Ru-Band Lu, MD.Shiou-Lan Chen, PhD.

National Cheng Kung University

Neuro-degeneration and

Novel Treatment in Heroin dependence

Neurodegeneration in Mental Illnesses

• Schizophrenia

• Bipolar disorders

• Substance use disorders (alcohol)

• Anxiety disorders?

• Personality disorders?

2

Neuronal degeneration in schizophrenia

Schizophrenia and bipolar disorders

– Decreased gray & white matter and lateral ventricular dilatation (Largen et al., 1984; De Peri l et al., 2012)

3

Neuronal degeneration in Bipolar Disorder (BP)

• Diffused gray and white matter loss, enlarged ventricles & mild prefrontal volume loss. (Wilde, et al., 1985)

• 17% larger lateral ventricles and 2.5 times in deep white matter hyperintensities. (Kempton, M.J., et al., 2008.)

4

MRI in middle-aged women

Neuronal degeneration in Substance Abuse

5

Neuronal degeneration in substance use?

Animal studies

•Opioid use – Reduction dopaminergic neurons in ventral tegmental area (VTA) of rats (Chu, 2008)

•Heroin and cocaine use– Showing neurotoxicity in rat (Cunha-Oliveira et al., 2010)

•Alcohol use– Reduced hippocampal neurogenesis

– Impaired long-lasting memory in monkey (Taffe, 2010).

6

Treatment of Neurodegeneration disease

• Current therapy treating symptoms, but few slowing or inhibiting the progress

• Without treatment in heroin dependence

• Development of novel therapy for central, peripheral degeneration illness

7

Microglia

Source for

neurotrophin production

Target for

anti-inflammation

Astroglia

Glial cells : Key roles in disease and prime targets for therapy

8

9

Hypothesis

1.Neuronal

Degeneration

2.White/Gray Matter Damage

Causing/Onset/Relapse of Mental Illness

The activation of Neuro-immune system

BrainBlood

Blood-Brain-Barrier

CytokinesCytokines

Qin et al. (2007)

0

20

40

60

80

100

120

0 .5h 1h 2h 3h 6h 9h 7 m 10 m

TN

F

(%

of

pea

k)

Time

A single IP LPS injection induced TNF-α production both in peripheral and brain in rat

Liver**** **

**

**

**

Brain** **

**** ****

**

**

Blood**

**

**

**

TNF-

LPS

Kupffer cell

Role of microglia in toxin-induced progressive neurotoxicity

Pro-inflammatory

Factors (ROS, NO., TNFa, IL-1,

PGs)

Reactive Microgliosis

(Self-propelling)

LPS (Extra Neurotoxin)

Ac

tiva

tio

n

MPTP (Intra Neurotoxin)

Microglia

(Working Model)

Dopaminergic

Neuronal Damage

11(Hong et al., 2005)

NADPH oxidase (PHOX)

Activated

O2O2 • –

NADPH NADP+ + H +

Resting

gp91

p22rap1a

p40

p47rac2

p67

MAC-1

LPS

out

in

PKC

P

P

Naloxone,Dextromethorphan

Memantine

12(Hong et al., 2005)

Transfection of gp91PHOX, a membrane subunit of PHOX,

increases [3H]-naloxone binding

0

100

200

300

[3H]-(-)-Nal

Bin

din

g C

ap

acit

y (

% W

T)

WT

[3H]-(+)-Nal

*

Gp91-transfected

400COS-7 Cells (WT)

Ligand Binding Assay

StableTransfectio

n

COS-7 gp91

(gp91)

gp91

*

13(Hong et al., 2007)

O2

O2 • –

Dual Functions of Superoxide Radicals

gp91

Microglia

MAC1

TLR4LBP

CD14

PHOX

ROS

H2O2

O2 • –

TNF- IL-1

NO •

ONOO -

2) Gene Expression

1) Neurotoxicity

NF-kB,AP-1

PKCLPS

LPS

DA neurons 14(Hong et al., 2005)

15

Memantine

• An non-comparative NMDA receptor, medication for Alzheimer's disease (20 mg/day)

• Low dosage having neuroprotective effects in vitro and in vivo (Wu et al., 2009)

LPS

Microglia

TNF-aIL-1b

Neuronal death

ONOO-

NO • O2 • -

Conventional Regimen:

• Aspirin, COX 2 inhibitors (PG)

• Anti-oxidants (Free radicals)

• Antibodies (TNF-α, IL-1)

• Receptor antagonists (TNF-α)

• Cortisone (toxic for long-term use)

Novel Strategy: Modulation of microglial activity

•Morphinans : Naloxone, D-Morphine

Dextromethorphan

•Dynorphins, Enkephalin, PACAP

•Memantine

Novel anti-inflammatory therapy

PG’s

16

(Hong et al., 2005)

Memantine

LPS

Dopaminergic Neuron

ONOO-

TNFαIL-1β

NO O2 • -

Microglia

PHOX

Inhibition of microglial PHOX (by binding to gp 91) is

associated with DM-, Memantine-elicited neuroprotective effect

17(wu et al., 2009)

18

Mechanism underlying Memantine induced increase in expression of neurotrophic factors in astroglia

HDACHATs

Astroglia

Memantine

mRNA of GDNF, BDNF

GDNF, BDNF

Nuclear

(Chen et al., 2009)

LPS

Acti

vati

on

Trophic factors:(GDNF, BDNF, NGF)

Pro-inflammatory factors: Superoxide, iROS NO, TNF, PGE2

Reactive Microgliosis

Astroglia

Microglia

DA neuron

Inhibition

Memantine

Stimulation

Memantine

Combined therapy for neurodegenerative diseases

(Self-propelling)

Neurogenesis

19(wu et al., 2009)

Hypotheses & Experimental Protocol in Human

• Neuro-inflammation inducing heroin dependence and progressive neurodegeneration

• Heroin dependence and neurondegeneration causing progression of heroin use, mental illness

20

Cytokines v.s. Substance Use in Animal

• Opioid

– Increased IL-1β, and IL-6 in vitro. (Kapasi, 2000) – Increased iNOS and cytokines in the locus coeruleus (Dyuizen I et al., 2009)

• Alcohol – Increased microglia in cerebellar and hippocampal of the rat . (Riikonen, 2002)

– Downregulated in the neocortex, hippocampus, and cerebellum of rats and conversed by COX-2. (Pascual et al., 2007)

• Methamphetamine – Increased free radical & overactivity of cytokines. (Imam, 2001)

21

Research Aims

• Analysis of plasma cytokine and BDNF levels– Cytokines and BDNF: through BBB (Pan et al., 1998)

– BDNF: a neuro-degeneration biomarker (Laske et al. 2006)

– Cytokines: an neuro-inflammation biomarker (Buckley, 2007)

• The relationship of plasma cytokine, BDNF levels with heroin dependence

• Using effective drugs in neuroprotective and neurogenesis

22

23

Human-

Heroin dependence treatment

Methods

• Methadone maintaining treatment

• Adjustment the methadone dosage

• Memantine (5mg/day) or placebo

• Measuring methadone plasma level weekly

• Measuring BDNF, cytokines (pre- & post-treatment)

24(Chen et al., submitted 2013)

Results

• Longer of years of heroin use ( 5 years, n = 57 or 6 years, n = 58)

– lower TGF-β1 and BDNF levels

– Higher MTD (methadone) dosage

• Compared to healthy controls at baseline

– Higher plasma TNF-, CRP, IL-1β, IL-6, and IL-8 levels

– Lower plasma TGF-β1 and BDNF levels25

(Chen et al., submitted, 2013)

Correlation of heroin used period with plasma

MTD dose, cytokines and BDNFat baseline

  Heroin used years vs

MTD dose IL-8 TGF-β1 BDNF

Spearman r 0.203 -0.083 -0.212 -0.255

95% confidence interval

0.003 to 0.388 -0.276 to

0.116

-0.396 to -

0.013

-0.430 to -

0.061

P value (two-

tailed)

0.0404 0.3988 0.0321 0.0087

26

MTD, methadone; transforming growth factor (TGF)-β1; A non-parametric analysis

(Chen et al., 2013)

Plasma cytokine, and brain-derived neurotrophic factor

baseline data    Heroin-dependent Patients p-value

 Healthy Controls

MTD+Placebo MTD+Mem  

  n = 72 n = 57 n = 58  

Gender (F/M) 10/62 10/47 10/48 0.817

Age (years) 31.64 ± 0.72 37.53 ± 0.93*** 37.24 ± 0.94*** < 0.0001

Heroin use (years) - 7.49 ± 0.49 8.36 ± 0.97 0.492

MTD dose (mg) - 36.23 ± 2.96 36.18 ± 2.70 0.991

Plasam TNF- (pg/mL) 1.57 ± 0.20 3.74 ± 0.47*** 3.62 ± 0.34*** < 0.0001

Plasma CRP (ng/mL) 1.66 ± 0.17 4.38 ± 0.44*** 3.60 ± 0.38*** < 0.0001

Plasma IL1- (pg/mL) 0.54 ± 0.09 1.42 ± 0.28** 1.97 ± 0.27*** < 0.0001

Plasma IL-6 (pg/mL) 1.23 ± 0.13 2.47 ± 0.35** 2.42 ± 0.28** 0.0004

Plasma IL-8 (pg/mL) 1.38 ± 0.28 4.66 ± 0.56** 6.53 ± 1.24*** < 0.0001

Plasma TGF-1 (ng/mL) 32.27± 2.0 24.16 ± 2.22** 23.02 ± 2.00** 0.0023

Plasma BDNF (ng/mL) 20.0 0 ± 1.09 11.40 ± 1.18*** 9.55 ± 0.83*** < 0.0001

27Mem, memantine; MTD, methadone; TGF, transforming growth factor One-way ANOVA for comparing cytokines and BDNF; age was used as a covariate.

(Chen et al., submitted 2013)

Results

After the 12-week treatment, compared with

baseline

•MTD (methadone) +Placebo group

– Increasing percentage of MTD dose after 4-week

•MTD+MM (memantine) group

– No change in MTD dosage

28(Chen et al., submitted 2013)

Heroin dependence treatment Methadone + Memantine (5 mg/placebo)

29(Chen et al., 2012)

Results

After the 12-week treatment, compared with both groups at baseline

•MTD with Placebo group

– No change in TNF- and IL-6, and BDNF levels

•MTD+MM group

– Decreasing TNF-, , IL-1β, IL-6 and IL-8 levels

– Increased tendency in BDNF level

30

(Chen et al., submitted, 2013)

Heroin dependence treatment Methadone + Memantine (5 mg/placebo)

31(Chen et al., 2012)

Heroin dependence treatment Methadone + Memantine (5 mg/placebo)

32(Chen et al., 2012)

Comments

• Longer heroin use (including MTD) increasing

inflammation and neurotrophic dysfunction.

• MTD with memantine treatment decreasing MTD-

induced inflammation and neurotrophic dysfunction

• MM inhibiting the tolerance for MTD

• MM decreasing cytokines & increasing BDNF levels

33

Comments

• Low dose Memantine– 5 mg/day of MM plasma level 10-50 ng/ml (0.05-0.2 M)

– No effect in NMDA receptor antagonist (IC50: 2-3 M)

(Parsons et al. 1999)

– Effective in heroin dependence treatment

– Benefit in neuron protect & decreasing neurodegeneration

34

Conclusions

• Over inflammation correlation with neurodegeneration, heroin

dependence & mental illness

• Treatment symptoms and treatment progress

• Regulation autoimmune & neuron (cell) protection or genesis,

benefits from central to peripheral

• New effective drugs & the novel treatment model breaking

through the difficulty of new drugs development

• Inhibiting the tolerance of heroin dependence, one of important factors

of substance dependence 35

Future Studies

• Treatment neuronal (cell) degeneration and developing

novel treatment in animal and human

• Developing related gene with the treatment effects

& predicted treatment response in central and

peripheral diseases

36

Acknowledgement

• Professor Jau-Shyong Hong, PhD.• Professor Pao-Luh Tao, PhD.• Hung-Ming Wu, MD. PhD.• Shiou-Lan Chen, PhD.• Shih-Heng Chen, PhD.• Chun-Hsien, Chu, PhD.• Kuei-Ying Hsu, MS.• Chen-Lin Wang, MS.• Yu-Ting Hung, BD.• Mei-Chun Wen, BD.• En-Jhen Chang, BD.

37

Thank you38

39

Potential beneficial effects of anti-inflammation-related drugs

• Addiction – Opiate abuse– Alcohol abuse – Smoking – Compulsive eating

disorder

• CNS diseases– Bipolar disorders– Depression– Schizophrenia– Alzheimer’s dis.– Brain ischemia – Parkinson’s dis.– MS – Spinal injury

• Peripheral diseases– Asthma– Arthritis – Arteriosclerosis– Cancer– Diabetes – Heart attack– Hepatitis – Inflammatory pain– Crohn’s dis. – Lupus – Sepsis

(endotoxemia)

40

Human peripheral effect

41

The lipid-rich atherosclerotic lesions were

identified with Oil-Red-O staining.

Dextromethorphan (DM) decreases high lipid diet-induced atherosclerotic lesion formation in apo-E-deficient mice

PBS 5 10 20 400

5

10

15

20

25

* *

*

DM (mg/kg)

Le

sio

n a

rea

(%

)

Control DM(10mg/kg)

DM(20 mg/kg) DM(40 mg/kg)

(Liu et al. 2009)

42

6.0

7.0

8.0

9.0

5.0

4.0

3.0

Placebo

DM

*

†

‡

0 3 6Time (months) after DM (60 mg/tablet, bid)

Flo

w m

edia

ted

dil

ata

tio

n r

atio

Clinical trial of dextromethorphan (DM) in heavy smokers

CR

P l

evel

s (m

g/d

l )

Time (months) after DM (60 mg/tablet, bid)

1

2

3

45

6

7

8

9

10

0 1 2 3 6

*

*

Placebo

DM

* *

Blood flow-mediated dilatation ratio C-reactive protein (CRP)

(Liu et al.)

43

Clinical trial of naltrexone in alcoholics

44(Chen et al., 2012)

Neurotrophic and neuroprotective effects of memantine

H.M. WU, H.L. CHEN, N.S. TZENG

J.S. HONG, R.B. LU* (2009)

45

Memantine has neurotrophic and neuroprotective effects to dopaminergic neurons

and against LPS-induced neurotoxicity46(Wu et al., 2009)

The neurotrophic effect of memantine is astrocyte-dependent.

47(Wu et al., 2009)

Memantine decreases the levels of pro-inflammatory factors induced by LPS. 48

(Wu et al., 2009)

Memantine has independent neuroprotective effects on LPS-induced

neurotoxcity.

49(Wu et al., 2009)

Memantine attenuates LPS-induced morphological activation of

microglia. 50(Wu et al., 2009)

In Summary

Pro-inflammatory Factors

Reactive Microgliosis

(Self-propelling)

Microglia

Neurons Trophic

factors:(GDNF, BDNF,

NGF)Neurogenesis

Astroglia(1)

(2)

(-)

Anti-inflammat

ion

Neuro-protection

Neuronalgenesis

(+)

51

Plasma Cytokine

52(Chen et al., 2012)