POSIPHEN® | THE MOLECULE

Neurotoxic Aggregating Proteins are involved in a series of neurodegenerative diseases and share common features

• Protein misfolding and aggregation underpin many common neurological and systemic diseases. On the neurological side, this diverse group of diseases includes Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), transmissible spongiform encephalopathies (TSEs) and amyotrophic lateral sclerosis (ALS). Despite obvious differences in the neural cell population impacted, clinical symptoms and disease progression, these disorders share common features and regulatory elements that are relevant to the mechanism of action of Posiphen®.
• Neurotoxic aggregating proteins such as APP, αSYN and tau share similar features relative to gene activation, protein synthesis, folding and misfolding, toxicity and aggregation, as described in Disease Focus:

mRNA translation of neurotoxic aggregating proteins is up-regulated by iron (Fe) and down-regulated by iron regulatory protein-1 (IRP1)

Posiphen® interferes with this second step of the common cascade of the aggregating proteins. It enhances the binding and/or activity of IRP1 to the iron response element (IRE) stem loop in the 5’UTR of the mRNAs of neurotoxic aggregating proteins, therefore specifically lowering their synthesis. By potentiating the IRE/IRP1 complex, Posiphen® lowers the level of free mRNA to be translated by the ribosome.

QR Pharma studied the mechanism of action by a total of four approaches to confirm the mechanism and to understand specificity of binding.

1.  RNA pulldown inhibition and shows that IRP1 specifically binds to IREs of neurotoxic aggregating proteins and that Posiphen® ipotentiates the binding of the IRE to IRP1, therefore, lowering protein translation. – Conducted by Jack Roger’s laboratory at Massachusetts General Hospital
2. Thermophoresis binding of cloned purified IRP1 to synthetic IREs of neurotoxic aggregating proteins shows that Posiphen® specifically binds to the IRE/IRP1 complexes of neurotoxic aggregating proteins and increases the stability of the complexes, therefore, lowering protein translation. – Conducted by Rodrigo Vasquez in Prem Reddy’s lab at Mt. SinaiHospital
3. Proteomics, to explore the breadth of Posiphen®’s effect on protein inhibition i. The data very clearly show that Posiphen® inhibits neurotoxic aggregating proteins very specifically. – Conducted in by Carlos Barrera in Salim Merali’s lab at Temple University
4. Activity in vitro and in vivo, measure inhibitory effect of Posiphen® on neurotoxic aggregating proteins and on unrelated proteins in tissue culture cells, mouse brains and human spinal fluid.

Alzheimer’s Disease is the most common Neurodegenerative Disease and anticipated to affect over 150 million people by 2050.
Posiphen® normalizes the overexpression of both APP (decreasing the levels of N-APP, Aβ and C31) and tau.
β amyloid (Aβ) is a hydrophobic, neurotoxic self-aggregating 40 to 42 amino acid peptide that accumulates preferentially within senile plaques in the brain and is the target of many therapeutic clinical trials.

Aβ, C31 and N-APP are neurotoxic fragments derived from Amyloid Precursor Protein (APP). Beyond the ample literature around Aβ, tau protein has also been implicated in the pathogenesis of AD.
By normalizing the expression of APP, Posiphen® interferes with a basic pathway that leads to dementia in Down Syndrome, AD and likely other conditions, such as head trauma and stroke.

APP/PS1 AD transgenic mice were treated for 3 months with Posiphen® and compared to control. Treated mice showed a dose-dependent improvement in cognitive function in the water maze and fear conditioning tests. Posiphen® rescued spatial memory in a 2-day radial arm water maze in an AD model (APP/PS1) at oral doses of 10mg/kg and 25 mg/kg (p = 0.0033). The drug also restores long term potentiation and brain function as measured by electrophysiology in hippocampal slices.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is estimated to occur in about 1% of the population over the age of 55, costing up to $23 billion annually to the health care community.

It is characterized by progressive motor and non-motor decline that substantially contributes to disability and loss of quality of life. There is no treatment that blocks the neurodegeneration and progression of the disease.

A large body of evidence indicates that α-synuclein (αSYN) is involved in the pathogenesis of PD and is a valid target for developing drugs to treat this disease. αSYN mutations and gene multiplications are known to cause familial PD and Lewy bodies, which are a neuropathological hallmark of PD, consist of aggregated αSYN in all PD patients, including the sporadic forms.

Untreated transgenic PD mice resemble pre-Parkinson’s patients, showing symptoms of constipation but not motor symptoms (MS). At five to six months of age, the mice show constipation. As shown below, Posiphen® reverses this effect and prevents its recurrence. Posiphen® does not act as a laxative, since when given to two different control mice breeds that do not develop constipation, it does not affect their gut motility.

Traumatic brain injury (TBI) causes severe cognitive and neurological impairment which can incapacitate the patient, reduce quality of life, and increase the risk of morbidity and mortality. TBI is known to increase the risk for neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. A number of studies have analyzed changes in the brain after TBI and identified up-regulation of neurotoxic aggregating proteins, such as amyloid-beta, tau, and alpha-synuclein. Since these proteins are known to cause neurodegeneration and neurological impairment, therapies targeting their inhibition may be useful in blunting the neurodegenerative consequences of TBI as well as other CNS diseases.

Rats were subjected to fluid percussion injury (FPI) or sham operation to one side of the brain and Posiphen® was administered 1 hour post injury. Three doses of Posiphen® plus saline were given intraperitonealy for 4 weeks. At the termination of the treatment, all the rats were sacrificed and sections of the brain were stained with tyrosine hydroxylase (TH), wherein TH stains only live cells. The amounts of TH immunoreactivity in the whole striatum of the brain slices were measured.

Because FPI can induce microglial activation, we checked whether Posiphen® would reverse this pathological. Microglial activation was assessed by quantitative measure of the diameter of IBA-1-positive cells (ionized calcium adaptor binding protein). Microglia with cell body diameters less than 5 µm had a resting morphology characterized by multiple ramified processes. Hyper-ramified microglia/partially activated microglia had a mean cell body diameter of 5-6 μm. Fully activated amoeboid microglia had a mean cell body diameter of 7-14 μm.

The sham operated group was compared with the vehicle group with two-way ANOVA, Bonferroni comparisons, wherein *p
<0.05. and the vehicle group was compared with the Posiphen®-treatment groups with one-way ANOVA, Bonferroni comparisons, wherein #p<0.05. In the Posiphen® group, the rats treated with 3 doses of Posiphen® showed a statistically significant increase over the vehicle treated animals in the number of surviving cells in the ipsilateral area of the brain at all 3 doses. Posiphen® protected the striatum in fluid perfusion injury at all 3 doses tested.