THERAPEUTIC ANTISENSE AGENTS AND APTAMERS

1. THERAPEUTIC ANTISENSE AGENTS AND APTAMERS By NARENDAR.D M.PHARM. II - SEMESTER Department of Pharmaceutics University College of Pharmaceutical Sciences, KAKATIYA UNIVERSITY Warangal - 506009

2. CONTENT INTRODUCTION ANTISENSE AGENTS DEFINITION MECHANISM ADVANTAGES CELLULAR ACTIVITY CELLULAR UPTAKE CLINICAL TRIAL SUBSTANCES APTAMERS DEVELOPMENT PROPERTIES ADVANTAGES AND DISADVANTAGES APPLICATIONS CONCLUSION REFERENCES

3. INTRODUCTION The term ‘Antisense Therapeutics’ or ‘Antisense Technology’ encompasses several types of nucleic acids that have the ability to modulate gene expression. The most common types of nucleic acids included in this term are antisense oligonucleotides (ODNs), ribozymes (RNA enzymes) and more recently, DNAzymes (DNA enzymes).

4. Definition: Antisense refers to the use of short, Single stranded synthetic ONs to inhibit gene expression. These compounds are designed to be complementary to the coding (sense) sequence of RNA inside the cell. After hybridization totarget sequences, translational arrest occurs viaone of several putative mechanisms.

5. OLIGONUCLEOTIDE • Also called oligos. • Sequence of DNA or RNA with a phosphate backbone but may have a sulfate, peptide, or morpholino backbone in place of phosphate one, to reduce or eliminate oligo degradation nucleases. Main backbone of ONs is the phospodiaste.

6. MECHANISM • First is the ribosomal blockade where the antisense molecule hybridizes to the sense sequence and prevents the ribosome from reading the mRNA code, resulting in production of a defective nonfunctional protein. • The second is the specific cleavage of RNA strand by activated RNAaseH following RNA-ON hybridization. This cleavage results in destruction of the coding message and inhibition of protein synthesis. • The third is the competition between the ribosome and the antisense ON for binding to the 5’untranslated region (.5’-UTR) of the mRNA.

7. Binding of the ON to the 5’-UTR can also result in activation of RNase H and subsequent cleavage of the mRNA. Finally, synthesis of fully mature mRNA in the cytosol can also be prevented at the level of RNA transcription, splicing, processing. Or transport across the nuclear membrane. • For example, ON can bind to the complementary sequence on nuclear DNA. forming triplex DNA which selectively inhibits DNA transcription.

8. ADVANTAGES: • Mature technology (20 years in development). • Drug discovery and research is faster and more predictable. • Compounds are potentially more selective, effective and less toxic. • Broad disease application. • Dosing advantages (route and frequency). • Specificity and is the relative simplicity in which the drugs can be rationally designed.

9. Antisense activity at barriers • First, the ONs must find their way to target cells where they must then penetrate the plasma membrane to reach their target site in the cytoplasm or nucleus. • Second, once inside the cell the ON must be able to withstand enzymatic degradation presented by various endogenous nucleases. • Third, the ON must be able to find and then bind specifically to its intended target site in order to inhibit expression of the disease-causing gene.

10. Stability and chemical modification • The initial successful demonstrations of the antisense strategy in cell culture employed the naturally occurring phosphodiester ONs. • Phosphodiester ONs are easily degraded in cell culture medium containing serum due to 3’-exonuclease digestion. Consequently, the antisense effects could only be observed if high ON concentrations (up to 100 PM) were used. • Protection from degradation can be achieved by the use of a “3’-end cap” strategy in which nuclease-resistant linkages are substituted for phosphodiester linkages at the 3’ end of the ON . Alternatively, ONs containing a 3’-terminal hairpin-like structure were found to exhibit improved resistance to exonuclease digestion.

11. Phosphodiester ONs enter cells, they can be further degraded by cellular endonucleases. Neither 3’-end caps nor 5’-end caps protect ONs from degradation in HeLa cell extracts. • Thus, phosphodiester ONs are poor candidates for use as therapeutic agents in vivo. Consequently a number of chemical modifications have been made to improve enzymatic stability of these compounds while preserving their ability to hybridize to cognate targets. • The most commonly used are the first-generation analogs that possess modifications of the phosphodiester backbone. Examples of these include the phosphorothioate and phosphorodithioate analogs which have sulfur substituted for one or both nonbridging oxygens.

12. Cellular uptake of aptamers • Cellular uptake of ONs is an energy-dependent process and can be inhibited by treating the cells with metabolic inhibitors or by lowering the temperature. • This transport across the membrane takes place in a saturable and sequence-independent manner. Any sequence or size of ribo- and deoxyribonucleotide was demonstrated to compete with labeled ON for uptake. The uptake is endocytic and appears to be mediated by membrane receptor proteins. • Several approaches have been developed to improve cellular uptake of ONs. These include inclusion of ONs into liposomes or attaching them covalently or electrostatically to specific or nonspecific carriers.

13. Liposome-mediated antisense delivery Cationic liposomes which can form stable complexes with the polyanionic ONs. These liposomes consist mainly of a positively charged lipid, most notably N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride or DOTMA, and a co-lipid. E.g. dioleylphosphatidylethanolamine, to aid cytoplasmic delivery of the polynucleotides. Recently, several different types of cationic lipids have been developed including lipofectin, quaternary ammonium compounds, cationic derivatives of cholesterol- diacyl glycerol and lipid derivative of polyamines

15. Polylysine- mediated antisense delivery • Poly( r_-lysine (PLL), a well-known polycationic drug carrier, has been used to facilitate cellular uptake of various drugs including antisense ONs. Using VSV-infected L929 cells as a model system, Lemaitre and Leonetti et al. demonstrated that ONs complementary to viral nucleocapsid initiation site or to viral genomic RNA sequences promoted a sequence-specific and dose-dependent antiviral activity when administered as PLL conjugates. • Antiviral activities of such conjugates were observed at concentrations below 1 PM while nonconjugated ONs were equally active when used at concentrations greater than 50 ,uM. Likewise, PLL-conjugated ONs complementary to a HIV-1 splice site inhibited cytopathic effects at much lower concentrations than non-conjugated phosphodiester or methylphosphonate ONs.

16. Other methods of antisense delivery • ON modifications reported to increase cellular uptake include the attachment of hydrophobic molecules, such as cholesterol and phospholipids, to the ONs. • Coupling of a single cholesterol moiety to an ON appears to increase its intracellular uptake by up to 1%fold. • Similarly, anti-HIV cholesteryl-conjugated ONs are more effective than their unmodified counterparts. • Similarly, conjugation of an ON to phospholipids was shown to promote its anti-tumor activity. It is not known from these studies whether endocytosis is involved in the uptake of modified ONs. • Avidin, a cationic protein known to internalized via an adsorptive endocytosis process, has been coupled to ONs Association of a biotin-conjugated ON with avidin is rapid and of high affinity. Cellular uptake of an avidin-biotin- ON complex was shown to be 4-fold more efficient than the biotin-ON conjugate alone.

17. Example clinical trials studies involving antisense oligonucleotides --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ODN Target Therapeutic Class Clinical Trial hase Company ----------------------------------------------------------------------------------------------------------------------------------------------------------- Vitravene CMV Retinitis Anti-viral in Approved Isis AIDS patients CIBAVision ISIS 2302 ICAM-1 Renal transplant rejection Phase II Isis Psoriasis (topical) Phase IIa Ulcerative colitis (enema) Phase IIa ISIS 3521 PKC-alpha Cancer Phase II Isis ISIS 5132 c-raf kinase Cancer Phase II Isis ISIS 2503 Ha-ras Cancer Phase II Isis ISIS 14803 HCV Hepatitis C Phase I / II Isis /Elan GEM231 Protein kinase A 1a Cancer Phase II Hybridon/methylgene MG98 DNA methyltransferas Cancer Phase 1 Hybridon/ methylgene

18. APTAMERS Aptamers are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acid by an iterative process of adsorption, recovery and reamplification. They have potential applications in analytical devices, including biosensors, and as therapeutic agents. Also defined as aptamers are oligonucleotide sequence that bind ligands or antigens in a way that is similar in may respects to antibody-ligand interactions.

19. Aptamers range in size from approximately 6 to 40 k Da and sometimes have complex three-dimensional structures, produced by a combination of Watson–Crick and non-canonical intramolecular interactions. More specifically, aptamers can be classified as: • DNA or RNA aptamers. They consist of (usually short) strands of oligonucleotides. • Peptide aptamers. They consist of a short variable peptide domain, attached at both ends to a protein scaffold.

20. DEVELOPMENT OF APTAMERS • Isolation of nucleic acids from artificial libraries on the basis of their biochemical properties were being widely discussed during 1988 and 1989, three groups independently published their results in 1990. • First, the Joyce group reported the use of in vitro mutation, selection and amplification to isolate RNAs that were able to cleave DNA. • Second, the Gold group described experiments designed to identify the sequence requirements of T4 DNA polymerase. • ‘SELEX’ (selective expansion of ligands by exponential enrichment), was able to identify the natural target of the enzyme as the predominant, high-affinity ligand, with one major variant emerging with similar affinity.

21. PROPERTIES OF APTAMERS • Structures • Aptamer Size • Aptamer Targets • Affinity

22. Structure: Determined by - enzymatic probing - chemical probing - NMR - X-ray crystallography NMR has disadvantages - small size and rigidity when complexed with target. - similariries between the interaction sites between protien ligands and their receptrors.

23. Aptamer size: • Size of aptamer depends on sequence family. • Minimum within VEGF aptamers was between 23 and 35 nt ; minimum xanthine and guanine aptamers were 32 nt long. • Solvent-exposed surface area for a typical aptamer expected to be in the range 50-60 nm2.

24. Aptamer targets: • Aptamers targeted against small ions, such as zinc, to nucleotides such as ATP, oligopeptides and large glycoproteins such as CD4, size range 65kDa-150kDa, with no theoretical upper limit.

25. Aptamer affinity: • Aptamers against small molecules have affinities in micromolar range. against aminoacids such as citrulline and arginine range from 0.3 to 65µM, and those against ATP and xanthine are 6 and 3.3µM respectively. • Aptamers to nucleic acid-binding molecules have affinities in the nanomolar range. against retroviral integrase – 10-800nM reverse transcriptase – 0.3-20nM

26. ADVANTAGES • Used To analyze the natural processes of nucleic acid–protein recognition. • To generate inhibitors of enzymes, hormones and toxins with potentially pharmacological uses. • To Detect the presence of target molecules in complex mixtures and to generate lead compounds for medicinal chemistry. • Their advantages over alternative approaches include the relatively simple techniques and apparatus required for their isolation, the number of alternative molecules that can be screened (routinely of the order of 1015) and their chemical simplicity.

27. Disadvantages of aptamers include: • their pleiomorphism, • their high molecular mass, • the restricted range of target sites that appear to be suitable.

28. IN VIVO APPLICATION OF APTAMERS • Aptamers targeting coagulation factors E.g against factor IXa • Aptamers targeting growth factors or hormones E.g against VEGF • Aptamers targeting antibodies involved in autoimmune diseases E.g. auto-antibodies against nicotinic AChRs (for m gravis)

29. Aptamers targeting inflammation markers E.g against elastase • Aptamers targeting neuropathological targets E.g against synthetic βA amyloid peptide (Alzheimer) • Aptamers against infectious diseases E.g against gp120 or HA • Aptamers targeting membrane biomarkers E.g against CTL-4 • Aptamers targeting whole organisms E.g against CMV

31. Conclusion

32. Conclusion

33. REFERENCES • Y. Rojanasakul - Advanced Drug Delivery Reviews 18 (1996) 115-131 • S. Akhtar et al - Advanced Drug Delivery Reviews 44 (2000) 3 –21 • R.J. Boado - Advanced Drug Delivery Reviews 15 (1Wf) 7% 107 • James swarbrick – Encyclopedia of pharmaceutical technology 3- edition, volume-2 935-936 • James swarbrick – Encyclopedia of pharmaceutical technology 3- edition, volume-3 1575-1576

34. William James; Encyclopedia of Analytical Chemistry; pp. 4848–4871 • S.P.Vyas and Roop K. Khar; targeted & Controlled drug delivery: Novel Carrier Systems, • www.pharmainfo.net.com • www.wikipedia.org • www.informaworld.com

35. Conclusion