Intranasal Delivery of Proteins Intranasal Delivery of Proteins Using Cationic Liposomes for the Treatment Using Cationic Liposomes for the Treatment

of Parkinson’s Disease and the Use of of Parkinson’s Disease and the Use of

BioquantBioquant®® Image Analysis Software Image Analysis Software

Presented by Mattia M. MigliorePresented by Mattia M. Migliore

April 20, 2007April 20, 2007

Introduction:Introduction: Parkinson’s disease (PD) is a Parkinson’s disease (PD) is a

progressive neurodegenerative progressive neurodegenerative disease, which interferes with disease, which interferes with normal motor function, and normal motor function, and eventually results in akinesia and eventually results in akinesia and death.death.

Results from the destruction of Results from the destruction of dopaminergic neurons of the A9 dopaminergic neurons of the A9 nigrostriatal pathway.nigrostriatal pathway.

Affects approximately 1.5 Affects approximately 1.5 million people in the US alone.million people in the US alone.

PD has no cure and current PD has no cure and current treatments only provide treatments only provide temporary symptomatic relief.temporary symptomatic relief.

Introduction (cont.):Introduction (cont.):

GDNF is a protein with therapeutic potential for GDNF is a protein with therapeutic potential for PD because it exerts neurotrophic and PD because it exerts neurotrophic and neuroregenerative effects of dopamine neurons.neuroregenerative effects of dopamine neurons.

GDNF levels are decreased by as much as GDNF levels are decreased by as much as 19.4% per SN neuron in PD patients 19.4% per SN neuron in PD patients (Chauhan et al., 2001; (Chauhan et al., 2001; Hurelbrink and Barker, 2004).Hurelbrink and Barker, 2004).

GDNF does not cross the blood-brain barrier GDNF does not cross the blood-brain barrier (BBB).(BBB).

GDNF administration requires invasive GDNF administration requires invasive intracerebral infusions to reach its site of action.intracerebral infusions to reach its site of action.

Introduction (cont.):Introduction (cont.):

The goal of this project is to develop a cationic The goal of this project is to develop a cationic liposomal drug delivery system to transport liposomal drug delivery system to transport GDNF to the brain using the intranasal route GDNF to the brain using the intranasal route of administration.of administration.

The intranasal route of administration was The intranasal route of administration was chosen because it is non-invasive, and it chosen because it is non-invasive, and it bypasses the BBB.bypasses the BBB.

Specific AIMS:Specific AIMS:

Specific AIM 1Specific AIM 1: To characterize and optimize a : To characterize and optimize a nanoparticle formulation for intranasal GDNF.nanoparticle formulation for intranasal GDNF.

Using first a model protein to optimize our cationic Using first a model protein to optimize our cationic liposomal formulation.liposomal formulation.

Specific AIM 2Specific AIM 2: : To determine brain delivery of To determine brain delivery of GDNF in rats following intranasal administration.GDNF in rats following intranasal administration.

Specific AIM 3Specific AIM 3: To determine the therapeutic : To determine the therapeutic efficacy of intranasal GDNF in a rat model of efficacy of intranasal GDNF in a rat model of Parkinson’s disease. Parkinson’s disease.

Using BioquantUsing Bioquant®® to Quantitate to Quantitate Protein Brain Delivery:Protein Brain Delivery:

Fluorescently tagged Fluorescently tagged ovalbumin was ovalbumin was intranasally administered intranasally administered to rats.to rats.

The fluorescent label, The fluorescent label, Alexa-488 was seen Alexa-488 was seen intracellularly in coronal intracellularly in coronal brain sections.brain sections.

Quantification was Quantification was accomplished by accomplished by thresholding the cells thresholding the cells that take up the protein that take up the protein and performing a pixel and performing a pixel count.count.

GDNF GDNF immunohistochemistry immunohistochemistry was performed w/ a was performed w/ a fluorescently tagged fluorescently tagged TxR secondary TxR secondary antibody following the antibody following the same procedure.same procedure.

Using Bioquant® to MapProtein Distribution in the

Brain

Olfactory Bulb

Using Bioquant® to determineco-localization of the administeredprotein with a dopamine neuronal

marker, tyrosine hydroxylase

Conditional FrequencyAnalysis

Using BioquantUsing Bioquant® ® to Determine If GDNF Can to Determine If GDNF Can Effectively Protect Against a 6-Hydroxydopamine Effectively Protect Against a 6-Hydroxydopamine

Lesion:Lesion: 6-Hydroxydopamine will be 6-Hydroxydopamine will be

injected into the MFB to injected into the MFB to create an animal model of create an animal model of PD.PD.

BioquantBioquant® ® will be used to will be used to quantify the extent of the quantify the extent of the lesion, with a goal unilateral lesion, with a goal unilateral lesion of 50-75%.lesion of 50-75%.

Following administration of Following administration of GDNF, we will quantify the GDNF, we will quantify the lesion to look for therapeutic lesion to look for therapeutic effectiveness.effectiveness.

Conclusion:Conclusion:

BioquantBioquant® ® will be used to qualitatively and will be used to qualitatively and quantitatively analyze the data in this project.quantitatively analyze the data in this project.