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“Therapeutic Delivery Agents for Improved
Treatment of Cardiovascular Diseases and Stroke
David D. McPherson, M.D., FACP, FACC, FAHA
Professor and Chairman, Department of Internal Medicine
Director, Division of Cardiology
Executive Director, Center for Clinical and Translational Sciences
University of Texas Health Science Center
Medical Director, Heart and Vascular Institute
Memorial Hermann Hospital
Houston, Texas
Investigators
• University of Texas-Houston
– ST Laing, YJ Geng, A Hamilton, P Kee, H Kim, SL Huang, M Klegerman, Yuejiao Zou,
DD McPherson, T Peng, M Moody
• University of Cincinnati
– CK Holland, K Haworth, K Bader, F Himanshu, T Abruzzo
• Northwestern University
– RC MacDonald
• NIH Funding
– R01 HL059586 NHLBI
“Targeted Liposomes for Acoustic Cardiovascular Imaging”
– R01 HL074002 NHLBI
“Echogenic Liposomes for Transfection/Drug Delivery”
– R01 NS047603 NINDS
“Ultrasound-Assisted Thrombolysis for Stroke Therapy”
– R21 NS067454 NINDS
“Novel Strategy Development for Neuroprotective Therapeutics Delivery in Stroke”
Conlicts – Zymo Pharma
Although the development of therapeutics (drugs,
genes, bioactive gases, stem cells) has rapidly
progressed, methods to deliver them to their
target sites has been slow.
Therapeutics often have to be given at near or
toxic doses or in massive numbers for efficacy to
occur. (i.e., chemotherapeutic drugs, stem cells).
Therapeutic Delivery is an Area of
Active Investigation
(Drug and Gene Delivery Section of NIH)
(Nanomedicine Study Section of NIH )
How Do You make a Delivery Agent?
1. Carrier – nanoparticles – generally lipid or protein
based but can be polymer or metal.
2. Delivery strategy
IV/IM/Transcutaneous
Inhalation
GI transit (least effective)
3. Targeting Strategy
4. Release and Effect Strategy
Carriers
•
•
•
•
Microbubbles
Drugs
Metals
Lipid/Protein based vesicles
Delivery
• Upregulate tissue
• Increase vascular delivery
(heating, permeability)
• Develop specialized delivery devices
(inhalation, vascular jets, scaffolds placed next to tissue of interest)
Targeting Strategy
• Upregulate antigens on pathologic tissue
• Target pathologic tissue
• Use therapeutics as a targeting moiety as well as a
therapeutic
Release and Effect Strategy
• Sonoporation (ultrasound)
• Electroporation (electricity)
• HIFU (high intensity focused ultrasound-heating and
sonoporation)
• Electromagnetic Fields
(or enzymatic cleavage of chemical bonds)
Chemical
Examples
1. Therapeutics attached to iron particles –
electromagnets take them to site of interest. EM field
turned off and therapeutic enters cell.
2. HIFU – increases vascular permeability over site of
interest and therapeutic localized to site of action
(pancreatic cancer).
3. Electrical gaskets over region of interest that causes
electroporation delivery. (Stem cell delivery to scarred
myocardium in cardiomyopathy).
4. Develop agents that have cavitation ability with
ultrasound. (Sonoporation with ultrasound contrast
agents)
WHAT IS CAVITATION?
“Nucleation and Pulsation of Bubbles”
Cavitation
Is being intensively investigated in fields of
contrast echo, as well as therapeutic delivery.
Cavitation
• Acoustic cavitation A low energy density
acoustic wave produces a much higher energy
density local effect
• Microcavitation Cavitation at the micron scale
What Happens?
With an acoustic wave the ultrasound causes the bubble
to expand (at the same outside temperature) and then
quickly collapse (with no time to change the outside
temperature). This rapid expansion/contraction causes
inertial cavitation- downward motion by inertia of fluid
around the bubble.
Our group has developed a series of liposomes which
by formulation and methodology retain echogenic
characteristics without the addition of extra air/gas.
They have conjugation abilities and can entrap and
release pharmaceutics and genes.
“Acoustically Reflective Liposomes and Methods
to Make and Use the Same.”
Patents 5,612,057 and 5,858,399
Ultrasound can be used to deliver therapeutics into
cells and also release the therapeutic without cell
death
LIPOSOMES HAVE THE ABILITY TO ENTRAP
DRUGS, GENES AND BIOACTIVE GASES
Acoustic liposomes have the ability to act as
cavitation agents to enhance therapeutic delivery
Ultrasound-mediated delivery to vascular
smooth muscle cells in vivo using
echogenic liposomes
Laing ST, Kim H, Parikh D, Huang SL, Klegerman ME,
McPherson DD. J. Liposome Res. 2010; 2: 160-167
No Ultrasound
With Ultrasound
Effect of Ultrasound on Calcein Delivery
140
*
Mean Gray Scale Value
120
N=8 pairs; x ± sd
*p<0.05 vs. No ultrasound
*
100
80
*
60
40
20
0
Intima
Media
Adventitia
GENE DELIVERY
Encapsulation and Delivery
requires different methodologies
(carriers, RNA-vector delivery, etc.)
Morphometric analysis of the carotid arteries 14 days after balloon
injury with or without sGC plasmid DNA / ultrasound treatment.
Intimal
area
Injury
n= 8
ELIP
n=6
Plasmid
DNA 1
Plasmid
DNA 2
SD
I/M
SD
0.43
0.09
0.96
0.39
0.45
0.09
1.06
0.34
0.27
0.42
0.21
0.38
1.2
1
Injury control
0.8
0.6
0.4
0.2
0
Plasmid DNA treated
1
2
Injury
Control
Liposome
only
3
Plasmid
DNA
Rabbit #1
4
Plasmid
DNA
Rabbit #2
Echogenic Liposomes for
Vasoactive Gas Delivery and
Inhibition of Intimal Hyperplasia in
Atheroproliferative Disease
Huang SL, et al. Nitric Oxide Loaded Echogenic Liposomes for
Nitric Oxide Delivery and Inhibition of Intimal Hyperplasia. J
Am Coll Cardiol. 2009(54):652-659.
Echogenic Liposomes for
Bioactive Gas Delivery
(Nitric Oxide, Argon, Xenon)
Bioactive gas delivery is presently limited due to inability
to deliver the area of interest and retain gas effect. ELIP
may provide the solution.
Enhanced Atheroma Penetration
•Nitric Oxide (NO) is a vasodilator
•Noble Gases are relatively inert, but can
stabilize reactive gases such as NO
•Noble Gases (helium, neon, argon, krypton,
xenon and radon)
•Liposomes have the ability to encapsulate
and release these gases
“Gas Containing Liposomes”
Patent Issued
U.S. Patent office
07/12/11
#7,976,743
NITRIC OXIDE
Echogenic liposomes that contain air can also
act as a vehicle to carry the bioactive gas to
a region of interest.
Results: NO encapsulation and release
The amount of released nitric oxide can be controlled by mixing
nitric oxide with other gases. Mean +/- SD, n=3
Relationship between the mangitude of pressure application and
The amount of released nitric oxide can be controlled by mixing
Total 0.5 ml
liposomes.
encapsulated
oxidegases.
of nitric
the amount
nitric oxide
with other
Mean +/-inSD,
n=3
solution. Lipid concentration: 10mg/ml. Mean+/-SD; n=4.
20% Nitric oxide gas + 80% Argon
Pure Nitric Oxide gas
501600
1400
Nitrite concentration
Gas encapsulation (l)
Nitrite concentration
1400
40
1200
1000
30
800
20 600
400
10
1200
1000
800
600
400
200
0
0
20% Nitric oxide gas + 80% Argon
Pure Nitric Oxide gas
1600
200
0
10
9 atm
Without
liposones
20
1atm
30
3 atm
Time
(min)
40
6 atm
50
9 atm
60
0
0
With liposomes
10
20
30
Time (min)
40
50
60
Nitric oxide delivery into cultured cells by liposomes
Nitric Oxide saturated solution Liposomes containing Nitric Oxide
in the absence of hemoglobin in the absence of hemoglobin
Nitric Oxide saturated solution
in the presence of hemoglobin
Liposomes containing Nitric Oxide
in the presence of hemoglobin
Huang, SL et al. Echogenic Liposomes for Nitric Oxide Delivery. Circulation. 2006, 114(18):II-436.
Bioactive Gas/Drug Co-encapsulation and
Release Improve Attenuation of Intimal
Hyperplasia
Following Acute Arterial Injury
Moody M, Huang S, Kim H, Chrzanowski S,
McPherson DD. Circulation 2008; 118:S573
These Agents Can Be Utilized for other
Vascular Diseases (Stroke)
In Vivo Therapeutic Gas Delivery for Neuroprotection With
Echogenic Liposomes. Britton G, Kim H, Kee P, Aronowski J, Holland CK, McPherson
D, Huang SL. Circulation, Oct 19, 2010; 122(16):1578-1587.
What is the treatment for ischemic stroke?
Thrombolytic stroke treatment includes tPA. rtPA should be
administered to eligible patients who can be treated in the
time period of 4.5 hours after stroke
However, TPA doesn't’t work well enough
•Only 1 of 3 patients improve, and less than half end up without
significant disability
•Only 20% of large clots lyse completely within 2 hours
SO, We need better treatment in association with tPA .
Thus, Most Strokes Cannot be
Completely Treated
If a neurologic (nerve cell) preservation agent
can be given in the field at the time a stroke
victim is found, this could stabilize neurons and
increase the window of treatment-potentially 23 times longer, allowing many many more
patients to undergo stroke treatment
Technologies and Patents
1.
Acoustically Reflective Liposomes for Targeted Molecular Imaging
•
# 5,612,057
•
#5,858,399
2.
3.
Gas Containing Liposomes
•
7/12/2011
•
#7,926,743
Neuroprotective Liposomes for Treatment of Stroke
• US Provisional
• Filed August 2012
• Nationalized (Australia, Canada, China, Europe, Hong Kong, Japan, United States)
4.
Neuroprotective Liposomes for Treatment of Hemorrhagic Stroke
• Chinese Provisional and Others
• Filed August 2012
5.
6.
Multifunctional Echogenic immunoliposomes for Directed Stem Cell Delivery to
atheroma United States Provisional
Other Bioactive Gas Patents for Cardiovascular Diseases (filed 2015)
Xenon has Neuroprotective effects
with minor side effects
Examples of Xenon Entrapment
Thrombotic Stroke
Xe-ELIP Stabilizes Neurons for Late tPA Administration
p< 0.001
20
15
10
p = 0.032
5
0
Stroke without treatment
Stroke + IV tPA
Stroke + Xe-ELIP + IV tPA
p=0.01
p< 0.001
Sham
Control
Stroke without
treatment
Stroke
+ IV tPA
Stroke
+ Xe-ELIP
+ IV tPA
Tunel positive cells / mm2
Normalized Infarct Volume (%)
25
Sham Control
700
600
500
Mean ± SEM ,
n=6/group
p=0.017
p=0.025
400
300
200
100
0
Sham
Control
Stroke without
treatment
Stroke
+ IV tPA
Stroke
+ Xe-ELIP
+ IV tPA
Xe-ELIP reduces tPA Induced Hemorrhage
Hemorrhage rate (%)
80
67%
60
40
27%
20
0
Stroke with tPA only
Stroke with tPA + Xe-ELIP
Stroke with Stroke with XetPA only
ELIP + tPA
Green = MMP9
Red = a-SM actin (microvessel)
Blue = nuclei
50 um
Stroke with tPA only
50 um
Stroke with tPA + Xe-ELIP
A potential mechanism of Xe-ELIP inhibition of tPA induced hemorrhage is by decreasing
MMP9 expression and improving integrity of the microvessels.
Neurologic Function
N=7 per group,
Mean  SEM
n = 7 per group,
*P<0.05
Mean  vs.
SEMStroke
+P<0.05 vs. tPA
*P<0.05 vs. Stroke
+P<0.05 vs. tPA
+
+
*
+
+
*
+
Co-Encapsulation of Hydrogen
or Hydrogen Sulfide into XE-ELIP in Ischemic Stroke
• Hydrogen Sulfide (H2S) is a neuroprotective compound with
anti-inflammatory and anti-apoptotic effects.
• Hydrogen (H2) can reduce infarct volume and cerebral
edema through its antioxidant effects.
Neuroprotective Effect of Xe/H2S or Xe/H2S ELIP on Ischemic Stroke
Xe/H2Ｓ ELIP
Summary of Infarct Volume
Mean ±SEM, n=5 per group
* p < 0.05 VS stroke control
Xe/H2 ELIP的脑保护作用
Stroke
control
XeELIP/US
H2/XeELIP/US
H2S/XeELIP/US
• H2S/Xe-ELIP reduced infarct volume.
Stroke
control
XeELIP/US
H2/XeELIP/US
H2S/XeELIP/US
Multifunctional Stem Cells
Stem Cell Delivery
Although stem cells have the potential for therapeutic
repair/regeneration, very few delivered stem cells
actually get to the target site for effect. Of 1 million
stem cells injected intravenously, about 2% get to the
site of action.
Targeting with ultrasound facilitated delivery has the
potential to focus stem cell effect.
M Klegerman Lab/YJ Geng Lab
(See Poster)
Delivery of Stem Cells to Vulnerable
Atheroma
• Use of bifunctional ELIP targeting both ICAM-1 (EC)
and CD34 (VSC)
• Permutations:
– NO delivery to increase vascular permeability
– Tetracycline delivery to activate therapeutic genes
transfected into stem cells
– Ultrasound-induced delivery
Therapeutic Protocol
Stem cell-ELIP delivery reduces atherosclerosis
We have developed novel carriers for therapeutic delivery
to better evaluate stabilize and importantly treat
cardiovascular disease and stroke
Many of these agents are in the final stages of development
prior to Phase I and Phase II clinical trials