**5. Therapeutic strategies**

424 Amyotrophic Lateral Sclerosis

Our study shows that mRNAs of TNF- were expressed at higher level in lymphocytes of sporadic ALS patients than in controls but there was not relationship with site of disease onset (spinal or bulbar), disease duration at the time of blood sample withdrawal or disease severity. We suppose that mRNA level increase may be due to an ALS disease's point common, as oxidative stress involvement, in all patients analyzed (Carrì et al., 1997; Ciriolo et al., 2002). Interaction between TNF-, oxidative stress and SOD1 will be better described

Several data support the hypothesis that TNF- and SOD1 may not only take part to a common cellular pathway but they may also regulate directly and indirectly their self. In fact, an inverse correlation between the expression of SOD1 and TNF- has been described: cytotoxic effects of TNF- can be reduced by increasing levels of SOD1 as inhibition of SOD1 mRNA and protein may result in a decrease in the protective effect of SOD1 against

In 2006 Afonso and collaborators demonstrated that, in U937 cells, TNF- down-regulated SOD1 protein expression in a time-dependent manner (Alfonso et al., 2006). Afonso and colleagues performed different experiments treating U937 cells with TNF- (10 ng/ml) for 1, 4 , 24 hours and their data showed a decline of SOD1 mRNA at 1 hour (22%), maximal

Although SOD1 activity is modified in some specific situations, the direct effect of the proinflammatory cytokine TNF- on SOD1 promoter has not been reported. Variable results were reported regarding SOD1 regulation by TNF- (Chovolou et al.,2003), confirmed by gene expression studies that show the same tendency of SOD1 and TNF- and suggest that these two genes may have a common system, or, at least, they may take part to the same

As concern SOD1 and TNF-, it is well documented that both TNF- and SOD1 pathways are regulated by reactive oxygen species (ROS) concentration and we suppose that oxidative

The important role of ROS is reported in TNF- signalling although it is unclear whether the TNF- action may be producer or reducer of ROS concentration. In fact, TNF- has been reported to increase ROS production from electron transport in mitochondria, plasma membrane NADPH oxidase and cytosolic phospholipase A2-linked cascade through signal transduction pathways triggered by TNFR-related proteins (Chandel et al., 2001; Micheau et al., 2003; Woo et al., 2000). Multimerization of TNFRs may lead to recruitment of TRAFs (TNFR-associated factors) by the receptors resulting in activation of kinases and

About the reducer role, in a mice model it was demonstrated that TNF- stimulation in mice deficient in TNF receptor-associated factor 2 (TRAF2) or p65 NF-kB subunit did not induce ROS accumulation, indicating that TRAF-mediated NF-kB activation normally suppresses the TNF-induced ROS accumulation (Sakon et al., 2003). ROS in lower concentrations may function as second messengers in mediating TNF- activated signal transduction pathways

As concern ROS role, Scott and collaborators demonstrated that ROS may up-regulate TACE activity and consequently, this increased activity may change TNF- cleavage by TACE

that regulate the NF-kB system (Grisham et al.,1998; Janssen-Heininger et al.,2000).

in the next paragraph.

one.

(Scott et al., 2011).

**4.3 TNF- and SOD1 pathways** 

inflammation (Meier et al.,1989; Wong & Goeddel ,1988).

stress is a common regulation point thought NF-kB activation.

transcription factors, such as c-Jun and NF-kB (Chandel et al., 2001).

suppressor at 4 h (54%) and lesser at 24h (38%).

### **5.1 Classic immunotherapy in ALS**

Several trials both controlled and uncontrolled using immunomodulating agents have been conduced in patients with ALS. These have included plasma exchange, steroids, azathioprine, cyclophosphamide, recombinant human IFN, cyclosporine, immunoglobulin, glatiramer acetate, minocycline.

High-dose therapy with intravenous immunoglobulins was used in ALS, the rationale was strengthened by observations that Ig was effective in improving the muscle strength of patients with a paraproteinemic or conduction block polyneuropathy and also in other autoimmune neuromuscular disease. The authors (Meucci et al. 1996, Dalakas et al. 1994) concluded that IVIg had no apparent therapeutic role in improving the symptoms or arresting the progression in ALS patients (Meucci et al. 1996). Meucci et al included in the study seven patients with a diagnosis of definite or probable ALS according to El Escorial criteria. All patients were treated with intravenous infusions of IVIg 0,4 g/kg/die for 5 consecutive days, followed by monthly, two day infusions at the same daily dosage for 4 to 13 months. All patients were concomitantly treated with oral cyclophosphamide, 1-2 mg/kg/die, as this therapy is effective delaying the frequency of IVIg maintenance infusions in other diseases. The response to treatment was assessed by the Medical Research Council rating scale for muscle strength on ten muscles per limb, a clinical bulbar function scale, a modified Rankin disability scale. The effect of treatment on the progression of the disease was evaluated by comparing the monthly rate of progression of upper and lower limb muscle weakness before and during treatment. All patients continued to deteriorate during treatment, reflected by the worsening of scores after treatment compared with the scores before therapy. The monthly rate of progression of limb weakness during therapy

The Role of TNF-Alpha in ALS: New Hypotheses for Future Therapeutic Approaches 427

Glatiramer acetate (GA) is a synthetic copolymer composed of four amino acids, used in MS for reduction of the frequency of relapses. GA induces a wide variety of actions on T-cells and leads to generalized, antigen-non-specific alterations of various types of antigen presenting cells in such a way that they stimulate Th2-like responses. It blocks the release of TNF- and interleukin in monocytes and dendritic cells. So GA has neuroprotective as well as immunomodulatory actions. Meininger and collaborators (Meininger et al., 2009) recruited patients with El Escorial definite, probable or laboratory probable ALS of less than three years duration. Patients were given 40 mg GA daily for a period of 52 weeks. The prospectively defined primary efficacy outcome was the slope of ALSFRS score, the secondary efficacy outcome was time to death, tracheostomy or positive pressure ventilation more than 23 h per day. Additional functional endpoints included mean change from baseline and across visits in ALSFRS score, manuasl muscle testing score and slow vital capacity. GA was shown to be safe and well tolerated, the most significant adverse event was the injection site reaction. This study suggested that glatiramer acetate didn't show any

Minocycline has anti-apoptotic and anti-inflammatory effects in vitro in CNS, so several trials are planned or are in progress to assess whether minocycline slows human neurodegeneration. Gordon (Gordon et al., 2007) did a multicentre trial in which patients with diagnosis of ALS (according to El Escorial criteria) received minocycline escalating doses of up to 400 mg/day for 9 months (started at 100 mg twice per day and increased every week by 50 mg twice per day to the highest dose of 400 mg). The primary outcome measures was the changes in ALSFRS-r, the secondary outcome measures were forced vital capacity, manual muscle testing, quality of life, time of tracheostomy, chronic assisted ventilation, survival and safety. ALSFRS-r score deterioration was faster in the minocycline group of patients and greater mortality during the 9-months treatment phase was registered in the same group. Adverse events were most commonly reported in the respiratory system, gastrointestinal

system (nausea, diarrhoea, costipation), neurological system (dizziness, fatigue).

The discovery, in 1988, of a naturally occurring TNF- inhibitor in human urine (Seckinger et al., 1990), which was identified as a soluble form of the TNF-receptor that acted by neutralizing the cytokine, opened the way to immunotherapy. Subsequently two TNFbinding proteins were purified that were capable of inhibiting the binding of TNF- to cells (Engelmann et al., 1990). The identification of soluble TNF- receptors paved the way for the development of soluble TNF- receptors antibodies currently used for the treatment of several systemic inflammatory diseases, including rheumatoid arthritis, juvenile polyarticular rheumatoid arthritis, inflammatory bowel diseases, psoriatic arthritis and

There are three anti-TNF- agents approved for clinical use: Etancercept, Infliximab, Adalimumab. The latter two are full-length bivalent IgG monoclonal antibodies specific for sTNF and tmTNF, whereas Etanercept is a genetically engineered Fc fusion protein generated from the extracellular domain of human TNF-R2 and functions as a decoy receptor to block sTNF, tmTNF and distinct ligands of lymphotoxin, a TNF-related protein

The important side effects that have been most extensively related to TNF- inhibitors include: lymphoma (hepatosplenic T-cell lymphoma in young patients being treated for

beneficial effect in ALS patients either for course or survival.

**5.2 TNF- and new approaches in immunotherapy** 

ankylosing spondylitis (Sfikakis et al., 2010).

(Tracey et al., 2005).

was not better and possibly worse than that estimated in the period before therapy. No major side effect was reported by the patients.

Dalakas and collaborators (Dalakas et al. 1994) used intravenous infusions of high-dose immunoglobulin administered once a month for 3 months (total dose 2g/kg, divided into two daily doses) in nine ALS patients (El Escorial criteria) with a rapidly progressive course of disease. The efficacy of treatment was assessed by objective measurement of maximum voluntary isometric contraction in all muscle groups of two limbs or with Medical Research Council sores, before and after therapy. All patients worsened during the study and, by the end of the third month, their mean total muscle scores had declined. The pace of progression did not change during the observation period. All patients tolerated the intravenous immunoglobulin infusions well and adverse effects were noted.

Another approach was designed by Drachman lab's group (Drachman et al., 1994), who assessed a more powerful and prolonged immunosuppression obtained by total lymphoid irradiation (TLI) in ALS patients. The discovery that TLI produces powerful immunosuppression in humans led to its use in the treatment of autoimmune diseases. The basic principles of TLI therapy involve the lymphoid organs while shielding non-lymphoid tissues and delivering the radiation in multiple small fractions. The study included thirty patients with ALS. The radiation field consisted of an extended mantle, a para-aortic field and an inverted-Y including the spleen. Patients received anterior and posterior irradiation 5 days/week at a rate of 1,8 Gy/day. Blood counts were obtained 1 to 3 times/week as needed to detect haematological toxicity. Four types of parameters of motor function were evaluated: quantitative dynamometry (4 pairs of muscles in the upper extremities and 5 pairs of muscles in the lower extremities), manual muscle testing, functional tests (swallowing, breathing), activity indexes. Tests of immune function were: leukocytes (absolute lymphocyte counts, decrease in CD4 cells, CD4/CD8 ratio), cell-mediated immunity (negative conversion of skin tests), humoral immunity (tetanus antibody response). To assess whether the effectiveness of immunosuppression had an influence on the course of ALS, they analysed the relationships between parameters of immunosuppression and the measures of progression of ALS. This analysis showed that evidence of more effective immunosuppression did not correlate with a more favourable disease course.

Another immunotherapy tried in ALS was liquorpheresis (Andrich et al. 1996; Finsterer et al., 1999) with no results both in sporadic and familial ALS.

IFNs alpha and beta cytokines can regulate the major histocompatibility complex and the presentation of antigens to T-cell receptors. They have been used in variable doses for up to 6 months in small trials in ALS patients with negative results, however the small sample size, the possibility inadequate dose and the short period of follow-up prevented definite conclusions about the efficacy of IFNs. For these reasons a study was undertaken in which recombinant IFNbeta-1a was used in a large patient population at a dose twice as large as that found to be effective in patient with MS. Beghi et al. (2000) recruited patients with 6 to 24 months history of confirmed ALS, that received 12 mIU of IFN subcutaneously three times a week for 6 months and were followed up for an additional 6 months. Medical Research Council scale, Norris scale, bulbar scores were used to assess disability; selected electrophysiologic measures were also used. There were no significant differences of disease progression and disability in patients treated with IFN. Common adverse events were flulike syndrome, local erythema, gastrointestinal symptomps.

was not better and possibly worse than that estimated in the period before therapy. No

Dalakas and collaborators (Dalakas et al. 1994) used intravenous infusions of high-dose immunoglobulin administered once a month for 3 months (total dose 2g/kg, divided into two daily doses) in nine ALS patients (El Escorial criteria) with a rapidly progressive course of disease. The efficacy of treatment was assessed by objective measurement of maximum voluntary isometric contraction in all muscle groups of two limbs or with Medical Research Council sores, before and after therapy. All patients worsened during the study and, by the end of the third month, their mean total muscle scores had declined. The pace of progression did not change during the observation period. All patients tolerated the intravenous

Another approach was designed by Drachman lab's group (Drachman et al., 1994), who assessed a more powerful and prolonged immunosuppression obtained by total lymphoid irradiation (TLI) in ALS patients. The discovery that TLI produces powerful immunosuppression in humans led to its use in the treatment of autoimmune diseases. The basic principles of TLI therapy involve the lymphoid organs while shielding non-lymphoid tissues and delivering the radiation in multiple small fractions. The study included thirty patients with ALS. The radiation field consisted of an extended mantle, a para-aortic field and an inverted-Y including the spleen. Patients received anterior and posterior irradiation 5 days/week at a rate of 1,8 Gy/day. Blood counts were obtained 1 to 3 times/week as needed to detect haematological toxicity. Four types of parameters of motor function were evaluated: quantitative dynamometry (4 pairs of muscles in the upper extremities and 5 pairs of muscles in the lower extremities), manual muscle testing, functional tests (swallowing, breathing), activity indexes. Tests of immune function were: leukocytes (absolute lymphocyte counts, decrease in CD4 cells, CD4/CD8 ratio), cell-mediated immunity (negative conversion of skin tests), humoral immunity (tetanus antibody response). To assess whether the effectiveness of immunosuppression had an influence on the course of ALS, they analysed the relationships between parameters of immunosuppression and the measures of progression of ALS. This analysis showed that evidence of more effective immunosuppression did not correlate with a more favourable

Another immunotherapy tried in ALS was liquorpheresis (Andrich et al. 1996; Finsterer et

IFNs alpha and beta cytokines can regulate the major histocompatibility complex and the presentation of antigens to T-cell receptors. They have been used in variable doses for up to 6 months in small trials in ALS patients with negative results, however the small sample size, the possibility inadequate dose and the short period of follow-up prevented definite conclusions about the efficacy of IFNs. For these reasons a study was undertaken in which recombinant IFNbeta-1a was used in a large patient population at a dose twice as large as that found to be effective in patient with MS. Beghi et al. (2000) recruited patients with 6 to 24 months history of confirmed ALS, that received 12 mIU of IFN subcutaneously three times a week for 6 months and were followed up for an additional 6 months. Medical Research Council scale, Norris scale, bulbar scores were used to assess disability; selected electrophysiologic measures were also used. There were no significant differences of disease progression and disability in patients treated with IFN. Common adverse events were flu-

major side effect was reported by the patients.

disease course.

immunoglobulin infusions well and adverse effects were noted.

al., 1999) with no results both in sporadic and familial ALS.

like syndrome, local erythema, gastrointestinal symptomps.

Glatiramer acetate (GA) is a synthetic copolymer composed of four amino acids, used in MS for reduction of the frequency of relapses. GA induces a wide variety of actions on T-cells and leads to generalized, antigen-non-specific alterations of various types of antigen presenting cells in such a way that they stimulate Th2-like responses. It blocks the release of TNF- and interleukin in monocytes and dendritic cells. So GA has neuroprotective as well as immunomodulatory actions. Meininger and collaborators (Meininger et al., 2009) recruited patients with El Escorial definite, probable or laboratory probable ALS of less than three years duration. Patients were given 40 mg GA daily for a period of 52 weeks. The prospectively defined primary efficacy outcome was the slope of ALSFRS score, the secondary efficacy outcome was time to death, tracheostomy or positive pressure ventilation more than 23 h per day. Additional functional endpoints included mean change from baseline and across visits in ALSFRS score, manuasl muscle testing score and slow vital capacity. GA was shown to be safe and well tolerated, the most significant adverse event was the injection site reaction. This study suggested that glatiramer acetate didn't show any beneficial effect in ALS patients either for course or survival.

Minocycline has anti-apoptotic and anti-inflammatory effects in vitro in CNS, so several trials are planned or are in progress to assess whether minocycline slows human neurodegeneration. Gordon (Gordon et al., 2007) did a multicentre trial in which patients with diagnosis of ALS (according to El Escorial criteria) received minocycline escalating doses of up to 400 mg/day for 9 months (started at 100 mg twice per day and increased every week by 50 mg twice per day to the highest dose of 400 mg). The primary outcome measures was the changes in ALSFRS-r, the secondary outcome measures were forced vital capacity, manual muscle testing, quality of life, time of tracheostomy, chronic assisted ventilation, survival and safety. ALSFRS-r score deterioration was faster in the minocycline group of patients and greater mortality during the 9-months treatment phase was registered in the same group.

Adverse events were most commonly reported in the respiratory system, gastrointestinal system (nausea, diarrhoea, costipation), neurological system (dizziness, fatigue).

### **5.2 TNF- and new approaches in immunotherapy**

The discovery, in 1988, of a naturally occurring TNF- inhibitor in human urine (Seckinger et al., 1990), which was identified as a soluble form of the TNF-receptor that acted by neutralizing the cytokine, opened the way to immunotherapy. Subsequently two TNFbinding proteins were purified that were capable of inhibiting the binding of TNF- to cells (Engelmann et al., 1990). The identification of soluble TNF- receptors paved the way for the development of soluble TNF- receptors antibodies currently used for the treatment of several systemic inflammatory diseases, including rheumatoid arthritis, juvenile polyarticular rheumatoid arthritis, inflammatory bowel diseases, psoriatic arthritis and ankylosing spondylitis (Sfikakis et al., 2010).

There are three anti-TNF- agents approved for clinical use: Etancercept, Infliximab, Adalimumab. The latter two are full-length bivalent IgG monoclonal antibodies specific for sTNF and tmTNF, whereas Etanercept is a genetically engineered Fc fusion protein generated from the extracellular domain of human TNF-R2 and functions as a decoy receptor to block sTNF, tmTNF and distinct ligands of lymphotoxin, a TNF-related protein (Tracey et al., 2005).

The important side effects that have been most extensively related to TNF- inhibitors include: lymphoma (hepatosplenic T-cell lymphoma in young patients being treated for

The Role of TNF-Alpha in ALS: New Hypotheses for Future Therapeutic Approaches 429

elaborate and promising data collected thus far to assign function to TNF- in neurodegeneration, surprisingly little is still known about the cellular and stage-specific

The data reported in this chapter also underline the importance of TNF- pathways in ALS

In fact, the data that demonstrated the down-regulation of SOD1 after treatment with TNF- (Afonso et al., 2006), related to the up-regulation of SOD1 mRNA expression in ALS patients suggest to carry on the studies about TNF- in ALS disease to better define the TNF function in neurodegeneration. A better understanding of SOD1 regulation related to TNF-

We are grateful to "our" ALS patients and their families that trust our work. Moreover we would like to thank National and International Research Agencies and Italian Health

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Aisen P.S., K.L. Davis, J.D. Berg, K. Schafer, K. Campbell, R.G. Thomas, M.F. Weiner, M.R.

Arnett H.A., J. Mason, M. Marino, K. Suzuki, G.K. Matsushima, J.P. Ting. (2001). TNF-alpha

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R.G. Thomas, L.J. Thal. (2003). Effects of rofecoxib or naproxen vs placebo on Alzheimer disease progression: a randomized controlled trial. JAMA, 289, 2819-

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promotes proligeration of oligdendrocyte progenitors and remyelination. Nat

(Sep 2009). Activation of caspase-8 by tumour necrosis factor receptor 1 is necessary for caspase-3 activation and apoptosis in oxygen-glucose deprived cultured cortical

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(Jun 1996) 27;334(26):1717-25.

2008 Sep 22.

function may permit to develop novel immunotherapy application in ALS disease.

roles of this cytokine.

**7. Acknowledgement** 

**8. References** 

2826.

Minister that found our research projects.

Neurosci, 4, 1116-1122.

pathology due to the interaction with SOD1 gene.

Chron disease and ulcerative colitis), infections (fungal infections such as histoplasmosis, coccidioidomycosis, blastomycosis and tuberculosis), congestive heart failure, demyelinating disease, a lupus-like syndrome, induction of auto-antibodies, injection site reactions and systemic side effects (Scheinfeld et al., 2006).

Clinical trials examining the effects of TNF- inhibition have been conducted on patients with Multiple Sclerosis (MS) and Alzheimer disease (AD).

Strategies to inhibit TNF- in MS seemed promising in preclinical applications but have widely failed in human clinical trials due to the lack of therapeutic selectivity. During an open-label phase I trial, a monoclonal TNF- antibody was infused into two human patients exhibiting rapidly progressing disease. Subsequently, in a double-blinded, placebo controlled, multicentered phase II study, 168 relapsing-remitting MS patients were administered Lenercept, a sTNF-R1 fusion protein that neutralizes TNF-. Lenercept-treated individuals experienced higher occurrence of relapse and increased neurological deficits (Van oosten et al., 1996). The ineffectiveness of anti-TNF- therapy in MS may be a consequence of divergent roles for the TNF receptors, considering that blocking TNF-R1 in mouse models dampens disease severity, while suppressing TNF-R2, the receptor that induces remyelination and harbors immunosuppressive properties, results in exacerbated disease (Arnett et al., 2001; Kassiotis et al., 2011). Recently, pharmacological agents selectively targeting TNF-R1 have been investigated. Using phage display technology, a TNF-R1 antagonist was developed and upon evaluation in mice it was found that administration of this selective antagonist improved clinical scores, reduced cerebral demyelination and suppressed the number of infiltrating inflammatory cells (Nomura et al., 2011).

TNF- intervention in AD has been evaluated in open-labeled phase I clinical trials where perispinal extrathecal administration of Etanercept was administered weekly to a small number of patients ranging from mild to severe AD for a short duration of 6 months that claimed substantial cognitive and behavioural improvements, including verbal fluency and aphasia (Tobinick et al., 2006; Tobinick et al., 2008). Currently a phase II study is recruiting to evaluate the safety and tolerability of Etanercept in AD. These results seem promising but conclusions regarding the promise of such a therapeutic strategy should be reserved until after extensive chronic suppression of TNF- activity is performed in preclinical models and double-blind human clinical trials have been conducted and results critically reviewed by the research community.

Trails on the immunological hypothesis in ALS are not yet established although TNF system implications have been described for a long time. This therapeutic approach was not considered using neither synthetic TNF--receptor inhibitors nor monoclonal anti-TNF antibody.
