Oligonucleotide Formulations

A. Physical-chemical Properties

Due to the presence of a mixture of diastereisomers, phospho-rothioate oligodeoxynucleotides are amorphous solids possessing the expected physical properties of hygroscopicity, low-bulk density, electrostatic charge pick up, and poorly defined melting point prior to decomposition. Their good chemical stability allows storage in the form of a lyophilized powder, spray-dried powder or a concentrated, sterile solution; more than 3 years of storage is possible at refrigerated temperatures.

Due to their polyanionic nature, phosphorothioate oligode-oxynucleotides are readily soluble in neutral and basic conditions. Drug-product concentrations are limited (in select applications) only by an increase in solution viscosity. The counter ion composition, ionic strength, and pH also influence the apparent solubility. Phosphorothioate oligodeoxynucleotides have an apparent pKa in the vicinity of 2 and will come out of solution in acidic environments (i.e., the stomach). This precipitation is readily reversible with increasing pH or by acid-mediated hydrolysis.

Instability of phosphorothioate oligodeoxynucleotides have been primarily attributed to 2 degradation mechanisms: oxidation and acid-catalyzed hydrolysis. Oxidation of the (P = S) bond in the backbone has been observed at elevated temperatures and under intense ultraviolet light, leading to partial phosphodiesters (still pharmacologically active) and are readily monitored by anion-exchange high-performance liquid chromatography. Under acidic conditions, hydrolysis reactions followed by chain-shortening depurination reactions have been documented by length-sensitive electrophoretic techniques.

B. Parenteral Injections

Given the excellent solution stability and solubility possessed by phosphorothioate oligodeoxynucleotides, it has been relatively straightforward to formulate the first-generation drug products in support of early clinical trials. Simple, buffered solutions have been successfully used in clinical studies by intravenous, intradermal, and subcutaneous injections. Recently, the intravitreal route was approved for the first antisense drug application.

C. Topical Delivery for Diseases of the Skin

The barrier properties of human skin have been an area of multidisciplinary research for a long time. Skin is one of the most difficult biological membrane to penetrate, primarily due to the presence of stratum corneum (SC), which is composed of corneocytes laid in a brick-and-mortar arrangement with layers of lipid. The corneocytes are partially dehydrated, an-uclear, metabolically active cells completely filled with bundles of keratin with a thick and insoluble envelope replacing the cell membrane (179). The primary lipids in the SC are ceramides, free sterols, free fatty acids, and triglycerides

(180), which form lamellar lipid sheets between the corneo-cytes. These unique structural features of SC provide an excellent barrier to penetration of most molecules.

Therefore, as the primary barrier to transport of molecules to the skin, physical alteration in SC can result in improved skin penetration. Tape stripping and abrasion by repeated brushing reduced the SC barrier sufficiently to allow penetration of naked plasmid DNA and produced gene expression in skin at a level comparable to that after intradermal injection of naked plasmid DNA (181). Other studies have also shown an increase in oligonucleotide penetration upon physical removal of SC barrier (182-184).

1. Altering the Thermodynamic Properties of the Molecules

Increasing lipid partitioning to improve skin penetration has been evaluated using 2 techniques that alter the thermody-namic properties of oligonucleotide molecules. A complex of phosphorothioate oligonucleotide with hydrophobic cations such as benzalkonium chloride resulted in increased penetration through isolated hairless mouse skin that was explained on the basis of greater partitioning in lipid phase (184). Chemical modification of oligonucleotides to eliminate the negative charges also resulted in a size-dependent increase in the penetration of oligonucleotide into the skin when used with chemical penetration enhancers such as ethanol and dimethyl sulfoxide (183).

2. Electrical Field for Alteration of Skin Permeability

Iontophoresis, which involves application of electric field across the skin to induce electrochemical transport of charged molecules, is studied extensively for transdermal delivery of phosphorothioate oligonucleotides (185,186). The transdermal delivery was shown to be size dependent with steady-state flux values ranging from 2 to 26 pmol/cm2 in isolated hairless mice skin. The steady-state flux also depended on the sequence, and not just the base composition, of the oligonucleotide. Molecular structure, therefore, is a key contributor to iontophoretically assisted transport of oligonucleotides (187-189). Electroporation a technique using much higher voltage than iontophoresis to cause formation of transient aqueous pathway in skin lipids, provides therapeutic levels (> 1 ^M) of oligonucleotides in the viable tissues of the skin (190).

3. Formulations for the Alteration of Skin Permeability

Chemical penetration enhancers have recently been studied for increasing transdermal delivery of oligonucleotides or other polar macromolecules. Chemical-induced transdermal penetration results from a transient reduction in the resistance of the SC barrier properties. The reduction may be attributed to a variety of factors such as opening of intercellular junctions due to hydration (191), solubilization of SC lipids (192,193) or increased lipid bilayer fluidization (194). Types of chemicals known to be penetration enhancers include alkyl esters (195), phospholipids (196), terpenes (197), nonionic surfactants (198), and laurocapram (Azone) (199). A combination of various surfactants and cosolvents can be used to achieve skin penetration with therapeutically relevant concentration of phosphorothioate oligonucleotides in the viable epidermis and dermis (200). The topical formulations produced significantly higher epidermal and dermal levels of oligonucleotide than those achieved by an intravenous injection at highest tolerated doses. This suggests that the topical route is more efficient in reaching all layers of the skin than systemic administration of phosphorothioate oligonucleotides.

Liposomes have been studied to transport oligonucleotides into the skin. They can increase the fluidity of skin lipid layers (similar to chemical enhancers) to facilitate transdermal permeation and can also carry encapsulated molecules through appendageal pathway (201,202). Mixture of a phosphorothio-ate oligodeoxynucleotide with a suspension of anionic or neutral lipids resulted in a slight increase in accumulation in epidermis and dermis (R. Mehta, unpublished, 1999). Using a combination of different delivery techniques and formulations, it appears to be feasible to deliver a therapeutically relevant amount of antisense oligonucleotide to the skin. In addition, preliminary results in our laboratory show a dose-dependent pharmacological effect consistent with the antisense mechanism of action of an ICAM-1 antisense oligonu-cleotide, ISIS 2302 (200). Studies are also underway to assess pharmacology and tissue kinetics of ISIS 2302 in human disease models.

D. Oral Delivery

Of the numerous barriers proposed by Nicklin and others (138) to the oral delivery of oligonucleotides, our experience has confirmed that 2 stand out as critical: instability in the gastrointestinal (GI) tract and low permeability across the intestinal mucosa. Given the formidable nature of these 2 barriers, it is not surprising that oral delivery of oligonucleotides has been considered impossible, or at best, difficult—as is the case with proteins, which has necessitated the latter's nonenteral administration in order to achieve systemic concentrations considered therapeutic. Nevertheless, progress has been made to address and/or understand each barrier with respect to oligonucleotides. (P = S)-oligonucleotides have a distinct advantage over proteins in that the former does not rely on secondary structure for activity. This provides freedom from concern over secondary structure destabilization and allows for (P = S)-oligonucleotide structural modifications to address both presystemic and systemic metabolism.

Natural DNA and RNA are rapidly digested by the ubiquitous nucleases found within the gut. As a consequence, oligo-nucleotides need to be stabilized in order to achieve a reasonable GI residence time to allow for absorption to occur. Surprisingly, phosphorothioate oligodeoxynucleotides were found to be rapidly degraded by nucleases found in the GI tract; therefore, additional protection from nuclease degradation is required to achieve significant oral bioavailability. Oli-gonucleotides that are uniformly modified or modified on the

3'-end (gapmers or 3'-hemimers) (Fig. 6) with nuclease-resis-tant modification have the potential to exhibit increased oral bioavailability. This was demonstrated for both backbone modifications (methylphosphonates) and for sugar-modified (2'-O-methyl) oligonucleotides (141,203). We have found that 2'-O-methoxyethyl-modified oligonucleotides also exhibit increased oral absorption compared with phosphorothioate oli-godeoxynucleotides (80,142).

The physicochemical properties of phosphorothioate oligo-deoxynucleotides present a significant barrier to their GI absorption into the systemic circulation or the lymphatics. These factors include their large size and molecular weight (i.e., up to 6.5 kDa for 20-mers), hydrophilic nature (log Do/wapproxi-mating -3.5) and multiple ionization pkas (e.g., G. Hardee, 1999, unpublished titration data, using a Sirius GlpKa instrument on a 20-mer sequence, noted over 17 pkas for phosphorothioate oligodeoxynucleotide and over 32 pkas for the 2'-O-methoxyethyl hemi-mer form). The use of formulations can improve upon GI permeability. Oligonucleotide drug formulations designed to improve oral bioavailability need to consider the mechanism of oligonucleotide absorption—either paracel-lular via the epithelial tight junctions, or transcellular via direct passage through the lipid membrane bilayer. By using paracellular and transcellular models appropriate for water-soluble, hydrophilic macromolecules, it was determined that oligonucleotides predominantly traverse GI epithelium via the paracellular route. In this regard, formulation design considerations involve the selection of those penetration enhancers (PEs) that facilitate paracellular transport and meet other formulation criteria, including suitable biopharmaceutics, safety considerations, manufacturability, physical and chemical stability, and practicality of the product configuration (i.e., regarding production costs, dosing regimen, and patient compliance, etc.). Work is in progress, optimizing oligonucleotide chemistry with various permeation enhancers (142,204,205). Preliminary data are encouraging and support continued investment of resources on this endeavor.

E. Liposome Formulations

Liposome formulations of antisense oligonucleotides offer several potential advantages over saline phosphorothioate oli-godeoxynucleotides, such as decreased toxicity, altered tissue and cellular distribution, and more convenient dose schedule for the patient. Interesting progress has been reported regarding the passive targeting of oligonucleotides to specific tissues using liposome-encapsulated therapeutics. Accumulation at sites of infection, inflammation, and tumor growth has been attributed to increased circulation times of these materials and the leaky vasculatures associated with these processes (206,207). One caution regarding these observations is worth noting. Because the mononuclear phagocyte system (MPS) is largely responsible for clearing these materials from circulation, misleading data regarding circulation time may be obtained in species with less-evolved systems (i.e., rodents).

Cationic liposomes bind to oligonucleotides due to the electrostatic interaction between positively charged head groups on lipids and negatively charged phosphates on oligonucleotides. Using the technique of complexation, all the oligonucleotide can be entrapped and purification is not required. The utility of in vivo delivery of oligonucleotide using cationic lipid is limited due to sequestration of material in lung and the RES system (144,208). In addition, interaction of the complex with blood components leads to serum sensitivity and cytotoxicity (209,210).

There are few examples of oligonucleotide delivery by anionic or charge-neutral liposomes. Oligonucleotides encapsulated into cardiolipin-containing anionic liposomes were shown to be taken up 7 to 18-fold more in human T leukemia and ovarian carcinoma cells in vitro. The intracellular release of oligonucleotides was also facilitated and the majority of oligonucleotide was delivered into liposomes (211,212). Methylphosphonate analogs were incorporated into DPPC-containing liposomes and targeted against the Bcr-abl neogene found in chronic myelogenous leukemia (CML). The liposom-al- encapsulated oligonucleotides inhibited the growth of CML cells (213). Cellular uptake of oligonucleotides against epidermal growth factor (EGF) encapsulated in DPPC:CHOL liposome containing folate was 9 times higher than nonfolate liposomes and 16 times higher than unencapsulated liposomes

(214). There are 2 limitations to intracellular delivery of oligo-nucleotides by anionic or neutral liposomes: (1) not all cells take up particulate matter, and (2) these liposomes have low encapsulation efficiency.

There is only 1 report of using anionic liposomes in vivo to deliver oligonucleotides. Ponnappa et al. described liposomes consisting DPPC:CHOL:DMPG targeted toward Kupffer cells

(215). In this study, greater than 65% of the liver-associated oligonucleotide was found in Kupffer cells.

Conjugation of antibodies to liposomes have been used for targeting of oligonucleotides to specific targets (216-220). Problems with the approach include the inhibition of cellular uptake by the high molecular weight antibody, cost, and poor encapsulation efficiency.

The primary mechanism for cell internalization of neutral liposomes is by endocytosis with the vesicles and their contents delivered to lysozomes (221). pH-sensitive liposomes have been designed to fuse with the endosomes at low endosomal pH and empty their content into cytosol. These pH-sensitive liposomes have been used to deliver antisense oligonucle-otides. pH-sensitive liposomes composed of oleic acid: DOPE:Chol-encapsulating antisense oligonucleotide targeted against friend retrovirus inhibited the viral spreading, whereas free oligonucleotide and non pH-sensitive liposomes were ineffective (222,223). pH-sensitive liposomes encapsulating the anti-env oligonucleotide were found to inhibit viral spread at low concentration in infected Dunni cells (224). The major limitation of pH-sensitive liposomes in vivo is their instability in plasma (225,226). This problem was overcome by adding polyethylene glycol-phosphatidylethanolamine (PEG-PE) into the formulation (227). PEG-PE is believed to coat the surface of liposomes, thereby preventing the interaction of liposomes with blood components. This reduced interaction leads to increased stability and plasma half-life of liposomes.

The pH-sensitive liposomes composed of CHEMS:DOPE: PEG-PE, when injected intravenously into rats, had similar pharmacokinetics parameters as non pH-sensitive sterically stabilized liposomes. The regular pH-sensitive liposomes without PEG-PE were cleared rapidly from the circulation.

Looking past the question of uptake, a novel approach to releasing endosomal contents into the cytoplasm after uptake has been recently reported (228-230). A 58-kDa protein isolated from Listeria monocytogenes was incorporated into pH-sensitive fluorescent dye. It could be determined that as soon as the endosome began to acidify, the liposome/endosome contents were released into the cytosol. As with the other delivery systems mentioned above, the eventual usefulness of a particular approach will be determined in the near future as we further define the mechanisms and governing restrictions for the inter- and intracellular trafficking of oligonucleotides.

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