NTX-010

NTX-010, the company’s lead product, advanced from discovery to IND in under 18 months. NTX-010 is currently being tested in the clinic in patients with cancers that have neuroendocrine properties including those with small cell lung cancer. NTX-010 has dramatic efficacy in mouse xenograft models, excellent in vitro and in vivo specificity, and an excellent safety profile as demonstrated in GLP toxicology studies.

The Company believes that NTX-010 has key desirable properties for commercial success, including:

• High Selectivity For Neuroendocrine Tumor Cells
• Targets Critical Cancer Biology Pathways Which May Provide Biomarker Development Possibilities
• Minimum Potential for Mutation and/or Recombination
• Anti-tumor Efficacy Despite Host Defenses
• Low Systemic Administered Dose Will Reach All Cancerous Sites
• Minimum Toxicity to Normal Tissues
• High Therapeutic Index
• Minimum Potential for Transmission
• Feasible Manufacturing and Distribution
• Formulation for Infusion Therapy
• Ongoing Clinical Trials

High Selectivity for Neuroendocrine Tumor Cells

In vitro screening of over 170 cell lines and 13 primary adult normal human cell cultures revealed that NTX-010 will infect and potently kill certain types of cancer cells that express one or more neuroendocrine markers. These data suggest that cancer indications that may be permissive for NTX-010 include the majority of small cell lung cancers (SCLC), endocrine cancers, some non-small cell lung cancers (NSCLC), neuroblastoma, Wilms, medulloblastoma, and retinoblastoma. Additionally, as many as 5-10% of all cancers express these markers and therefore represent potential target markets.

NTX-010 has an extremely high selectivity index of greater than 10,000. The selectivity index measures the number of tumor cells killed per normal cell killed. NTX-010’s index is 1,000 fold higher than traditional chemotherapeutics which are known to be very toxic toward normal cells. Importantly, NTX-010 does not infect 13 out of 13 human adult normal cell cultures tested, even when those cells are exposed to 10,000 viral particles per cell, which is 10,000 times more than the dose necessary to kill the majority of permissive lines.

Cancer Biology Pathways and Early Biomarker Development

The Company has identified key components of the mechanism of tropism (selectivity) and one or more of these may be very useful as biomarkers to be utilized in ongoing or planned clinical trials. Neotropix has also developed a unique antibody that recognizes a key, surface-expressed protein involved in the mechanism of NTX-010 tropism. The virus targets key pathways in cancer known to be involved in metastases and invasion. The Company is in the process of filing patents on mechanism of tropism, biomarker and related discoveries. Further studies are ongoing to potentially utilize the biomarker(s) in clinical trials. Identifying the mechanism of tropism and associated biomarker early in the drug development process has numerous key advantages; development of critical reagents to screen normal tissues to see if any potential target organs of toxicity exists; screen a wide array of cancer indications to determine which cancers are best able to respond and at what percentages of each indication, and more efficiently, rapidly, and cost-effectively perform clinical trials with a high probability of FDA approval. In addition, some of these key reagents and molecules identified in the process may be novel and promising cancer
targets as well as potential therapeutics, and as such are candidates for out-licensing and/or partnering. The Company believes its system for discovering the key components of the mechanism of viral tropism (BioScreen) is a unique asset that can advance the therapeutic potential of newly discovered viruses that have oncolytic potential.

Minimum Potential for Mutation and/or Recombination

Unlike viruses with the ability to rapidly mutate, such as the influenza virus of the family Orthomyxoviridae, members of the Picornaviridae family to which NTX-010 belongs cannot undergo genetic reassortment because a single copy of non-segmented RNA functions as its genome. Although all viruses evolve and change their antigenicity over time, these changes in picornaviruses rarely produce a change of tropism, i.e. the ability to infect cell types heretofore uninfectable. Neotropix has tested this premise by attempting to generate viral mutations using selective pressure by sequentially passing NTX-010 on multiple non-permissive lines. No mutants were generated that had acquired the ability to replicate in non-permissive cells. In other tests, no altered NTX-010 was isolated when intentionally passed in a permissive line in the presence of neutralizing antibodies. These observations indicate a low rate, if any, of antigenic drift. Finally, all production lots of NTX-010 have passed all CMC release tests.

Anti-tumor Efficacy Despite Host Defenses

NTX-010 has been shown to produce long-term, durable tumor eradication in pre-established xenograft-based SCLC tumors in mice following systemic administration at doses as low as 1E7 viral particles per kg. Even extremely large (2000 mm3), pre-established tumors can be completely eradicated by a single intravenous dose. NTX-010 has demonstrated significant systemic efficacy in 11 of 11 different models tested, albeit not always eliciting complete eradication, including xenograft, syngeneic, orthotropic and metastatic tumor models. Cancer indications tested include SCLC, NSCLC, neuroblastoma, retinoblastoma, medulloblastoma and rhabdoid tumors. Three of these 11 models were tested at St. Jude Children’s Research Hospital as part of NCI’s Pediatric Preclinical Testing Program (PPTP). The preclinical models developed and utilized in the PPTP have been shown to predict the activity of agents in the clinic. NTX-010 induced durable complete responses in each of these three models.

Unlike other oncolytic viruses, NTX-010 is not inhibited by any component of human blood, thereby permitting systemic administration and treatment of metastatic disease. NTX-010 does not bind to red blood cells or cause them to bind together. Virtually no pre-existing antibodies have been found in humans. It has been shown that the host immune response does not eliminate ongoing replication or active therapy within the tumor itself. Following a single systemic administration of NTX-010 in syngeneic tumor-bearing mice, large amounts of virus are observed in the blood stream until day 4, indicating significant replication within the tumor. This signal lasts until neutralizing antibodies appear after which infectious virus in serum is no longer detectable. Despite the presence of these antibodies, the levels of NTX-010 observed within tumors is over one billion-fold higher than that detectable in normal tissue. Additional analysis indicated ongoing virus replication occurs in the tumor after serum antibodies are detectable. These results suggest that a single intravenous administration provides for long term therapy and that the host immune response does not eliminate active therapy within the tumor. This hypothesis is now supported by human clinical data that demonstrate active ongoing
replication only within tumor masses 28 days following a single systemic dose, despite the presence of high levels of circulating antibodies appearing as early as day 8 following administration.

Low Systemic Administered Dose Will Reach All Cancerous Sites

A single, low systemic administration of the oncolytic virus should infect all metastatic tumor masses, including those undetectable by traditional diagnostic methods. At 27 nm, the virus molecule is extremely small compared to other viruses and may have a distinct advantage in reaching and infecting tumor masses throughout the body. NTX-010’s size also enables better penetration and spread within solid tumors as compared to larger molecule competitors, such as the adenovirus. The importance of the size of the viral molecule has been borne out by experiments that demonstrate that NTX-010 can infect tumor xenografts following a systemic administration but an adenovirus cannot, despite similar infection rates of tumor cells in vitro.

Following infection at the tumor site, the virus will replicate in the initially infected tumor cells, amplify, and infect all permissive cells at those cancer sites. The in vivo amplification effect should increase potency despite a single low systemic administered dose. Competitive therapeutics can require multiple doses over a period of time.

Minimum Toxicity to Normal Tissues

The virus exhibits no overt toxicity in multiple different strains of mice following systemic administration of 1E14 viral particles/kg. This dose is one million-fold higher than that required to eradicate pre-established SCLC xenograft tumors in mice. Importantly, Neotropix has demonstrated that mice are a relevant animal model for toxicity since transient low level replication does occur in normal mice and murine tumor cells expressing neuroendocrine markers are as permissive as human tumor cells expressing similar properties. This is also a distinct advantage over other oncolytic viruses as mice usually do not support replication are thus not a relevant model. Additionally, murine tumor cells expressing neuroendocrine markers are as permissive as human tumor cells expressing similar properties. Intravenous delivery of up to 1E11 viral particles/kg (the highest dose tested) revealed minimum toxicity in swine and non-human primates.

To test selectivity for cancerous cells over normal cells, following systemic administration of NTX-010 in mice, a panel of normal tissues and control tumors were examined by NTX-010 capsid immunohistochemistry to determine if any normal tissue supported replication of the virus. A positive immunohistochemistry signal was clearly observed in all tumor sections examined. However, no staining was observed in any of the normal tissues from 20 organs examined. These results indicate that only a small number of dispersed cells, which cannot be detected by this assay, are responsible for the low-level viral replication observed in non-cancerous mice. These results correlate with the lack of overt toxicity exhibited in extensive studies. Finally, these data further support the dramatic selectivity, or tropism, of this virus toward tumor cells even in vivo.

High Therapeutic Index

The Therapeutic Index helps to predict whether an efficacious dose can be safely administered in humans and is defined as the maximum amount of drug delivered without toxicity vs. the
effective dose. Efficacy is observed in several mouse models at 1E7 viral particles/kg, while no toxicity was observed even at 1E14 viral particles/kg. This translates to a therapeutic index of
over 1 million
. This is significantly higher (by orders of magnitude) then most if not all cancer therapeutics used to treat metastatic disease, and is a key feature of NTX-010.

The data and conclusions discussed above have been reproduced and extended in part by investigators at the National Cancer Institute, Johns Hopkins University, St. Jude Children’s Research Hospital, and Baylor College of Medicine. As an example, a collaborator at Baylor College of Medicine has demonstrated that after establishment of an orthotopic metastatic retinoblastoma model in the eye (these metastases normally spread to neighboring brain cells), systemic administration of NTX-010 killed metastasized cancer cells. Collaborators at St. Jude Children’s Research Hospital have also demonstrated significant efficacy following systemic administration in 3 of 3 pre-established pediatric cancer models that are believed to be highly predictive of a response in the clinic.

Minimal Potential for Transmission

To elucidate whether NTX-010 could be transmitted by the host following administration, Neotropix performed a study whereby mice were injected with NTX-010 and then co-mingled with naïve mice. After 30 days no naïve mice were found to have neutralizing antibodies, demonstrating a lack of viral transmission. In contrast, all control injected mice developed neutralizing antibodies.

To date there has been no evidence of transmission of the virus among regularly tested employees or among a limited number of caregivers tested for the NTX-010 antibody. In other tests, the Company did not observe any effects on fetal development, birth or early development when injected mice were commingled with naïve pregnant mice.

Feasible Manufacturing and Distribution

Unlike most oncolytic viruses, NTX-010 is naturally occurring and does not require re-engineering or genetic modification to demonstrate the desired therapeutic effect. As a result, NTX-010 can be produced to extremely high levels in multiple production systems, has a rapid life cycle, and is easily manipulated. The FDA has extensive experience with picornaviruses, the class of virus to which NTX-010 belongs. the Picornavirus class is used in polio vaccines that have been in production for nearly 50 years. These properties confer significant advantages in manufacturing.

With current technology, the Company has produced the product in serum containing media at levels as high as 200,000 viral particles per cell. The Company is switching to non-serum containing media as it is preferred by regulatory bodies. Based upon current process development studies the Company believes that it can produce a desirable product with yields of 5,000-50,000 viral particles per cell in FDA-approved producer cell lines. CMC Testing has successfully been completed for the toxicology lot, master virus bank, and Phase I lot. Downstream processing, formulation development, and assay qualification are ongoing.

Formulation for Infusion Therapy

NTX-010 is distributed as a single dose vial consisting of virus and excipients, suspended in saline. It is administered as a one hour intravenous infusion. The entire treatment regimen for any one patient consists of this single infusion, as compared to chemotherapy, radiation, and competitive oncolytic viral therapies that may require multiple treatments over a period of months or, in some cases, for the remainder of the patient’s life.

Ongoing Clinical Trials

Following submission of the Company’s IND for NTX-010, the Phase I trial opened in May, 2006. The Phase I trial will evaluate safety in a dose escalation study from 1E7 to 1E11 viral particles per kg administered intravenously to patients with advanced cancers and with at least one neuroendocrine marker suggesting the possibility of susceptibility to the virus. Depending on the data, it is expected that approximately 30-50 patients who have advanced cancers will be enrolled, with the last patients entered late in 2007. As of November, 2006, no dose limiting toxicity has been encountered, and dose escalation continues.

In the current dose-escalation phase of the clinical trial any patient that has one of three neuroendocrine markers (synaptophysin, chromogranin A, CD56) are permitted to enter the trial. This is due to the fact that these markers are classically utilized to diagnose SCLC, our primary target and our finding that additional cancer indications that are not SCLC also have similar neuroendocrine markers expressed. These markers are tested on previously fixed histologic or cytologic specimens. Not all patients with such tumors will achieve an objective tumor response, which is defined as the measurable reduction in size of a tumor. The Company’s current clinical goal is for at least 25% of NTX-010 treated patients to have a measurable or detectable reduction in tumor size (objective response) lasting at least 2 months. If the observed response rate is greater, then there will be an opportunity to accelerate the development plan.