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Pharmaceutical Delivery of Antisense Oligonucleotides (ODNs) Aws Alshamsan, RPh Teaching Assistant Department of Pharmaceutics College of Pharmacy – King Saud University Nucleic Acid Structure Antisense ODNs? They are sequenceselective nucleotide sequences that can bind to a target mRNA to inhibit gene expression i.e. to inhibit protein synthesis. Protein Synthesis Antisense Strategy Antisense Approach vs. Conventional Therapy Disadvantages of Conventional Therapy Requires the non-rational approach i.e. screening thousands of compounds to find an active molecule. Lacks specificity of action. Why don’t we have an ODN for every disease? The main barrier to antisense strategy is now achieving delivery of the ODNs in sufficient quantities to the correct target sites of action and for the desired time frame to achieve the desired level of gene inhibition. ODNs are polyanionic macromolecule (large and charge). Designing Biologically Stable ODNs Designing Biologically Stable ODNs Cellular Uptake The cellular uptake of naked ODNs is generally inefficient. ODNs enter cells in vitro through pino- and endocytosis. The mechanisms of uptake depend on factors such as ODN chemistry, length, conformation and concentration, cell type, stage of cell cycle, degree of cell differentiation and cell culture condition. Delivery Strategies 1. 2. 3. 4. 5. 6. Liposomes and Lipoplexes. Dendrimers. Receptor-mediated endocytosis. Local delivery to the brain. Polymers. Oral delivery. Liposomes Cationic liposomes are the most widely used system for ODNs delivery. They afford ODNs protection and enhance cellular uptake. A significant optimization in terms of charge ratio between cationic lipid and ODNs at a given dose is essential for effective delivery and activity. Lipoplexes They are lipid complexes with ODNs. Cationic lipoplexes are preferable for ODNs delivery. The toxicity and serum sensitivity of ODNLipoplex systems might limit their widespread use in vivo. Liposomes and Lipoplexes In vivo studies have shown that they are rapidly cleared from the circulation by the macrophages. Modification with PEG (Stealth Liposomes) reduces opsonization and phagocytosis and resulted in long circulating drugs. Such systems may allow enhanced passive targeting of tumours, which have leaky vasculature that is permeable to small-size liposomes. Dendrimers (DEN) An advantage of DEN is the very defined polymerization reactions yielding monodisperes, reproducible products. Most of studies to date regarding dendrimer delivery of ODNs have been done using the polyamidoamine (PAMAM) starburst DEN. DEN-ODNs complex have shown reduce metabolic degradation and enhance cellular uptake. Percentage intact in serum was 10% naked ODNs to 85% DENODNs complex. Receptor-Mediated Endocytosis This process has the potential for the effective targeting of ODNs. Tumour cells has been a good candidate for such targeting due to its increased requirement for essential nutrition. Conjugating ODNs to a transferrin receptor antibody demonstrated a 3-fold increase in cellular uptake compared to naked ODNs. Transferrin receptor is highly expressed in brain capillary endothelium, this technique is useful for ODNs CNS delivery. Local Delivery to the Brain The in vitro success of the previous technique targeting the brain has not been transferable to the in vivo model. This is may be due to the significant binding of ODNs to plasma proteins forming a high molecular mass complex which will be easily excluded from crossing BBB. Direct intracerebroventricular injection is considered for such cases but brain tissue toxicity may occur. A promising development is the use of biodegradable microspheres, which may overcome the toxicity problems and provide region-specific and sustained delivery of ODNs. Polymers Advantages of Polymers Usage for ODNs Delivery: 1. 2. 3. 4. Afford protection to ODNs. Control the rate of the release of the encapsulated drug. Can be fabricated as implantable devices for local application. Could be administered parenterally. Factors Controlling Release Profile: 1. 2. 3. The size of the microsphere. The length of ODNs. The molecular weight of the polymer. Polymers Polylactides and co-polymers of lactic acid and glycolic acid P(LA-GA) have been evaluated for ODNs delivery. Entrapment of ODNs within P(LA-GA) microsphere matrix, provides nuclease protection. Release of ODNs from the microspheres in vitro showed that ODNs were still able to function. Polymers The release profile has been shown to be “triphasic”. Phase 1: “burst effect” after 48h. Phase 2: More sustained release. Phase 3: Additional increased release results from bulk degradation of the microsphere. Polymers Polyalkylcyanoacrylate (PACA) nanoparticles have been investigated for ODNs delivery. ODNs are adsorbed onto the charged (cationic) surface of the PACA nanoparticles providing efficient protection and cellular uptake. The toxicity of hydrophobic cation and the production of formaldehyde after degradation may limit the in vivo use of this system. Oral Delivery ODNs are rapidly degraded in GIT. However, some studies have shown that some P.O. ODNs are as effective as I.P. ODNs. Some studies suggest slow passive diffusion and transcytosis as possible mechanisms for oral bioavailability. Oral Delivery ODNs are rapidly degraded in GIT. However, some studies have shown that some P.O. ODNs are as effective as I.P. ODNs. Some studies suggest slow passive diffusion and transcytosis as possible mechanisms for oral bioavailability. Fomivirsen Sodium (Vitravene)® For the local treatment of CMV retinitis in AIDS patients. Dose 150-330 μg intravitreal injection. Cleared locally by exonucleases 1-2 hr after injection.