• Development of Spray Dried Microparticulate Delivery Systems for Vaccines and Oligonucleotides

      Ubale, Ruhi V; College of Pharmacy
      In recent years, research related to the development of biotherapeutics has gained tremendous interest in industry as well as academia. There are a number of biologics available in the market and many more currently being evaluated in clinical trials. Their development has gained momentum due to the range of advantages they offer over traditional small molecule drugs. However, there still remain challenges with their delivery mainly due to the poor stability of these molecules in harsh physiological conditions. A number of strategies are being devised for the delivery of biomolecules by non-invasive routes to make delivery easier and more patient compliant. Microparticles are one of the options being explored as they offer protection, sustained release and enhanced bioactivity to the biomolecule. In this project, we have studied the use of microparticles for the development of formulations for a meningococcal polysaccharide vaccine and an antisense oligonucleotide to NF-kB. Meningitis occurs majorly in children and is caused by Neisseria meninigitidis. Infection by this bacterium spreads rapidly through the body resulting in loss of limbs, hearing and can also be fatal. Hence, vaccination is considered as the only means prevention for meninigitis. Marketed vaccines use protein conjugated bacterial capsular polysaccharides as the antigen. Polysacchatides are considered inherently weak antigens with a T- independent immune response. With the development of a microparticulate vaccine using a protien matrix, we expected to stimulate the T-dependent immune pathway for these antigens avoiding the use of a conjugated Diphtheria toxoid. We have studied the enhancement of innate immune responses by the microparticulate vaccine. The antigen used N. meningitidis polysaccharide A, aslo an adjuvant- kdtA(unglecosylated lipid A)- was studied for its response. The microparticulate vaccine was characterized for its physicochemical characteristics like size, charge and toxicity. Also it was studied for the innate immune responses the microparticles can elicit after incubation with murine and human macrophages. Release of innate immune markers like TNF-a, IL-1B, IL-8, nitric oxide and reactive oxygen species from macrophages was studied and the microparticulate vaccine was observed to elicit a desirable innate immune response. Antigen presentation by macropaphages stimulated with the microparticulate vaccine was also studied by visualizing autophagy. Lung inflammation occurs doe to an infection or a damaging agent that irritates the lung lining or pleura. NF-kB is a nuclear transcription factor activated by stimuli like endotoxin and bacteria that initiates the synthesis of pro inflammatory cytokines such as TNF-a, IL-1, IL-6, etc. Antisense are single stranded RNA complementary to a chosen sequence that offer great potential to inhibit the synthesis of individual proteins by interfering with protein translation. As intracellular penetration of antisence compounds has proven to be a limiting factor in their effectiveness, we have proposed to develop a microparticulate formulation for pulmonary delivery of antisense to NF-kB and evaluate their efficacy. The microparticulate formulation of antisense to NF-kB was characterized for its physiochemical characteristics like size, charge, antisense content and release. Since enhancing uptake into cells was the crux of the study, we also visualized and quantified uptake of microparticles by microphages. We looked at the period of lung resistance and the biodistribution of the microparticles using a near infrared bioimager. Serum levels of proinflammatory cytokines were studied after induction of lung inflammation in a rat model. Animals that were delivered antisense microparticles to the lung showed reduced levels of TNF-a and IL-1b.
    • Enhancement Technologies for Delivery of Small Molecules Into and Across Skin

      Kale, Madhura; College of Pharmacy
      Transdermal drug delivery is a non-invasive route of drug administration through the skin. It offers several benefits, such as bypassing the first-pass metabolism, avoiding fluctuations in plasma concentrations, and easy termination of therapy. Furthermore, local and systemic delivery via this non-invasive route makes it patient-friendly, and this route avoids compliance issues, especially for pediatric and geriatric populations. However, although unionized drugs, having molecular weight <500 Da and moderate lipophilicity (log P 1-3) can passively permeate through the skin, larger molecules and hydrophilic drugs cannot pass through the stratum corneum, the tightly packed lipophilic layer which acts as a barrier to drug delivery. Thus, to overcome this barrier, physical enhancement techniques are applied to achieve permeation into and across the skin. Physical enhancement techniques like iontophoresis, skin microporation using microneedles or an ablative laser, and microdermabrasion have been explored for their drug delivery potential. In this study, we evaluated the topical delivery of nordihydroguaretic acid (NDGA), a compound having the potential to attenuate arsenic toxicity. Studies showed that this compound has the potential to get retained in the skin, which is the target site of delivery. Also, skin exposed to UV radiation too showed similar delivery with that of normal skin. Thus, topical delivery of NDGA is feasible for reducing arsenic toxicity. We also evaluated the topical delivery of kinetin, an antiaging phytohormone that inhibits senescence in plants and helps increase catalase activity. We enhanced the topical delivery using two approaches: increasing the drug loading in the formulation and using microdermabrasion. Our studies showed that increasing the drug loading as well as abrading the skin led to enhancement in the topical delivery of kinetin. We also evaluated the transdermal delivery of donepezil, a cholinesterase inhibitor that is used for managing Alzheimer’s. Physical enhancement were used alone and in combination to control the delivery of donepezil. We observed that the flux profiles could be tailored successfully using a combination of skin microporation and iontophoresis, and therapeutic levels of donepezil can be delivered. In this project, we also developed an iontophoretic patch for the transdermal delivery of zanamivir. A conducting crosslinked chitosan membrane was formulated that, upon hydration, can conduct current. Prophylactic levels of zanamivir were delivered transdermally with this iontophoretic patch. Thus, we successfully delivered pharmaceutical actives across the skin using passive and physical enhancement techniques that have the potential to manage diseases and help patients lead better lives.