Pharmacokinetic study of dexamethasone disodium phosphate using intravitreal, subconjunctival, and intravenous delivery routes in rabbits.

Dexamethasone is a corticosteroid with proven efficacy for treating both anterior- and posterior-segment ocular diseases. Delivery of drugs to the back of the eye has always been a challenge, with dexamethasone being no exception. There are multiple delivery routes to the retina, with each exhibiting different pharmacokinetics, depending on the drug molecule and specific route of administration. In this study, we used intravenous (IV), subconjunctival (SC), and intravitreal (IVT) injections in rabbits to determine the pharmacokinetics of dexamethasone phosphate and its metabolic product, dexamethasone, at low (25 microg/kg) and high (250 microg/kg) doses. Plasma samples were collected from each group of animals at different time points up to 24 h after the injection. Using a liquid chromatographic mass spectrometric method with a limit of detection of 0.5 ng/mL, the plasma concentration for dexamethasone and its prodrug compound were quantified. IV delivery showed the fastest plasma elimination, followed by SC delivery. IVT delivery exhibited a depot effect, with very low plasma levels throughout the 24-h time course. At 24 h postinjection, only the high-dose IVT and low- and high-dose SC dexamethasone injections were still detectable in the plasma.

Accelerated immune response to DNA vaccines.

DNA vaccines offer considerable promise for improvement over conventional vaccines. For the crucial step of delivering DNA vaccines intracellularly, electroporation (EP) has proven to be highly effective. This method has yielded powerful humoral and cellular responses in various species, including nonhuman primates. In an attempt to further improve DNA vaccination we used micron-size gold particles (which do not bind or adsorb DNA) as a particulate adjuvant which was coinjected with DNA intramuscularly into mice, followed by EP of the target site. The presence of gold particles accelerated the antibody response significantly. Maximum titers against hepatitis B surface antigen (HBsAg) were reached after one boost in 6 weeks, whereas 8 weeks were required without particles. These immunizations were effective in protecting mice against tumor challenge with cancer cells expressing HBsAg as a surrogate cancer antigen. Computer modeling of electric fields and gene expression studies indicate that gold particles do not stimulate EP and subsequent antigen expression. The particles may act as an attractant for immune cells, especially antigen presenting cells. We conclude that particulate adjuvants combined with DNA vaccine delivery by EP reduces the immune response time and may increase vaccine efficacy. This method may become valuable for developing prophylactic as well as therapeutic vaccines. The rapid response may be of particular interest in countering bio-terrorism.

Increased gene expression and inflammatory cell infiltration caused by electroporation are both important for improving the efficacy of DNA vaccines.

One potential reason for the enhancement of immune responses to DNA vaccines following electroporation is increased gene expression. However, the inflammatory response and accompanying cellular infiltration stimulated by electroporation may also be essential for enhancing immune responses to DNA vaccines. These parameters were investigated in pigs, using different electroporation conditions to induce different levels of gene expression and inflammation. Results indicated that the least effective strategy was conventional intramuscular injection where there was low gene expression and low inflammatory cell infiltration. The most efficacious strategy was plasmid administration immediately followed by electroporation. This latter set of conditions elicited a combination of high gene expression and high cellular infiltration. This indicates that electroporation enhances immune responses to DNA vaccines through increased gene expression and inflammatory cell infiltration.

Enhancement of the effectiveness of electroporation-augmented cutaneous DNA vaccination by a particulate adjuvant.

DNA vaccines are attracting increased attention due to multiple advantages over conventional vaccines. Attempts to improve these vaccines focus on enhancing DNA delivery and employing novel immunoadjuvants. Electroporation (EP) has emerged as an effective method for delivering DNA vaccines, significantly enhancing humoral and cellular responses. To further improve EP-augmented DNA vaccination, we used micron-size gold particles as a particulate adjuvant. DNA is not bound, or adsorbed, to the particles. Gold particles were coinjected intradermally with plasmid DNA encoding the hepatitis B virus surface antigen (HBsAg) into mice, both in the absence and presence of noninvasive EP. The particles enhanced the percentage of responding animals, and shortened the time for reaching maximal antibody titers by 2 weeks. Subtyping of the produced antibodies revealed a predominantly Th1-like response which did not change significantly with the absence or presence of particles. The particles likely function as an attractant for antigen-presenting cells (APCs), and probably do not affect EP or antigen expression to a significant extent. We conclude that micron-size gold particles injected intradermally together with DNA followed by EP give rise to an accelerated, potent immune response with a strong cellular component. This method may become important for the development of fast-acting therapeutic and prophylactic vaccines.

Enhancement of antibody and cellular immune responses to malaria DNA vaccines by in vivo electroporation.

We evaluated the effectiveness of in vivo electroporation (EP) for the enhancement of immune responses induced by DNA plasmids encoding the pre-erythrocytic Plasmodium yoelii antigens PyCSP and PyHEP17 administered intramuscularly and intradermally to mice. EP resulted in a 16- and 2-fold enhancement of antibody responses to PyCSP and PyHEP17, respectively. Immunization with 5 microg of DNA via EP was equivalent to 50 microg of DNA via conventional needle, thus reducing by 10-fold the required dose to produce a given effect. Moreover, IFN-gamma responses were increased by approximately 2-fold. Data demonstrate the potential of EP to enhance immune responses to DNA vaccines against infectious agents.

Immunogenicity of hybrid DNA vaccines expressing hepatitis B core particles carrying human and simian immunodeficiency virus epitopes in mice and rhesus macaques.

An effective HIV vaccine will likely need to induce broad and potent CTL responses. Epitope-based vaccines offer significant potential for inducing multi-specific CTL, but often require conjugation to T helper epitopes or carrier moieties to induce significant responses. We tested hybrid DNA vaccines encoding one or more HIV or SIV CTL epitopes fused to a hepatitis B core antigen (HBcAg) carrier gene as a means to improve the immunogenicity of epitope-based DNA vaccines. Immunization of mice with a HBcAg-HIV epitope DNA vaccine induced CD8(+) T cell responses that significantly exceeded levels induced with DNA encoding either the whole HIV antigen or the epitope alone. In rhesus macaques, a multi-epitope hybrid HBcAg-SIV DNA vaccine induced CTL responses to 13 different epitopes, including 3 epitopes that were previously not detected in SIV-infected macaques. These data demonstrate that immunization with hybrid HBcAg-epitope DNA vaccines is an effective strategy to increase the magnitude and breadth of HIV-specific CTL responses.

Effect of delivery parameters on immunization to ovalbumin following intracutaneous administration by a coated microneedle array patch system.

Immunization to the model antigen ovalbumin was investigated using a novel intracutaneous delivery system consisting of antigen-coated microneedle arrays. The influence of the following parameters on the resulting immune responses was investigated: depth of vaccine delivery, dose of vaccine delivered, density of microneedles on the array, and area of application. The immune response was found to be dose dependent, and mostly independent of depth of delivery, density of microneedles, or area of application. Our studies show that the shortest, most tolerable microneedle arrays can be used for achieving consistent and high antibody titers. Overall, the microneedle array proves to be a very versatile delivery technology, allowing easy and reproducible antigen delivery to skin for efficient vaccination without the use of a needle and syringe.

Electroporation improves the efficacy of DNA vaccines in large animals.

It is generally recognized that DNA vaccines are often less effective in large animals than in mice. One possible reason for this reduced effectiveness may be transfection efficiency and the low level of expression elicited by plasmid vectors in large animals. A possible way to improve plasmid gene expression in vivo is electroporation. To determine whether we could enhance immune responses in pigs by electroporation, we used plasmids encoding two different genes (bovine herpesvirus glycoprotein D (gD) and hepatitis B surface antigen (HBsAg)) and two different electrodes, a single-needle electrode and a six-needle electrode. Electroporation significantly enhanced immune responses to both antigens. In addition, we demonstrated that co-administration of plasmids coding for two different antigens (pgD and pHBsAg) did not result in significant interference between the plasmids. We also incorporated a DNA prime/protein boost strategy to examine the effect of DNA priming with electroporation on the immune response after a protein boost.

Needle-free topical electroporation improves gene expression from plasmids administered in porcine skin.

Electroporation has been shown to increase the potency of DNA vaccines that have demonstrated significant potential in mice. However, there is a need to develop noninvasive or minimally invasive vaccination methods. In pigs, in vivo gene expression was assessed to compare intradermal needle injection to a needle-free dermal BioJect as a means of delivery of plasmids. Each administration method was further tested with and without surface electroporation. Experiments with plasmid DNA encoding luciferase demonstrated that needle-free administration results in higher gene expression levels than needle injection. Electroporation enhanced gene expression for both intradermal delivery methods. Needle-free plasmid injection in combination with electroporation led to a more rapid induction of immune responses compared to other methods of plasmid administration. It was concluded that needle-free topical electroporation significantly enhances gene expression, possibly by improving cellular uptake of plasmid DNA.

Enhancement of DNA vaccine potency in rhesus macaques by electroporation.

The potency of an HIV DNA vaccine was enhanced in rhesus macaques by in vivo electroporation, as judged by increased onset, magnitude and duration of antibody and cell-mediated immune responses against both components of a combination Gag and Env vaccine. These data demonstrate the utility of the electroporation technology for use in large animals.