The Future: Targeted Microbubbles - SD

Introduction

I am Evan Unger,
and I'm a board certified radiologist.
I'm a professor of radiology
and biomedical engineering at the University of Arizona,
and I invented the ultrasound contrast Agent Definit.

In my presentation, I'm going
to discuss the potential of developing targeted,
specific contrast agents
for molecular imaging with ultrasound.
I'm gonna describe the future for targeted microbubbles.
I'm gonna discuss the background behind this field.
Design considerations
for making targeted microbubbles some
of the potential clinical applications,
and particularly discuss angiogenesis in cancer
inflammation, the potential to combine therapeutic
and diagnostic applications into this new field
called theranostics.
And then describe the summary for this field.

Background on Ultrasound Contrast Agents

Current ultrasound contrast agents are vascular blood
pool tracers, and they provide valuable
information about blood flow.
Blood flow is an imperfect marker of disease.
Targeted microbus afford the potential to detect
and monitor molecular markers of disease.
Theranostics is this field that combines the potential
of diagnostic imaging with therapy.

Microbubbles are micron sized,
and they're too large to extravasate out of the vasculature.
If we're going to design targeted microbubbles,
we're limited to endothelial based targets
and circulating immune cells,
and potentially also labeling of stem cells.

Design Considerations for Targeted Microbubbles

Some of the considerations are the concentration
of the target on the cells, the presence of flow
and shear stress in the vasculature,
which may wash the bubbles away, the strength
of the ligand target interaction
and how quickly the microbubbles bind to the target,
referred to as the on-off rate.

Antibodies are large molecules and they're strong binders,
but they have a slow on-off rate,
which may make them difficult to bind
in conditions of high flow
where the microbubbles are flowing quickly against the
targets, the microbubbles need to be humanized.
You can use FAB, which is a fragment of the antibody,
or smaller structures such
as min bodies can be used.

Peptides are small molecules.
They have the advantages
of being relatively non immunogenic.
They have a fast on off rate,
but they tend to have relatively weak binding.
They also need to be optimized for hydrolysis
so that they are not dissolved too quickly in the bloodstream.
This can be accomplished by using de amino acids,
and changing the structure of the peptides
to make peptide mimetics.

One of the things that is in our favor in making the
targeted microbubbles is valency
and hetero valency can be used as well.
Valency refers to having many of the ligands on the surface of the bubble.
And hetero valency refers to having two
or more different kinds of ligands, which bind
to different sites on the same epitope
or to different epitopes.
And this is a way of increasing the strength of the binding.

Generally, we use a linker such as polyethylene glycol
to provide freedom of movement to the ligands to attach these to the microbubbles.
Avid and biotin has often been used in exploratory studies
to bind ligands to microbubbles.
It's a useful tool for target validation,
but it is not translatable.

This drawing shows a biotinylated antibody
that is then bound to the avadon.
And so we can attach avadon, for example, to microbubbles,
and then biotinylated antibodies or other molecules
and label the microbubbles in this manner.
Allergies will develop to avadon.
So this is not clinically translatable,
but it's a good thing to do in the lab.
We did this more than 10 years ago with microbubbles.
And this is an example.

Targeted Imaging Examples

Inflammation Imaging

This is targeted imaging
of TNF alpha induced inflammation in a mouse
using antibodies to CD 62,
which is a marker
that is upregulated on the endothelial cells
with inflammation.

This is the image of the mouse hind paw using a non-targeted agent such as definit.
And here we have the targeted tissue specific agent binding to the CD 62.
And so we can see the degree of expression,
and then we can apply a high mi pulse
and we can quantitate the degree of binding.
So this allows us with ultrasound to quantitate the degree of molecular expression.

Microbubble Design

One of the designs of microbubbles for targeted imaging is shown here.
This would be a lipid coated phospholipid coated microbubble.
There's polyethylene glycol coating some of the lipids, and then some of them are deriv
to have the targeting ligand exposed at the end.
And so this can be achieved by using a lipid anchor,
a polyethylene glycol spacer.
And then the in this case,
peptide based targeting ligand.
And the one we're using here is A-C-R-G-D analog,
which is a cyclist relatively small peptide,
which will bind to angiogenesis
and also to activated receptor on platelets.

Clinical Applications of Targeted Microbubbles

Where can we use these targeted microbus?
Well, there are many applications, inflammation such
as inflammatory bowel disease, arthritis, uveitis,
diabetic retinopathy, hepatitis and nephritis.
We can use them for white blood cell trafficking,
for stem cell therapy to follow the migration of stem cells,
potentially to label the stem cells
and then use ultrasound to direct their localization.
We can image vascular thrombosis
or vulnerable plaque,
potentially do treatments in those areas,
which I will discuss.
We can use them to follow angiogenesis in cancer and inflammation.
And in theranostics, we can do drug
and gene delivery and thrombolysis.

Angiogenesis and Cancer Imaging

There are a number of molecular targets in angiogenesis of cancer
that have been described.
These include platelet derived derived growth factor,
vascular endothelial growth factor, and others.
But one of the very important markers
that has emerged is vg.
GF and a number of different drugs are either
in development or clinically used that target various
parts of VGF.

Many different drugs are currently in clinical trials
to treat different kinds of cancer targeting angiogenesis in particular targeting vgf.
If we could image VEGF follow its expression over time,
this could have a great clinical benefit
because we might be able to use diagnostic imaging
to select the patients who would be most apt to respond
to anti-VEGF therapy.
And then over time, we could monitor the effects.

BRACO has developed
a targeted agent to vgf.
It's a phospholipid coated per fluoro
butane microbubble.
There's a phospholipid anchor,
a polyethylene glycol spec spacer,
and a hetero DME peptide.
The peptide has two different parts, each of which bind
to different epitopes on vegf.
So this is an example
of the hetero polyvalent cooperative binding
that I was discussing earlier.
And this particular agent has a very high affinity
with a KD of 0.5 nan mo per liter, which is
a very strong binding, the kind of binding
that we might expect with an antibody.

And here what we see is a tumor in a mouse pre using a non-targeted agent
and then using the targeted agent, which is binding to the VEGF.
And so this allows us to monitor the expression of vegf.
And this is shown using labels
for CD 31, a vascular endothelial marker, VEGF,
and colocalization showing expression
of VEGF in the tumor tissue.

Now, in another mouse, which has been treated with B 20,
which is an anti-VEGF agent,
what we see is downregulation of vgf.
And so when these animals are treated with the B 20,
here we have the tumor size, the tumor size stays relatively constant and doesn't grow much at all.
Whereas the animal, which is untreated, has continued growth of tumor as the VEGF is expressed.
And the investigators working
with this new agent have shown that they can monitor
VEGF expression over time.

Inflammation Imaging: Crohn's Disease

Crohn's disease, inflammatory bowel disease is another
very important target.
Currently we do far too much CT scanning in these often
young patients and expose them to radiation.
One of the epitopes
that has been described in Crohn's disease is mad cam one.
And this is work by investigators that studied mad cam one in an experimental model
of a Crohn's disease.
They made microbubbles with an antibody to MadCAM one,
wherein the avadon technique was used to couple
the antibody to the microbubbles,
and they showed that the microbubbles bound
to the endothelial cells in the NC two video microscopy,
they showed the expression
of the mad cam one in the tissue specimens in the
animals with disease.

And here we see the ex vivo specimen
with thickening of the terminal ileum in this model
of Crohn's disease and an inflamed mesenteric lymph node.
On the contrast enhanced images,
we can see the enhancement of the terminal ileum
and the mesenteric lymph node.

Here what we have is a normal animal using a nonspecific agent,
the targeted agent showing no specific binding,
the tissue showing a normal appearing epithelium of the terminal ileum.
Here is the non-specific agent.
Here is the targeted agent in the mesenteric lymph node showing intense enhancement.
And here we see the inflamed thickened epithelium in the terminal ileum.
And the investigators showed that they were able
to monitor the degree of inflammation over time.
And here we see the animal with disease.
This is the video intensity from the
targeted agent, the non-targeted agent compared
to the normal mice with the targeted
and non-targeted agent.
And they showed that the degree of enhancement
of video intensity with the targeted agent had a great,
high correlation with the total inflammatory score.

Theranostics: Combining Imaging and Therapy

Theranostics is this new field that combines imaging
and therapy, and there are a number
of potential applications.
And there are a couple of important mechanisms underlying this potential.
Microbubbles lower the threshold
of energy for cavitation.
This can be used for cell permeability, for example,
to open the blood-brain barrier drug and gene delivery.
And for sono thrombolysis radiation force is the pushing
force of the ultrasound, which can improve penetration
of materials into cells.
There's a potential for targeted microbubbles
to be used in theranostics for site cell specific therapy.

Now, there are some practical considerations,
however, microbubbles have only a limiting care
and capacity for drugs.
So this can be used for highly active drugs,
such as genes or sir NA, which are very active medications
and require only a small dose.
We can do co-administration of drugs with microbubbles,
where we might use a less active drug.
And then we use the microbubbles with ultrasound,
for example, to increase the vascular permeability,
for example, to open the blood brain barrier.
This might be a strategy to coadminister the drugs.

Microbubbles also are highly efficient carriers
of oxygen and other gases.
And we're working on this in our lab. Now.
This shows flowing microbubbles in blood,
and there is a plate that is biotinylated,
and now the ultrasound is turned on,
and the microbubbles are beginning to bind
to the plate, do the Aden biotin interaction.
And so what this is showing in flowing blood is
that the combination of the ultrasound,
the radiation force of the ultrasound increases the binding
of the microbubbles to a target.
And this same thing can be shown in vivo,
as I will show you in the subsequent side slides.

Drug and Gene Delivery with Nano Droplets

Now, we had a grant from the National Cancer Institute,
and we made acoustically active nano droplets.
They had a layer of fluorocarbon an oil layer with drug,
and had polyethylene glycol on the surface
of the lipids and a targeting ligand in this case to an integrin that is upregulated in prostate cancer
that our co-investigators discovered.
And we used a combinatorial library to identify peptides
and then medicinal chemistry to make appropriate peptides to bind to that target.

And so what we're going to see here is
prostate cancer cells expressing that rin after they have been incubated with the nano droplets
and ultrasound has been applied.
And so here, what we see on these tomographic optical images is the fluorescent
nano droplets containing the paclitaxel
that has been delivered into the cells.
And we did not see that unless ultrasound was applied.
When things bind to this particular rine, they'll stick
to the surface of the cells,
but will not be internalized
until the ultrasound is applied.
And so on these fluorescence tomographic images,
what we see are nano droplets internalized into the cytosol of the cells,
prostate cancer cells due to the ligament interaction,
but then the radiation force of ultrasound pushing the nano droplets into the cells.

And in vivo, we saw the same phenomenon.
This again, is using the fluorescently labeled
paclitaxel in the acoustically active lipo spheres
with the ligand to the integrin, upregulated
and prostate cancer.
And here we have the xenografts of the tumors in the flanks
of the animals on each side.
And so here, we see the scale
red being the highest uptake.
And this is the side which had the ultrasound exposed,
and we have very high level of uptake in the tumor corresponding to the region that was exposed
to the ultrasound.
And we also showed a tumor growth delay
that was significant in the animals that were treated
with this technique.

Sono Thrombolysis

Now, this is a movie showing cavitation
of a lipid shelled microbubble in response
to a single acoustic pulse of ultrasound
with ultra high speed video microscopy.
And so here the microbubble expands, collapses,
the daughter fragments undergo one more expansion cycle,
and then if we had the next frame, it would show that they disappear.
So when this occurs, this creates a local shockwave
that is probably on the order of some number of microns
and might be used to increase cell permeability
or potentially for other therapeutic processes such
as sono thrombolysis.

And this cartoon is meant to show an occlusive thrombus in an artery
and a distal to this region.
There will be a relative absence of blood flow.
And so when we give an intravenous dose of the microbubbles,
they are smaller than red blood cells
and will permeate the blood clot.
And then when ultrasound is applied,
these microbubbles will be cavitated,
and this can then be used to restore the blood flow.

I helped introduce this into stroke,
which is a promising area,
and merits further research and,
and people are continually work in that field.
And I've been fortunate to have a longstanding collaboration with Dr. Thomas Porter
and his colleagues at the University of Nebraska.
And we've been looking at ultrasound to treat acute myocardial infarction.
And Dr. Porter has a thrombotic occlusion
of the left anterior descending coronary artery model in pigs.
And he's moved on to treat hyperlipidemic pegs,
wherein they have a plaque in the LID
and they have an occlusive thrombus
and are administered in IV infusion of microbubbles.
And then ultrasound is applied to the at-risk area.
The animals are studied with EKG angiography,
wall motion, radio labeled microspheres
to determine blood flow
and postmortem measurements of mi.

And here is an angiogram showing an occlusion
of the left anterior descending coronary artery.
After successful sono thrombolysis giving an intravenous infusion
of the microbubbles
and transthoracic application of ultrasound,
there has been restoration
of blood flow in the left anterior descending coronary
artery, and myocardial contrast enhanced echo.
During the infarction, you see thinning
of the LV wall and absence of perfusion.
And then after successful sono thrombolysis,
we see restoration of myocardial thickening, restoration
of wall motion, and myocardial blood flow is restored.

And rather complicated slide here,
but here are the animals treated
with sono thrombolysis.
With microbubbles. Here are the control animals only given standard lytic therapy.
And we see that in all six of the animals there is complete angiographic restoration
of blood flow,
and only in 50% of the animals treated with conventional therapy.
So this shows a non-targeted microbubble making a very great difference in
restoring myocardial blood flow,
and restoring patency
of the left anterior descending coronary artery.

Now in the future, we would hope that we could introduce targeted microbubbles into this paradigm.
And these are images of human blood clot with targeted microbubbles
and control microbubbles.
And notice that the targeted microbubbles,
which are designed to bind to the clot, have a peptide
that is specific for clot,
have exuberant binding to the clot
and very little binding at all
of the control microbubbles to the clot.
And when we look at this by counting the amount
of bubbles, two different kinds
of targeted microbubbles here, one binds to platelets,
the other binds to fibrin,
and we see significant increase in binding
for either compared to the control microbubbles
and the fibrin microbubbles appear to outperform.

And when we do sono thrombolysis in vitro, we do see
enhanced lysis of clot with the targeted bubbles
and the preliminary in vivo studies
that have been performed support this hypothesis
that the targeted microbubbles are going
to be more effective.
They also may allow us to identify the clot
and target the ultrasound more precisely.

Summary

In summary, clinically translatable targeted microbials are feasible.
And the VEGF agent,
which I described is currently under clinical development.
There's potential
to provide valuable clinical information in a variety
of conditions, and including angiogenesis
and cancer, inflammation, vascular thrombosis,
and vulnerable plaque.
There's this new field of theranostics,
which may allow us
to combine diagnostic imaging with therapy.
Successful clinical development of targeted microbus
and theranostics will depend upon the clinical need
and the market potential.
Thank you very much for your attention.