers were not used. Convincing evidence of an antisense mechanism in the in vivo targeting of firefly luc gene in the brain of transgenic mice was obtained using a 125I-labeled peptide nucleic acid (PNA) and quantitative autoradiography. Higher accumulations of the antisense oligomer was reported compared to control oligomer (16). Autoradiography had also earlier been used to show higher in vivo accumulations of a 11Clabeled AS DNA against a target mRNA expressed in rat tumor compared to a mismatched control DNA (17). A more recent report presents whole body images of tumored rats obtained with 68Ga-AS DNA directed against the K-ras mRNA (18). While a control tumor not expressing the target mRNA was used and accumulation of labeled AS DNA compared to the study tumor, a control DNA was apparently not used in the imaging studies. In another study, a careful investigation of antisense targeting of c-myc mRNA in mice using 99mTc-labeled PNAs showed a significantly higher accumulation in tumor of the AS compared to mismatched control oligomer at 4 h but not 24 h post i.v. administration (19). Despite the encouraging results of this study, a positive biodistribution in a tumored animal that may be attributed to antisense targeting has not yet been reported. In a pioneer study, Dewanjee et al. reported surprisingly positive results in a mouse tumor model using an AS DNA of unspecified sequence complementary to the initiation codon site of the c-myc oncogene mRNA (20). However, these favorable results have yet to be reproduced elsewhere. The results obtained in this in vivo investigation are encouraging. In the absence of additional studies, it is not possible to claim that the mechanism of tumor accumulation observed herein involved in vivo antisense targeting. However, the significantly higher levels of AS DNA compared to control S DNA accumulated in tumor both following i.t. and i.v. administrations in a well

Bioconjugate Chem., Vol. 15, No. 6, 2004 1479

characterized AS DNA/tumor cell system (4) provides what may be convincing, albeit not yet definitive, evidence for antisense tumor targeting. These results would appear to satisfy two of the four criteria described above that may be necessary and sufficient to claim evidence of an antisense mechanism. First, tumor accumulation of AS DNA reached higher levels compared to control DNA. Second, tumor accumulation appeared to increase with increasing target mRNA levels in that the accumulations in Pgp++ KB-G2 tumors exceeded that of Pgp+ KB-31 tumor following i.t. administration. That no difference between tumor types was observed following i.v. administration may be attributed to the low dosage of DNAs delivered via this route that may have been so far below saturation levels that differences in mRNA target concentrations became irrelevant. In conclusion, evidence for tumor targeting in vivo by an antisense mechanism has been obtained. The successful localization of AS DNA in tumor demonstrates that in vivo antisense targeting of tumor is feasible although improvements in tumor delivery and clearance from normal tissues are needed for practical antisense imaging. ACKNOWLEDGMENT

The preparation of this report was supported in part by the Office of Science (BER), U.S. Department of Energy, Grant Nos. DE-FG02-99ER62781 and DE-FG0203ER63602. One of us (K.N.) was supported by a Grantin-Aid for Scientific Research (13670965) from the Japanese Ministry of Education, Science, Sports and Culture. LITERATURE CITED (1) Hnatowich, D. J. (1999) Antisense and Nuclear Medicine. J. Nucl. Med. 40, 693-703. (2) Zhang, Y. M., Wang, Y., Liu, N., Zhu, Z. H., Rusckowski, M., and Hnatowich, D. J. (2001) In vitro investigations of tumor targeting with 99mTc-labeled antisense DNA. J. Nucl. Med. 42, 1660-1669. (3) Zhang, Y., Rusckowski, M., Liu, N., Liu, C., and Hnatowich, D. J. (2001) Cationic liposomes enhance cellular/nuclear localization of 99mTc-antisense oligonucleotides in target tumor cells. Cancer Biother. Radiopharm. 16, 411-419. (4) Nakamura, K., Liu, G., Zhang, Y., Kubo, A., and Hnatowich, D. J. (2003) Antisense targeting of P-glycoprotein MDR expression in tissue culture. J. Nucl. Med. 44, 304 (Abstract). (5) Alahari, S. K., DeLong, R., Fisher, M. H., Dean, N. M., Viliet, P., and Juliano, R. L. (1998) Novel chemically modified oligonucleotides provide potent inhibition of P-glycoprotein expression. J. Pharm. Exp. Ther. 286, 419-428. (6) Winnard, P., Jr., Chang, F., Rusckowski, M., Mardirossian, G., and Hnatowich, D. J. (1997) Preparation and use of NHSMAG3 for technetium-99m labeling of DNA. Nucl. Med. Biol. 24, 425-432. (7) Liu, G., Zhang, S., He, J., Zhu, Z., Rusckowski, M., and Hnatowich, D. J. (2002) Improving the labeling of S-acetyl NHS-MAG3-conjugated morpholino oligomers. Bioconjugate Chem. 13, 893-897. (8) Chen, C., and Smith, B. J. (2003) Utility of Mdr1-gene deficient mice in assessing the impact of P-glycoprotein on pharmacokinetics and pharmacodynamics in drug discovery. Curr. Drug Metab. 4, 272-291. (9) Hnatowich, D. J. (2000) Antisense and Nuclear Medicine. Where are we now? Cancer Biother. Radiopharm. 15, 447-457. (10) Lewis, M. R., and Jia, F. (2003) Antisense imaging: and miles to go before we sleep? J. Cell Biochem. 90, 464-474. (11) Younes, C. K., Boisgard, R., and Tavitian, B. (2002) Labeled oligonucleotides as radiopharmaceuticals: pitfalls, problems and perspectives. Curr. Pharm. Des. 8, 1451-1466. (12) Wang, J., Chen, P., Mrkobrada, M., Hu, M., Vallis, K. A., and Reilly, R. M. (2003) Antisense imaging of epidermal growth factor-induced p21WAF-1/CIP-1 gene expression in MDA-

1480 Bioconjugate Chem., Vol. 15, No. 6, 2004 MB-468 human breast cancer xenografts Eur. J. Nucl. Med. Mol. Imaging 30, 1273-1280. (13) Lewis, M. R., Jia, F., Gallazzi, F., Wang, T., Zhang, J., Shenoy, N., Lever, S. Z., and Hannink, M. (2002) Radiometallabeled peptide-PNA conjugates for targeting bcl-2 expression: preparation, characterization, and in vitro mRNA binding. Bioconjugate Chem. 13, 1176-1180. (14) Sato, N., Kobayashi, H., Saga, T., Nakamoto, Y., Ishimori, T., Togashi, K., Fujibayashi, Y., Konishi, J., and Brechbiel, M. W. (2001) Tumor targeting and imaging of intraperitoneal tumors by use of antisense oligo-DNA complexed with dendrimers and/or avidin in mice. Clin. Cancer Res. 7, 3606-3612. (15) Wang, L., Prakash, R. H., Stein, C. A., Koehn, R. K., and Ruffner, D. E. (2003a) Progress in the delivery of therapeutic oligonucleotides: Organ/cellular distribution and targeted delivery of oligonucleotides in vivo. Antisense Nucleic Acid Drug Dev. 13, 169-189. (16) Lee, H. J., Boado, R. J., Braasch, D. A., Corey, D. R., and Partridge, W. M. (2002) Imaging gene expression in the brain in vivo in a transgenic mouse model of Huntington’s disease with an antisense radiopharmaceutical and drug-targeting technology. J. Nucl. Med. 43, 948-956.

Technical Notes (17) Kobori, N., Imabori, Y., Mineura, K., Ueda, S., and Fujii, R. (1999) Visualization of mRNA expression in CNS using 11C-labeled phosphorothioate oliogdeoxynucleotide. Neuroreport. 10, 2971-2974. (18) Roivainen, A., Tolvanen, T., Salomake, S., Lendvai, G., Velikyan, I., Numminen, P., Valila, M., Sipila, H., Bergstrom, M., Harkonen, P., Lonnberg, H., and Langstrom, B. (2004) 68Ga-labeled oligonucleotides for in vivo imaging with PET J. Nucl. Med. 45, 347-355. (19) Rao, P. S., Tian, X., Ojn, W., Aruva, M. R., Sauter, E. R., Thakur, M. L., and Wickstrom, E. (2003) 99mTc-peptidepeptide nucleic acid probes for imaging oncogene mRNAs in tumors. Nuc. Med. Commun. 24, 857-863. (20) Dewanjee, M. K., Ghafouripour, A. K., Kapodvanjwala, M., Dewanjee, S., Sarafini, A. N., Lopez, D. M., and Sfakianakis, G. N. (1994) Noninvasive imaging of c-myc oncogene messenger RNA with indium-111-antisense probes in a mammary tumor-bearing mouse model. J. Nucl. Med. 35, 1054-1063.

BC0499073