In the June 10, 2011 edition of Cell, there is a Leading Edge Preview (short review) and a research Article on a surprising new potential therapeutic strategy for neurodegenerative disease. The Preview is by Peter H Reinhart (Proteostasis Therapeutics, Cambridge MA) and Jeffery W Kelly (Skaggs Institute and Scripps Research Institute, La Jolla CA), and the the Article (Zwilling et al.) is by Paul J Muchowski (Gladstone Institute of Neurological Disease, University of California at San Francisco) and his colleagues. In addition to Dr. Muchowski’s academic collaborators, researchers from the Novartis Institutes for BioMedical Research in Basel, Switzerland participated in that work.
In previous studies, the kynurenine pathway (KP) of tryptophan degradation has been linked to such neurodegenerative diseases as Huntington’s disease (HD) and Alzheimer’s disease (AD). The kynurenine pathway (KP) is the most important pathway for degradation of the amino acid tryptophan in humans. Patients with HD and AD have elevated levels of two metabolites in the KP–quinolinic acid (QUIN) and 3-hydroxykynurenine (3-HK)–in their blood and brains. Studies in rodents have implicated both of these metabolites in pathophysiological processes in the brain. QUIN, which is a selective N-methyl-D-aspartate (NMDA) agonist, has been implicated in excitotoxicity, which is a mechanism by which excessive stimulation of glutamate receptors causes neuronal dysfunction and cell death. (NMDA receptors constitute a major type of glutamate receptor.) 3-HK is a free radical generator that can mediate neuronal cell death. Intrastriatal injection of QUIN in experimental animals duplicated many of the pathological features of HD. Administration of QUIN to other areas of the brain of experimental animals also duplicated features of AD, such as destruction of basal forebrain cholinergic neurons projecting to the cortex and memory deficits.
In contrast, kynurenic acid (KYNA), which is formed in a side arm of the KP by conversion of kynurenine by the enzyme kynurenine aminotransferase, appears to be neuroprotective. KYNA is an antagonist of ionotropic excitatory amino acid receptors. In particular, KYNA blocks the neuropathological effects of QUIN. Kynurenine aminotransferase is found in the brain, and is thus capable of transforming kynurenine (which is actively transported into the brain by a neutral amino acid transporter) to KYNA in that organ. The concentration of brain KYNA is often decreased in HD and AD.
All of these studies in rodents were done in the 1980s or 1990s. However, no therapies based on that research have yet been advanced into the clinic.
Studies with JM6, a prodrug small-molecule inhibitor of kynurenine 3-monooxygenase, in wild type mice
In the Zwilling et al. study, researchers studied inhibition of kynurenine 3-monooxygenase (KMO) as a strategy for inducing a more favorable ratio of KYNA to QUIN in vivo. KMO is the enzyme in the KP that converts kynurenine to 3-hydroxykynurenine, which is further converted in three steps to QUIN. KMO is found at high levels in peripheral blood macrophages and other immune cells in the blood. Inhibition of KMO results in elevation of kynurenine levels in the blood. This kynurenine can then enter the CNS, where it is converted to the neuroprotective metabolite KYNA.
In 1996, researchers at Roche published the synthesis and characterization of a KMO inhibitor, 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]benzenesulfonamide, known as Ro 61-8048. Subsequent studies showed that Ro 61-8048 was neuroprotective in rodent models of brain ischemia and cerebral malaria, and in a model of levodopa-induced dyskinesias (movement disorders) in parkinsonian monkeys.
However, Zwilling et al found that Ro 61-8048 was metabolically unstable. They therefore developed an orally bioavailable “slow-release” prodrug of Ro 61-8048, 2-(3,4-dimethoxybenzenesulfonylamino)-4-(3-nitrophenyl)-5-(piperidin-1-yl)methylthiazole (JM6). JM6 was designed to be converted to Ro 61-8048 in the gut. However, when JM6 was administered orally to wild type mice, the researchers found high levels of both JM6 and Ro 61-8048 in the blood. However, neither JM6 nor Ro 61-8048 accumulated to any great extent in the brain, and the brain concentration of both drugs was insufficient to inhibit KMO. Thus neither JM6 nor Ro 61-8048 appeared to cross the blood-brain barrier.
Despite the failure of JM6 and Ro 61-8048 to cross the blood-brain barrier, oral administration of JM6 results in increased brain levels of KYNA. Administration of an inhibitor of kynurenine aminotransferase to the brain inhibits the increase in levels of KYNA in that organ. This is consistent with the hypothesis that Ro 61-8048 inhibition of KMO in the blood results in elevated blood levels of kynurenine, which is transported into the brain. Kynurenine aminotransferase in the brain converts the kynurenine to KYNA. In addition, elevation of KNYA levels in the brain coincides with a decrease in extracellular concentrations of brain glutamate. This is consistent with earlier studies that showed that increases in brain KYNA, via inhibition of presynaptic α7 nicotinic receptors, reduce extracellular brain glutamate levels. Blocking of presynaptic α7 nicotinic receptors results in inhibition of glutamate release from neurons that bear these receptors. Reduction in extracellular brain glutamate levels may be responsible for KYNA’s neuroprotective effects, via reduction of excitotoxicity. However, at high local concentrations of KYNA, this metabolite may also block glutamate receptors directly.
Studies with JM6 in mouse models of Alzheimer’s and Huntington’s disease
After performing these studies in wild type mice, the researchers then tested the effects of JM6 in mouse models of AD and HD. Transgenic J20 mice that express a mutant form of the human amyloid precursor protein (hAPP) develop spatial memory deficits and synaptic loss starting at 4-5 months of age. Oral administration of JM6 starting at 2 months of age gave significant improvement in spatial memory in mice tested at 6 months of age, as compared to untreated J20 APPtg (transgenic APP) mice. JM6 treatment also prevented synaptic loss in J20 APPtg mice. However, JM6 treatment had no effect on beta amyloid (Aβ) plaque load, which was increased in the hippocampus and cortex of JM6-treated and untreated J20 mice. Under the amyloid hypothesis of AD pathogenesis, Aβ plaques are central to the causation of AD.
J20 APPtg mice had lower brain KYNA levels than wild type littermate controls, consistent with findings in AD patients. Treatment of J20 APPtg mice with oral JM6 (over a 120 day period) increased brain and plasma levels of KYNA. KMO activity, and 3-HK and QUIN levels in the brains and QUIN levels in the plasma of J20 APPtg mice treated with JM6 were not significantly different from levels in wild type controls.
The researchers also tested the effects of oral JM6 administration in R6/2 mice, the best characterized mouse model of HD. HD is a trinucleotide repeat disorder in which a cytosine-adenine-guanine (CAG) repeat segment in exon one of the huntingtin gene (HTT) (encoded in the germline of the individual) exceeds a normal range. The HD CAG repeat region encodes 36 or greater repeated glutamines in the polyQ region of the huntingtin protein (Htt); people without the disease have fewer than 36 glutamines. The disease-associated huntingtin protein is neurotoxic.
In the R6/2 mouse model, mice are transgenic for the 5′ end of the human HD gene carrying a large CAG repeat expansion. These mice develop a progressive neurologic disease, including motor deficits, weight loss, and premature death. The researchers started oral JM6 administration at 4 weeks of age, which is an early symptomatic stage in R6/2 mice. JM6 administration had a dramatic dose-dependent effect on survival. JM6 treatment did not affect the weight of the mice, but it did modestly improve performance on an accelerating rotarod (a measure of motor performance). JM6 treatment also prevented synaptic loss and reduced CNS inflammation in R6/2 mice.
JM6 treatment of R6/2 mice did not influence the size or abundance of neuronal inclusion bodies in these mice. These inclusion bodes are related to those seen in HD in humans. Thus in mouse models of both AD and HD, JM6 treatment did not affect the aggregated proteins (Aβ plaques and mutated Htt inclusion bodies, respectively) that are thought to cause the diseases; nevertheless, they ameliorated disease symptoms.
In chronically JM6-treated J20 APPtg (AD model) and R6/2 (HD model) mice, although JM6 and Ro 61-8048 accumulated in plasma, brain levels of these compounds were nil. Thus JM6 treatment of both neurodegenerative disease models resulted in increased brain levels of KYNA and neurodegenerative disease amelioration, despite the inability of JM6 and R0 61-8048 to cross the blood-brain barrier.
JM6 treatment is a surprising therapeutic strategy for neurodegenerative diseases for three reasons.
- JM6 cannot cross the blood-brain barrier, which is almost always a sine qua non of CNS disease therapy.
- JM6 ameliorates disease without affecting the protein aggregates that are usually thought to cause the diseases.
- JM6 ameliorates multiple neurodegenerative diseases.
How might this novel therapeutic strategy be moved into the clinic?
Clinical trials in AD are notoriously long and expensive. Therefore, Drs. Reinhart and Kelly in their Preview suggest that it might be best to first conduct clinical trials in HD, since the cause of HD is much better understood than for AD, and disease progression in placebo controls is better characterized than for AD.
The results of the mouse model studies suggest that JM6 will ameliorate, but not cure, HD and AD. However, since there are no disease-modifying therapies for either disease, demonstrating amelioration of HD comparable to that seen in the mouse models (provided the drug is proven to be safe in humans) will almost certainly gain approval for the drug. However, in the long run JM6 would need to be combined with other disease-modifying drugs to more effectively treat diseases such as HD and AD. Since other drugs developed for neurodegenerative diseases will almost always act directly in the brain, combining them with JM6, which does not enter the brain, may help maximize the clinical benefit of a combination therapy. It may also aid in minimizing the toxicity of combination therapies (since the two drugs would not interact in the brain).
Lennart Mucke, MD, the director of the Gladstone Institute, suggested that Dr. Muchowski and his colleagues might begin testing JM6 in patients within the next two years.
The ability of JM6/Ro 61-8048 to ameliorate neurodegenerative diseases in animal models also raises questions as to the mechanisms by which it does so, and how these mechanisms might interact with mechanisms thought to be central to the pathobiology of neurodegenerative diseases (e.g., the amyloid and Tau pathways in AD, huntingtin inclusions, inflammatory pathways, apoptotic pathways, etc.). What are the molecular mechanisms downstream from KYNA elevation? Is JM6 treatment prophylactic, or is it efficacious in animals (and in humans) that are already suffering disease symptoms and that have pathogenic protein aggregates?
Research to answer these questions may lead to still newer therapeutic strategies, including potentially more effective combination therapies for neurodegenerative diseases that include JM6.
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