**4. Potential therapeutics targeted at the kynurenine pawthay for ALS**

In 1995, riluzole became the first drug, and remains the only drug, approved by the FDA (USA) for treatment of ALS. The approval was based on two large placebo controlled clinical studies where riluzole decreased the rate of muscle deterioration and modestly improved the survival rate of ALS patients (Bensimon *et al.* 1994; Lacomblez *et al.* 1996). Though the precise mechanism of riluzole remains unclear, it appears to interfere with excitatory amino acid signalling, perhaps through the inhibition of glutamate release (Mizoule *et al.* 1985; Cheramy *et al.* 1992; Martin *et al.* 1993), blockade of inactivated sodium channels (Benoit and Escande 1991) and interaction with guanosine triphosphate (GTP)-binding proteins (Doble *et al.* 1992). 16 years on, there is still a lack of effective treatment available and an intense search is on going to discover better treatments for ALS.

In developing therapeutic agents aimed at modulating the kynurenine pathway, two approaches may be taken: (1) to develop analogues of the neuroprotective kynurenines; (2) to inhibit the synthesis of the neurotoxic QUIN. Figure 4 summarizes the drugs targeting the kynurenine pathway that could be potential candidates for ALS.

### **4.1 IDO inhibitors**

As the first enzyme in the kynurenine pathway, suppression of IDO would lead to decrease QUIN production. Although it has not been specifically tested in neurodegenerative disorders, it is a novel therapeutic target in cancer research and the results have been positive. Using transgenic mouse model of breast cancer, IDO-1 inhibitors, 1-MT and methyl-thiohydantoin-tryptophan, were able to potentiate the efficacy of chemotherapy drugs, promoting tumour regression without increasing the side effects (Muller *et al.* 2005).

### **4.2 4-chlorokynurenine**

QUIN neurotoxicity can be prevented by blocking the glycine modulatory site of the NMDA receptor (Foster *et al.* 1990; Hartley *et al.* 1990). 7-chlorokynurenate, a synthetic derivative of KYNA, is such an NMDA receptor antagonist (Kemp *et al.* 1988) but has difficulty crossing the BBB (Rao *et al.* 1993). On the other hand, its precursor, 4-chlorokynurenine, is rapidly transported across the BBB (Hokari *et al.* 1996). Intracerebral and intraperitoneal administration of 4-chlorokynurenine with QUIN showed successful enzymatic transamination of 4-chlorokynurenine into the neuroprotective 7-chlorokynurenate (Wu *et al.* 1997; Wu *et al.* 2000).

The Kynurenine Pathway 365

1999) and prevents Th1 cell activation while promoting Th2 cell differentiation (Dimitrova *et al.* 2002). The exact mechanism of action is still unclear though it has been shown to

In 1998, leflunomide was approved by the FDA (USA) for the treatment of rheumatoid arthritis. Leflunomide has also been successful in inhibiting disease progression in animal models of autoimmune diseases, such as experimental autoimmune neuritis (Ogawa *et al.* 1990), EAE (Bartlett *et al.* 1993) and experimental myasthenia gravis (Vidic-Dankovic *et al.* 1995). In a phase II trial recently, teriflunomide proved to be well tolerated and effective in reducing active lesions in patients with relapsing MS

Tranilast (Rizaben®) is a synthetic anthranilic acid derivative drug with several inhibitory actions. It has the ability to inhibit the release of chemical mediators, such as histamine, during hypersensitivity reactions and from mast cells and also suppresses the release of TGF-β and inhibits angiogenesis (Suzawa *et al.* 1992; Isaji *et al.* 1997). Thus, it is effective against many diseases, including allergic rhinitis, atopic dermatitis, bronchial asthma, hypertrophic scar formation and keloid. Recently, tranilast showed promising results against EAE, shifting the cytokine profile towards favouring Th2 cells, inhibiting the actions of Th1 cells and promoting the generation of IL-10 producing Th2 cells, an effect similar to

The synthesis of QUIN can also be blocked by inhibiting either KYNU or KMO activity, thus diverting the kynurenine pathway towards the synthesis of KYNA. Nicotinylalanine is one such agent (Decker *et al.* 1963). When administered together with probenecid (to allow for the accumulation of KYNA by inhibiting the organic acid transport system), nicotinylalanine increased the amount of KYNA produced in the brain and protected the brain from induced seizures (Connick *et al.* 1992; Russi *et al.* 1992) and QUIN induced striatal damage (Harris *et* 

Another alanine derivative capable of inhibiting KMO is meta-nitrobenzoylalanine (Pellicciari *et al.* 1994). The inhibition of KMO results in an increase in brain KYN and KYNA, which is associated with sedation and anticonvulsant effects (Chiarugi and Moroni 1999) and reduction in neuronal loss from brain ischemia (Cozzi *et al.* 1999). In immune activated mice, meta-nitrobenzoylalanine also significantly reduced the formation of QUIN

Ro61-8048 (3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl] benzenesulfon-amide) is another potent KMO inhibitor (Rover *et al.* 1997). In addition to raising brain KYNA level, Ro61-8048 also reduces glutamate concentration in the extracellular spaces of the basal ganglia in rats without impairing the learning or memory process typically associated with glutamate receptor antagonists (Moroni *et al.* 2005). In rats with EAE, administration of Ro61-8048 significantly reduces the neurotoxic levels of 3-hydroxykynurenine and QUIN in the CNS (Chiarugi *et al.* 2001). Like meta-nitrobenzoylalanine, Ro61-8048 also decreases neuronal loss

attenuate EAE independent of pyrimidine depletion (Korn *et al.* 2004).

(O'Connor *et al.* 2006).

**4.6 Alanine derivatives** 

*al.* 1998).

**4.7 Ro61-8048** 

that of natural TRP catabolites (Platten *et al.* 2005).

in the periphery and CNS (Chiarugi and Moroni 1999).

due to brain ischemia (Cozzi *et al.* 1999).

**4.5 Tranilast** 

Fig. 4. Potential drug candidates targeting the kynurenine pathway for ALS. 1-MT, methylthiohydantoin-tryptophan, nicotinylalanine, meta-nitrobenzoylalanine and Ro61-8048 are kynurenine pathway inhibitors, while 4-chlorokynurenine, laquinimod, leflunomide, teriflunomide and tranilast are analogues of kynurenines.

### **4.3 Laquinimod**

Laquinimod (ABR-215062) is a novel synthetic quinoline with high oral bioavailability. In preclinical trials, the compound exhibited immunomodulatory properties without immunosuppression (Brunmark *et al.* 2002; Zou *et al.* 2002; Yang *et al.* 2004). In rats with experimental autoimmune encephalomyelitis (EAE), a widely used animal model for MS, laquinimod inhibited disease progression and infiltration of CD4+ T cells and macrophages into the CNS (Yang *et al.* 2004). It also shifted the cytokine profile towards Th2/Th3 cytokines IL-4, IL-10 and transforming growth factor β (TGF-β) (Yang *et al.* 2004). Furthermore, laquinimod is able to act synergistically with IFN-β, though the mechanism of action is currently unknown but is independent of IFN-β (Runstrom *et al.* 2006). In addition, laquinimod has also successfully reduced the development of active lesions in patients with relapsing MS (Polman *et al.* 2005).

### **4.4 Leflunomide**

Leflunomide (Avara®) is an immunosuppressive and anti-inflammatory pro-drug, which is converted *in vivo* to its active open-ring metabolite, teriflunomide (A771726), an inhibitor of mitochondrial dihydroorotate dehydrogenase, an essential enzyme for *de novo* pyrimidine synthesis (Williamson *et al.* 1995). Leflunomide is a potent inhibitor of the nuclear factor *kappa*-light-chain-enhancer of activated B cells (NF-κB) activation (Manna and Aggarwal

Fig. 4. Potential drug candidates targeting the kynurenine pathway for ALS. 1-MT, methylthiohydantoin-tryptophan, nicotinylalanine, meta-nitrobenzoylalanine and Ro61-8048 are kynurenine pathway inhibitors, while 4-chlorokynurenine, laquinimod, leflunomide,

Laquinimod (ABR-215062) is a novel synthetic quinoline with high oral bioavailability. In preclinical trials, the compound exhibited immunomodulatory properties without immunosuppression (Brunmark *et al.* 2002; Zou *et al.* 2002; Yang *et al.* 2004). In rats with experimental autoimmune encephalomyelitis (EAE), a widely used animal model for MS, laquinimod inhibited disease progression and infiltration of CD4+ T cells and macrophages into the CNS (Yang *et al.* 2004). It also shifted the cytokine profile towards Th2/Th3 cytokines IL-4, IL-10 and transforming growth factor β (TGF-β) (Yang *et al.* 2004). Furthermore, laquinimod is able to act synergistically with IFN-β, though the mechanism of action is currently unknown but is independent of IFN-β (Runstrom *et al.* 2006). In addition, laquinimod has also successfully reduced the development of active lesions in patients with

Leflunomide (Avara®) is an immunosuppressive and anti-inflammatory pro-drug, which is converted *in vivo* to its active open-ring metabolite, teriflunomide (A771726), an inhibitor of mitochondrial dihydroorotate dehydrogenase, an essential enzyme for *de novo* pyrimidine synthesis (Williamson *et al.* 1995). Leflunomide is a potent inhibitor of the nuclear factor *kappa*-light-chain-enhancer of activated B cells (NF-κB) activation (Manna and Aggarwal

teriflunomide and tranilast are analogues of kynurenines.

**4.3 Laquinimod** 

**4.4 Leflunomide** 

relapsing MS (Polman *et al.* 2005).

1999) and prevents Th1 cell activation while promoting Th2 cell differentiation (Dimitrova *et al.* 2002). The exact mechanism of action is still unclear though it has been shown to attenuate EAE independent of pyrimidine depletion (Korn *et al.* 2004).

In 1998, leflunomide was approved by the FDA (USA) for the treatment of rheumatoid arthritis. Leflunomide has also been successful in inhibiting disease progression in animal models of autoimmune diseases, such as experimental autoimmune neuritis (Ogawa *et al.* 1990), EAE (Bartlett *et al.* 1993) and experimental myasthenia gravis (Vidic-Dankovic *et al.* 1995). In a phase II trial recently, teriflunomide proved to be well tolerated and effective in reducing active lesions in patients with relapsing MS (O'Connor *et al.* 2006).

### **4.5 Tranilast**

Tranilast (Rizaben®) is a synthetic anthranilic acid derivative drug with several inhibitory actions. It has the ability to inhibit the release of chemical mediators, such as histamine, during hypersensitivity reactions and from mast cells and also suppresses the release of TGF-β and inhibits angiogenesis (Suzawa *et al.* 1992; Isaji *et al.* 1997). Thus, it is effective against many diseases, including allergic rhinitis, atopic dermatitis, bronchial asthma, hypertrophic scar formation and keloid. Recently, tranilast showed promising results against EAE, shifting the cytokine profile towards favouring Th2 cells, inhibiting the actions of Th1 cells and promoting the generation of IL-10 producing Th2 cells, an effect similar to that of natural TRP catabolites (Platten *et al.* 2005).

### **4.6 Alanine derivatives**

The synthesis of QUIN can also be blocked by inhibiting either KYNU or KMO activity, thus diverting the kynurenine pathway towards the synthesis of KYNA. Nicotinylalanine is one such agent (Decker *et al.* 1963). When administered together with probenecid (to allow for the accumulation of KYNA by inhibiting the organic acid transport system), nicotinylalanine increased the amount of KYNA produced in the brain and protected the brain from induced seizures (Connick *et al.* 1992; Russi *et al.* 1992) and QUIN induced striatal damage (Harris *et al.* 1998).

Another alanine derivative capable of inhibiting KMO is meta-nitrobenzoylalanine (Pellicciari *et al.* 1994). The inhibition of KMO results in an increase in brain KYN and KYNA, which is associated with sedation and anticonvulsant effects (Chiarugi and Moroni 1999) and reduction in neuronal loss from brain ischemia (Cozzi *et al.* 1999). In immune activated mice, meta-nitrobenzoylalanine also significantly reduced the formation of QUIN in the periphery and CNS (Chiarugi and Moroni 1999).

### **4.7 Ro61-8048**

Ro61-8048 (3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl] benzenesulfon-amide) is another potent KMO inhibitor (Rover *et al.* 1997). In addition to raising brain KYNA level, Ro61-8048 also reduces glutamate concentration in the extracellular spaces of the basal ganglia in rats without impairing the learning or memory process typically associated with glutamate receptor antagonists (Moroni *et al.* 2005). In rats with EAE, administration of Ro61-8048 significantly reduces the neurotoxic levels of 3-hydroxykynurenine and QUIN in the CNS (Chiarugi *et al.* 2001). Like meta-nitrobenzoylalanine, Ro61-8048 also decreases neuronal loss due to brain ischemia (Cozzi *et al.* 1999).

The Kynurenine Pathway 367

Ball, H. J., A. Sanchez-Perez*, et al.* (2007). Characterization of an indoleamine 2,3 dioxygenase-like protein found in humans and mice. *Gene* 396(1): 203-13. Bartlett, R. R., H. Anagnostopulos*, et al.* (1993). Effects of leflunomide on immune responses and models of inflammation. *Springer Semin Immunopathol* 14(4): 381-94. Baverel, G., G. Martin*, et al.* (1990). Glutamine synthesis from aspartate in guinea-pig renal

Bendotti, C., N. Calvaresi*, et al.* (2001). Early vacuolization and mitochondrial damage in

Bensimon, G., L. Lacomblez*, et al.* (1994). A controlled trial of riluzole in amyotrophic lateral

Bi, M., C. Naczki*, et al.* (2005). ER stress-regulated translation increases tolerance to extreme

sclerosis. ALS/Riluzole Study Group. *N Engl J Med* 330(9): 585-91.

hypoxia and promotes tumor growth. *Embo J* 24(19): 3470-81.

cytochrome oxidase histochemical reactivity. *J Neurol Sci* 191(1-2): 25-33. Benoit, E. and D. Escande (1991). Riluzole specifically blocks inactivated Na channels in

myelinated nerve fibre. *Pflugers Arch* 419(6): 603-9.

motor neurons of FALS mice are not associated with apoptosis or with changes in

CNSCentral nervous system CSFCerebrospinal fluid

GTPGuanosine triphosphate HDHuntington's disease

IFN-γInterferon gamma

ILInterleukin

KYNKynurenine KYNAKynurenic acid

HIVHuman immunodeficiency virus IDOIndoleamine 2,3-dioxygenase

KMOKynurenine 3-monooxygenase

mRNAMessenger ribonucleic acid

NMDA*N*-methyl *D*-aspartate

NRNMDA receptor PICPicolinic acid QUINQuinolinic acid ROSReactive oxygen species SOD1Superoxide dismutase 1 TDOTryptophan 2,3-dioxygenase TGF-βtransforming growth factor β

TRPTryptophan

**7. References** 

MCPMonocyte chemoattractant protein MIPMacrophage inflammatory protein MPTPMethyl-4-phenyl-1,2,3,6-tetra-pyridine

cortex. *Biochem J* 268(2): 437-42.

NF-κBNuclear factor *kappa*-light-chain-enhancer of activated *B* cells

D-1-MTD-1-methyl-tryptophan

EAEExperimental autoimmune encephalomyelitis GCN2General control non-derepressible-2 kinase
