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STABILIZATION OF STABILIZATION OF VULNERABLE PLAQUESVULNERABLE PLAQUES

using

Ultra Super Paramagnetic Iron Oxideand

Ferromagnetic Hyperthermia

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Vulnerable Atherosclerotic Vulnerable Atherosclerotic PlaquePlaque

Large lipid core

Thin Fibrous Cap

Macrophage Infiltration (inflammation)

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Vulnerable PlaqueVulnerable Plaque

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Factors Responsible for Factors Responsible for Plaque InstabilityPlaque Instability

Increased macrophage infiltrationSMC apoptosisCollagen degradationOxidized LDLPlaque angiogenesisInfection (CP, HSV, CMV)Systemic inflammation / infections

(influenza)

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Factors Responsible for Factors Responsible for Plaque StabilityPlaque Stability

Reduced Inflammation (Macrophage Apoptosis?)

SMC ProliferationCollagen ProductionLipid Lowering (lipid core)

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Methods of Stabilizing / Methods of Stabilizing / Treatment of PlaquesTreatment of Plaques

Lipid loweringStent PlacementAnti inflammatoryPhotodynamic therapyGene therapy

Beta blockersACE inhibitorsAntioxidantsEstrogen therapyAntibiotics

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Photodynamic TherapyPhotodynamic TherapyPDT is based on the use of light-sensitive

compounds, photo sensitizers (PS), which can be activated by light of a specific wavelength that is matched to the absorption characteristic of the particular PS. Photo activation in the presence of tissue oxygen leads to the formation of cytotoxic-reactive oxygen species such as singlet oxygen. Photo activation of PS therefore may result in the damage of cellular components, eventually leading to cell death. (Levy, 1994).

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The rationale for using PDT in atherosclerosis is based on the observation that various PS, such as porphyrins, phthalocyanines, and texaphyrins accumulate in the atherosclerotic plaque as compared with the adjacent normal vessel wall. Local intra-arterial application of light of the appropriate wavelength to the atherosclerotic vessel therefore may reduce the narrowing of the vessel.

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Substances Used As Substances Used As PhotosensitizersPhotosensitizers

porphyrins, Phthalocyanines(2.6 times more in plaques after 4 hours)

Gadolinium texaphyrinLutetium texaphyrinTetracyclinPheophorbideMono-L-aspartyl chlorin e 6

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Texaphyrins in PDTTexaphyrins in PDT( Lutetium Texaphyrins) ( Lutetium Texaphyrins)

These are aromatic and highly colored compounds related to porphyrins.

Lutetium texaphyrin is a second generation PS. It possesses a low energy maximum in the far red

portion of the visible spectrum. This provides for activation of these compounds by light

that is capable of penetration through tissue and blood. It is retained selectively in the macrophages in the

atherosclerotic plaque in spite of rapid clearance from blood.

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……ContinuedContinued

It can be used in photo angioplasty and reduction of atherosclerotic plaque.

The efficacy of Lutetium Texaphyrin has been demonstrated in animal models

It is in phase I clinical trial for coronary artery procedure and in phase II for peripheral arteries.

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Left: Pretreatment angiogram of the femoral artery of a patient Left: Pretreatment angiogram of the femoral artery of a patient showing the severe narrowing of this vessel caused by showing the severe narrowing of this vessel caused by atherosclerosis. Right: One month after a single treatment with atherosclerosis. Right: One month after a single treatment with Lu-Tex-based ANTRINLu-Tex-based ANTRIN(TM)(TM) photosensitizer-based angioplasty, photosensitizer-based angioplasty, shows a 50% increase in the diameter of the arterial opening and shows a 50% increase in the diameter of the arterial opening and increased blood flow. Reproduced with permission. increased blood flow. Reproduced with permission. 98 Pharmacyclics Inc.98 Pharmacyclics Inc.

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Biodistribution of Lutetium Biodistribution of Lutetium Texaphyrin Texaphyrin (10mg/kg)(10mg/kg)

Time Plasmag/g Muscleg/g

Tumorg/g

3 hours 5.07 0.60 5.25

5 hours 4.72 0.46 1.96

12 hours 3.91 0.41 0.48

24 hours 2.79 0.40 0.18

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Uptake of Porphyrins in Uptake of Porphyrins in arteries and plaquesarteries and plaques

Hsiang (1993) found that porphyrins are taken up by human arteries in in-vitro studies.

In in-vivo studies in miniswine these are taken up by the plaques and normal arteries in a ratio of 1.7-3.5 with a dose of 2.0mg/kg.

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Disadvantages of PDTDisadvantages of PDTCoagulation necrosis, cell cytotoxicity

Burning plaque

Skin photosensitivity and severe skin burns upon exposure to sun light

Even LT is not highly plaque specific.

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Selective Targeting of PSSelective Targeting of PSSelective targeting of PS to atherosclerotic

plaque may reduce its possible side effects, such as skin hypersensitivity to sunlight

Photo activation of cells loaded with Ox LDL-AlPc(aluminum phthalocyanine chloride) complexes resulted in selective cell death that correlated with the time of illumination. The cytotoxic effect of OxLDL-AlPc on leukemic murine macrophages is concentration- and incubation time-dependent.[Vries 1999]

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Effects of Gentle HeatingEffects of Gentle Heating

Anti-Proliferative Effect of Heat

Anti-inflammatory Effects of Heat

Other Effects of Heat

Mitra Rajabi:Mitra Rajabi:

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Anti-Proliferative Effect of Anti-Proliferative Effect of HeatHeat

Heat as well as other stressors (such as withdrawal of growth factors, lack of oxygen or glucose) can induce apoptosis, in a wide variety of cell types.

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AntiAnti--inflammatory Effects of inflammatory Effects of HeatHeat

Reduced expression of pro inflammatory cytokines: TNF-, IL-1 and IL-6 and many others cytokines that are important mediators of inflammation.

Temperatures between 40- 45 C or chemical inducers of heat shock proteins cause inhibition of both TNF alpha and IL-1 expression.

Inducing selective macrophage apoptosis.

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Effects of Heat on Effects of Heat on CollagenCollagen(cap strengthening)(cap strengthening)

Heat can increase expression of TGF- 1 and VGEF which are well known for their effects on collagen and matrix production.

Low grade heat therapy has been shown to help the process of wound healing by increasing collagen production in several studies. Heated wounds showed increased collagen fibers which were denser, thicker, better arranged and more continuous, and the mean tensile strength of them was significantly greater than control group.

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Effects of Heat on LipidEffects of Heat on Lipid

It has been shown that rise in temperature to 41ºC causes partial melting of the lipid crystals in rabbit atherosclerotic plaque

At 45ºC most of the crystals melt.

Small, Arterosclerosis 1988

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Effect of Heat on MMP ActivityEffect of Heat on MMP Activity

Heat shock at 42C preferentially suppresses the production and expression of MT1-MMP and thereby inhibits pro MMP-2 (gelatinase) activation, events which subsequently inhibits tumor invasion

Sato, 1999

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Disadvantages of Non-selective Disadvantages of Non-selective HyperthermiaHyperthermia

Partial damage of the endothelial cell membrane (at 54ºC for 5-10 minutes) [Gabbiani,1984]

Swelling of the endothelial cell nuclei (at43ºC for 12 minutes) [Emami, 1984]

Sticking of the WBC’s to the vessel walls when heated, [Emami, 1984]

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Methods of Interstitial Methods of Interstitial HyperthermiaHyperthermia

Radiofrequency MicrowaveInfrared Laser Focused Ultra SoundExtra corporeal heating of blood.Whole Body Hyperthermia

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Types of Hyperthermia Types of Hyperthermia

Invasive Catheters Ferromagnetic

Hyperthermia Surgical implantation

of NeedleThermo seed Extra corporeal

heating of blood.

Non Invasive. Inductive HT

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RF & Ferromagnetic HyperthermiaRF & Ferromagnetic Hyperthermia

Because the layers of tissues within the human body have different water content and dielectric properties, deep pattern of exogenous energy absorption is highly non-uniform. To make hyperthermia clinically useful and feasible, it is essential that the heat delivery techniques be able to provide a sufficient heat dose within the whole site of interest while leaving surrounding normal tissue unaffected.

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Use of Ferromagnetic Micro Particles Use of Ferromagnetic Micro Particles for Interstitial / Intracellular for Interstitial / Intracellular

HyperthermiaHyperthermiaHeat production by metal devices implanted

directly into the tissuesThermoseeds absorb energy from an

externally applied EM field in a non contact manner.

Ferromagnetic microparticles and colloidal magnetic particles act like thermoseeds but in submicron and intracellualr level.

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Ferromagnetic particles act like self-contained heaters for which no cable connections are needed.

They dissipate energy by various kinds of energy losses (eddy currents, hysteresis and residual loss) and cause heat on exposure to external alternating magnetic field.

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Amount of heat produced by ferromagnetic particles is a function of frequency (HZ) and power (W) of currents created by RF generator.

It varies from gentle heating (1 -2 C rise) to aggressive heating and melting tissues (50-70 C rise )

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Ex Vivo StudiesEx Vivo Studies

1989-94 Dextran Magnetite Complex was used to generate heat by exposing them to AC magnetic field.

In 1994 Mitsumori compared heat generation in DM/Lipiodal emulsion and DM/Degradable starch micro sphere emulsions.

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…….continued.continued1999-Shinkai, used agar containing

magnetite particles and heated them with MHz-RF capacitative device. It resulted in an increase in the temperature of the agar by 9ºC at 5minutes and by 24ºC at 9 minutes.

2000-Hilger, heated magnetite particles placed in cow muscle tissue with 400 kHz RF. Temperature elevation of up to 87ºC was observed at a distance of 15mm from the magnetite deposition area.

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In Vivo Studies Using Iron In Vivo Studies Using Iron Oxide ParticlesOxide Particles

1981-Gordon used Iron particles with diameter of less than 1 in rats and rabbits and exposed them to external high frequency electromagnetic field for 8- 10 minutes.

The treated animals showed in a marked decrease in the lipid accumulation and intimal hyperplasia

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Submicron colloidal carbon iron particles could be observed immediately Submicron colloidal carbon iron particles could be observed immediately under the endothelium 5 hours after the administration, in mesenteric under the endothelium 5 hours after the administration, in mesenteric

artery, in a ratartery, in a rat

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……ContinuedContinued

1994- Mitsumori injected DM containing embolic material to renal arteries of rabbits and selectively heated the embolized kidney by exposure to AC magnetic field.

The temperature of the kidney was raised 12.7º3.1 during the initial 10 minutes.

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Iron Particles in the KidneyIron Particles in the Kidney

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……continuedcontinued

1999-Shinkai, injected CMC-Magnetite (carboxy methyl complex) into the femur of pigs. 8 MHz-RF was used to heat the magnetite complex.

The temperature in the CMC-Mag complex was increased to 43ºC after 7 minutes.

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……continuedcontinued

1998 Merkle and associates, injected 6 male NZ normal rabbits with ferumoxide and applied RF field for 8 minutes.

Coagulative lesions were produced in the liver of the rabbits which were detected by MR imaging.

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Nonenhanced T1-weighted SE image (480/26 with three signals acquired) allows better visualization of the hypointense RF-induced lesion (arrows) [Merkle et al 1999-

Radiology]

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SPIO, USPIOSPIO, USPIOMagnetic resonance imaging contrast

medium with a central core of iron oxide generally coated by a polysaccharide layer

Shortening MR relaxation time

Phagocytosed and accumulated in cells with phagocytic activity

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SPIO in Macrophages

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SPIOs AND USPIOsSPIOs AND USPIOs

These biocompatible particles are injectable and nontoxic agents that have the ability to dissipate the energy of radio frequency electro magnetic field in the form of heat.

Can be useful in the site / cell specific hyperthermia treatment.

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In Vivo Hyperthermia Using In Vivo Hyperthermia Using CMIOCMIO(Colloidal Magnetic Iron Oxide)(Colloidal Magnetic Iron Oxide)

Dr Chan and associates evaluated the effect of RF hyperthermia with CMIO injected mice.

Mice were implanted with three types of human lung carcinoma cells.

Tumors were exposed to RF field at 0.85 MHz for 6-10minutes.

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……continuedcontinued

Body vs tumor temperature differential of 6.5º-8.5ºC was reached.

Dr. Chan’s studies demonstrated the ability to achieve clinically significant, site specific heating of the tumors.

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Uptake of Ferromagnetic particles by macrophages in the plaque.

Heating of the Ferromagnetic particles with low frequency magnetic field.

Increasing the temperature of the particles in the macrophages to within the febrile range (38º-43ºC)

Apoptosis of the macrophages in the plaques leading to decreasing inflammation and increasing stability of the plaque.

SPIO Heating of Vulnerable SPIO Heating of Vulnerable PlaquesPlaques

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Our preliminary findings confirmed selective accumulation of ferromagnetic particles in the atherosclerotic plaque, RES system of the liver and spleen as well as lymph nodes.

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Apo-e Mice 1 and 3 Days Apo-e Mice 1 and 3 Days After Injection of FeridexAfter Injection of Feridex

Pearl’s Staining

Day1Day3

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Hypotheses Hypotheses Radiofrequency heating of USPIO injected

animals for a short period of time (15-20 minutes at 42-43C) triggers apoptosis in macrophages containing these particles and therefore helps to stabilize the plaque by reducing inflammation.

We also seek other beneficial effects of gentle heating on collagen production as seen in wound healing process, which may further help to stabilize vulnerable plaque by cap strengthening.

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Study DesignStudy Design

Non-Invasive Using inductive RF heatingInvasive Using Intravascular cooled tip RF

catheter

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Using Intravascular catheter for applying radiofrequency

For Non-Invasively heating the plaque we need to improve the bio-distribution of SPIOs to make them more plaque specific so that hyperthermia can be induced non-invasively

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Non-invasive Arm of the Non-invasive Arm of the Study Study

1 5 w ith R F H y p e r th e rm ia 5 w ith o u t R F H y p e r th e rm ia

2 0 m ic e U S P IO in je c t io n

5 w ith R F H y p e r th e rm ia 5 w ith o u t R F H y p e r th e rm ia

1 0 w ith o u t in je c t io n T y p e t it le h e re

3 0 M ic e

All animals sacrificed & then samples will be send for Pathologic evaluation for apoptosis 

5 Days Later

12hours later

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Invasive Arm of the StudyInvasive Arm of the Study

5rabb it with R F 5rabb it withou t R F

10rabbit with U SPIO in jection

5 rabbits with R F 5rabbits withou t R F

10rabb it without in jection

20 W H H L rabb it

After 5 days

All animals sacrificed & then samples will be send for Pathologic evaluation for apoptosis

After 12 hours

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Future PlanFuture PlanTargeting of vulnerable plaque Ox-LDL

– USPIO tagged with FC receptor for MDA epitope of Ox-LDL

Adhesion Molecules-VCAM / ICAM- P-Selectin- CD39

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