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Pharmacokinetics and biodistribution of phosphorodiamidate morpholino antisense oligomers
Pharmacokinetics and biodistribution of phosphorodiamidate morpholino antisense oligomers Adams Amantana and Patrick L Iversen The concept of using antisense oligonucleotides to interfere with gene expression offers a new therapeutic strategy for the treatment of diseases resulting from overexpression or dysfunction of certain genes. Phosphorodiamidate morpholino oligomers (PMOs) represent a neutral class of antisense agents that interfere with target gene expression either by binding and sterically blocking the assembly of translation machinery, resulting in inhibition of translation, or by altering splicing of pre-mRNA. Studies in animal models and human clinical trials have demonstrated a high degree of functional bioavailability in several target organs. Preclinical and clinical studies have shown that PMOs demonstrate improved efficacy, excellent kinetic behavior, biological stability, and a good safety profile. We conclude from the emerging data that PMOs display advantageous pharmaceutical properties in comparison with other antisense strategies. Addresses Research and Development, AVI BioPharma Inc, 4575 SW Research Way, Suite 200, Corvallis, OR 97333, USA Corresponding author: Amantana, Adams ([email protected])
Current Opinion in Pharmacology 2005, 5:550–555 This review comes from a themed issue on New technologies Edited by Patrick Iversen Available online 8th August 2005 1471-4892/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2005.07.001
Introduction
of translation of the target protein [1], resulting in reduced protein levels, or splice altering of target mRNA [2,3], which ultimatelyleadstoalteredproteinstructureorfunction.The fundamental requirements for effective antisense activity include sufficient concentration of antisense agent at the target site, an ability to hybridize to the target mRNA sequence, the capacity of the oligonucleotide/mRNA duplex to interfere with gene expression, and sufficient biological stability of the antsense agent. Unlike natural (phosphorodiester) DNA molecules, antisense oligonucleotides can contain chemically altered backbone, sugar or nucleotide portions or a combination of these modifications [4]. These modifications are aimed at improving water solubility, nuclease resistance, cellular uptake, target affinity and kinetic behaviour of antisense agents. There are presently several antisense strategies, with phosphorothioate oligonucleotides (PS-ODNs) representing the earliest generation. PS-ODNs possess a chemically modified backbone in which the non-bridging oxygen atoms in the phosphodiester bond are replaced by sulfur. VitraveneTM (ISIS 2922, fomivirsen), the first antisense drug to be approved by the US FDA, is a PS-ODN designed for the treatment of cytomegalovirus-induced retinitis in AIDS patients [5,6]. The mode of action of PS-ODNs involves the recruitment of RNase H through the formation of regular Watson–Crick base pairs with the target RNA, which results in cleavage of the target RNA [7–9]. Pronounced cardiovascular response [10] and activation of the complement cascade [11] are some of the undesirable physiological changes triggered by these biological interactions between PS-ODNs and certain biological proteins. PS-ODN-related mild-to-moderate thrombocytopenia, hyperglycermia or hypotension have also been observed in clinical trials [12,13].
Antisense technology is currently in use for the therapeutic application and validation of molecular medicine, the confirmation of therapeutic strategies and analysis of gene function. Antisense agents are highly selective inhibitors or modulators of gene expression and range from plasmid vectors that express antisense RNA molecules several nucleotides long to short single-stranded oligonucleotides consisting of 10–50 nucleotide residues. Their ability to inhibit or modulate gene expression in a sequence-specific manner offers the potential for high specificity in immediate therapeutic applications. Several preclinical and clinical trials are currently ongoing to evaluate the use of antisense oligonucleotides as therapeutic agents.
Apart from PS-ODNs, RNA interference (RNAi) and ribozymes represent other emerging antisense approaches that are currently at various stages of development; however, their therapeutic potential has yet to be established. RNAi strategy involves the use of small interfering (si)RNA to suppress gene expression. Although this approach has been shown to be effective against target genes in vivo [14,15], the gene silencing effect is transient [16,17]. Sustained gene knockdown can be achieved with stably transfected plasmid vectors that express siRNA, but this requirement places a limitation on its clinical application [18,19]. Another limitation associated with this approach is the lack of flexibility in the chemistry and target site design [20,21].
The mechanism of action of antisense oligonucleotides in downregulating gene expression involves either inhibition
Hammerhead ribozymes are the most widely studied class of ribozymes. Similar to PS-ODNs, ribozyme
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Pharmacokinetics and biodistribution of phosphorodiamidate morpholino antisense oligomers Amantana and Iversen 551
susceptibility to nuclease activity and in vivo delivery requirements represent some of the major obstacles associated with the therapeutic applications of this technology [22]. Phosphorodiamidate morpholino oligomers (PMOs) — a neutral class of antisense agents —possess desirable characteristics and promise an excellent therapeutic alternative to the above-mentioned antisense agents. This review summarizes the biodistribution, stability, efficacy, safety and kinetic behaviour of PMOs in preclinical studies on their way to becoming therapeutically relevant agents.
Phosphorodiamidate morpholino oligomers
The neutral chemistry of PMOs confers on these compounds more desirable properties over their PS-ODN, ribozyme and siRNA counterparts. The non-ionic character of PMOs avoids potential non-specific drug interactions with cellular components (except for the target RNA sequence) observed with PS-ODNs. In addition, the modified chemistry of PMOs provides excellent resistance to nuclease and protease activity [27], which is the basis for the enhanced stability in plasma, tissues, cerebrospinal fluid and urine. The neutral character of PMO chemistry not only guarantees a high safety profile but also sufficient tissue concentrations required for effective PMO oligonucleotide/RNA duplex formation, thus enhancing their affinity for the target RNA sequence [28] and hence increasing efficacy.
Chemistry and mechanism of action
PMOs are a novel class of antisense agents that seem to offer a better therapeutic alternative to other antisense strategies. PMOs possess a non-ionic backbone at physiological pH in which the ribose sugar is replaced by a 6-membered morpholine moiety and the phosphorodiester intersubunit bonds with phosphorodiamidate linkages [23] (Figure 1).
PMOs have been evaluated to confirm their antisense effect in several animal models, as well as in human clinical trials, and have proven to be efficacious with an excellent safety profile [29–36]. The antisense activity of several PMOs in animal models have been reviewed, some of which are presently in various stages of human clinical trials [37].
Unlike PS-ODNs, PMOs do not elicit RNase H activity [24]; instead, the mechanism by which they alter gene expression involves binding to the target RNA sequence and sterically blocking ribosomal assembly or intron–exon splice junction sites, leading to translational arrest or splice-altering effects [25,26].
Delivery and cellular uptake
Figure 1
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Structure of phosphorodiamidate morpholino oligomers. www.sciencedirect.com
Despite the encouraging prospects that oligonucleotidebased chemistry offers, the ability of PMOs to reach the target RNA remains a fundamental challenge particularly in cell culture studies. PMOs must be present in the cells of the target organ in sufficient amounts to ensure effective modulation of target gene expression. Many cell types in culture require assisted delivery techniques, both physical and charged-based delivery procedures. These delivery techniques have been shown to deliver antisense oligonucleotides efficiently into the cytosolic/nuclear compartment of the cells in culture. Charge-based procedures involving the formation of a non-ionic morpholino/carrier complex [38] have been used to facilitate delivery. The leash-duplex method, which involves the annealing of the PMO to a cDNA/RNA molecule or ‘leash’, has also been used in studies to aid delivery of PMOs [38,39]. In addition, the use of short arginine-rich peptides (ARPs) conjugated to PMOs has demonstrated enhanced delivery of PMOs into cultured cells [40,41]. However, the effect of these ARPs on PMO delivery in the in vivo setting as well as on the pharmacokinetics of PMOs has yet to be determined. Physical methods such as scrape loading, syringe loading, and osmotic loading have been used to successfully deliver PMOs in cell culture [42,43]. Although several of these methods are now available for delivery purposes into the cytosolic or nuclear compartments of cultured cells, none of the above methods are suitable for in vivo application. Unlike established cell lines, significant PMO uptake in primary cell cultures can be achieved without assisted delivery [30,44]. It is posCurrent Opinion in Pharmacology 2005, 5:550–555
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sible that established cells in culture may lack transport systems required for PMO uptake present in primary cell lines [45]. Significant tissue PMO concentrations can be achieved as early as 24 hours following administration, indicating rapid distribution of the compound. This might be a result, at least in part, to poor plasma protein binding characteristics and low ability to accumulate in erythrocytes. The fact that for several of the PMOs examined in animals models and human clinical trials a modest 10-30% of the administered dose is eliminated through urine [37] strongly supports efficient tissue accumulation. In the absence of cellular uptake, renal excretion equivalent to the given dose would be observed within minutes following administration, but no such observation has been made.
Pharmacokinetics of oligonucleotides Pharmacokinetic studies primarily require a sensitive and reliable procedure for quantifying the test article in tissues and biological fluids such as blood, urine and cerebrospinal fluid. PS-ODNs have in the past been analysed using radiolabeled material [46–48]. The limitation associated with this method of analysis is that it is unable to discriminate between intact and modified PSODNs. For instance, the plasma elimination half-life of several PS-ODNs has been reported to be in the range of 20–60 h (for review, see [37]) using radioactive techniques for quantitative analysis. However, the plasma elimination half-life of the PS-ODN trecovirsen was shown to be much shorter, with a range of 38–75 min when the unchanged oligonucleotide was analysed by high performance liquid chromatography instead of a radioactive technique [49]. Thus, the method of quantitative analysis is crucial to the pharmacokinetics of a given antisense oligonucleotide.
this procedure is effective in achieving maximum PMO extraction, it remains that the PMO extract is impure owing to the presence of proteins from the tissue homogenate. Currently, acetonitrile extraction is the preferred protocol, which tends to address the limitation associated with methanol extraction. The non-polar nature of acetonitrile not only guarantees maximal PMO extraction but also minimizes the levels of protein contaminants in the organ extract. Although several of the PMOs studied are similar in size and base composition, they demonstrate significant differences in pharmacokinetic parameters despite the same extraction and quantification techniques being used for the pharmacokinetic evaluations. Emerging data involving several PMOs have shown that the pharmacokinetic profile of each PMO is unique to the base sequence. This could result from variations in the 3-dimensional structure of each oligomer, their protein binding characteristics or their affinity for target RNA, which may serve as temporary depot for the PMO. Pharmacokinetics and biodistribution of PMOs
Comprehensive pharmacokinetic analyses of several PMO antisense agents in multiple species have been reported [28]. A similar pharmacokinetic profile is observed following administration via intravenous, oral, intraperitoneal, transdermal, pulmonary and subcutaneous routes [31,32,50–52].
Recently, more sophisticated techniques such as capillary gel electrophoresis and liquid chromatography have been developed for detailed analysis. The ‘high performance liquid chromatography–duplex’ method, which is best suited to evaluate PMO levels in various biological matrices, employs the use of a 50 -fluorescein-labeled DNA probe that is complementary to the sequence of the analyte PMO. Both the analyte and internal standard are capable of forming a stable duplex with the 50 -fluorescein-labeled DNA under chromatographic conditions. Quantification is achieved by the use of an internal standard, the sequence of which is derived from a truncated analyte.
Like PS-ODNs, the plasma kinetics of PMOs best fit with a two compartmental model. The estimated plasma clearance of most PMOs ranges from 1–6 ml/min [37], which coincides with a rapid decline in plasma levels following administration. The distribution phase takes about 1–4 h to complete and the number of sample points within the detectable range of the quantitation procedure is small for the lower dose administered. The rapid decline in plasma concentrations can be attributed primarily to the rapid distribution from plasma to tissues and not to renal clearance or metabolism, as PMOs are bioenzymatically stable and the parent compound can be detected in tissues even after six half-lives. Pharmacokinetic evaluations of several PMOs administered via intravenous and subcutaneous routes in rats have demonstrated dosedependent increases in plasma elimination half-life ranging from 1–9 h [53]. The half-life of AVI-4126, a c-myc antisense PMO that entered clinical trials in 2000, is estimated to be 10 h [37]. Therefore, we conclude that PMOs are cleared from plasma with half-lives ranging from 1–20 h.
In addition to finding and employing very sensitive methods of quantitation, identifying an effective extraction procedure is also vital in the evaluation of pharmacokinetics of PMOs. Until recently, methanol extraction was the preferred protocol for PMO extraction. Although
The area under the plasma concentration time curve (AUC), which is a measure of exposure, is observed to increase with dose. Human pharmacokinetic studies for AVI-4126 show a strong linear relationship between AUC and dose administered, indicating non-saturable kinetic
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Pharmacokinetics and biodistribution of phosphorodiamidate morpholino antisense oligomers Amantana and Iversen 553
behavior [37]. An estimated total systemic subcutaneous bioavailability of approximately 80% is achieved following subcutaneous injections, with the peak plasma concentration occurring within minutes after administration [53]. Furthermore, significant concentrations of intact PMO considered to be therapeutically relevant are retained in numerous target organs 24 h following a single dose injection, with the kidney and liver being the sites of maximum accumulation followed by the spleen, lung, heart, skeletal muscle and thymus, and minor concentrations (usually below the limit of detection) occurring in the brain [37] (Amantana A et al., unpublished). Like PSODNs, the pattern of distribution of PMOs is independent of sequence and length of PMO, as well as of route of administration. To date, no detectable PMO metabolite has been observed in whole blood, plasma, tissues, cerebrospinal fluid or urine. Whereas PS-ODNs degrade in a time- and tissue-dependent manner [54], PMOs demonstrate sufficient biological stability irrespective of the route of administration [27,31]. PMOs accumulate rapidly in target organs shortly after administration in a dose-dependent manner [37]. Tissue concentration has also been demonstrated to be dependent on the route of administration. Increased tissue concentration in the range of 10–50% has been achieved by injection via the subcutaneous route rather than the intravenous route for a single equivalent dose [53]. PMOs have been shown to be eliminated mainly in urine and fecal matter in a dose- and time-dependent manner. For most PMOs, urinary excretion seems to be the major route of elimination, although in some cases fecal excretion has been estimated to be about 30% of the dose administered [37]. Unlike PS-ODN counterparts, safety evaluation studies show that PMOs are well tolerated with no mortality, clinical signs or gross lesions, even at doses several times greater than the anticipated therapeutic dose for clinical application [28] The emerging data are encouraging in that not only do PMOs demonstrate specificity, efficacy and stability but they also offer the promise of an excellent safety profile.
this review demonstrate enhanced stability as well as an excellent safety profile, in that therapeutic doses can be achieved over a long period of time following administration with no adverse effects. Also, emerging preclinical pharmacokinetic data demonstrate that PMOs are rapidly distributed in a wide range of target sites at therapeutically relevant concentrations via several routes of administration. Although the route of administration is not a factor in determining the pattern of PMO distribution in tissues, it can be vital in predicting plasma concentration and tissue burden. The data presented in this review not only demonstrate the advantage that PMOs have in addressing pharmaceutical limitations associated with other technologies but also provide a useful guide in establishing future therapeutic development plans. A number of PMO antisense molecules are presently in various phases of human clinical trials, demonstrating that this strategy is effective against various diseases with no apparent toxicity. The novel chemistry associated with PMO antisense strategy has moved the concept of antisense technology far beyond the first-generation PS-ODNs, and continued development promises even more efficient delivery, an improved safety profile and increased efficacy.
Acknowledgements The authors wish to thank the analytical, synthesis and purification groups at AVI BioPharma Inc. We also acknowledge the technical assistance provided by Melissa Cate and Ariel Eberle.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Fulford AJ, Harbuz MS, Jessop DS: Antisense inhibition of pro-opiomelanocortin and proenkephalin A messenger RNA translation alters rat immune cell function in vitro. J Neuroimmunol 2000, 106:6-13.
2.
Mann CJ, Honeyman K, Cheng AJ, Ly T, Lloyd F, Fletcher S, Morgan JE, Partridge TA, Wilton SD: Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse. Proc Natl Acad Sci USA 2001, 98:42-47.
3.
Vacek M, Sazani P, Kole R: Antisense-mediated redirection of mRNA splicing. Cell Mol Life Sci 2003, 60:825-833.
4.
Kurreck J: Antisense technologies. Improvement through novel chemical modifications. Eur J Biochem 2003, 270:1628-1644.
5.
Detrick B, Nagineni CN, Grillone LR, Anderson KP, Henry SP, Hooks JJ: Inhibition of human cytomegalovirus replication in a human retinal epithelial cell model by antisense oligonucleotides. Invest Ophthalmol Vis Sci 2001, 42:163-169.
6.
Geary RS, Henry SP, Grillone LR: Fomivirsen: clinical pharmacology and potential drug interactions. Clin Pharmacokinet 2002, 41:255-260.
7.
Zamaratski E, Pradeepkumar PI, Chattopadhyaya J: A critical survey of the structure-function of the antisense oligo/RNA heteroduplex as substrate for RNase H. J Biochem Biophys Methods 2001, 48:189-208.
8.
Giles RV, Tidd DM: The direction of ribonucleases H by antisense oligodeoxynucleotides. Methods Mol Biol 2001, 160:157-182.
Conclusions and future directions Numerous studies have demonstrated that PMOs are capable of interfering with gene expression both in vitro and in vivo in a sequence-specific manner, indicating that PMOs have huge potential for being developed as drugs. Data characterizing the specificity, toxicology, biological stability, efficacy and kinetic behaviour of PMOs represent a major improvement in the development of antisense agents and might be vital to addressing therapeutically relevant issues. First, data presented in www.sciencedirect.com
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9.
Ten Asbroek AL, van Groenigen M, Nooij M, Baas F: The involvement of human ribonucleases H1 and H2 in the variation of response of cells to antisense phosphorothioate oligonucleotides. Eur J Biochem 2002, 269:583-592.
10. Sereni D, Tubiana R, Lascoux C, Katlama C, Taulera O, Bourque A, Cohen A, Dvorchik B, Martin RR, Tournerie C et al.: Pharmacokinetics and tolerability of intravenous trecovirsen (GEM 91), an antisense phosphorothioate oligonucleotide, in HIV-positive subjects. J Clin Pharmacol 1999, 39:47-54. 11. Levin AA: A review of the issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides. Biochim Biophys Acta 1999, 1489:69-84. 12. Chi KN, Gleave ME, Klasa R, Murray N, Bryce C, Lopes de Menezes DE, D’Aloisio S, Tolcher AW: A phase I dose-finding study of combined treatment with an antisense Bcl-2 oligonucleotide (Genasense) and mitoxantrone in patients with metastatic hormone-refractory prostate cancer. Clin Cancer Res 2001, 7:3920-3927. 13. Waters JS, Webb A, Cunningham D, Clarke PA, Raynaud F, di Stefano F, Cotter FE: Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin’s lymphoma. J Clin Oncol 2000, 18:1812-1823. 14. Song E, Lee S-K, Wang J, Ince N, Ouyang N, Min J, Chen J, Shankar P, Lieberman J: RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 2003, 9:347-351. 15. Xia H, Mao Q, Paulson H, Davidson BL: siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 2002, 20:1006-1010. 16. Cerutti H: RNA interference: traveling in the cell and gaining functions? Trends Genet 2003, 19:39-46. 17. Schwarz DS, Hutvagner G, Haley B, Zamore PD: Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol Cell 2002, 10:537-548. 18. Scherr M, Morgan MA, Eder M: Gene silencing mediated by small interfering RNAs in mammalian cells. Curr Med Chem 2003, 10:245-256. 19. Shi Y: Mammalian RNAi for the masses. Trends Genet 2003, 19:9-12. 20. Grunweller A, Wyszko E, Bieber B, Jahnel R, Erdmann VA, Kurreck J: Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 20 -O-methyl RNA, phosphorothioates and small interfering RNA. Nucleic Acids Res 2003, 31:3185-3193. 21. Thompson JD: Applications of antisense and siRNAs during preclinical drug development. Drug Discov Today 2002, 7:912-917. 22. Khan AU, Lal SK: Ribozymes: a modern tool in medicine. J Biomed Sci 2003, 10:457-467. 23. Summerton J, Weller D: Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev 1997, 7:187-195. 24. Summerton J: Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta 1999, 1489:141-158. 25. Ghosh C, Stein D, Weller D, Iversen P: Evaluation of antisense mechanisms of action. Methods Enzymol 2000, 313:135-143. 26. Sazani P, Kole R: Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing. J Clin Invest 2003, 112:481-486. 27. Hudziak RM, Barofsky E, Barofsky DF, Weller DL, Huang SB, Weller DD: Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nucleic Acid Drug Dev 1996, 6:267-272. 28. Kurreck J, Wyszko E, Gillen C, Erdmann VA: Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Res 2002, 30:1911-1918. Current Opinion in Pharmacology 2005, 5:550–555
29. Crooke ST (Ed): In Antisense Drug Techcology- Principles, Strategies and Applications. Marcel Dekker, Inc; 2001. 30. Arora V, Cate ML, Ghosh C, Iversen PL: Phosphorodiamidate morpholino antisense oligomers inhibit expression of human cytochrome P450 3A4 and alter selected drug metabolism. Drug Metab Dispos 2002, 30:757-762. 31. Arora V, Knapp DC, Reddy MT, Weller DD, Iversen PL: Bioavailability and efficacy of antisense morpholino oligomers targeted to c-Myc and cytochrome P-450 3A2 following oral administration in rats. J Pharm Sci 2002, 91:1009-1018. 32. Arora V, Hannah TL, Iversen PL, Brand RM: Transdermal use of phosphorodiamidate morpholino oligomer AVI-4472 inhibits cytochrome P450 activity in male rats. Pharm Res 2002, 19:1465-1470. 33. McCaffrey AP, Meuse L, Karimi M, Contag CH, Kay MA: A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice. Hepatology 2003, 38:503-508. 34. Kipshidze N, Moses J, Shankar LR, Leon M: Perspectives on antisense therapy for the prevention of restenosis. Curr Opin Mol Ther 2001, 3:265-277. 35. Kipshidze NN, Kim HS, Iversen P, Yazdi HA, Bhargava B, New G, Mehran R, Tio F, Haudenschild C, Dangas G et al.: Intramural coronary delivery of advanced antisense oligonucleotides reduces neointimal formation in the porcine stent restenosis model. J Am Coll Cardiol 2002, 39:1686-1691. 36. Iversen PL, Arora V, Acker AJ, Mason DH, Devi GR: Efficacy of antisense morpholino oligomer targeted to c-myc in prostate cancer xenograft murine model and a Phase I safety study in humans. Clin Cancer Res 2003, 9:2510-2519. 37. Arora V, Devi GR, Iversen PL: Neutrally charged phosphorodiamidate morpholino antisense oligomers: uptake, efficacy and pharmacokinetics. Curr Pharm Biotechnol 2004, 5:431-439. This report establishes that even at very low doses PMOs could be efficiently delivered to several target sites in the in vivo setting by intraperitoneal administration. The report also makes available literature demonstrating PMO activity in different animal models. 38. Morcos PA: Achieving efficient delivery of morpholino oligos in cultured cells. Genesis 2001, 30:94-102. 39. Gebski BL, Mann CJ, Fletcher S, Wilton SD: Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle. Hum Mol Genet 2003, 12:1801-1811. 40. Moulton HM, Hase MC, Smith KM, Iversen PL: HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers. Antisense Nucleic Acid Drug Dev 2003, 13:31-43. 41. Moulton HM, Nelson MH, Hatlevig SA, Reddy MT, Iversen PL: Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides. Bioconjug Chem 2004, 15:290-299. This study demonstrates that conjugating short ARPs to PMOs significantly enhanced delivery of PMO to target sites within the cell. The authors establish that PMO delivery required conjugation rather than complexation of peptide to the PMO. 42. Amantana A, London CA, Iversen PL, Devi GR: X-linked inhibitor of apoptosis protein inhibition induces apoptosis and enhances chemotherapy sensitivity in human prostate cancer cells. Mol Cancer Ther 2004, 3:699-707. In this study, the authors establish that a significant reduction in XIAP gene expression and apoptosis in DU145 human prostate cancer cells is achieved by the scrape-loading delivery technique. This study also demonstrates the specific antisense activity of a PMO against XIAP, which is one of the cancer survival genes. In addition, the study shows that PMOs might have the potential of sensitizing refractory cancer cells to chemotherapy. 43. Ghosh C, Iversen PL: Intracellular delivery strategies for antisense phosphorodiamidate morpholino oligomers. Antisense Nucleic Acid Drug Dev 2000, 10:263-274. 44. Suwanmanee T, Sierakowska H, Lacerra G, Svasti S, Kirby S, Walsh CE, Fucharoen S, Kole R: Restoration of human beta-globin gene expression in murine and human IVS2-654 www.sciencedirect.com
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thalassemic erythroid cells by free uptake of antisense oligonucleotides. Mol Pharmacol 2002, 62:545-553. 45. Hanss B, Leal-Pinto E, Teixeira A, Christian RE, Shabanowitz J, Hunt DF, Klotman PE: Cytosolic malate dehydrogenase confers selectivity of the nucleic acid-conducting channel. Proc Natl Acad Sci USA 2002, 99:1707-1712. 46. Agrawal S, Temsamani J, Galbraith W, Tang J: Pharmacokinetics of antisense oligonucleotides. Clin Pharmacokinet 1995, 28:7-16. 47. Zhang R, Diasio RB, Lu Z, Liu T, Jiang Z, Galbraith WM, Agrawal S: Pharmacokinetics and tissue distribution in rats of an oligodeoxynucleotide phosphorothioate (GEM 91) developed as a therapeutic agent for human immunodeficiency virus type-1. Biochem Pharmacol 1995, 49:929-939. 48. Srinivasan SK, Iversen P: Review of in vivo pharmacokinetics and toxicology of phosphorothioate oligonucleotides. J Clin Lab Anal 1995, 9:129-137. 49. Sereni D, Tubiana R, Lascoux C, Katlama C, Taulera O, Bourque A, Cohen A, Dvorchik B, Martin RR, Tournerie C et al.: Pharmacokinetics and tolerability of intravenous trecovirsen
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(GEM 91), an antisense phosphorothioate oligonucleotide, in HIV-positive subjects. J Clin Pharmacol 1999, 39:47-54. 50. Arora V, Iversen PL: Redirection of drug metabolism using antisense technology. Curr Opin Mol Ther 2001, 3:249-257. 51. Iversen PL, Arora V, Acker A, Mason DH, Devi GR: Efficacy of antisense morpholino oligomer targeted to c-myc in prostate cancer xenograft murine model and a Phase I safety study in humans. Clin Cancer Res 2003, 9:2510-2519. 52. Qin G, Taylor M, Ning YY, Iversen P, Kobzik L: In vivo evaluation of a morpholino antisense oligomer directed against tumor necrosis factor-alpha. Antisense Nucleic Acid Drug Dev 2000, 10:11-16. 53. Amantana A, Cate M, Reddy M, Weller D, Iversen P: Pharmacokinetics and Toxicokinetics of Phosphorodiamidate morpholino oligomers against hepatitis C virus after single intravenous injection. 44th Annual Meeting of the Society of Toxicology, March 2005, New Orleans, LA. [Abstract 1246]. 54. Geary RS, Watanabe TA, Truong L, Freier S, Lesnik EA, Sioufi NB, Sasmor H, Manoharan M, Levin AA: Pharmacokinetic properties of 20 -O-(2-methoxyethyl)-modified oligonucleotide analogs in rats. J Pharmacol Exp Ther 2001, 296:890-897.
Current Opinion in Pharmacology 2005, 5:550–555
Current Opinion in Pharmacology 2005, 5:550–555 This review comes from a themed issue on New technologies Edited by Patrick Iversen Available online 8th August 2005 1471-4892/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2005.07.001
Introduction
of translation of the target protein [1], resulting in reduced protein levels, or splice altering of target mRNA [2,3], which ultimatelyleadstoalteredproteinstructureorfunction.The fundamental requirements for effective antisense activity include sufficient concentration of antisense agent at the target site, an ability to hybridize to the target mRNA sequence, the capacity of the oligonucleotide/mRNA duplex to interfere with gene expression, and sufficient biological stability of the antsense agent. Unlike natural (phosphorodiester) DNA molecules, antisense oligonucleotides can contain chemically altered backbone, sugar or nucleotide portions or a combination of these modifications [4]. These modifications are aimed at improving water solubility, nuclease resistance, cellular uptake, target affinity and kinetic behaviour of antisense agents. There are presently several antisense strategies, with phosphorothioate oligonucleotides (PS-ODNs) representing the earliest generation. PS-ODNs possess a chemically modified backbone in which the non-bridging oxygen atoms in the phosphodiester bond are replaced by sulfur. VitraveneTM (ISIS 2922, fomivirsen), the first antisense drug to be approved by the US FDA, is a PS-ODN designed for the treatment of cytomegalovirus-induced retinitis in AIDS patients [5,6]. The mode of action of PS-ODNs involves the recruitment of RNase H through the formation of regular Watson–Crick base pairs with the target RNA, which results in cleavage of the target RNA [7–9]. Pronounced cardiovascular response [10] and activation of the complement cascade [11] are some of the undesirable physiological changes triggered by these biological interactions between PS-ODNs and certain biological proteins. PS-ODN-related mild-to-moderate thrombocytopenia, hyperglycermia or hypotension have also been observed in clinical trials [12,13].
Antisense technology is currently in use for the therapeutic application and validation of molecular medicine, the confirmation of therapeutic strategies and analysis of gene function. Antisense agents are highly selective inhibitors or modulators of gene expression and range from plasmid vectors that express antisense RNA molecules several nucleotides long to short single-stranded oligonucleotides consisting of 10–50 nucleotide residues. Their ability to inhibit or modulate gene expression in a sequence-specific manner offers the potential for high specificity in immediate therapeutic applications. Several preclinical and clinical trials are currently ongoing to evaluate the use of antisense oligonucleotides as therapeutic agents.
Apart from PS-ODNs, RNA interference (RNAi) and ribozymes represent other emerging antisense approaches that are currently at various stages of development; however, their therapeutic potential has yet to be established. RNAi strategy involves the use of small interfering (si)RNA to suppress gene expression. Although this approach has been shown to be effective against target genes in vivo [14,15], the gene silencing effect is transient [16,17]. Sustained gene knockdown can be achieved with stably transfected plasmid vectors that express siRNA, but this requirement places a limitation on its clinical application [18,19]. Another limitation associated with this approach is the lack of flexibility in the chemistry and target site design [20,21].
The mechanism of action of antisense oligonucleotides in downregulating gene expression involves either inhibition
Hammerhead ribozymes are the most widely studied class of ribozymes. Similar to PS-ODNs, ribozyme
Current Opinion in Pharmacology 2005, 5:550–555
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Pharmacokinetics and biodistribution of phosphorodiamidate morpholino antisense oligomers Amantana and Iversen 551
susceptibility to nuclease activity and in vivo delivery requirements represent some of the major obstacles associated with the therapeutic applications of this technology [22]. Phosphorodiamidate morpholino oligomers (PMOs) — a neutral class of antisense agents —possess desirable characteristics and promise an excellent therapeutic alternative to the above-mentioned antisense agents. This review summarizes the biodistribution, stability, efficacy, safety and kinetic behaviour of PMOs in preclinical studies on their way to becoming therapeutically relevant agents.
Phosphorodiamidate morpholino oligomers
The neutral chemistry of PMOs confers on these compounds more desirable properties over their PS-ODN, ribozyme and siRNA counterparts. The non-ionic character of PMOs avoids potential non-specific drug interactions with cellular components (except for the target RNA sequence) observed with PS-ODNs. In addition, the modified chemistry of PMOs provides excellent resistance to nuclease and protease activity [27], which is the basis for the enhanced stability in plasma, tissues, cerebrospinal fluid and urine. The neutral character of PMO chemistry not only guarantees a high safety profile but also sufficient tissue concentrations required for effective PMO oligonucleotide/RNA duplex formation, thus enhancing their affinity for the target RNA sequence [28] and hence increasing efficacy.
Chemistry and mechanism of action
PMOs are a novel class of antisense agents that seem to offer a better therapeutic alternative to other antisense strategies. PMOs possess a non-ionic backbone at physiological pH in which the ribose sugar is replaced by a 6-membered morpholine moiety and the phosphorodiester intersubunit bonds with phosphorodiamidate linkages [23] (Figure 1).
PMOs have been evaluated to confirm their antisense effect in several animal models, as well as in human clinical trials, and have proven to be efficacious with an excellent safety profile [29–36]. The antisense activity of several PMOs in animal models have been reviewed, some of which are presently in various stages of human clinical trials [37].
Unlike PS-ODNs, PMOs do not elicit RNase H activity [24]; instead, the mechanism by which they alter gene expression involves binding to the target RNA sequence and sterically blocking ribosomal assembly or intron–exon splice junction sites, leading to translational arrest or splice-altering effects [25,26].
Delivery and cellular uptake
Figure 1
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Structure of phosphorodiamidate morpholino oligomers. www.sciencedirect.com
Despite the encouraging prospects that oligonucleotidebased chemistry offers, the ability of PMOs to reach the target RNA remains a fundamental challenge particularly in cell culture studies. PMOs must be present in the cells of the target organ in sufficient amounts to ensure effective modulation of target gene expression. Many cell types in culture require assisted delivery techniques, both physical and charged-based delivery procedures. These delivery techniques have been shown to deliver antisense oligonucleotides efficiently into the cytosolic/nuclear compartment of the cells in culture. Charge-based procedures involving the formation of a non-ionic morpholino/carrier complex [38] have been used to facilitate delivery. The leash-duplex method, which involves the annealing of the PMO to a cDNA/RNA molecule or ‘leash’, has also been used in studies to aid delivery of PMOs [38,39]. In addition, the use of short arginine-rich peptides (ARPs) conjugated to PMOs has demonstrated enhanced delivery of PMOs into cultured cells [40,41]. However, the effect of these ARPs on PMO delivery in the in vivo setting as well as on the pharmacokinetics of PMOs has yet to be determined. Physical methods such as scrape loading, syringe loading, and osmotic loading have been used to successfully deliver PMOs in cell culture [42,43]. Although several of these methods are now available for delivery purposes into the cytosolic or nuclear compartments of cultured cells, none of the above methods are suitable for in vivo application. Unlike established cell lines, significant PMO uptake in primary cell cultures can be achieved without assisted delivery [30,44]. It is posCurrent Opinion in Pharmacology 2005, 5:550–555
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sible that established cells in culture may lack transport systems required for PMO uptake present in primary cell lines [45]. Significant tissue PMO concentrations can be achieved as early as 24 hours following administration, indicating rapid distribution of the compound. This might be a result, at least in part, to poor plasma protein binding characteristics and low ability to accumulate in erythrocytes. The fact that for several of the PMOs examined in animals models and human clinical trials a modest 10-30% of the administered dose is eliminated through urine [37] strongly supports efficient tissue accumulation. In the absence of cellular uptake, renal excretion equivalent to the given dose would be observed within minutes following administration, but no such observation has been made.
Pharmacokinetics of oligonucleotides Pharmacokinetic studies primarily require a sensitive and reliable procedure for quantifying the test article in tissues and biological fluids such as blood, urine and cerebrospinal fluid. PS-ODNs have in the past been analysed using radiolabeled material [46–48]. The limitation associated with this method of analysis is that it is unable to discriminate between intact and modified PSODNs. For instance, the plasma elimination half-life of several PS-ODNs has been reported to be in the range of 20–60 h (for review, see [37]) using radioactive techniques for quantitative analysis. However, the plasma elimination half-life of the PS-ODN trecovirsen was shown to be much shorter, with a range of 38–75 min when the unchanged oligonucleotide was analysed by high performance liquid chromatography instead of a radioactive technique [49]. Thus, the method of quantitative analysis is crucial to the pharmacokinetics of a given antisense oligonucleotide.
this procedure is effective in achieving maximum PMO extraction, it remains that the PMO extract is impure owing to the presence of proteins from the tissue homogenate. Currently, acetonitrile extraction is the preferred protocol, which tends to address the limitation associated with methanol extraction. The non-polar nature of acetonitrile not only guarantees maximal PMO extraction but also minimizes the levels of protein contaminants in the organ extract. Although several of the PMOs studied are similar in size and base composition, they demonstrate significant differences in pharmacokinetic parameters despite the same extraction and quantification techniques being used for the pharmacokinetic evaluations. Emerging data involving several PMOs have shown that the pharmacokinetic profile of each PMO is unique to the base sequence. This could result from variations in the 3-dimensional structure of each oligomer, their protein binding characteristics or their affinity for target RNA, which may serve as temporary depot for the PMO. Pharmacokinetics and biodistribution of PMOs
Comprehensive pharmacokinetic analyses of several PMO antisense agents in multiple species have been reported [28]. A similar pharmacokinetic profile is observed following administration via intravenous, oral, intraperitoneal, transdermal, pulmonary and subcutaneous routes [31,32,50–52].
Recently, more sophisticated techniques such as capillary gel electrophoresis and liquid chromatography have been developed for detailed analysis. The ‘high performance liquid chromatography–duplex’ method, which is best suited to evaluate PMO levels in various biological matrices, employs the use of a 50 -fluorescein-labeled DNA probe that is complementary to the sequence of the analyte PMO. Both the analyte and internal standard are capable of forming a stable duplex with the 50 -fluorescein-labeled DNA under chromatographic conditions. Quantification is achieved by the use of an internal standard, the sequence of which is derived from a truncated analyte.
Like PS-ODNs, the plasma kinetics of PMOs best fit with a two compartmental model. The estimated plasma clearance of most PMOs ranges from 1–6 ml/min [37], which coincides with a rapid decline in plasma levels following administration. The distribution phase takes about 1–4 h to complete and the number of sample points within the detectable range of the quantitation procedure is small for the lower dose administered. The rapid decline in plasma concentrations can be attributed primarily to the rapid distribution from plasma to tissues and not to renal clearance or metabolism, as PMOs are bioenzymatically stable and the parent compound can be detected in tissues even after six half-lives. Pharmacokinetic evaluations of several PMOs administered via intravenous and subcutaneous routes in rats have demonstrated dosedependent increases in plasma elimination half-life ranging from 1–9 h [53]. The half-life of AVI-4126, a c-myc antisense PMO that entered clinical trials in 2000, is estimated to be 10 h [37]. Therefore, we conclude that PMOs are cleared from plasma with half-lives ranging from 1–20 h.
In addition to finding and employing very sensitive methods of quantitation, identifying an effective extraction procedure is also vital in the evaluation of pharmacokinetics of PMOs. Until recently, methanol extraction was the preferred protocol for PMO extraction. Although
The area under the plasma concentration time curve (AUC), which is a measure of exposure, is observed to increase with dose. Human pharmacokinetic studies for AVI-4126 show a strong linear relationship between AUC and dose administered, indicating non-saturable kinetic
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behavior [37]. An estimated total systemic subcutaneous bioavailability of approximately 80% is achieved following subcutaneous injections, with the peak plasma concentration occurring within minutes after administration [53]. Furthermore, significant concentrations of intact PMO considered to be therapeutically relevant are retained in numerous target organs 24 h following a single dose injection, with the kidney and liver being the sites of maximum accumulation followed by the spleen, lung, heart, skeletal muscle and thymus, and minor concentrations (usually below the limit of detection) occurring in the brain [37] (Amantana A et al., unpublished). Like PSODNs, the pattern of distribution of PMOs is independent of sequence and length of PMO, as well as of route of administration. To date, no detectable PMO metabolite has been observed in whole blood, plasma, tissues, cerebrospinal fluid or urine. Whereas PS-ODNs degrade in a time- and tissue-dependent manner [54], PMOs demonstrate sufficient biological stability irrespective of the route of administration [27,31]. PMOs accumulate rapidly in target organs shortly after administration in a dose-dependent manner [37]. Tissue concentration has also been demonstrated to be dependent on the route of administration. Increased tissue concentration in the range of 10–50% has been achieved by injection via the subcutaneous route rather than the intravenous route for a single equivalent dose [53]. PMOs have been shown to be eliminated mainly in urine and fecal matter in a dose- and time-dependent manner. For most PMOs, urinary excretion seems to be the major route of elimination, although in some cases fecal excretion has been estimated to be about 30% of the dose administered [37]. Unlike PS-ODN counterparts, safety evaluation studies show that PMOs are well tolerated with no mortality, clinical signs or gross lesions, even at doses several times greater than the anticipated therapeutic dose for clinical application [28] The emerging data are encouraging in that not only do PMOs demonstrate specificity, efficacy and stability but they also offer the promise of an excellent safety profile.
this review demonstrate enhanced stability as well as an excellent safety profile, in that therapeutic doses can be achieved over a long period of time following administration with no adverse effects. Also, emerging preclinical pharmacokinetic data demonstrate that PMOs are rapidly distributed in a wide range of target sites at therapeutically relevant concentrations via several routes of administration. Although the route of administration is not a factor in determining the pattern of PMO distribution in tissues, it can be vital in predicting plasma concentration and tissue burden. The data presented in this review not only demonstrate the advantage that PMOs have in addressing pharmaceutical limitations associated with other technologies but also provide a useful guide in establishing future therapeutic development plans. A number of PMO antisense molecules are presently in various phases of human clinical trials, demonstrating that this strategy is effective against various diseases with no apparent toxicity. The novel chemistry associated with PMO antisense strategy has moved the concept of antisense technology far beyond the first-generation PS-ODNs, and continued development promises even more efficient delivery, an improved safety profile and increased efficacy.
Acknowledgements The authors wish to thank the analytical, synthesis and purification groups at AVI BioPharma Inc. We also acknowledge the technical assistance provided by Melissa Cate and Ariel Eberle.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Fulford AJ, Harbuz MS, Jessop DS: Antisense inhibition of pro-opiomelanocortin and proenkephalin A messenger RNA translation alters rat immune cell function in vitro. J Neuroimmunol 2000, 106:6-13.
2.
Mann CJ, Honeyman K, Cheng AJ, Ly T, Lloyd F, Fletcher S, Morgan JE, Partridge TA, Wilton SD: Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse. Proc Natl Acad Sci USA 2001, 98:42-47.
3.
Vacek M, Sazani P, Kole R: Antisense-mediated redirection of mRNA splicing. Cell Mol Life Sci 2003, 60:825-833.
4.
Kurreck J: Antisense technologies. Improvement through novel chemical modifications. Eur J Biochem 2003, 270:1628-1644.
5.
Detrick B, Nagineni CN, Grillone LR, Anderson KP, Henry SP, Hooks JJ: Inhibition of human cytomegalovirus replication in a human retinal epithelial cell model by antisense oligonucleotides. Invest Ophthalmol Vis Sci 2001, 42:163-169.
6.
Geary RS, Henry SP, Grillone LR: Fomivirsen: clinical pharmacology and potential drug interactions. Clin Pharmacokinet 2002, 41:255-260.
7.
Zamaratski E, Pradeepkumar PI, Chattopadhyaya J: A critical survey of the structure-function of the antisense oligo/RNA heteroduplex as substrate for RNase H. J Biochem Biophys Methods 2001, 48:189-208.
8.
Giles RV, Tidd DM: The direction of ribonucleases H by antisense oligodeoxynucleotides. Methods Mol Biol 2001, 160:157-182.
Conclusions and future directions Numerous studies have demonstrated that PMOs are capable of interfering with gene expression both in vitro and in vivo in a sequence-specific manner, indicating that PMOs have huge potential for being developed as drugs. Data characterizing the specificity, toxicology, biological stability, efficacy and kinetic behaviour of PMOs represent a major improvement in the development of antisense agents and might be vital to addressing therapeutically relevant issues. First, data presented in www.sciencedirect.com
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554 New technologies
9.
Ten Asbroek AL, van Groenigen M, Nooij M, Baas F: The involvement of human ribonucleases H1 and H2 in the variation of response of cells to antisense phosphorothioate oligonucleotides. Eur J Biochem 2002, 269:583-592.
10. Sereni D, Tubiana R, Lascoux C, Katlama C, Taulera O, Bourque A, Cohen A, Dvorchik B, Martin RR, Tournerie C et al.: Pharmacokinetics and tolerability of intravenous trecovirsen (GEM 91), an antisense phosphorothioate oligonucleotide, in HIV-positive subjects. J Clin Pharmacol 1999, 39:47-54. 11. Levin AA: A review of the issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides. Biochim Biophys Acta 1999, 1489:69-84. 12. Chi KN, Gleave ME, Klasa R, Murray N, Bryce C, Lopes de Menezes DE, D’Aloisio S, Tolcher AW: A phase I dose-finding study of combined treatment with an antisense Bcl-2 oligonucleotide (Genasense) and mitoxantrone in patients with metastatic hormone-refractory prostate cancer. Clin Cancer Res 2001, 7:3920-3927. 13. Waters JS, Webb A, Cunningham D, Clarke PA, Raynaud F, di Stefano F, Cotter FE: Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin’s lymphoma. J Clin Oncol 2000, 18:1812-1823. 14. Song E, Lee S-K, Wang J, Ince N, Ouyang N, Min J, Chen J, Shankar P, Lieberman J: RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 2003, 9:347-351. 15. Xia H, Mao Q, Paulson H, Davidson BL: siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 2002, 20:1006-1010. 16. Cerutti H: RNA interference: traveling in the cell and gaining functions? Trends Genet 2003, 19:39-46. 17. Schwarz DS, Hutvagner G, Haley B, Zamore PD: Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol Cell 2002, 10:537-548. 18. Scherr M, Morgan MA, Eder M: Gene silencing mediated by small interfering RNAs in mammalian cells. Curr Med Chem 2003, 10:245-256. 19. Shi Y: Mammalian RNAi for the masses. Trends Genet 2003, 19:9-12. 20. Grunweller A, Wyszko E, Bieber B, Jahnel R, Erdmann VA, Kurreck J: Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 20 -O-methyl RNA, phosphorothioates and small interfering RNA. Nucleic Acids Res 2003, 31:3185-3193. 21. Thompson JD: Applications of antisense and siRNAs during preclinical drug development. Drug Discov Today 2002, 7:912-917. 22. Khan AU, Lal SK: Ribozymes: a modern tool in medicine. J Biomed Sci 2003, 10:457-467. 23. Summerton J, Weller D: Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev 1997, 7:187-195. 24. Summerton J: Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta 1999, 1489:141-158. 25. Ghosh C, Stein D, Weller D, Iversen P: Evaluation of antisense mechanisms of action. Methods Enzymol 2000, 313:135-143. 26. Sazani P, Kole R: Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing. J Clin Invest 2003, 112:481-486. 27. Hudziak RM, Barofsky E, Barofsky DF, Weller DL, Huang SB, Weller DD: Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nucleic Acid Drug Dev 1996, 6:267-272. 28. Kurreck J, Wyszko E, Gillen C, Erdmann VA: Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Res 2002, 30:1911-1918. Current Opinion in Pharmacology 2005, 5:550–555
29. Crooke ST (Ed): In Antisense Drug Techcology- Principles, Strategies and Applications. Marcel Dekker, Inc; 2001. 30. Arora V, Cate ML, Ghosh C, Iversen PL: Phosphorodiamidate morpholino antisense oligomers inhibit expression of human cytochrome P450 3A4 and alter selected drug metabolism. Drug Metab Dispos 2002, 30:757-762. 31. Arora V, Knapp DC, Reddy MT, Weller DD, Iversen PL: Bioavailability and efficacy of antisense morpholino oligomers targeted to c-Myc and cytochrome P-450 3A2 following oral administration in rats. J Pharm Sci 2002, 91:1009-1018. 32. Arora V, Hannah TL, Iversen PL, Brand RM: Transdermal use of phosphorodiamidate morpholino oligomer AVI-4472 inhibits cytochrome P450 activity in male rats. Pharm Res 2002, 19:1465-1470. 33. McCaffrey AP, Meuse L, Karimi M, Contag CH, Kay MA: A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice. Hepatology 2003, 38:503-508. 34. Kipshidze N, Moses J, Shankar LR, Leon M: Perspectives on antisense therapy for the prevention of restenosis. Curr Opin Mol Ther 2001, 3:265-277. 35. Kipshidze NN, Kim HS, Iversen P, Yazdi HA, Bhargava B, New G, Mehran R, Tio F, Haudenschild C, Dangas G et al.: Intramural coronary delivery of advanced antisense oligonucleotides reduces neointimal formation in the porcine stent restenosis model. J Am Coll Cardiol 2002, 39:1686-1691. 36. Iversen PL, Arora V, Acker AJ, Mason DH, Devi GR: Efficacy of antisense morpholino oligomer targeted to c-myc in prostate cancer xenograft murine model and a Phase I safety study in humans. Clin Cancer Res 2003, 9:2510-2519. 37. Arora V, Devi GR, Iversen PL: Neutrally charged phosphorodiamidate morpholino antisense oligomers: uptake, efficacy and pharmacokinetics. Curr Pharm Biotechnol 2004, 5:431-439. This report establishes that even at very low doses PMOs could be efficiently delivered to several target sites in the in vivo setting by intraperitoneal administration. The report also makes available literature demonstrating PMO activity in different animal models. 38. Morcos PA: Achieving efficient delivery of morpholino oligos in cultured cells. Genesis 2001, 30:94-102. 39. Gebski BL, Mann CJ, Fletcher S, Wilton SD: Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle. Hum Mol Genet 2003, 12:1801-1811. 40. Moulton HM, Hase MC, Smith KM, Iversen PL: HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers. Antisense Nucleic Acid Drug Dev 2003, 13:31-43. 41. Moulton HM, Nelson MH, Hatlevig SA, Reddy MT, Iversen PL: Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides. Bioconjug Chem 2004, 15:290-299. This study demonstrates that conjugating short ARPs to PMOs significantly enhanced delivery of PMO to target sites within the cell. The authors establish that PMO delivery required conjugation rather than complexation of peptide to the PMO. 42. Amantana A, London CA, Iversen PL, Devi GR: X-linked inhibitor of apoptosis protein inhibition induces apoptosis and enhances chemotherapy sensitivity in human prostate cancer cells. Mol Cancer Ther 2004, 3:699-707. In this study, the authors establish that a significant reduction in XIAP gene expression and apoptosis in DU145 human prostate cancer cells is achieved by the scrape-loading delivery technique. This study also demonstrates the specific antisense activity of a PMO against XIAP, which is one of the cancer survival genes. In addition, the study shows that PMOs might have the potential of sensitizing refractory cancer cells to chemotherapy. 43. Ghosh C, Iversen PL: Intracellular delivery strategies for antisense phosphorodiamidate morpholino oligomers. Antisense Nucleic Acid Drug Dev 2000, 10:263-274. 44. Suwanmanee T, Sierakowska H, Lacerra G, Svasti S, Kirby S, Walsh CE, Fucharoen S, Kole R: Restoration of human beta-globin gene expression in murine and human IVS2-654 www.sciencedirect.com
Pharmacokinetics and biodistribution of phosphorodiamidate morpholino antisense oligomers Amantana and Iversen 555
thalassemic erythroid cells by free uptake of antisense oligonucleotides. Mol Pharmacol 2002, 62:545-553. 45. Hanss B, Leal-Pinto E, Teixeira A, Christian RE, Shabanowitz J, Hunt DF, Klotman PE: Cytosolic malate dehydrogenase confers selectivity of the nucleic acid-conducting channel. Proc Natl Acad Sci USA 2002, 99:1707-1712. 46. Agrawal S, Temsamani J, Galbraith W, Tang J: Pharmacokinetics of antisense oligonucleotides. Clin Pharmacokinet 1995, 28:7-16. 47. Zhang R, Diasio RB, Lu Z, Liu T, Jiang Z, Galbraith WM, Agrawal S: Pharmacokinetics and tissue distribution in rats of an oligodeoxynucleotide phosphorothioate (GEM 91) developed as a therapeutic agent for human immunodeficiency virus type-1. Biochem Pharmacol 1995, 49:929-939. 48. Srinivasan SK, Iversen P: Review of in vivo pharmacokinetics and toxicology of phosphorothioate oligonucleotides. J Clin Lab Anal 1995, 9:129-137. 49. Sereni D, Tubiana R, Lascoux C, Katlama C, Taulera O, Bourque A, Cohen A, Dvorchik B, Martin RR, Tournerie C et al.: Pharmacokinetics and tolerability of intravenous trecovirsen
www.sciencedirect.com
(GEM 91), an antisense phosphorothioate oligonucleotide, in HIV-positive subjects. J Clin Pharmacol 1999, 39:47-54. 50. Arora V, Iversen PL: Redirection of drug metabolism using antisense technology. Curr Opin Mol Ther 2001, 3:249-257. 51. Iversen PL, Arora V, Acker A, Mason DH, Devi GR: Efficacy of antisense morpholino oligomer targeted to c-myc in prostate cancer xenograft murine model and a Phase I safety study in humans. Clin Cancer Res 2003, 9:2510-2519. 52. Qin G, Taylor M, Ning YY, Iversen P, Kobzik L: In vivo evaluation of a morpholino antisense oligomer directed against tumor necrosis factor-alpha. Antisense Nucleic Acid Drug Dev 2000, 10:11-16. 53. Amantana A, Cate M, Reddy M, Weller D, Iversen P: Pharmacokinetics and Toxicokinetics of Phosphorodiamidate morpholino oligomers against hepatitis C virus after single intravenous injection. 44th Annual Meeting of the Society of Toxicology, March 2005, New Orleans, LA. [Abstract 1246]. 54. Geary RS, Watanabe TA, Truong L, Freier S, Lesnik EA, Sioufi NB, Sasmor H, Manoharan M, Levin AA: Pharmacokinetic properties of 20 -O-(2-methoxyethyl)-modified oligonucleotide analogs in rats. J Pharmacol Exp Ther 2001, 296:890-897.
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