Post on 15-Oct-2020
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.