Mechanizmy účinku dostupných imunomodulačních léků Pavel Štourač Neurologická klinika FN Brno

MS je chronické progresivní onemocnění CNS Relaps-remise Sekundární progrese lKlinická disabilita Klinický práh Objem mozku Zánět Inflammation and Axonal Loss in MS The above figure illustrates the course of MS.1-5 During the relapsing-remitting phase, the disease is characterized by frequent inflammation, demyelination, axonal transection, and remyelination. Relapses are more frequent and complete recovery from disability generally occurs.2-5 During the secondary-progressive phase of the disease, inflammation and relapses occur infrequently, axonal loss is increased and disability progresses. MRI-defined plaque burden and clinical impairment accumulate over time. 1. Compston A, Coles A. Multiple sclerosis. Lancet. 2002;359:1221- 1231. Erratum in: Lancet. 2002;360:648. 2. Lublin FD, Reingold SC. Defining the clinical course of multiple sclerosis: results of an international survey. Neurology. 1996;46:907-911. 3. Olerup O, Hillert J, Fredrikson S, et al. Primary chronic progressive and relapsing/remitting multiple sclerosis: two immunogenetically distinct disease entities. Proc Natl Acad Sci U S A. 1989;86:7113-7117. 4. Thompson AJ, Kermode AG, MacManus DG, et al. Patterns of disease activity in multiple sclerosis: clinical and magnetic resonance imaging study. BMJ. 1990;300:631-634. 5. Revesz T, Kidd D, Thompson AJ, Barnard RO, McDonald WI. A comparison of the pathology of primary and secondary progressive multiple sclerosis. Brain. 1994;117:759-765. Axonální ztráta Převaha zánětu, demyelinizace,axonální transekce, plasticita, a remyelinizace Pokračující zánět, přetrvávající demyelinizace Minimální zánět, chronická axonální degenerace, glióza Compston A, Coles A. Lancet. 2002;359:1221-1231.

Signál neboli spouštěč Patogeneze MS Signál neboli spouštěč aktivace, diferenciace, klonální expanze T autoreaktivní T - lymfocyty T T adheze T VLA-4 VCAM-1 periferie transmigrace produkce cytokinů aktivace makrofágů protilátky B M NO IFN- TNF- Hemato-encefalická bariera Lokální reaktivace T APC CNS Demyelinizace a axonální ztráta Prof. R. Hohlfeld.

Demyelinizace a axonální degenerace Clinical Disability Clinical Threshold Brain Volume Inflammation Inflammation and Axonal Loss in MS The above figure illustrates the course of MS.1-5 During the relapsing-remitting phase, the disease is characterized by frequent inflammation, demyelination, axonal transection, and remyelination. Relapses are more frequent and complete recovery from disability generally occurs.2-5 During the secondary-progressive phase of the disease, inflammation and relapses occur infrequently, axonal loss is increased and disability progresses. MRI-defined plaque burden and clinical impairment accumulate over time. 1. Compston A, Coles A. Multiple sclerosis. Lancet. 2002;359:1221- 1231. Erratum in: Lancet. 2002;360:648. 2. Lublin FD, Reingold SC. Defining the clinical course of multiple sclerosis: results of an international survey. Neurology. 1996;46:907-911. 3. Olerup O, Hillert J, Fredrikson S, et al. Primary chronic progressive and relapsing/remitting multiple sclerosis: two immunogenetically distinct disease entities. Proc Natl Acad Sci U S A. 1989;86:7113-7117. 4. Thompson AJ, Kermode AG, MacManus DG, et al. Patterns of disease activity in multiple sclerosis: clinical and magnetic resonance imaging study. BMJ. 1990;300:631-634. 5. Revesz T, Kidd D, Thompson AJ, Barnard RO, McDonald WI. A comparison of the pathology of primary and secondary progressive multiple sclerosis. Brain. 1994;117:759-765. Axonal Loss Waxman SG, NEJM. 1998;338:323.

Patologie sclerosis multiplex Zánět Demyelinizace Normální MS Axonální ztráta Glióza

Axonální poškození u SM Axonální zakončení Nefosforylovaná neurofilamenta Axonal Damage in MS These confocal micrographs from postmortem tissue of MS patients were obtained in a very elegant study by Trapp and colleagues and show axonal damage and demyelination in active MS lesions.1 The sections were taken from MS lesions and were stained with fluorescent antibody to myelin (red) and nonphosphorylated neurofilaments (green), which are usually abundant only in neuronal cell bodies and dendrites. The presence of nonphosphorylated neurofilaments in the axon indicates demyelination. Panel A is from the center of an active lesion. The arrows point to terminal axonal ovoids with single axonal connections, and the arrowhead points to an axonal ovoid with two axonal connections. The scale bar equals 64 microns. Panel B shows three large axons that stained positively for nonphosphorylated neurofilaments and are undergoing active demyelination (arrowheads). The arrow points to a large terminal ovoid at the end of an axon. The scale bar equals 45 microns. 1. Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, Bo L. Axonal transection in the lesions of multiple sclerosis. N Engl J Med. 1998;338:278-285. 64 m 45 m Trapp BD et al. N Engl J Med. 1998;338:278-285.

Axonální ztráta korelující s disabilitou

Souhrn MS má 2 aspekty Fokální Akutní Rekurentní Klinický relaps Zánět Neurodegenerace Fokální Akutní Rekurentní Klinický relaps Méně vyjádřená progrese onemocnění Difuzní Časný Chronický Progresivní Vyjadřuje progresi onemocnění

Protizánětli-vá léčba Mechanismy léčby SM Klasický léčebný přístup Nový léčebný přístup CNS Proti zánětlivý Axon protektivní Zánět demyelinizace zánět demyelinizace axonální ztráta NEURODEGENERACE AXONÁLNÍ ZTRÁTA Protizánětli-vá léčba RELAPS DISABILITA ? Krátkodobě RELAPS DISABILITA Dlouhodobě RELAPS DISABILITA

Neuronální dysfunkce v MRI Mozková atrofie Normal T1W obrazy Patient Sub-kortikální atrofie Kortikální atrofie Atrofie bílé hmoty Rozšíření komor Mozková atrofie = ztráta mozkové tkáně

Ovlivnění axonální degenerace Copaxone redukuje atrofii mozku –2.0 –1.5 –1.0 –0.5 0.5 Brain Volume (%) –0.6% –1.8% P = 0.0078 Změna/rok Glatiramer acetat Placebo GA Targeting Axonal Degeneration GA Reduces Brain Atrophy In the pivotal US trial of glatiramer acetate, a fully automated MRI technique with a 3-mm-slice thickness and fuzzy connectivity segmentation algorithm was used to evaluate data on brain atrophy in 27 patients over 2 years. These patients had RRMS and were from the original cohort.1 A Bonferroni correction for multiple hypothesis tests was used to compare two cohorts, to ensure type I-error rate no greater then 5%. Specifically, with respect to each variable, the treatment groups were declared significantly different at 5% only if the probability value for the relevant statistical test was < 0.05/6 = 0.0083 (six variables evaluated overall). The loss of brain volume per year was significantly lower in patients on glatiramer acetate than in patients in the placebo cohort (0.6% vs 1.8%, respectively; P = 0.0078). After 2 years, the glatiramer acetate group had a threefold smaller rate of brain volume loss per year. 1. Ge Y, Grossman RI, Udupa JK, et al. Glatiramer acetate (Copaxone) treatment in relapsing-remitting MS: quantitative MR assessment. Neurology. 2000;54:813-817. Ge Y et al. Neurology. 2000; 54:813-817.

Ovlivnění axonální degenerace GA & mozková atrofie EU/Kan studie – SIENA M9-M0 M18-M9 0.0 -0.2 GA p = 0.015 placebo -0.4 původní placebo PBVC [%] -0.6 -0.6 -0.8 -0.8 -0.9 -1.0 -1.0 -1.2 p = 0.037 Sormani et al., Neurology (2004) Accepted

Histopatologické koreláty “ Black Holes” T1- hypointenzity přímo korelují se stupněm axonální ztráty axonální denzita 1. Silně hypointenzní 20% 2. Slabě hypointenzní 60% 3. NAWM 100% Histopathologic Correlates of Black Holes T1 Hypointensity Directly Correlates to Degree of Axonal Loss Combined histopathologic and MRI studies have provided the most compelling evidence that marked hypointense T1 lesions reflect severe tissue damage.1,2 These studies have demonstrated a strong correlation between lesion hypointensity on T1-weighted images and the percentage of residual axons. In the van Waesberghe study,1 the histopathology of postmortem tissue samples from 17 MS patients was evaluated on the basis of T2-weighted imaging, including NAWM and T1 hypointense lesions. In each sample, MTRs, T1 contrast ratio, myelin, and axonal density was measured. “Postmortem tissue sampling by MRI revealed a range of pathology, illustrating the high sensitivity and low specificity of T2-weighted imaging. T1 hypointensity and MTR were strongly associated with axonal density, emphasizing their role in monitoring progression in multiple sclerosis.”1 1. van Waesberghe JH, Kamphorst W, De Groot CJ, et al. Axonal loss in multiple sclerosis lesions: magnetic resonance imaging insights into substrates of disability. Ann Neurol. 1999;46:747-754. 2. Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG. Multiple sclerosis. N Engl J Med. 2000;343:938-952. Other Studies Histopathology: severe myelin and axon loss. Brück W, Bitsch A, Kolenda H, Brück Y, Stiefel M, Lassmann H. Inflammatory central nervous system demyelination: correlation of magnetic resonance imaging findings with lesion pathology. Ann Neurol. 1997:42:783-793. van Waesberghe JH, Kamphorst W, De Groot CJ, et al. Axonal loss in multiple sclerosis lesions: magnetic resonance imaging insight into substrates of disability. Ann Neurol. 1999;46:747-745. MTI: markedly reduced MTR. van Walderveen MA, Barkhof F, Pouwels PJ, van Schijndel RA, Polman CH, Castelijns JA. Neuronal damage in T1-hypointense multiple sclerosis lesions demonstrated in vivo using proton magnetic resonance spectroscopy. Ann Neurol. 1999;46:79-87. MRS: markedly reduced NAA. Loevner LA, Grossman RI, McGowan JC, Ramer KN, Cohen JA. Characterization of multiple sclerosis plaques with T1-weighted MR and quantitative magnetization transfer. AJNR Am J Neuroradiol. 1995;16:1473-1479. DTI: markedly increased diffusivity and reduced fractional anisotropy. Filippi M, Cercignani M, Inglese M, Horsfield MA, Comi G. Diffusion tensor magnetic resonance imaging in multiple sclerosis. Neurology. 2001;56:304-311. van Waesberghe JH, van Walderveen MA, Castelijns JA, et al. Patterns of lesion development in multiple sclerosis: longitudinal observations with T1-weighted spin-echo and magnetization transfer MR. AJNR Am J Neuroradiol. 1998;19:675-683. Noseworthy JH et al. N Engl J Med. 2000;343:938-952. van Waesberghe JH et al. Ann Neurol. 1999;46:747-754.

Léze přecházející do Black Holes (%) Ovlivnění axonální degenerace GA redukuje poměr nových lézí přecházejících do permanentních ”Black Holes” 5 10 15 20 25 30 35 7 8 Stáří lézí (měsíce) Léze přecházející do Black Holes (%) P = 0.002 P = 0.04 31.4% 15.6% Glatiramer acetát Placebo 50% Targeting Axonal Degeneration GA Reduces the Proportion of New Lesions Evolving Into Permanent Black Holes Evidence for the neuroprotective action of glatiramer acetate in MS comes from various sources. This slide shows the results of a study following the evolution of new lesions into black holes. Black holes represent lesions where severe tissue disruption has occurred. In the European/Canadian MRI study, 1,722 new lesions from 239 MS patients enrolled in a placebo-controlled study were monitored with monthly MRI. The percentage of black holes on follow-up MRI was lower in the glatiramer acetate group than in the placebo group at each time point.1 This difference achieved statistical significance 7 months after lesion appearance (P = 0.04). Of the 275 new lesions that were identified as T1 hypointense lesions at month 1 and followed for up to 8 months, 21 (15.6%) in the glatiramer acetate group and 44 (31.4%) in the placebo group remained hypointense and could be classified as permanent black holes. The difference between treatment groups was statistically significant (P = 0.002). 1. Filippi M, Rovaris M, Rocca MA, et al. Glatiramer acetate reduces the proportion of new MS lesions evolving into “black holes”. Neurology. 2001;57:731-733. Filippi M et al. Neurology. 2001; 57:731-733.

NAA imunohistochemické barvení Neuronální dysfunkce v MRI 1H-MRS a SM-klinické studie NAA imunohistochemické barvení Coyle et al., 1989 NAA Normální SM pacient Arnold et al., Ann Neurol 1992, etc 4.10 ppm 0.78 NAA Cr Cho MS Lesion NAWM

Mechanismy působení neurotrofického faktoru - BDNF aktivované T, B-lymfocyty a monocyty produkují BDNF, podporující neuronální přežití in vitro v SM lézích je BDNF přítomný v imunitních buňkách a v astrocytech BDNF receptor je exprimován v neuronech a přilehlých astrocytech SM lézí Kerschensteiner and colleagues then showed that activated human T- cells, B-cells and monocytes secrete BDNF that supports neuronal survival in vitro. Furthermore, BDNF is present in the inflammatory infiltrate of patients with MS. Later these observations were extended (Stadelmann et al 2002) to show that in MS lesions BDNF is primarily present in immune cells (such as T-cells) and astrocytes, and there are high concentrations of BDNF-positive immune cells in lesions with ongoing demyelination. The BDNF receptor is expressed in neurones near the lesion and astrocytes within the lesion. This suggests that in MS, immune cells secrete BDNF which mediates neuroprotective actions. Kerschensteiner M et al. Activated human T-cells, B-cells and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J Exp Med 1999; 189(5): 865-870. Stadelmann C et al. BDNF and gp145trkB in multiple sclerosis brain lesions: neuroprotective interactions between immune and neuronal cells? Brain 2002; 125: 75-85. Kerschensteiner M et al. J Exp Med 1999; 189(5): 865-870. Stadelmann C et al. Brain 2002; 125: 75-85.

BDNF exprese v aktivních MS lézích Mikroglie T buňky Perivaskulární infiltrát BDNF Expression in Active MS In MS lesions, BDNF is primarily present in immune cells such as T cells and astrocytes, and there are high concentrations of BDNF-positive immune cells in lesions with ongoing demyelination.1 The BDNF receptor is expressed in neurons near the lesion and in astrocytes within the lesion. This suggests that in MS, BDNF and its receptor are involved in neuroprotective interactions that are mediated by immune cells. The top row shows BDNF-immunoreactivity present in neurons, inflammatory cells, and astrocytes in brains with MS. Serial sections demonstrate a perivascular inflammatory infiltrate immunoreactive for BDNF (a), CD68 (b; Ma—microphages/microglia) and CD3 (c; T cells). Panel (a) can overlay Panels (b) and (c) to show the presence of BDNF in immune cells. The bottom row shows hippocampal neurons containing BDNF in the subiculum (d) and the dentate gyrus (e). Reactive astrocytes within MS lesions display weak to moderate BDNF immunoreactivity. 1. Stadelmann C, Kerschensteiner M, Misgeld T, Brück W, Hohlfeld R, Lassmann H. BDNF and gp145trkB in multiple sclerosis brain lesions: neuroprotective interactions between immune and neuronal cells? Brain. 2002;125:75-85. BDNF barvení Neurony Astrocyty Stadelmann C et al. Brain. 2002;125:75-85.

BDNF produkce u pacientů s SM nestimulovaná MBP stimulovaná 100 200 300 400 500 600 700 800 900 1000 stabilní relaps remise BDNF hladiny (pg/mL) P < 0.01 BDNF Production in Patients With MS In RRMS, the production of BDNF appears to increase during periods of disease activity evidenced by clinical relapses. Sarchielli and colleagues recently studied the production of BDNF by PBMCs in 20 RRMS patients, 15 SPMS patients, and 20 matched control subjects.1 In the RRMS patients, BDNF values were significantly (P < 0.01) higher in the supernatants of PBMCs collected after the onset of relapse compared with the stable phase of the disease 3 months after relapse. These levels also remained higher during the following recovery phase.1 As shown on the slide, the mean BDNF levels in patients with MS during stable periods was 31.06 pg/mL ± 8.3 pg/mL (unstimulated) and 236.9 pg/mL ± 31.7 pg/mL (MBP stimulated); during relapse periods, it was 127.6 pg/mL ± 26.4 pg/mL (unstimulated) and 851.5 pg/mL ± 100 pg/mL (MBP stimulated); and during recovery periods, it was 79.7 pg/mL ± 20.9 pg/mL (unstimulated) and 644.9 pg/mL ± 46.3 pg/mL (MBP stimulated). The values come from Table 3 in the article by Sarchielli et al (2002).1 1. Sarchielli P, Greco L, Stipa A, Floridi A, Gallai V. Brain-derived neurotrophic factor in patients with multiple sclerosis. J Neuroimmunol. 2002;132:180-188. Adapted with permission from Sarchielli P et al. J Neuroimmunol. 2002;132:180-188

Aharoni, R. et al. PNAS. 2003;100(42)14157-14162. GA-specifické T buňky se koncentrují v mozku a produkují IL10, TGF beta a BDNF in situ BDNF IL-10 TGFß IFN Glatiramer acetate specific T cells accumulate in the brain and secrete interleukin 10 transforming growth factor beta and brain-derived neurotrophic factor (BDNF) in situ. Pictures come from figures 1-4 of Aharoni, R. et al. Glatiramer acetate-specific T cells in the brain express T helper 2/3 cytokines and brain-derived neurotrophic factor in situ. PNAS,2003;100(24):14157-14162. Study investigated whether GA-specific cells can function as suppressor cells with therapeutic potential in the target organ by in situ expression of T helper 2/3 cytokines and neurotrophic factors. A two-stage system of double flourescent labeling was employed: I. labeling of the GA specific T cell, before their adoptive transfer; II. Detection of the secreted factors by immunohistological methods after brain harvesting. This procedure was developed in order to detect the specific subset of GA specific T cells as well as their in situ secretion of cytokines and BDNF, on the level of whole brain tissue. Top left panel on BDNF comes from figure 1. Adoptively transferred GA-specific cells (see methods below) that have been accumulated in the brain expressed BDNF in situ. GA-specific cells expressing BDNF were observed in various regions in the brain, mainly in tissue surrounding the ventricles and in the perivascular cuffs (area shown in picture). Only marginal BDNF staining was observed within the CNS of control EAE-induced and normal mice into which no GA-specific cells were injected. Bottom left panel on IL-10 comes from figure 2. This in situ IL-10 expression by GA specific cells was clearly demonstrated on a single cell level in Hoechst-labeled (shown in picture) and PK-labeled (not shown) cells. Clusters of GA-labeled cells expressing IL-10 were abundant in various regions throughout the brain. Moreover, unlabeled cells within the vicinity of these clusters also expressed IL-10. These IL-10 positive cells had elongated astrocyte-like morphology consistent with activated microglia (confirmed with GFAP staining). Intense IL-10 secretion was observed only in brains with adoptively transferred GA cells, whereas control mice expressed low to moderate IL-10 levels. IL-10 expression in brains of EAE-induced mice was slightly higher than that observed in brains of normal mice. Top right panel on TGF comes from figure 3. Adoptively transferred GA-specific cells in the brain manifested extensive TGF-B expression with strong cytoplasmic and membranal staining of individual cells. The staining pattern was similar to the pattern observed for IL-10, including the positive staining of surrounding elongated cells of astrocyte morphology. In contrast to the strong TGF-B secretion in the brains with adoptively transferred GA cells, control mice expressed low to moderate TGF-B levels which were higher in brains in EAE-induced mice than those of normal mice. Bottom right panel on IFN- comes from figure 4. GA-specific cells that had been accumulated in the brain did not express IFN-y in situ. It was not possible to detect any IFN-y in the brains of mice injected with GA-labeled cells, neither in EAE-induced nor in normal mice. On the other hand, intense staining for IFN-y was found in corresponding brain regions of EAE-induced mice that were not injected with GA-specific cells. The presence of GA specific T cells in the brain 7 days after their injection to the periphery, as well as their ability to secrete anti-inflammatory cytokines and neurotrophic factor in the organ in which the pathological processes can occur, is of considerable therapeutic significance. A direct linkage can be drawn between the therapeutic activity of GA in EAE/MS and an in situ immunomodulatory effect, namely the anti-inflammatory bystander suppression and neuroprotection induced by GA specific T cells in the brain. Závěr: studie prokázala přítomnost GA specifických T-buněk v mozku a jejich schopnost produkovat protizánětlivé cytokiny a BDNF Aharoni, R. et al. PNAS. 2003;100(42)14157-14162.

COPAXONE reguluje zánětlivý proces a vytváří neuroprotekci Periferie CNS HEB MHC TCR antigen- presentující buňka makrofág TH 2 GA GA-terapie CNS Ag MHC microglie protizánětlivé- cytokiny supresorický efekt IL-4 IL-10 TCR GA- specifická T-buňka TH 1 TCR TH 2 Neurotrofní faktor neuroprotekce BDNF

Léčba zánětu GA léčba reguluje Th1-Th2 přesmyk IL-12 IL-4 TH1 TH2 GA GA GA terapie pro-zánětlivé IL-2 IL-12 IFN- proti-zánětlivé IL-4 IL-5 IL-10 IL-13 TGF- A wealth of data indicates that glatiramer acetate is presented as an antigen to generate GA-specific T cells. The GA-specific T cells are polarized in the Th2 direction (reviewed in 1 and 2). Thus, glatiramer acetate treatment results in the generation of GA-specific Th2-biased cells which elaborate anti-inflammatory cytokines.  1. Neuhaus O, Farina C, Wekerle H, Hohlfeld R. Mechanisms of action of glatiramer acetate in multiple sclerosis. Neurology 2001; 56:702-708. 2. Sela M, Teitelbaum D. Glatiramer acetate in the treatment of multiple sclerosis. Expert Opin Pharmacother 2001;2:1149-1165. Ziemssen T. Drugs in Focus, 2004