Future Targets

Trinity J. Bivalacqua MD PhDJohns Hopkins Hospital

The James Buchanan Brady Urological InstituteBaltimore, MD USA

Pathways activating cGMP

cGMP

GMP

sGuanylatecyclase

NO

Relaxation

Smooth Muscle

CO

HO‐1

Membrane

NEP

Opiorphin

xCNP

pGuanylatecyclase

Natriuretic peptides

Arginase/ NOS Pathway

cAMP signaling pathways

ATP cAMP

Ca2+PKA

Relaxation

Adenylatecyclase

PGE1R

Alprostadil

A2br

Adenosine

AdenosineAdenosineDeaminase(ADA)

S‐adenosyl‐homocysteineHydrolase (SAHH)

Inosine S‐adenosyl‐homocysteine

AMP

ADP

ATP

ADK

CD39CD73

ATPAdenosine ADP

Adenosine

Adenosine is generated both intra‐ and extracellularly. 

PDE5

Hif1

Endothelial Cell

Smooth Muscle Cell

K+

K-ATP-Channels

cystathionineβ‐synthase (CBS) and CSE

Nerve

H2S

CSE

L‐cysteineL‐cysteine

Cystathionine γ‐lyase (CSE)

L‐cysteine

Ca2+

cAMP/cGMP?

H2S involvement in erectile physiology  

Vasoconstrictive Pathways

MLC

KC

a2+-C

aM SMM

P

RELAXATIONMyosinLC20

MyosinLC20.PCONTRACTION

ROCK

RhoA‐GTP

Endothelin‐1NorepinepherineSerotoninAngiotensin

DAGPLC

PKC

CPI‐17

Future Targets

• Rho‐kinase• Arginase• Adenosine• sGC activators• Stem Cells/Tissue engineering

Future Targets

• Rho‐kinase *• Arginase• Adenosine• sGC activators

• Stem Cells/Tissue engineering *

* Most promising targets at this time.

MLC PHOSPHATASE(ACTIVE)

MLC PHOSPHATASE~P(INACTIVE)

RHO-KINASE

RHOA~GDP(INACTIVE)

RHOA~GTP(ACTIVE)

++ATP

HTN, DM, Hypoxia, NE, ET-1STIMULATE

MLC ~P(CONTRACTION)

MLC(RELAXATION)

Fibrosis/Apoptosis

MLC-KINASE

BINDS GTP

MIGRATES TO MEMBRANE

MODIFICATIONS

PHOSPHATASEPHOSPHATASE (?)

eNOS ProteineNOS mRNA stability

Activation of RhoA / Rho‐Kinase Inhibits Corporal Relaxation

MLC PHOSPHATASE(ACTIVE)

MLC PHOSPHATASE~P(INACTIVE)

RHO-KINASE

RHOA~GDP(INACTIVE)

RHOA~GTP(ACTIVE)

NO/cGMP/PKGINHIBIT

MLC ~P(CONTRACTION)

MLC(RELAXATION)

ERECTION)

MLC-KINASE

PHOSPHATASEPHOSPHATASE (?)

eNOS ProteineNOS mRNA stability

Inhibition of RhoA / Rho‐Kinase Enables Corporal Relaxation

Y-27632(30 nmol)

160

140

120

100

80

60

40

20

0

Pres

sure

(mm

Hg)

ICP

SAP

Time (min)0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Effect of Intracavernous Injection of the Rho-kinase Inhibitor, Y-27632

Rho‐KINASE

Human CC

Sinusoid

DAPI

ROCK1

αSMA

Diabetes 40%

Vascular Disease30%

Spinal Cord Injury8%

Radical Surgery13%

Endocrine Disorders6%

MultipleSclerosis3%

Zonszein, Urol Clin North Am, 1995.

Common Systemic Diseases Associated with ED

Rho-Kinase Inhibition Reduces Apoptosis and Preserves SMC content in Diabetic Rats

WJ Li et al., JSM 2011

Rho-Kinase Inhibition Reduces Intimal Thicking in Internal Iliac Artery

K Park, et al. JSM 2006

Control Atherosclerosis

Fasudil

K Park, et al. JSM 2006

Rho-Kinase Inhibition Improves Endothelial NOS and Prevents ED

Peripheral Nerve Injuries

Cell Body

Basement Membrane

Myelin Sheath

Axon

Normal Compressed/Stretch

Sheath Loss Disconnection Degeneration

Strategies to promote axon regeneration

3. Neutralization of the inhibitory factors in the injured PNS A therapeutic vaccine approach Anti-scarring treatment (inhibition of fibroblast

proliferation) Anti-inflammatory / Antioxidants Immune modulators Anti-apoptotic agents Axonal outgrowth inhibitory neutralizers

1. Stimulation of axon regeneration by modulating the neuronalsignaling responses:

- Treatment with neurotrophic factors (NGF, BDNF, NT-3,GDNF, LIF, FGF-2)

2. Cell transplantation: e.g. embryonic stem cells, adipose-muscle-, bone marrow derived -mesenchymal stem cells.

• RhoA mediates growth inhibitor signals which stiffens the actincytoskeleton, thereby inhibiting axonal elongation.

• RhoA is a key inhibitory regulator of axonal regeneration in the CNS, however, little is known in the PNS.

Bilateral Cavernous Nerve Injury (BCNI)

Right CN

Major PelvicGanglion (MPG)

Major PelvicGanglion (MPG)

Cavernous Nerve Injury with forceps 15 sec x 3

http://www.pelvipharm.com

Left CN

Cellular responses to peripheral nerve injury

Major Pelvic

Ganglion (MPG)

Penis

Erectile Dysfunction

0.0 2.0 4.0 6.00.0

0.2

0.4

0.6

0.8

1.0

ShamBCNI 48hBCNI 7d

BCNI 14dBCNI 30dBCNI 60d

Volts

****

*

***** **

***

ICP/

MA

P

Major Pelvic

Ganglion (MPG)

PenisInflammatory

Cytokines upregulate

RhoA/Rho-kinase

↑ Apoptosis(via caspase)

↓ neuronal nitric oxide synthase

WallerianDegeneration

Erectile Dysfunction

Hypothesis

BCNI significantly elevated RhoA/Rho‐kinase

Y-27632 → Rho-kinase (ROCK) inhibitor

5 mg/kg i.p. BID (dose selective for ROCK isoforms)

Effect of ROCK Inhibition on the MPG?

Day 0: Sham/BCNI Y-27632

Day 14: ICP, tissue collection

ROCK inhibition preserves cavernous nerve structure

Magnification 40x

• Increased myelinated axons • Strong presence of Schawnn cells

Sham BCNI BCNI+Y-27632

223.52 µm

275.35 µm

314.11 µm

179.21 µm

185.25 µm

Incubation 24h

10xIncubation 48h

10x

Incubation 72h

10x

A

C

B

D

Hannan JL et al., Molecular Neuroscience 2014

48h incubation

Sham BCNI14d BCNI14d+Y276320

100

200

300

400

500

Neu

rite

Len

gth

( m

) **

Inhibition of ROCK: Effect onMPG Neuritogenesis

Sham Y27632

1.0 2.0 4.0 8.0 16.0 32.0 48.0 64.00

50

100

150

200

250

SHAM (n=6)BCNI (n=6)BCNI + Y-27632 (n=8)

* * *

*

Frequency (Hz)

Con

trac

tion

(%M

ax K

Cl)

Improved nerve-mediated responses after ROCK inhibition

1.0 2.0 4.0 8.0 16.0 32.0 48.0 64.00

25

50

75

100

** *

* **

*

Frequency (Hz)

Rel

axat

ion

(%M

ax P

E)

* p<0.05 vs Sham; Ψ p<0.05 vs Sham and BCNI,;δ P<0.05 vs BCNI

Sham BCNI BCNI + Y-276320.0

0.5

1.0

1.5

*

n=6

**

nNO

S/G

APD

H

Sham BCNI BCNI + Y-276320.0

0.5

1.0

1.5

*

n=4-6

m-e

NO

S/G

APD

H

m‐eNOS

GAPDH

a b

Figure 3

nNOS

GAPDH

Sham BCNIBCNI +Y‐27632 Sham BCNI

BCNI +Y‐27632

†

†

J Hannan et al, J Urol 2012

2.0 4.0 6.0 8.00

25

50

75

100

SHAM (n=8)CNI (n=8)CNI + Y-27632 (n=7)

**

**

**

Voltage

Peak

ICP

(mm

Hg)

1 2 3 40.0

0.2

0.4

0.6

0.8

1.0

2.0 4.0 6.0 8.0

**

**

Voltage

ICP/

MAP

1 2 3 40

2000

4000

6000

*

2.0 4.0 6.0 8.0

**

*

*

Voltage

Tota

l IC

P(A

rea

Und

er th

e C

urve

; mm

Hg*

s)

2.0 4.0 6.0 8.00

10

20

30

40

* * *

*

Voltage

T80

(s)

a b

c d

Figure 1

†

† †† ††

†

†

† †† †

†

†† †

J Hannan et al, J Urol 2012

Stem cells

• Clinical need• Types of stem cells• Efficacy and mechanisms of action

– ED• cavernous nerve injury• (aging)• (diabetes and metabolic syndrome)

– Peyronie’s disease

STEM CELL TREATMENT FOR ERECTILE DYSFUNCTION

Medline Search –Stem Cells and Erectile Dysfunction

Stem Cells and Erectile Dysfunction

• Animal Models used to study stem cell-based therapies.– Aging– Hypogonadism– Hypercholesterolemia, Arterial insufficiency.– Diabetes mellitus type 1 and 2 (Metabolic Syndrome)– Cavernous nerve injury and resection.

Stem Cell Biology

Stem cell research in ED

LacZ (gene)GFP (gene)BrdU (DNA-incorporation)EdU (DNA-incorporation)CM-DiI (cytoplasm)PKH26 (cell membrane)

Ex-Vivo Gene Modifications (eNOS, VEGF)

Stem cells1) diseased/lost cell replacement2) paracrine modification of natural coarse of disease

or paracrine stimulation of trophic processes

Dual effects: neural tissues vs penile tissues

Primary:

CAVERNOUS NERVE INJURYAxonotmesis

neuro-inflammation

Wallerian degeneration

NO bioavailability

TNF-αTGF-β1

chemokines

Dual effects: neural tissues vs penile tissues

Secondary:

CAVERNOSAL DENERVATION

hypoxia

smooth muscle apoptosiscollagen deposition (fibrosis)

veno-occlusive dysfunction

TGF-β1

Sham

BC

NI 1

4dB

CN

I 30d

2x 10x

adipose tissue-derived stem cells (ADSC)

bone marrow derived stem cells (BMDSC)

J Sex Med 2010;7:3331–3340

ADSC recovers erectile function in cavernous nerve injured rats

J Sex Med 2010;7:3331–3340

Preserved nNOS expression in dorsal penile nerve

Proteomic profile angiogenesis array analysis of human ADSCs

Guihua Liu et al, PLOS One 8: 2013

Mesenchymal Stem Cells (ADSC) demonstrate neurotrophic effects on 

cavernous nerve regeneration

Nerve Grafts to replace CN

Hannan JL, Mao M, Bivalacqua TJ

Biomaterials with Schwann cells,Growth factors, Rho-kinase inhibitors,Neurotrophic factors, stem cells

Stem Cell Based Clinical Trials• Treatment of Diabetic Impotence with Umbilical Cord

Blood Stem Cell Intracavernosal Transplant –negative trial (Jong Yoon Bahk et al. Experimental and Clinical Transplantation 8:150-160, 2010)

• Clinical trial in France using IC MSCs – ongoing.• Industry sponsored IC ADSCs for post-RP ED.

5/7 clinicalresponse

Stem Cell Based ED Clinical Trial at Johns Hopkins: PRIMES 2014

• PRIMES (PReventing Impotence with MEsenchymalStem Cells) trial for post-radical prostatectomy ED.

• PI: Medical Oncology – Sam Denmeade M.D. Ph.D. and Johns Isaacs Ph.D

• PI: Urology – Trinity J. Bivalacqua M.D. Ph.DAlan Partwin M.D. Ph.D.

• Protocol – Intravenous injection of MSCs prior to RP to prevent degeneration of cavernous nerves and identification in prostate cancer.

Conclusion

• Development of pharmacological agents targeting a number of molecular pathways are necessary for treatment of PDE5 inhibitor non‐responders.– However, is there interest to invest in their development for treatment of ED.

• Stem cell based therapies have support in pre‐clinical animal models and clinical trials results are forthcoming to determine efficacy in men with ED.

AcknowledgmentsArthur L. Burnett, M.D. MBAJohanna Hannan Ph.D.Ahmet Hoke, M.D.Maarten Albersen MD PhD.Xiaopu Liu, B.S.Joseph WatsonHotaka Matsui M.D.