AEOL 10150 in ALS "Lou Gehrig's Disease"

Amyotrophic Lateral Sclerosis, the most common motor neuron disease, results from progressive degeneration of both upper and lower motor neurons. The incidence is 1-2 per 100,000. ALS occurs twice as often in men as women, with typical onset between 50 and 70 years of age. ALS is progressive and always fatal. Approximately 80% of ALS patients die within 5 years of diagnosis, with only 10% living more than 10 years. The average life expectancy is 3 years after diagnosis, with death from respiratory and/or bulbar muscle failure. The International Alliance of ALS/MND Associations reports there are over 350,000 patients with ALS/MND worldwide and 120,000 cases diagnosed each year. In the United States there are approximately 30,000 patients with ALS with 5,000 new patients diagnosed each year.

Sporadic (i.e., of unknown origin) ALS (SALS) is the most common form, accounting for 80-90% of cases. The etiology of SALS is unknown. Familial ALS (FALS) comprises the remainder of cases, at least 20% of which are associated with mutation in the SOD1 gene. More than 90 point mutations have been identified, all of which appear to associate with ALS, and result in motor neuron disease in corresponding transgenic mice. The nature of the dysfunction produced by the SOD1 mutation remains unclear. Though the association of SOD with SALS is not clear, the clinical and pathological manifestations of FALS and SALS are indistinguishable, suggesting common elements in the pathogenesis in both types of ALS.

Rilutek® (riluzole) is the only approved treatment for ALS. Its mechanism of action in ALS is unknown, but it inhibits glutamate release, blocks excitatory amino acid pathways and inactivates voltage-dependent sodium channels. Administration of Rilutek prolongs survival of ALS patients by an average of 60-90 days, but has little or no effect on the progression of muscle weakness or on quality of life. Rilutek was approved in the US in 1995, and in 2001 in the EU. It is estimated that in 2002 Rilutek was used in the treatment of approximately 3,800 ALS patients in the US, accounting for only 13% of all US ALS patients. The product achieved approximately $35 million in US sales in 2002 and $106 million worldwide in 2001. It is reported that the product should achieve annual worldwide sales of approximately $129 million in 2006.

The study of ALS has changed in recent years with the development of transgenic mice that express the mutant human SOD1, facilitating the search for new ALS treatments. These mice exhibit a motor neuron disease that presents initially as hind limb weakness, at about 100-120 days of age, and progresses to respiratory failure within 10-15 days of symptom onset. To date, virtually all reported studies in this model initiate treatment substantially prior to symptom onset, e.g. at 30-60 days of age.

The use of catalytic antioxidants such as AEOL 10150 is a novel approach to ALS therapy. To test this hypothesis, the effect of AEOL 10150 on the survival of G93A SOD1 transgenic mice was examined (Table 1). The transgenic mouse is the only in vivo model available to test potential ALS therapies. Robust positive effects in transgenic mice are generally viewed as sufficient to warrant clinical investigation.

Table 1. Summary of experiments with AEOL 10150
on survival in the G93A SOD1 mouse model of ALS

Effect of AEOL 10150 treatment at symptom onset on survival of G93A transgenic ALS mice

Twenty-one confirmed transgenic mice were alternately assigned to control or AEOL 10150 treatment on the day of symptom onset, defined as a noticeable hind-limb weakness, to more accurately reflect the clinical treatment of ALS patients. The initial dose of AEOL 10150 was 5 mg/kg intraperitoneally (IP), with continued treatment at a dose of 2.5 mg/kg/day IP until death or moribundity.

Downlaod Table: Effect of AEOL 10150 on Survival of G93A SOD1 Transgenic Mice

The table above shows G93A SOD1 mice treated with AEOL 10150, starting at symptom onset, survived for 2.5 times as long after symptom onset as did untreated G93A SOD1 mice (32.2 ± 2.73 days vs. 12.8 ± 0.79 days, p<0.0001). In addition, symptom progression (as assessed by visual observation) was markedly slowed in the mice treated with AEOL 10150 until just a few days before sacrifice, but the condition of the untreated control mice declined in an almost linear fashion.

A second study was initiated to confirm and expand upon these findings. This study included four treatment groups as follows:

• Control
• AEOL 10150 5 mg/kg IP at onset, followed by 2.5 mg/kg/day IP
• Rofecoxib + creatine PO (diet admixture) initiated at onset and continued until death
• Rofecoxib + creatine diet PO plus AEOL 10150 IP as per above regimens

Both rofecoxib and creatine have been reported to delay symptom onset and extend survival in this model when treatment begins prior to symptom onset.

The results of the second study, shown in Table 3 (next page), confirm the earlier findings of extended survival associated with AEOL 10150 treatment alone. Survival was also extended by AEOL 10150 in combination with rofecoxib + creatine PO. Mice treated with AEOL 10150 alone lived approximately 3 times as long after onset of symptoms as did untreated controls (40.0 + 3.25 vs. 13.5 + 0.45 days). The extension of survival post-onset by rofecoxib and creatine was less than that seen previously when they were administered prior to symptom onset. The effect of AEOL 10150 is the same with or without rofecoxib and creatine, indicating that the effects are not additive.

Downlaod Table: Effect of AEOL 10150, Rofecoxib, and Creatine on Survival of G93A SOD1 Transgenic Mice

In a third experiment in 13 transgenic mice, AEOL 10150 was administered by the subcutaneous route at the same dose that was found to be efficacious by intraperitoneal administration. Determination of symptom onset, endpoints and analysis were as described for the IP studies.

Treatment with AEOL 10150, initiated at symptom onset by the subcutaneous route, markedly increased the survival time of G93A SOD1 mice (Table 4). Survival after symptom onset was 2.3 times as long in the AEOL 10150-treated group (41.3 + 0.84 days) than in untreated controls (17.7 days + 1.12 days). In addition, symptom progression (as assessed by visual observation) was markedly slowed in the mice treated with AEOL 10150 until just a few days before sacrifice, but the condition of the untreated control mice declined in an almost linear fashion.

Downlaod Table: Effect of AEOL 10150 Treatment by the Subcutaneous Route on Survival of G93A SOD1 Transgenic Mice

A dose-response study was conducted with AEOL 10150 in the G923A mouse model as well. Both the 0.5 mg/kg and 2.5 mg/kg doses produced significant improvement in survival.

In a recent peer-review publication regarding these results, and with respect to overall motor activity of the G93A mice, the authors noted that there was a "marked slowing in the rate of disease progression, as well as the absence of specific deficits in the AEOL 10150-treated mice." As elucidated by the study authors, "untreated control mice almost always experienced development of complete paralysis of one or both hind limbs within 10 to 14 days of symptom onset. By contrast, complete hind-limb paralysis was virtually absent in AEOL 10150-treated mice, despite the fact that they lived up to three times longer after symptom onset. [AEOL 10150]-treated mice retained their ability to walk, run, cage-climb, groom, eat, and drink throughout the period of extended survival. Rather than a steady decline in motor function over time (such as with control mice), [AEOL 10150]-treated mice typically maintained global function and retained the use of hind limbs until a rapid decline at end-stage disease." The article notes that the only US FDA-approved treatment for ALS is ‘riluzole’, which extends survival of ALS patients by 2 to 3 months, but does not alter the rate of disease progression or preserve muscle strength." John P. Crow, PhD, Noel Y. Calingasan, PhD, Junyu Chen, MA, PhD, Julie Lynch Hill, CVT, M. Flint Beal, MD, Ann Neurol 2005;58:258-265.

Histopathology analysis performed on spinal cord tissue from AEOL 10150- treated G93A mice and untreated G93A mice comparatively evidenced that in the AEOL 10150-treated mice, there was a statistically significant increase in motor neuron survival. Immunohistochemistry analysis on the spinal cord tissue from these animals evidenced a decrease in gliosis (a marker for neuronal injury) in the AEOL 10150-treated mice, as well as a decrease in markers of oxidative injury. Based on the results of these analyses, the study authors further concluded that the "neuroprotective effect was related, at least in part, to the novel antioxidant properties of [AEOL 10150]." Crow et al.
AEOL 10150 was also tested in the G93A mouse model in the laboratory of M. Flint Beale. In his laboratory AEOL 10150 was administered both upon symptom onset and prior to symptom onset. AEOL 10150 showed no effect when administered prior to symptom onset. The compound did produce a statistically significant improvement of survival when administered upon symptom onset (Figure 2).

In the laboratory of Robert Brown the compound produced no effect in the G93A mouse model of ALS when administered (IP) at symptom onset.

In addition to these survival studies, a small study was initiated to determine the histopathologic effects of AEOL 10150 on degeneration of spinal motor neurons. Eleven transgenic mice were randomly assigned to AEOL 10150 treatment (n = 6) at the same dosing used previously or control treatment (n = 5) administered beginning at symptom onset. Mice from both treatment groups were euthanized after 10 days of treatment, and spinal cord and brain tissues were preserved for evaluation. The number of neurons per spinal cord section (20 sections per mouse) was determined. As shown in Table 6, sections from AEOL 10150 treated mice had 63% more neurons than those from control mice, suggesting that AEOL 10150 treatment was associated with a preservation of spinal neurons.

In summary, treatment with the catalytic antioxidant AEOL 10150, beginning at symptom onset, has been shown to prolong survival and delay symptom progression in the G93A SOD1 mouse model of ALS.