Therapeutic Angiogenesis: Protein and Gene Therapies offer Hope to Patients with Myocardial Ischemia Ryan McAuley Dept. of Biology Furman University Greenville, SC

Palo Alto Veterans Affairs Healthcare System Stanford University School of Medicine Department of Cardiology

Palo Alto Veterans Affairs Healthcare System

Stanford University School of Medicine Department of Cardiology

Outline Overview of Myocardial Ischemia: etiology, diagnosis, symptoms, and conventional treamtents Angiogenesis: history and development Overview of current studies Results from clinical trials Future Studies

Overview of Myocardial Ischemia: etiology, diagnosis, symptoms, and conventional treamtents

Angiogenesis: history and development

Overview of current studies

Results from clinical trials

Future Studies

Myocardial Ischemia Gr. ischein “to hold back” + haima “blood” Caused by: -Stenosis of coronary arteries -Acute blockage Coronary blood flow inadequate for maintaining cardiac function Result: cardiac muscle is deprived of essential nutrients and gas exchange Symptoms: most common is angina pectoris

Gr. ischein “to hold back” + haima “blood”

Caused by:

-Stenosis of coronary arteries

-Acute blockage

Coronary blood flow inadequate for maintaining cardiac function

Result: cardiac muscle is deprived of essential nutrients and gas exchange

Symptoms: most common is angina pectoris

Diagnosis Electrocardiogram (ECG) Exercise Tolerance Test (ETT) Myocardial Perfusion Imaging

Electrocardiogram (ECG)

Exercise Tolerance Test (ETT)

Myocardial Perfusion Imaging

Treatment Complications include: myocardial infarction, cardiac arrhythmias, CHF, and low quality of life Pharmacotherapy: combination of drugs -antiplatelet agents -antithrombotic drugs -lipid-lowering drugs -anti-anginal drugs Invasive Therapies: -CABG and PCI

Complications include: myocardial infarction, cardiac arrhythmias, CHF, and low quality of life

Pharmacotherapy: combination of drugs

-antiplatelet agents

-antithrombotic drugs

-lipid-lowering drugs

-anti-anginal drugs

Invasive Therapies:

-CABG and PCI

Limitations Symptoms not relieved by drugs Patient is not good candidate for invasive procedures

Symptoms not relieved by drugs

Patient is not good candidate for invasive procedures

Angiogenesis Offers hope to these “no-option” patients. Current clinical trials to assess safety and efficacy for FDA approval Definition: extension of already formed primitive blood vessels by budding of new capillaries through proliferation and migration of endothelial cells Takes place during embryonic development and combined with vasculogenesis, is responsible for development of the circulatory system Naturally-occurring process in adults that is prompted by hypoxia or ischemia after occlusion of an artery

Offers hope to these “no-option” patients.

Current clinical trials to assess safety and efficacy for FDA approval

Definition: extension of already formed primitive blood vessels by budding of new capillaries through proliferation and migration of endothelial cells

Takes place during embryonic development and combined with vasculogenesis, is responsible for development of the circulatory system

Naturally-occurring process in adults that is prompted by hypoxia or ischemia after occlusion of an artery

Meet the Growth Factors Fibroblast Growth Factor (FGF) -Peptide Family -Cross-species homology -Targets: endothelial cells, smooth muscle cells, fibroblasts, myocytes, and some tumor cells Vascular Endothelial Growth Factor (VEGF) -Glycoprotein Family -Targets endothelial cells exclusively

Fibroblast Growth Factor (FGF)

-Peptide Family

-Cross-species homology

-Targets: endothelial cells, smooth muscle cells, fibroblasts, myocytes, and some tumor cells

Vascular Endothelial Growth Factor (VEGF)

-Glycoprotein Family

-Targets endothelial cells exclusively

The Process Hypoxic conditions VEGF upregulation occurs within 6 hours due to: -stabilization of mRNA coding -increased transcription due to activation of Hypoxia-Inducible Factor-1 (HIF-1) in the promoter region of VEGF If this is a naturally-occurring process, why do pts. still have disabling chest pain??

Hypoxic conditions

VEGF upregulation occurs within 6 hours due to:

-stabilization of mRNA coding

-increased transcription due to activation of Hypoxia-Inducible Factor-1 (HIF-1) in the promoter region of VEGF

If this is a naturally-occurring process, why do pts. still have disabling chest pain??

The Problem Animal studies have shown impaired angiogenesis and reduced endothelial cell viability in older animals Decreased angiogenic activity also noted in diabetic and hypercholesterolemic mice Since many patients. with myocardial ischemia have other health problems such as these, angiogenesis does not sufficiently improve coronary blood flow Don’t be sad…

Animal studies have shown impaired angiogenesis and reduced endothelial cell viability in older animals

Decreased angiogenic activity also noted in diabetic and hypercholesterolemic mice

Since many patients. with myocardial ischemia have other health problems such as these, angiogenesis does not sufficiently improve coronary blood flow

Don’t be sad…

BE GLAD!!! In all of these cases VEGF supplementation produced favorable results with regards to and Endothelial Cell Response Growth

In all of these cases VEGF supplementation produced favorable results with regards to

and

Early Work Discovered by Folkman in early 1970’s Link between vascular GF’s and neovascularization associated with tumor growth In 1983, Kumar et al. studied the presence of an “angiogenesis factor” in the human heart following MI Mid-1980’s: several polypeptide growth factors associated with angiogenesis identified and purified As a result, animal and human studies could be expanded Pre-clinical animal studies used an ameroid constrictor to gradually occlude one of the coronary arteries. Pigs, dogs, and rabbits have been used for models of therapeutic angiogenesis

Discovered by Folkman in early 1970’s

Link between vascular GF’s and neovascularization associated with tumor growth

In 1983, Kumar et al. studied the presence of an “angiogenesis factor” in the human heart following MI

Mid-1980’s: several polypeptide growth factors associated with angiogenesis identified and purified

As a result, animal and human studies could be expanded

Pre-clinical animal studies used an ameroid constrictor to gradually occlude one of the coronary arteries.

Pigs, dogs, and rabbits have been used for models of therapeutic angiogenesis

Animal Studies VEGF and FGF administered in various amounts and by different routes Effectiveness measured by many means including: -size and number of vessels -measurement of coronary blood flow -quantitation of endothelial cell markers Positive results for protein and genes, but a few problems

VEGF and FGF administered in various amounts and by different routes

Effectiveness measured by many means including:

-size and number of vessels

-measurement of coronary blood flow

-quantitation of endothelial cell markers

Positive results for protein and genes, but a few problems

Current Studies Endpoints Gene Therapy Vs. Protein Therapy Administration Route Dosage -Placebo?

Endpoints

Gene Therapy Vs. Protein Therapy

Administration Route

Dosage

-Placebo?

Common Endpoints Change in total ETT time at baseline and after treatment Frequency of angina: # of doses of nitro per week Perfusion Imaging: MRI, angiography

Change in total ETT time at baseline and after treatment

Frequency of angina: # of doses of nitro per week

Perfusion Imaging: MRI, angiography

ETT Evaluation

ST Segment

ST Segment Depression = ST segment depression Normal Myocardial Ischemia

Data Points from ETT Time to onset of angina Time to >1mm change in ST segment Measurement of HR, BP, and ST segment depression at maximal exercise (angina pain rated as a 3 out of 4 or exhaustion) Measurement of ST segment depression at 1, 3, and 5 minutes recovery

Time to onset of angina

Time to >1mm change in ST segment

Measurement of HR, BP, and ST segment depression at maximal exercise (angina pain rated as a 3 out of 4 or exhaustion)

Measurement of ST segment depression at 1, 3, and 5 minutes recovery

Overview of Current Studies Comparisons: -Protein Therapy Vs. Gene Therapy -Administration Route -Dosage FDA approval: -Phase I to determine safety/feasibility -All subsequent phases must include placebo group to determine efficacy

Comparisons:

-Protein Therapy Vs. Gene Therapy

-Administration Route

-Dosage

FDA approval:

-Phase I to determine safety/feasibility

-All subsequent phases must include placebo group to determine efficacy

Physical Properties of GF GOAL: High exposure to coronary vessels, Low systemic exposure Protein Therapy: -recombinant form of FGF or VEGF Gene Therapy: - VEGF or FGF inserted into a viral vector -Naked DNA plasmid encoding for transcription of VEGF or FGF

GOAL: High exposure to coronary vessels, Low systemic exposure

Protein Therapy:

-recombinant form of FGF or VEGF

Gene Therapy:

- VEGF or FGF inserted into a viral vector

-Naked DNA plasmid encoding for transcription of VEGF or FGF

Exposure to GF Need for repeat dose Readministration Exposure to foreign genetic material Systemic Exposure Protein Therapy Gene Therapy Characteristic Short-lived Prolonged More likely Less likely Easier Potential risk for inflammatory response if viral vector used No Yes Short-term, Long-term, but high level but low level

Exposure to GF

Need for repeat dose

Readministration

Exposure to foreign genetic material

Systemic Exposure

Administration Routes GOAL: Least invasive procedure that allows for Optimal uptake of GF’s Many have been used Most common are Intracoronary and Intramyocardial

GOAL: Least invasive procedure that allows for Optimal uptake of GF’s

Many have been used

Most common are Intracoronary and Intramyocardial

Dosage GOAL: Dose is Large enough to be effective in coronary arteries, but Small enough that systemic exposure is not a concern Protein Therapy: µg/kg or ng/kg Gene Therapy: -number of viral particles -DNA plasmids in units of µg Escalating dose groups to determine how side effects and effectiveness are related to the amount of GF administered Placebo group shows objective comparison to treatment group and randomization removes physician bias

GOAL: Dose is Large enough to be effective in coronary arteries, but Small enough that systemic exposure is not a concern

Protein Therapy: µg/kg or ng/kg

Gene Therapy:

-number of viral particles

-DNA plasmids in units of µg

Escalating dose groups to determine how side effects and effectiveness are related to the amount of GF administered

Placebo group shows objective comparison to treatment group and randomization removes physician bias

Results of Phase I Studies In all studies, favorable results were reported Increased myocardial perfusion shown on MRI and angiography, increased ETT time compared to baseline, and decreased angina However, small sample size, lack of placebo group, and nonrandomization result in poor predictive value

In all studies, favorable results were reported

Increased myocardial perfusion shown on MRI and angiography, increased ETT time compared to baseline, and decreased angina

However, small sample size, lack of placebo group, and nonrandomization result in poor predictive value

Rosengart et al. 1999 n=21 Randomized: No Angiogenic Factor: VEGF 121 viral vector Administration Route: Intramyocardial Results: Improved angiography results, increased exercise time, decreased angina

n=21

Randomized: No

Angiogenic Factor: VEGF 121 viral vector

Administration Route: Intramyocardial

Results: Improved angiography results, increased exercise time, decreased angina

Results of Phase II Studies Not consistently significant Dramatically demonstrate normalizing effect of placebo group

Not consistently significant

Dramatically demonstrate normalizing effect of placebo group

Kleiman and Califf 2000 FIRST multicenter study n=337 total in 3:1 ratio of active agent to placebo Randomized: Yes Angiogenic Factor: FGF-2 Administration Route: Intracoronary Results: -No significant improvement in exercise time or stress nuclear perfusion imaging at 90 days - Less angina in treatment group (P=0.057) - Trend toward greater improvement in older and more symptomatic pts.

n=337 total in 3:1 ratio of active agent to placebo

Randomized: Yes

Angiogenic Factor: FGF-2

Administration Route: Intracoronary

Results:

-No significant improvement in exercise time or stress nuclear perfusion imaging at 90 days

- Less angina in treatment group (P=0.057)

- Trend toward greater improvement in older and more symptomatic pts.

What Happened? Small sample size Insensitive end-points Single-administration of GF Acute myocardial ischemia in animal models Vs. chronic myocardial ischemia in humans

Small sample size

Insensitive end-points

Single-administration of GF

Acute myocardial ischemia in animal models Vs. chronic myocardial ischemia in humans

So… While angiogenesis has great potential, more research needed Short term goal: prove efficacy in large-scale, placebo-controlled trials Determine long-term safety by addressing concerns…

While angiogenesis has great potential, more research needed

Short term goal: prove efficacy in large-scale, placebo-controlled trials

Determine long-term safety by addressing concerns…

Some Clinical Concerns Cancer Abnormal vascular growth in non-target tissues Immune consequences of using viral vectors with foreign genetic material Risks associated with local myocardial delivery Note: these concerns have not all been validated in research and the list will most likely evolve in the future

Cancer

Abnormal vascular growth in non-target tissues

Immune consequences of using viral vectors with foreign genetic material

Risks associated with local myocardial delivery

Note: these concerns have not all been validated in research and the list will most likely evolve in the future

The Future Variations on the theme that increased exposure to GF’s yields optimal vascularization Multiple doses and/or sustained release of recombinant proteins Administration of multiple GF’s Administration of HIF-1 Autologous bone marrow injection

Variations on the theme that increased exposure to GF’s yields optimal vascularization

Multiple doses and/or sustained release of recombinant proteins

Administration of multiple GF’s

Administration of HIF-1

Autologous bone marrow injection

THE END Special thanks to Dr. Thompson for her support and guidance through this entire project. To Victor Froelicher, MD and Jonathan Myers, PhD for allowing me the opportunity to work with them and for their help with my paper. To my uncle Paul McAuley, PhD for the “referral” to the aforementioned Docs. To Soon-to-be-Dr. Schammel for her encouragement and technological assistance. To Dr. Turgeon for her help and enthusiasm. And finally, to Dean Charles Brock, PhD for allowing Furman students the opportunity to participate in internships such as these through Furman Advantage funding.

Special thanks to Dr. Thompson for her support and guidance through this entire project.

To Victor Froelicher, MD and Jonathan Myers, PhD for allowing me the opportunity to work with them and for their help with my paper.

To my uncle Paul McAuley, PhD for the “referral” to the aforementioned Docs.

To Soon-to-be-Dr. Schammel for her encouragement and technological assistance.

To Dr. Turgeon for her help and enthusiasm.

And finally, to Dean Charles Brock, PhD for allowing Furman students the opportunity to participate in internships such as these through Furman Advantage funding.