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Ru-Band Lu, MD. Shiou-Lan Chen, PhD. National Cheng Kung University

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1 Ru-Band Lu, MD. Shiou-Lan Chen, PhD. National Cheng Kung University
Neuro-degeneration and Novel Treatment in Heroin dependence Ru-Band Lu, MD. Shiou-Lan Chen, PhD. National Cheng Kung University

2 Neurodegeneration in Mental Illnesses
Schizophrenia Bipolar disorders Substance use disorders (alcohol) Anxiety disorders? Personality disorders?

3 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)

4 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.)

5 Neuronal degeneration in Substance Abuse
MRI in middle-aged women

6 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).

7 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

8 Astroglia Microglia Glial cells :
Key roles in disease and prime targets for therapy Astroglia Microglia 1.The major advance in research in neurobiology for the last few decades has been the understanding of the role of neuronal systems in brain function.. 2.As a consequence, most of the drugs to treat neurodegenerative disorders has been developed that work by altering chemical neurotransmission in the brain. 3. Curiously, little attention has been paid to the nonneuronal cellular components of the brain, which outnumber neurons by a factor of 10 to 1. 4.Glia, a heterogeneous population of cells, have largely been viewed as passive handmaidens to the neurons, which have been considered the primary arbiters of information processing in the brain. 5. Recent research, however, has demonstrated that glia are far from passive but actively participate in the communicational processes of the brain. 6.Disturbances of glial function could conceivably contribute to psychopathology, and glial cells may, therefore, serve as appropriate targets for therapeutic interventions. Source for neurotrophin production Target for anti-inflammation

9 The activation of Neuro-immune system
Hypothesis The activation of Neuro-immune system Blood 1.Neuronal Degeneration 2.White/Gray Matter Damage Brain Causing/Onset /Relapse of Mental Illness Cytokines Cytokines Blood-Brain-Barrier

10 A single IP LPS injection induced TNF-α production both in peripheral and brain in rat
120 Liver ** Brain ** Blood ** 100 TNF- 80 Kupffer cell TNFa (% of peak) 60’’ 1.We tried to understand the mechanism why a single injection is capable of producing this long-delay and progressive effects. Move liver to this slide. 2. Since it is known that very few amount of systemically injected LPS can reach the brain tissue, we predicted that proinflammatory factors produced in the peripheral system may mediate these actions. 3. We measured various cytokines and obtained evidence that is consistent with our thinking. Her I use TNFa, one of the major pro-inflammatory cytokines as an example. 4. LPS injected produced a rapid increase in blood TNFa, which is mainly came from the macrophages in the liver. The half-life are relatively short. 5. The most striking finding is the long-lasting elevation of TNFa- in the brain. 6. This long-lasting of these cytokines suggests a chronic neuro-inflammation and leads to neurodegeneration and motor deficits. 60 40 20 LPS .5h 1h 2h 3h 6h 9h 7 m 10 m Time

11 Role of microglia in toxin-induced progressive neurotoxicity
(Working Model) LPS (Extra Neurotoxin) Activation MPTP (Intra Neurotoxin) Reactive Microgliosis Dopaminergic Neuronal Damage Microglia The progressive nature of PD is a well-described phenomenon, however, very little is known about its mechanism. 2. Our working model of the progressive nature centers on microglia. 3. Earlier work from our lab using LPS-induced inflammation as a PD model indicate activation of microglia by LPS is responsible for the damage of dopaminergic neurons. 4. Later on we found that the microglial dependence is not only limited to the action of LPS, MPTP, is known to exert direct and selective neurotoxic to DA neurons. 5. We have recently found that the presence of microglia is necessary for the expression of the full potency of MPTP. 6.This is mediated from reactive microgliosis which is different from LPS. 7. The way LPS and MPTP kills DA neurons are different, however, the death of DA neurons would further activate microglia through “reactive Microgliosis”, which in turn will release more proinflammatory factors to cause further damage of DA neurons 8.Thus,it appears that there exists a vicious cycle which form a self –propelling force to cause a progressive DA neuronal degeneration even LPS or MPTP disappear from the brain long time before PD occurs. (Self-propelling) Pro-inflammatory Factors (ROS, NO., TNFa, IL-1, PGs) (Hong et al., 2005)

12 NADPH oxidase (PHOX) a  LPS out in Activated Resting MAC-1 PKC O2
Naloxone, Dextromethorphan Memantine O2 O2 • – NADPH NADP+ + H + LPS MAC-1 out a gp91 in rap1a p22 PKC p40 p47 rac2 p67 P P (Hong et al., 2005) Activated Resting

13 Transfection of gp91PHOX, a membrane subunit of PHOX,
increases [3H]-naloxone binding 400 [3H]-(-)-Nal [3H]-(+)-Nal COS-7 Cells (WT) * * 300 Stable Transfection Binding Capacity (% WT) gp91 200 COS-7 gp91 (gp91) 100 WT Gp91-transfected Ligand Binding Assay (Hong et al., 2007)

14 Dual Functions of Superoxide Radicals
gp91 Microglia MAC1 TLR4 LBP CD14 PHOX LPS O2 O2 • – LPS PKC ROS H2O2 NF-kB,AP-1 O2 • – NO • 2) Gene Expression TNF- IL-1 ONOO - 1) Neurotoxicity (Hong et al., 2005) DA neurons

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)

16 Novel anti-inflammatory therapy
(Hong et al., 2005) LPS Novel Strategy: Modulation of microglial activity Morphinans : Naloxone, D-Morphine Dextromethorphan Dynorphins, Enkephalin, PACAP Memantine Microglia This slide illustrates the strategies for developing novel anti-inflammatory drugs. The conventional anti-inflammatory drugs shown here, such as aspirin, COX 2 inhibitors are designed to inhibit a single pro-inflammatory factor. For example, COX-2 inhibitor inhibit the production of PGs by inhibiting COX 2 enzyme. These types of anti-inflammatory drugs are limited in their efficacy, and this figure explains the reason. When the immune cells are over-activated, they secret a large number of factors. Thus, by targeting only one or two factors, these anti-inflammatory drugs are not able to contain the inflammation. We took a different approach at the upstream by preventing the over-activation of immune cells. We hypothesized if we can contain the over-activation of microglia, which will not cause the secretion of down stream factors, then we do not need to deal with the individual factors. Over the last few year, we do find some old drugs which can serve this kind of purpose and turn out to be very potent and safe anti-inflammatory drugs and excellent nreuroprotective agents. TNF-a IL-1b 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) PG’s ONOO- Neuronal death

17 Inhibition of microglial PHOX (by binding to gp 91) is
associated with DM-, Memantine-elicited neuroprotective effect LPS Microglia PHOX Memantine ONOO- TNFα IL-1β NO O2 • - Dopaminergic Neuron (wu et al., 2009)

18 Mechanism underlying Memantine induced increase in
expression of neurotrophic factors in astroglia HATs HDAC Memantine The second approach is targeting astroglia by releasing neurotrophic factors. Recently, we found a series of widely anti-convulsants, such as valproate or VPA, are neruoprotective. Mechanistic studies showed that VPA inhibits Histone deactylase and increase the acetylation of histone protein, leading to chromation rearrangement and increase the expression of triphic factors. C A C A C A C A Nuclear C A C A C A C A Astroglia mRNA of GDNF, BDNF (Chen et al., 2009) GDNF, BDNF

19 Combined therapy for neurodegenerative diseases
LPS Activation Stimulation Memantine Astroglia Inhibition Memantine Trophic factors: (GDNF, BDNF, NGF) Neurogenesis Reactive Microgliosis Microglia (Self-propelling) DA neuron Pro-inflammatory factors: Superoxide, iROS NO, TNF, PGE2 (wu et al., 2009)

20 Hypotheses & Experimental Protocol in Human
Neuro-inflammation inducing heroin dependence and progressive neurodegeneration Heroin dependence and neurondegeneration causing progression of heroin use, mental illness

21 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)

22 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 ) Cytokines: an neuro-inflammation biomarker (Buckley, 2007) The relationship of plasma cytokine, BDNF levels with heroin dependence Using effective drugs in neuroprotective and neurogenesis The major theme of our research is to test the hypothesis that------ Why is it important? Although progression of PD is so obvious in PD patients, but little is known about the mechanism. Thus, so far no effective treatment for halting the disease progression. We speculate that neuroinflammation play a major role in driving the disease progression. Three aims are inter-related.

23 Human- Heroin dependence treatment

24 Methods Methadone maintaining treatment
Adjustment the methadone dosage Memantine (5mg/day) or placebo Measuring methadone plasma level weekly Measuring BDNF, cytokines (pre- & post-treatment) (Chen et al., submitted 2013)

25 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 levels (Chen et al., submitted, 2013)

26 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 to 0.116 to to P value (two-tailed) 0.0404 0.3988 0.0321 0.0087 MTD, methadone; transforming growth factor (TGF)-β1; A non-parametric analysis (Chen et al., 2013)

27 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*** < 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*** Plasma CRP (ng/mL) 1.66 ± 0.17 4.38 ± 0.44*** 3.60 ± 0.38*** Plasma IL1- (pg/mL) 0.54 ± 0.09 1.42 ± 0.28** 1.97 ± 0.27*** 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*** Plasma TGF-1 (ng/mL) 32.27± 2.0 24.16 ± 2.22** 23.02 ± 2.00** 0.0023 Plasma BDNF (ng/mL) ± 1.09 11.40 ± 1.18*** 9.55 ± 0.83*** Mem, 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)

28 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 (Chen et al., submitted 2013)

29 Heroin dependence treatment Methadone + Memantine (5 mg/placebo)
(Chen et al., 2012)

30 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 (Chen et al., submitted, 2013)

31 Heroin dependence treatment Methadone + Memantine (5 mg/placebo)
(Chen et al., 2012)

32 Heroin dependence treatment Methadone + Memantine (5 mg/placebo)
(Chen et al., 2012)

33 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

34 Comments Low dose Memantine
5 mg/day of MM plasma level ng/ml ( 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

35 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

36 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

37 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.

38 Thank you


40 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)

41 Human peripheral effect

42 Dextromethorphan (DM) decreases high lipid diet-induced
atherosclerotic lesion formation in apo-E-deficient mice DM(10mg/kg) Control 25 20 Lesion area (%) 15 * 10 * * 5 PBS 5 10 20 40 DM(20 mg/kg) DM(40 mg/kg) DM (mg/kg) The lipid-rich atherosclerotic lesions were identified with Oil-Red-O staining. (Liu et al. 2009)

43 Clinical trial of dextromethorphan (DM) in heavy smokers
Blood flow-mediated dilatation ratio C-reactive protein (CRP) 10 Placebo 9.0 DM 9 * * 8 DM 8.0 * 7 * * 7.0 Fig 1 FMD was calculated as the percentage change in diameter compared with the baseline. 6 Flow mediated dilatation ratio 6.0 CRP levels (mg/dl ) 5 4 5.0 Placebo 3 4.0 2 3.0 1 3 6 1 2 3 6 Time (months) after DM (60 mg/tablet, bid) Time (months) after DM (60 mg/tablet, bid) (Liu et al.)

44 Clinical trial of naltrexone in alcoholics
(Chen et al., 2012)

45 Neurotrophic and neuroprotective effects of memantine
H.M. WU, H.L. CHEN, N.S. TZENG J.S. HONG, R.B. LU* (2009)

46 Memantine has neurotrophic and neuroprotective effects to dopaminergic neurons and against LPS-induced neurotoxicity (Wu et al., 2009)

47 The neurotrophic effect of memantine is astrocyte-dependent.
(Wu et al., 2009)

48 Memantine decreases the levels of pro-inflammatory factors induced by LPS.
(Wu et al., 2009)

49 Memantine has independent neuroprotective effects on LPS-induced neurotoxcity.
(Wu et al., 2009)

50 Memantine attenuates LPS-induced morphological activation of microglia.
(Wu et al., 2009)

51 In Summary Anti-inflammation (+) (-) (2) (1) Neuro-protection Neuronal
genesis Anti-inflammation (+) (-) Neurons (1) Trophic factors: (GDNF, BDNF, NGF) (2) Astroglia Microglia 2.Two approaches are: 1. So far most of PD drugs developed are neuron-based. Our strategies are glia-based by halting the self-propelling cycle. anti-inflammation by reducing microglial activation. and release of trophic factors from astroglia. 3. We have made significant progress in this field of research. Due to the time limit, I will only be able to show a few summary slide. Reactive Microgliosis (Self-propelling) Neurogenesis Pro-inflammatory Factors

52 Plasma Cytokine (Chen et al., 2012)

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