Gregory E. Hardee

Transdermal Delivery of Antisense Oligonucleotides with Microprojection Patch (macroflux®) Technology

In recent years, antisense oligodeoxynucleotide (ODN) technology has emerged as one of the most promising functional genomic therapies. Several clinical trials have demonstrated its therapeutic value and low toxicity (1). To date, parenteral infusion has been the primary mode of ODN delivery. However, efforts to develop more convenient routes of administration are being explored. Transdermal iontophoresis increased ODNs across the skin; however, delivery of a therapeutically relevant dose was not achieved (2–5). The major barrier for transdermal delivery is the stratum corneum, the outermost “dead” layer of the skin. In human skin, the stratum corneum is 10–20 m thick, whereas in mice and rats it is significantly thinner. Removal of the stratum corneum by mechanical abrasion, tape stripping, or chemical treatment has been shown to significantly enhance permeation through the skin for a wide range of pharmaceuticals, including phosphorothioated (PS) ODNs (5–8). However, these approaches may be limited due to the lack of control and reproducibility, as well as the irritancy potential (9). Microprojection patch is a novel microfabrication technology for controlled transdermal drug delivery. The patch system incorporates a stainless steel or titanium microprojection array. When applied onto the skin manually or by an applicator, microprojections penetrate and create superficial pathways through the skin barrier layer to allow drug delivery. The array can be combined either with passive or iontophoretic delivery systems. In this study, we demonstrate that microprojection patch technology can facilitate the controlled transdermal ODN delivery. MATERIALS AND METHODS

Improvement of the encapsulation efficiency of oligonucleotide-containing biodegradable microspheres

The objective of this study was to encapsulate an oligonucleotide drug within poly(lactide) microparticles with high encapsulation efficiencies at high theoretical drug loadings by the solvent evaporation method. With the conventional W/O/W method, the encapsulation efficiency decreased with increasing internal water content, increasing stirring time prior to filtration of the microparticles and increasing drug loading. The encapsulation was improved by replacing methylene chloride with ethyl acetate, by using micronized drug powder instead of an internal aqueous phase or by adding electrolytes or nonelectrolytes to the external phase. With ethyl acetate, a pre-emulsification step into a smaller volume of external aqueous phase was necessary in order to avoid premature polymer precipitation and to obtain microparticles. The addition of salts (NaCl or MgCl(2)) or sorbitol to the external aqueous phase significantly improved the encapsulation efficiency, even at high theoretical drug loadings. The microparticles had a denser structure with a smooth, pore-free surface.

Topical Delivery of Anti-sense Oligonucleotides Using Low-Frequency Sonophoresis

No HeadingPurpose.Topical delivery of oligonucleotides, though attractive for the treatment of skin disorders, is limited by the low permeability of the stratum corneum. Herein, we assessed the potential of low-frequency ultrasound (20 kHz, 2.4 W/cm2) in delivering therapeutically significant quantities of anti-sense oligonucleotides into skin.Methods.Dermal penetration of oligonucleotides penetration was quantified in vitro using radiolabeled oligonucleotides.Results.Estimated concentrations of oligonucleotides (ODNs) in the superficial layers of the skin ranged from ~0.5% to 5% of the donor concentration after a 10-min application of ultrasound and ODN. Microscopic evaluations using fluorescently labeled oligonucleotides and sulforhodamine B revealed heterogeneous penetration into the skin. Heterogenous penetration led to the formation of localized transport pathways (LTPs), which occupied about 5% of the total exposed skin area. Immuno-histochemical studies using an oligonucleotide that reacts specifically with an antibody also confirmed penetration of ODNs into LTPs. Histologic studies revealed that no gross structural changes were induced in the skin due to ultrasound application.Conclusions.These results show successful delivery of anti-sense oligonucleotides using low-frequency ultrasound.

Characterization of alginate/poly-l-lysine particles as antisense oligonucleotide carriers

The gel forming characteristics of alginate in the presence of calcium ions and further crosslinking with poly-L-lysine led to the formation of sponge-like nano- and microparticles. The particle size was varied by adjusting the final concentrations of and proportions between the components. The region for particle formation was from 0.04 to 0.08% (w/v) of alginate in the final formulation, the change from the nm to microm size range occurred at a concentration of approx. 0.055% (w/v). Oligonucleotide-loaded microparticles were prepared by two different methods, either by absorption of the drug into the crosslinked polymeric matrix or by incorporation of an oligonucleotide/poly-L-lysine complex into a calcium alginate pre-gel. The release of oligonucleotide from microparticles prepared by the first method was higher. The addition of increasing amounts of poly-L-lysine resulted in larger particles, higher oligonucleotide loading and slower drug release. An increase in the final solid content of the formulation led to larger particles, especially with high concentrated calcium alginate pre-gels. Microparticles based on alginate and poly-L-lysine are potential carriers for antisense oligonucleotides.

Alginate/Poly-L-Lysine Microparticles for the Intestinal Delivery of Antisense Oligonucleotides

AbstractPurpose. A microparticle carrier based on alginate and poly-L-lysine was developed and evaluated for the delivery of antisense oligonucleotides at the intestinal site. Formulations of oligonucleotide-loaded microparticles having differences in the carrier molecular weight and composition were characterized in vitro and in vivo.Methods. Polymeric microparticles were prepared by ionotropic gelation and crosslinking of alginate with calcium ions and poly-L-lysine. The loading of the antisense oligonucleotide into the microparticles was achieved by absorption in aqueous medium. The association capacity, loading and particle size of the microparticles were characterized. The in vivo performances of various formulations after intrajejunal administration were studied in rat and in dog models. Results. Microparticles had a sponge-like structure and an oligonucleotide loading of 27-35%. The composition of the medium affected the particle size and the in vitro release profiles. The oligonucleotide bioavailability after intrajejunal administration to rats in the presence of permeation enhancers was good for most of the tested systems. The application of microparticles in powder form compared to an equivalent suspension improved the intrajejunal bioavailability of the oligonucleotide (25% and 10% respectively) in rats. On the contrary, the intrajejunal administration to dogs resulted in poor oligonucleotide bioavailability (0.42%). Conclusions. The formulation of antisense oligonucleotides within alginate and poly-L-lysine microparticles is a promising strategy for the oral application.

Characterization of complexes of an antisense oligonucleotide with protamine and poly-l-lysine salts

The objectives of this work were to study the interaction of an antisense oligonucleotide (ISIS 2302) with poly-L-lysine (PLL) and protamine salts, to determine the physico-chemical characteristics of the resulting complex systems and to analyze the influence of permeation enhancers (Na-chenodeoxycholate and Na-caprate) on the dissociation of the complexes. Specific conductivity, zeta potential, particle size distribution and dialysis studies of the resulting complex systems were performed. Conductometric titration defined the molar ratios between the ionic species in the complex. Zeta potential data confirmed the conductometric equivalence points and explained the good physical stability of charged complexes when compared to neutral complexes (+/-40 mV for PLL-based complexes and +/-25 mV for protamine sulfate complexes). The particle size was less than 175 nm for most systems. The incorporation of Na-chenodeoxycholate promoted complex dissociation, while Na-caprate gave opposite results. An increase in the ionic strength of the environment had a destabilizing effect and promoted dissociation of the complexes.

Stability of polycationic complexes of an antisense oligonucleotide in rat small intestine homogenates

Presystemic degradation in the gastrointestinal tract is one of the major problems contributing to the poor oral absorption of antisense oligonucleotides. Complexes between the antisense phosphorothioate oligodeoxynucleotide ISIS 2302 and the polycationic carriers protamine sulfate grade X, protamine chloride grade V, protamine phosphate grade X, poly-L-lysine hydrobromide (PLL), spermidine phosphate salt, spermine diphosphate salt, and Protasan G113 and CL113 were formulated in order to increase stability against intestinal nucleolytic degradation. Specific conductivity measurements were carried out to determine the charge ratio of the complex systems. Nuclease stability assays were performed in a rat small intestine homogenate model, which displayed significant exo- and endonuclease activity. Full-length oligonucleotide and metabolites were analyzed by capillary gel electrophoresis with UV detection at 260 nm. Most of the complexes of ISIS 2302 and the polycationic materials, except PLL-based systems, showed a better protection against enzymatic metabolism than free oligonucleotide. Protamine sulfate and protamine chloride considerably enhanced the nuclease stability of the phosphorothioate antisense oligonucleotide. The association of oligonucleotides with several polycationic substances proved to be an alternative to chemical modification in order to stabilize oligonucleotides in the gastrointestinal tract against nucleolytic degradation.

Formation of Oligonucleotide Adducts in Pharmaceutical Formulations

During preformulation studies, we observed that oligonucleotide extracted from topical formulations contained considerable amounts of covalently modified oligonucleotide adducts. In this report, we describe the identification and characterization of reaction products that form when PS-oligodeoxyribonucleotide ISIS 2302 (1) is brought into contact with aqueous solutions of glycerol-derived excipients. Compatibility tests showed that the presence of certain glycerides in the formulation lead to adduct formation (1 + 58x amu, 1 + 72x amu, 1 + 58x + 72y amu, x, and y are the number of modifications on one oligonucleotide strand). No adduct formation was observed in the presence of triglycerides or propylene glycol–derived excipients used in the study. Using nucleosides as model compounds, two modifications of deoxyguanosine were isolated by preparative reversed phase (RP)-high pressure liquid chromatography (HPLC) and characterized by nuclear magnetic resonance (NMR) and HPLC-mass spectrometry (MS). Modifications were identified as N2-(1-carboxymethyl)- and N2-(1-carboxyethyl) derivatives of 2′-deoxyguanosine. The mechanism of formation of these adducts may involve advanced glycation reactions possibly caused by excipient impurities or degradation products such as glyceraldehyde or glyceraldehyde derivatives.