AEOL 10150 in Other Diseases

In general, drug development tends to focus on selecting a disease state of interest and challenging that disease state with a drug candidate of interest. In the case of AEOL 10150 (as well as the other Aeolus compounds), as with any clinical compound, the requirement of evaluating the safety and tolerability of the compound in humans is an absolute first-step requirement. However, in terms of efficacy (Phase 2 and / or Phase 3 clinical evaluation), the opportunities for AEOL 10150 are substantially broader than most drug candidate compounds. This allows for selection of efficacy based clinical evaluation that relies upon the substantial and diverse disease models in which the compound has evidenced efficacy. In addition, once a first FDA-approved label for the compound is secured, the various opportunities available or AEOL 10150 can allow for more rapid label expansion under a Supplemental New Drug Application, as well as the possibility of off-label uses for the compound.

AEOL 10150 has shown efficacy in models of ALS, spinal cord injury, stroke and radiation induced lung fibrosis, as well as chronic obstructive pulmonary disease (AEOL 10150 decreases the adverse effects of exposure to tobacco smoke; Free Radical Biology & Medicine 33(8):1106, 2002), pancreatic islet cell preservation (apoptosis and necrosis associated with human islet isolation for use in allotransplantation reduced using AEOL 10150, resulting in higher cell survival and enhanced insulin release; Diabetes 53:2559, 2004); and inflammation (AEOL 10150 has been shown to: diminish the pro-inflammatory transcription activation factor NF-kB during isolation-stress in human pancreatic islet cells; decrease the cleavage of PARP, a pro-inflammatory marker of cell damage and death during isolation-stress in human pancreatic islet cells, and to attenuate the release of pro-inflammatory cytokine (IL-6) and chemokine (MCP-1) during isolation-stress in human pancreatic islet cells; Diabetes 53: 2559, 2004. AEOL 10150 has also been shown to diminish the pro-inflammatory transcription activation factor NF-kB during hemorrhage-induced acute lung injury in mice; Am J Physiol Cell Mol Physiol 284:L680, 2003, and to decrease lung leukocyte influx associated with cigarette smoke exposure in rats, to decrease the release of the chemokine MIP-2 into the airways of rats exposed to cigarette smoke, and to decrease the lung's expression of the pro-inflammatory adhesion molecule ICAM-1 in rats exposed to cigarette smoke. Free Radical Biology & Medicine 33(8): 1106, 2002).

Some of the effects observed with AEOL 10150 may be mediated by inhibition of redox-sensitive pro-inflammatory pathways or by preventing the generation of reactive oxygen species by interacting with endogenous flavin-dependent oxido-reductases. Accordingly, the potential therapeutic opportunities for AEOL 10150 are quite extensive. The Company believes that although the various compounds in its library may have attributes which provide specific disease treatment opportunities, other compounds will also evidence the type of opportunity breadth as AEOL 10150.

AEOL 10150 in Stroke

The reactive nature of ROS is exploited in the inflammatory response to a foreign substance in the body but excessive amounts of ROS damage cellular components, interfering with cellular function. For example, in stroke excitatory amino acids and inflammatory mediators initiate a series of biochemical events collectively called the ischemic cascade. Reactive oxygen species such as superoxide, peroxynitrite and the hydroxyl radical are formed that react with lipids, proteins and nucleic acids resulting in breakdown of cellular constituents and ultimately, in neuronal death (below).

In animal models of stroke in which a blood vessel in the brain is blocked, free radical damage to protein and DNA is markedly elevated in the affected hemisphere. Overexpression of various forms of SOD reduces brain cell death in these models; knockout mice deficient in SOD are more susceptible to experimental stroke.

Preclinical Pharmacology

AEOL 10150 has proven efficacious by multiple routes (ICV and IV), and in multiple species (rat and mouse), multiple models (permanent and temporary middle cerebral artery occlusion) and in multiple laboratories. The results of several of the studies with AEOL 10150 are summarized in the effect of AEOL 10150 in preclinical stroke models table below.

Rat Temporary Middle Cerebral Artery Occlusion (TMCAO) Model, ICV Administration AEOL 10150 has been tested in a rat model of ischemic stroke in which the middle cerebral artery was occluded for 90 minutes followed by reperfusion. After one week of recovery, infarct volume was measured. AEOL 10150 (300 ng, ICV) showed a marked and statistically significant protective effect on infarct volume when administered as late as 7.5 hours after the onset of ischemia (Figure 4).

Figure 4. The effect of AEOL 10150 (300 ng, ICV) on infarct volume in the rat TMCAO model when administered at 7.5 hours after the onset of ischemia.

Mouse Temporary Middle Cerebral Artery Occlusion (TMCAO) Models, IV Administration
AEOL 10150 has been tested in two intravenous mouse models of temporary MCAO occlusion. Both studies have shown similar results. In one of these experiments, the middle cerebral artery was occluded by a filament for 60 minutes. A bolus injection of 2.5 mg/kg of AEOL 10150 was administered at 5 minutes after reperfusion (65 minutes after the start of ischemia) followed by an intravenous infusion of 1 mg/kg/hour continued until 24 hours after the onset of ischemia, when the animals were sacrificed.

AEOL 10150 administered by the IV route produced a significant decrease in infarct volume (Figure 5). Neuroscores were reduced from 3 + 0 with saline to 2 + 1.75 with AEOL 10150 (median + interquartile range; adjusted p= 0.003), on a 0-4 scale where 0 = no observable deficit and 4 = animal cannot walk spontaneously. Values ranged from 3-4 with saline treatment and from 1-4 with AEOL 10150.

Figure 5. The effect of intravenously administered AEOL 10150 on infarct volume in the mouse temporary MCAO model. Infarct volume was evaluated at 24 hours.

Mouse Permanent Middle Cerebral Artery Occlusion (PMCAO) Models, ICV Administration:
The effect of AEOL 10150 has also been examined in two models of mouse permanent occlusion, in which the MCA is occluded for 24 hours. As shown in Table 8 (next page), 1500 ng of AEOL 10150 administered by the ICV route at 90 minutes after the onset of occlusion in a filament occlusion model significantly reduced infarct volume, improved neurologic function, and tended to reduce mortality.

The effect of AEOL 10150 by the ICV route in the mouse permanent MCAO (filament) model. Compound was administered either before or 90 minutes after onset of ischemia (see table below).

AEOL 10150 was also efficacious in another mouse model of permanent occlusion of the middle cerebral artery, which was conducted twice by another laboratory. In these experiments, the artery was occluded by electrocoagulation, and AEOL 10150 (1500 ng, ICV) or vehicle was administered at 90 minutes after occlusion. AEOL 10150 significantly reduced infarct volume in these two independent experiments using this model (Figure 6).

Figure 6. The effect of AEOL 10150 (1500 ng, ICV) in the mouse electrocoagulation PMCAO model when administered at 90 minutes after occlusion. Evaluation was at 24 hours after occlusion.

AEOL 10150 in Spinal Cord Injury

AEOL 10150 has been studied in a model of spinal cord compression (SCC) injury in the mouse. In this model, a compression tube (suture silk encased in a silicon tube) was used to compress the spinal cord at T11 for 60 minutes in C57BL/6J mice. Rotarod performance was evaluated at 3, 7, 14 and 21 days post-SCC in both an accelerating (3-38 rpm) and a fixed-speed (20 rpm) Rotarod. Screen grasping performance was assessed at 21 days post-SCC. Neither AEOL 10150 (0.5 mg/kg bolus followed by 1.0 mg/kg/hr for 24 hours) nor methylprednisolone (30 mg/kg bolus followed by 5.4 mg/kg/hr for 24 hours) was effective in this model when given by the intravenous route. AEOL 10150 given intrathecally improved both histological damage score (Table 9) and Rotarod performance (Figure 7).

The effect of intrathecally administered AEOL 10150 on spinal cord damage as assessed histopathologically and on screen grasping performance at 21 days post-SCC (see table below).

Figure 7. The effect of intrathecally administered AEOL 10150 (Mn III TDE-1-3-Imp5+) on Rotarod performance after spinal cord compression (SCC). In both accelerated and fixed Rotarod tests, AEOL 10150 increased the latency to fall (P= 0.05).

Radiation-Induced Lung Fibrosis

It has been recognized for many years that radiation therapy produces oxygen free radicals in the body that react with cellular components to kill cancer cells. These free radicals also harm normal healthy tissue, limiting the dose of radiation that can be given in cancer therapy and causing toxicities such as oral mucositis, lung inflammation and fibrosis. The ability of radiation therapy to treat tumors involving the chest such as lung or breast cancer is often limited by injury to the normal lung caused by radiation. Currently, radiation-related pulmonary symptoms occur in up to 30% of patients irradiated for lung cancer, breast cancer, lymphoma or thymoma. Laboratory analysis of AEOL 10150 evidenced that breathing rates (an indirect measure of lung restriction) increased beginning at eight weeks and reached a peak 18 weeks after radiation treatment. The average breathing rates in irradiated groups treated with AEOL 10150 (10 and 30 mg/kg/day) were 15% lower. Lung histology 20 weeks after RT resulted in significant amount of the lung fibrosis (70%) that was significantly decreased (40%) in animals receiving 10 and 30 mg/kg/day. AEOL 10150 treatment was also associated with a significant reduction in macrophage presence after lung irradiation.