Synthesis and Properties of Ester-Linked Peptide Nucleic Acid

Nadia Bendifallah, Frank Winther Rasmussen, Vladimir Zachar, Peter Ebbesen, Peter ... Audrey Di Giorgio , Roger Condom , Virendra N. Pandey , Nadia Pa...
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Bioconjugate Chem. 2003, 14, 588−592

Synthesis and Properties of Ester-Linked Peptide Nucleic Acid Prodrug Conjugates Nadia Bendifallah,†,§ Edward Kristensen,‡ Otto Dahl,§ Uffe Koppelhus,† and Peter E. Nielsen*,†,‡ Center for Biomolecular Recognition, Department of Medical Biochemistry and Genetics, University of Copenhagen, The Panum Institute, Blegdamsvej 3c, DK-2200 N Copenhagen, Denmark, Pantheco A/S, Bøge Alle´ 3, 2970 Hørsholm, Denmark, and Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark. Received October 7, 2002; Revised Manuscript Received February 13, 2003

A Boc-protected amino acid containing an ester function, 2-([N-Boc-glycyl]oxymethyl)benzoic acid, has been synthesized and incorporated into peptide nucleic acid (PNA) oligomers. In model experiments it is found that the ester is fairly stable in aqueous solution at pH 7.4 and 37 °C (t1/2 ) 6 h), whereas it is rapidly cleaved in mouse serum and in kidney and liver homogenates (t1/2 ) 0.1-0.5 min). Furthermore, ester-linked fatty acid PNA conjugates targeted to an aberrant splice site in luciferase mRNA were prepared and shown to be twice as potent for inducing active luciferase as the corresponding conjugate not containing the linker. Thus, a PNA prodrug approach may be useful for both ex vivo as well as in vivo applications.

INTRODUCTION

Esterases with highly relaxed substrate specificity are ubiquitous in mammalian cells and tissues as well as in the blood stream. Therefore ester-linkages are exploited with great success in the design of medical produgs (1, 2). Peptide nucleic acids are pseudopeptide DNA mimics based on amide chemistry. High biological (and chemical) stability combined with very favorable RNA (and DNA) hybridization properties have made PNAs attractive candidates for development of gene therapeutic antisense (and antigene) drugs, and recently a wide range of results have reemphasized this potential (3, 4). Cellular delivery of PNA is an area of active research, and several methods have been devised relying on conjugation to peptide transporters (5-8) or accomplished via cationic liposome carriers (9-11). In the latter case either PNA-DNA hybrids or PNA-fatty acid conjugates were employed. We reasoned that it would be advantageous to develop chemistry that would enable incorporation of an enzyme cleavable ester function between the PNA and the carrier. For instance this should allow cellular (and animal) delivery of lipophilic PNA prodrugs that could transverse the lipid bilayer of the cell with subsequent intracellular enzymatic release of the free PNA. However, PNA oligomerization (and especially cleavage from the resin) involves rather harsh acid and/or alkaline treatment, which is not a priori compatible with ester stability. Nonetheless, we now describe the synthesis of such an ester containing amino acid monomer and demonstrate that this is easily incorporated into PNA oligomers using standard solid-phase polymerization, * Corresponding author: E-mail: [email protected]; Fax: +4535396042. † Department of Medical Biochemistry and Genetics, University of Copenhagen. ‡ Pantheco A/S. § Department of Chemistry, University of Copenhagen.

deprotection, and cleavage conditions. We also show that this ester is a substrate for enzymes (esterases) present in plasma and tissue extracts. Finally, we present preliminary cellular antisense data on ester-linked PNAfatty acid conjugates. EXPERIMENTAL SECTION 1 H and 13C NMR spectra were obtained at either 250 MHz (Bruker AMX 250) or at 400 MHz (Varian Unity 400) in 5 mm tubes. FAB mass spectra were recorded on a JEOL Hx 110/110 mass spectrometer. MALDI-TOF mass spectra were recorded on a Kratos Compact Maldi II instrument operating in the positive ion mode, using 3,5-dimethoxy-4-hydroxycinnamic acid as the matrix. Dichloromethane (LAB-SCAN) and diethyl ether (LAB-SCAN) were dried over molecular sieves. The following chemicals were used as received: phthalide (Aldrich), 1,3-dicyclohexylcarbodiimide (DCC, Aldrich), NBoc-glycine (Sigma), 4-(dimethylamino)pyridine (DMAP, Aldrich), 4-(1-pyrenyl)butyric acid (Aldrich), decanoic acid (Aldrich), (1-adamantyl)acetic acid (Aldrich). TLC was performed on silica 60 (Merck 5554 aluminum sheet). 2-(Hydroxymethyl)benzoic Acid. A modification of the procedure of Gilman et al. (12) was used: Phthalide (5.0 g, 37.3 mmol) was dissolved in methanol (20 mL) and water (20 mL). NaOH (10 M, 8 mL) was added, and the mixture was refluxed for 90 min. The reaction mixture was then cooled to room temperature, and the free acid was precipitated with 4 M HCl (ca. 10 mL). The resulting precipitate was filtered off, washed with icecold water, and finally dried in vacuo to give a white crystalline product (5.03 g, 89%). Mp 101-105 °C (lit. 120.5-121.5 °C). 1H NMR (DMSO-d6) δ 8.12 (dd, 1H), 7.61 (m, 1H), 7.47 (m, 2H), 4.86 (s, 2H), 13C NMR (DMSOd6) δ 133.79, 132.01, 130.32, 128.00, 64.61 (it was not possible to see the CdO signal because of the low sample concentration). FAB+MS: 153.12 (M + H+). 2-([N-Boc-glycyl]oxymethyl)benzoic Acid. A symmetrical anhydride was formed from N-Boc-glycine (7.72 g, 44.1 mmol) and DCC (4.55 g, 22.0 mmol) in dry

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Ester-Linked Peptide Nucleic Acid Prodrug Conjugates Chart 1. Structures of Ester-Linked Pyrene and Adamantyl PNA Conjugates

dichloromethane (25 mL). The mixture was stirred at 0 °C for 30 min. The resulting N,N′-dicyclohexylurea (DCU) was filtered off, and the filtrate was added directly to a suspension of freshly prepared 2-(hydroxymethyl)benzoic acid (2.34 g, 15.4 mmol) in dry dichloromethane (30 mL). DMAP (0.19 g, 1.5 mmol) was added, and the mixture was refluxed overnight. The reaction mixture was washed with saturated aqueous NaHCO3 (3 × 50 mL). The combined aqueous extracts were acidified with 4 N HCl to pH 2, and the resulting precipitate was filtered off, washed with ice-cold water, and finally dried in vacuo to give a white crystalline product (2.98 g, 63%). Mp: 122124 °C. 1H NMR (DMSO-d6) δ 7.93 (d, 1H, J ) 7.5 Hz), 7.55 (m, 2H), 7.44 (m, 1H), 7.28 (t, 1H, J ) 6.1 Hz), 5.49 (s, 2H), 3.79 (d, 2H, J ) 6.1 Hz), 1.38 (s, 9H). 13C NMR (DMSO-d6) δ 170.33, 168.0, 155.99, 137.38, 132.21, 130.60, 129.28, 127.83, 127.53, 78.38, 64.18, 42.12, 28.24. FAB+HRMS: 310.1265 (M + H+, calc. for C15H19NO6 + H+ 310.1291). PNA Conjugates. The following PNA conjugates were synthesized (cf. Chart 1): PNA-1: Pyr-GMB2-TJ-eg1-eg1-eg1-CTTTCTT-NH2* PNA-2: Deca-eg1-FlLys-eg1-CATAGTATAAGT-LysNH2 PNA-3: Ada-eg1-FlLys-eg1-CATAGTATAAGT-LysNH2 PNA-4: Deca-GMB2-eg1-FlLys-eg1-CATAGTATAAGTLysNH2 PNA-5: Ada-GMB2-eg1-FlLys-eg1-CATAGTATAAGTLysNH2 PNA-6: Deca-GMB2-eg1-CCTCTTACCTCAGTTACANH2 PNA-7: Deca-eg1-CCTCTTACCTCAGTTACA-NH2 PNA-8: Ada-GMB2-eg1-CCTCTTACCTCAGTTACANH2 PNA-9: Ada-eg1-CCTCTTACCTCAGTTACA-NH2 PNA-10: Deca-eg1-CTTCTAACCTCTGTTACA-NH2 PNA-11: Deca-GMB2-eg1-CTTCTAACCTCTGTTACANH2 *[(ada: 1-adamantyl acetyl, GMB2: 2-(glycyl-oxymethyl)benzoyl, deca: decanoyl, eg1: 8-amino-3,6-dioxaoctanoyl (”ethyleneglycol linker”), FlLys: fluorescein-lysyl, pyr: 4-(1-pyrenyl)butanoyl), J: pseodoisocytosine]. PNA-1 was synthesized on a Boc-T-4-methylbenzhydrylamine resin (loading 0.12 mmol/g) using the standard synthetic protocol (13). For the incorporation of 4-(1pyrenyl)butyric acid and 2-([N-Boc-glycyl]oxymethyl)benzoic acid, the coupling was allowed to proceed for 60 min. PNAs 2 through 5 were synthesized on a Boc-L-Lys(2-chlorobenzyloxycarbonyl)-4-methylbenzhydrylamine resin (loading 0.15 mmol/g) using the standard synthesis protocol (13). PNAs 6 through 11 were synthesized similarly on a Boc-A-4-methylbenzhydrylamine resin. For the incorporation of decanoic acid, 1-adamantylacetic acid, 2-([N-Boc-glycyl]oxymethyl)benzoic acid, and the

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fluorescein-lysin monomer (6-O-[N-(5-Boc-amino-5-carboxypentyl)carbamoylmethyl]fluorescein ethyl ester) (14), the coupling was allowed to proceed for 60 min. The resulting PNA conjugates were deprotected and cleaved from the resin with a cocktail composed of m-cresol/thioanisole/trifluoromethanesulfonic acid/TFA (1/1/2/6, v/v), and the crude material was precipitated and washed with anhydrous (crucial to ensure ester stability) diethyl ether. The PNA conjugates were then purified by reversed-phase high performance liquid chromatography (HPLC) using a C18 column and an acetonitrile gradient in 0.1% TFA in H2O and characterized by MALDI-TOF mass spectroscopy. Tissue Extracts and Stability Analyses. The HPLC system (Waters) consisted of an Alliance 2690 (pump, autosampler, and degasser), a PDA UV absorbance detector Model 996 (195 nm - 600 nm), and Millennium32 Chromatography software version 3.2. HPLC separation was performed on a Waters Symmetry 300 C18, 2.1 × 150 mm (3.5 µm particles with 300 Å pore size) analytical column (Waters) equipped with a Zorbax Eclipse XDBC18 (5 µm particles with 80 Å pore size) guard column (Agilent) using a linear gradient of solvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile) from 2% to 75% solvent B over 8 min. The column was operated at 50 °C. Samples were kept in the autosampler at 5 °C. Solvent flow was 0.4 mL/min. Polypropylene (PP) test tubes and containers (Sarsted) were generally used for all solutions containing the test PNA. Samples were centrifuged in a Hettich Rotina 46R centrifuge and evaporated to dryness in a SpeedVac AES 2010 from Savant. NMRI mice (female, 25 g body weight) were obtained from M&B (Denmark). Plasma and tissue from sacrificed animals were used for in vitro stability/metabolism studies. Preparation of Tissue Homogenates for in Vitro Studies. The animal was sacrificed and the relevant tissues (liver, kidneys) rapidly excised. The tissue was immediately placed in 0.25 M sucrose at 0 °C for rapid cooling and removal of external blood. After cooling (3-5 min), the tissue was dried by blotting with paper and subsequently weighed and transferred to clean polypropylene test tubes. To each tissue was added 0.25 M sucrose in water to a final concentration of 150 mg tissue/ mL, using a density of 1 mg/mL for the tissue. The tissue was cut into pieces and homogenized for 1-5 min (depending on tissue) in an Ultra-Turrax T25 homogenizer (IKA) followed by centrifugation of the homogenate in a refrigerated centrifuge (4 °C) for 30 min at 3000 rpm (corresponding to approximately 1000 × g) to isolate subcellular fractions. The supernatant was carefully decanted (i.e., the postmitochondrial supernatant), transferred to polypropylene containers, and stored at -80 °C pending use. In Vitro Metabolism Studies. A mixture of 0.025 mL of 0.1 M Tris buffer pH 7.4, 0.135 mL of water, and 0.025 mL of the tissue homogenate (or plasma) was preincubated at 37 °C for 2 min; then 0.015 mL of the PNA stock solution (1 mg/mL) was added. After incubation for the desired time period, the enzymatic reactions were stopped by adding 0.300 mL of 16.6% acetonitrile in 0.1% TFA in water, and the sample immediately transferred to an ice-water bath (0 °C). The mixture was frozen at minus 18 °C for 30 min. For analysis, the homogenate mixtures were thawed at 4 °C and centrifuged at 3000 rpm for 10 min (approximately 1000 × g) at 4 °C. The supernatant (0.200 mL) was transferred directly to the HPLC autosampler vials, and 0.010 mL aliquots were injected into

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the HPLC system. Blind samples were prepared by replacing the PNA test compound with water. The blind samples were incubated and analyzed as described for the test samples. Recovery in the incubated samples was calculated from the HPLC areas using a reference solution. To determine whether the ester monomer was in fact cleaved, a sample was analyzed by LC-MS, giving the intact PNA-1 (m/z 3264.3 (calcd: 3266.9)) and the resulting PNA from which the pyrene was cleaved off (m/z 2939.7 (calcd: 2939.5)). Cellular Uptake. The IMR-90 cell line (human fetal fibroblast) were obtained from American Type Culture Collection and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin/streptomycin (10 µg/mL) (antibiotics) and 10% fetal calf serum (Gibco, Invitrogen, Denmark). Cell cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2 (standard conditions). For uptake analysis of the PNA conjugates, the cells were cultivated on four-well chamber slides (Nunc, Invitrogen, Denmark). The cells were seeded in a concentration of 20 × 103/mL. The next day the semiconfluent cells were prepared for transfection. This was done by washing the cells twice in fresh DMEM containing antibiotics and 2% fetal calf serum. After this procedure, to the cells was added fresh DMEM containing antibiotics and 2% fetal calf serum, and the slides were incubated 40 min at the standard conditions before subsequent replacement of the media with the transfection mixture. The transfection mixture was prepared as follows: 4 µL of lipofectAMINE (2 mg/mL) (Life Technologies) was diluted in 46 µL of DMEM (without serum) and left 5 min at room temperature. In a separate tube, PNA was diluted in 50 µL of DMEM (without serum) to a final concentration of 4 µM (approximately 18 µg/mL). Subsequently the lipofectAMINE dilution and the PNA dilution were gently mixed and left for equilibration at room temperature for 30 min. Thereafter, 400 µL of DMEM containing antibiotics and 2% fetal calf serum was added to the combined PNA/lipofectAMINE solution. The resulting transfection mixture was then gently layered upon the cells. After 14-16 h of incubation at the standard conditions, the cells were washed thoroughly in phosphate-buffered saline (PBS) and fixed on ice for 30 min in 3.7% formaldehyde, washed once more in PBS, and mounted with Slowfade Antifade Kit (Molecular Probes, PoortGebouw, The Netherlands) according to the manufacturer’s instructions. Counterstaining of the cells with propidium iodide (PI) (Sigma) was performed by adding PI to a final concentration of 5 µg/ mL to the cells for 15 min at the end of the incubation with the test substances. Counterstaining with Hoechst 33258 (Sigma) was performed, after formaldehyde fixation of the cells, by 20 min incubation at room temperature in PBS containing Hoechst 33258 (2.5 µg/mL). No detergent was used. Cells were examined by fluorescence microscopy on an Olympus BX61 microscope. Image analysis was performed using MetaMorph 4.6 (Universal Imaging Corporation) and Adobe Photoshop 4.0 (Adobe Systems Inc.). Antisense Activation of Luciferase in the Reporter Cell Line, pLuc705 HeLa. The pLuc705 HeLa cell line was purchased from Gene Tools (Philomath, Oregon). pLuc 705 HeLa cells are derived by stable integration of the pLuc705 plasmid into HeLa S2 cells as described in ref 15. The plasmid has luciferase downstream and out-of-frame with a dominant splicemutation in the β-globin intron position 705. The recombinant luciferase gene is under control of the immediate

Bendifallah et al. Scheme 1a

a (a) NaOH, MeOH aq, 4, 90 min; (b) 4N HCl (89%); (c) [BocGly]2-O, DMAP, DCM, ∆, overnight; (d) 4 N HCl (63%).

early cytomegalovirus promoter. Binding of PNA (of sequence H-CCT CTT ACC TCA GTT ACA-NH2) to a region covering the splice mutation results in restoration of wild-type splicing which generates a transcript with luciferase in-frame (15, 16). pLuc 705 HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin/ streptomycin (10 µg/mL) (antibiotics) and 10% fetal calf serum (Gibco, Invitrogen, Denmark). Cell cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2 (standard conditions). For transfection with the PNA conjugates, the cells were seeded in a 96-well microtiterplate (Nunc, Invitrogen, Denmark) in a volume of 200 µL and a concentration of 50-70 × 103 cells/mL. The next day the semiconfluent cells were prepared for transfection. This was performed by washing the cells once in fresh DMEM containing antibiotics but no serum (serum-free DMEM) and adding 200 µL of the medium to the cells afterward. The cells were then incubated at standard conditions before subsequent replacement of the medium with the transfection mixture. The transfection mixture was prepared as follows: 0.5 µL of lipofectAMINE (2 mg/mL) (Life Technologies) was diluted in 4.5 µL of DMEM (without serum) and left 5 min at room temperature. In a separate tube, PNA was diluted in 18 µL of DMEM (without serum) to a final concentration of 5.5 µM (approximately 23 µg/mL). Subsequently the lipofectAMINE dilution and the PNA dilution was gently mixed and left for equilibration at room temperature for 30 min. Thereafter, 77 µL of fresh DMEM (without serum) was added to the combined PNA/lipofectAMINE solution. The resulting transfection mixture was then gently layered upon the cells. After 2-4 h of incubation at the standard conditions, the transfection mixture was supplemented with 100 µL of DMEM containing penicillin/streptomycin (10 µg/mL) and 20% fetal calf serum. Control experiments were performed exactly as described here with the exception that addition of PNA and lipofectAMINE or just PNA was omitted. After incubation at standard conditions for 14-18 h, the luciferase activity was measured by use of the Steady-Glo Luciferase Assay System (Promega, Ramcon A/S, Birkerød, Denmark) and a Victor2 Multilabel Counter (Wallac, Perkin-Elmer Danmark A/S, Allerød, Denmark) according to the manufacturer’s instructions. RESULTS AND DISCUSSION

The ester-monomer, 2-([N-Boc-glycyl]oxymethyl)benzoic acid, was prepared as outlined in Scheme 1. A successful synthesis was dependent on preventing the intermediate 2-(hydroxymethyl)benzoic acid from reverting to phthalide. This occurred when 2-(hydroxymethyl)benzoic acid was kept in humid air or dissolved in solvents such as DMF or ethyl acetate. However, a suspension in dry dichloromethane worked well and gave the product in 63% yield. Analogously, we also prepared the 4-([N-Boc-glycyl]oxymethyl)benzoic acid.

Ester-Linked Peptide Nucleic Acid Prodrug Conjugates

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Figure 2. Kinetic plot of PNA-1 stability in mouse liver homogenate. Table 1. Half-Lives of PNA-1 in Various “Tissue Homogenates” As Determined by HPLC Analysisa “tissue”

t1/2 min

dilution

estimated t1/2 in “tissue”

none (control) plasma liver kidney

363 100 61 37

1:1000 1:140 1:280

360 0.1 0.5 0.15

a The half-life in “tissue” is extrapolated from the homogenate dilution factor.

Figure 1. HPLC (a) and MALDI-TOF mass spectrometric (b) analysis of PNA-5.

Incorporation of the 2-(glycyloxymethyl)benzoic acid ester-monomer into PNA oligomers proceeded under standard PNA oligomerization conditions and yielded products that were readily purified by reversed phase HPLC (Figure 1). The only necessary precaution taken was the employment of carefully dried ether for precipitation of the product after cleavage from the resin in order to avoid hydrolysis of the ester. (In contrast, we were not able to isolate correct oligomer products when using 4-([N-Boc-glycyl]oxymethyl)benzoic acid, presumably due to degradation during deprotection and/or cleavage from the resin.) To assess the utility of the 2-([N-Boc-glycyl]oxymethyl)benzoic acid monomer in a prodrug approach, we measured the stability of PNA-1 in mouse plasma and in mouse kidney and liver homogenates. PNA-1 was incubated with tissue homogenates at dilutions of the homogenates that resulted in convenient stability for analysis. The analysis was performed by reversed phase HPLC which resolved the intact PNA-1, the resulting PNA from which the pyrene was cleaved off as well as the released pyrene. Quantification was performed on the basis of peak area of the intact PNA-1 as compared to a standard curve of PNA-1. Samples were taken at various time points, and t1/2 was calculated from the [PNA-1] versus time plots (Figure 2). Taking the homogenate dilution factor into account, the stability in undiluted tissue homogenates could be estimated (Table 1). These results

clearly demonstrate that the ester is up to 2000-3000 times less stable in these tissue homogenates as compared to the stability in buffer, pH 7.4, at 37 °C. The results also show a reasonable although somewhat limited stability in buffer (t1/2 ) 6 h) of this ester linkage. We have previously found that fatty acid-PNA conjugates are more readily taken up by eukaryotic cells as compared to ”naked” PNA (10). In particular, such PNA conjugates can be delivered via cationic liposomes (10, 11). However, it could be an advantage if the PNA oligomers were released from the fatty acid once inside the cell, especially because PNA-fatty acid conjugates have limited solubility in an aqueous environment. Thus, they may easily become entrapped in lipid compartments inside the cell, such as various (endosomal, nuclear, and other) membranes which could limit their access to the intended mRNA (or DNA) targets. We therefore synthesized the ester-containing PNA conjugates PNA-4 and PNA-5 and the corresponding nonester controls PNA-2 and PNA-3. Preliminary studies of the uptake of these PNAs in IMR-90 cells indicated a different intracellular distribution of the ester linked conjugates (PNA-4 and PNA-5) as compared to the noncleavable conjugates (PNA-2 and PNA-3). The ester-linked PNA seemed to exhibit a less dispersed intracellular localization with a higher tendency to compartmentalize around the nuclei of the cells than their nonester-linked analogues. However, repeated experiments revealed that the differences were subtle and difficult to reproduce (results not shown). Although the cellular uptake experiments did not demonstrate a clear advantage of the ester-linked PNA conjugates, we designed a series of PNA conjugates for antisense experiments. We chose a PNA sequence (PNAs 6-9) that is known to up-regulate luciferase activity in a reporter cell line by restoring correct splicing through blockage of an intron-exon junction (for further details, see Experimental Section). The results presented in Figure 3 clearly demonstrate that the ester-linked decanoic acid PNA conjugate is twice as potent as the corresponding conjugate not containing the ester linkage, whereas only a smaller enhancement was seen for the adamantyl conjugates, and a two-base mismatch control (PNA-10 and PNA-11) did not show any activity. Transfection of naked PNA as well as simple addition of the PNA conjugates resulted only in a slight increase in

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Figure 3. Effect of lipofectamine mediated tranfection of different PNA conjugates on the expression of luciferase in pLuc 705 HeLa cells. The transfections were performed as described in the methods section. All transfection were done in triplicates and error bars indicate the standard deviations. All results were normalized to the average value of the nontreated control cultures.

luciferase activity (approximately two times the background, results not shown). Clearly we cannot at this stage ascribe the different uptake pattern of PNA-4 and PNA-5 and the improved cellular antisense potency of PNA-6 (and PNA-8) to cleavage of the ester linkage, but the results do encourage further studies of this type of PNA prodrugs for both ex vivo cellular as well as for future in vivo animal applications. This new monomer can of course also be incorporated into other peptides in which an esterase sensitive linkage is desired. ACKNOWLEDGMENT

This work was supported by Association for International Cancer Research (AICR), The Danish Cancer Society and the Lundbeck Foundation. LITERATURE CITED (1) Mizen, L., and Burton, G. (1998) The use of esters as prodrugs for oral delivery of beta-lactam antibiotics. Pharm. Biotechnol. 11, 345-365. (2) He, G., Massarella, J., and Ward, P. (1999) Clinical pharmacokinetics of the prodrug oseltamivir and its active metabolite Ro 64-0802. Clin. Pharmacokinet. 37, 471-484.

Bendifallah et al. (3) Nielsen, P. E. (2000) Antisense peptide nucleic acids. Current Opinion in Mol. Therapeutics 2, 282-287. (4) Braasch, D. A., and Corey, D. R. (2002) Novel antisense and peptide nucleic acid strategies for controlling gene expression. Biochemistry 41, 4503-4510. (5) Aldrian-Herrada, G., Desarmenien, M. G., Orcel, H., BoissinAgasse, L., Mery, J., Brugidou, J., and Rabie, A. (1998) A peptide nucleic acid (PNA) is more rapidly internalized in cultured neurons when coupled to a retro-inverso delivery peptide. The antisense activity depresses the target mRNA and protein in magnocellular oxytocin neurons. Nucleic Acids Res. 26, 4910-4916. (6) Pooga, M., Soomets, U., Hallbrink, M., Valkna, A., Saar, K., Rezaei, K., Kahl, U., Hao, J. X., Xu, X. J., Wiesenfeld-Hallin, Z., Hokfelt, T., Bartfai, T., and Langel, U. (1998) Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo. Nat. Biotechnol. 16, 857-861. (7) Cutrona, G., Carpaneto, E. M., Ulivi, M., Roncella, S., Landt, O., Ferrarini, M., and Boffa, L. C. (2000) Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal. Nat. Biotechnol. 18, 300-303. (8) Good, L., Awasthi, S. K., Dryselius, R., Larsson, O., and Nielsen, P. E. (2001) Bactericidal antisense effects of peptidePNA conjugates. Nat. Biotechnol. 19, 360-364. (9) Hamilton, S. E., Simmons, C. G., Kathiriya, I. S., and Corey, D. R. (1999) Cellular delivery of peptide nucleic acids and inhibition of human telomerase. Chemistry & Biology 6, 343351. (10) Ljungstrøm, T., Knudsen, H., and Nielsen, P. E. (1999) Cellular uptake of adamantyl conjugated peptide nucleic acids. Bioconjugate Chem. 10, 965-972. (11) Mologni, L., Marchesi, E., Nielsen, P. E., and GambacortiPasserini, C. (2001) Inhibition of promyelocytic leukemia (PML)/retinoic acid receptor-alpha and PML expression in acute promyelocytic leukemia cells by anti-PML peptide nucleic acid. Cancer Res. 61, 5468-5473. (12) Gilman, H., Brown, G. E., Webb, F. J., and Spatz, S. M. (1940) J. Am. Chem. Soc. 62, 977. (13) Christensen, L., Fitzpatrick, R., Gildea, B., Petersen, K. H., Hansen, H. F., Koch, T., Egholm, M., Burchardt, O., Nielsen, P. E., Coull, J. and Berg, R. H. (1995) Solid-phase synthesis of peptide nucleic acids (PNA). J. Peptide Sci. 3, 175-183. (14) Lohse, J., Nielsen, P. E., Harrit, N., and Dahl, O. (1997) Fluorescein-Conjugated Lysine Monomers for Solid-Phase Synthesis of Fluorescent Peptides and PNA Oligomers. Bioconjugate Chem. 8, 503-509. (15) Schmajuk, G., Sierakowska, H. and Kole, R. (1999) Antisense oligonucleotides with different backbones. Modification of splicing pathways and efficacy of uptake. J. Biol. Chem. 274(31), 21783-21789. (16) Sazani, P., Kang, S. H., Maier, M. A., Wei, C., Dillman, J., Summerton, J., Manoharan, M., and Kole, R. (2001) Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogues. Nucleic Acids Res. 29(19), 3965-3974.

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