New Areas of R&D

Pulmonary Fibrosis: WilVent

Introduction

Idiopathic pulmonary fibrosis (PF) is a progressive interstitial lung disease with no known etiology.  There is no known cure for PF, and current treatment modalities are minimally effective.  PF may be an autoimmune disorder, or the after effects of a viral infection.

PF is characterized by excessive deposition of intracellular matrix and collagen in the lung interstitium and gradual replacement of the air sacs or "alveoli" of the lungs by scar tissue as a result of inflammation and fibrosis.  As the disease progresses the increase in scar tissue interferes with the ability to transfer oxygen from the lungs to the bloodstream.  The act of breathing becomes more difficult with the passage of time leading to a condition of general breathlessness or dyspnea, and progressive hypoxemia.  PF patients eventually succumb to either heart or respiratory failure.  Even in the absence of a complicating disease, the median survival after the diagnosis of biopsy-confirmed PF is less than three years.

Market Overview

There are five million people worldwide that are affected by PF ([1]).  In the U.S. alone 200,000 suffer from this malady.  Of these more than 40,000 die annually.  The same number die from Breast Cancer, more than those who die from Ovarian or Prostate Cancer and forty times more than those who die from Cystic Fibrosis (CF). The debilitating impact of PF is equally severe as these types of cancer or CF and is usually fatal. 

WilVent

WilVent is a proprietary agent of Pharmagenesis.  It is a prodrug of PG490 and PG490 is a natural product supplied by Pharmagenesis.  The unusual in vivo effectiveness of WilVent in bleomycin-induced fibrosis holds promise for the inhibition of further fibrosis and the attendant morbidity and mortality of the disease of Pulmonary Fibrosis.

Immune Suppression

When incubated in human plasma, WilVent reduces viability in Jurkat T lymphoid cells and suppresses IL-2 production by stimulated Jurkat cells.  PG490 suppresses T lymphocyte activation and proliferation by Jurkat cells and human and mouse lymphocytes, and suppresses the production of proinflammatory cytokines. In addition to effects upon immune cells, PG490 suppresses IL-8 expression by bronchial epithelial cells.  The inhibition by PG490 of cytokine gene expression by human lymphocytes and bronchial epithelial cells supports the use of WilVent as an immunosuppressant in inflammatory lung disease.

Pulmonary Fibrosis

WilVent exerts an anti-fibrotic effect in a bleomycin model of mouse lung fibrosis and prevents fibrosis if administered the same day or 5 days after bleomycin.  This treatment also markedly reduces the number of lung myofibroblasts  of bleomycin-treated mice, the proliferation of which is thought to be a hallmark of lung fibrosis.  TGF-b is the most potent profibrotic cytokine, and plays a central role in the pathogenesis of PF.  WilVent produces a significant decrease in the level of TGF-b in the bronchoalveolar lavage fluid, and blocks the bleomycin-induced increase in TGF-b mRNA in cultured normal human lung fibroblasts.  WilVent administered systemically has a demonstrated therapeutic effect in preventing fibrosis development in the bleomycin model with the lung as a target organ.  The inhibition of fibrosis by WilVent when treatment was given late after bleomycin instillation supports the utility of WilVent in the serious therapeutic area of PF.

PF is a chronic disease, whereas other diseases of current significance involving the lung  [e.g., Severe Acute Respiratory Syndrome (SARS) and acute respiratory distress syndrome (ARDS)] are more acute. SARS is a serious, contagious, rapidly progressing viral lung disease. A virus-induced hyperimmune response appears to lead to the overproduction of proinflammatory cytokines. Immunopathological damage leads to ARDS, early stages of PF, and a fatal outcome.  PG490 suppresses in vitro production of proinflammatory cytokines such as IFN-g, TNF-a, IL-1b and IL-6 that are stimulated by viral infections and which contribute to ARDS. PG490 has potent immunosuppressive and antiinflammatory properties.  The effectiveness of PG490 in suppressing proinflammatory cytokines is consistent with the use of WilVent to prevent the morbidity and mortality of SARS.

Clinical Development

The strategy for the development of WilVent will be an intravenous formulation for pulmonary fibrosis, with the possibility of an oral formulation at a later time.  Pharmagenesis will seek a partner for later stages of development of WilVent in PF.

Neuroprotective Agents: NeuroTriptin

Introduction

Glutamate, the most important excitatory neurotransmitter in the central nervous system (CNS),  plays an important role in the pathophysiology of different neurological and psychiatric diseases.  Glutamate has a toxic action resulting from an activation of specific glutamate receptors, which leads to acute or chronic death of nerve cells.  Glutamate neurotoxicity has been implicated in many neurological disorders.  Such mechanisms are associated with acute neuronal death within the context of hypoxia, ischemia and trauma, as well as in the chronic neurodegenerative diseases of  Alzheimer's,  Parkinson’s and Huntington's Diseases and others.  A compound that antagonizes the neurotoxic action of glutamates may constitute a novel therapy for these diseases.

Brain inflammation plays a role in the pathogenesis of chronic neurodegenerative disorders like Alzheimer's and Parkinson’s Diseases.  Neurodegeneration caused by inflammation involves activation of the brain’s resident immune cells, the microglia, which produce large numbers of proinflammatory factors.  New insights into the pathogenesis of neurodegeneration in several diseases suggest potential utility of agents that can 1.) Suppress the production or activity of cytokines and proinflammatory mediators in the CNS, 2.) Inhibit the response to proinflammatory stimuli of neural cells and 3.) Exert neurotrophic effects upon neural tissue to enhance outgrowth and survival. 

Alzheimer’s Disease

Alzheimer’s Disease (AD) is a progressive neurodegenerative disease causing a gradual loss of cognitive function and characterized by beta-amyloid deposition, senile plaques with intimately associated reactive microglial cells, neurofibrillary tangles and dystrophic neurites.  Glutamate neurotransmission, an important process in learning and memory, is severely disrupted in patients with AD.  Loss of glutamatergic function in AD may be related to the increase in oxidative stress associated with the beta-amyloid peptide that is found in the brains of individuals who have the disease.  The toxic cellular response to aggregates of beta-amyloid, oxidative stress and energy metabolic disturbances promote neuronal sensitivity to glutamate-induced excitotoxic injury to an extent that even normal amounts of glutamate may become excitotoxic.  Neurodegeneration caused by inflammation involves microglia activated by the aberrant beta-amyloid proteins to produce large numbers of proinflammatory factors mediated via the NF-kB pathway.

Parkinson’s Disease

Like AD, Parkinson’s Disease (PD) is a progressive neurodegenerative disease.  PD is characterized by resting tremor, slowness of movement, rigidity and postural instability as a result of progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) region of the brain.  The cause for this loss of neurons is largely unknown, but considerable evidence supports the suggestion that brain inflammation participates in the pathogenesis of PD.  The striatal dopaminergic regions and spinal fluid of PD patients show a marked increase in the level of proinflammatory cytokines.  Microglial-mediated inflammation has been implicated in this CNS inflammatory process. 

Other Neurodegenerative Diseases

Recent evidence from experimental models of multiple sclerosis (MS) and studies with MS patients in relapse suggests an altered glutamate homeostasis in the brain of patients with MS.  Neurotoxic events occur in MS, and they can be responsible for oligodendrocyte and neuronal cell death in patients with this demyelinating disease.  Antagonizing glutamate receptor-mediated excitotoxicity may have therapeutic implications in MS patients.

Neurodegeneration can occur as a result of traumatic brain injury.  Acute brain insults are linked to inflammation that contributes to the propagation of the neuropathological events, and also trigger increased neurogenesis.  Microglia activation associated with inflammation impairs both the basal continuous formation of new neurons and the increased neurogenesis in response to a brain insult.  The deleterious effects of microglia are most likely mediated by the production of cytokines IL-1b or IL-6, TNF-a, NO and reactive oxygen species (ROS), which are neurotoxic.

Market Overview of Major Neurodegenative Diseases

Nearly five million Americans now have AD ([2]), which is predicted to affect 14 million Americans and 22 million persons totally worldwide in just a few decades.  One in 10 people over 65 have AD and nearly half of those over 85 have AD.  AD is the seventh leading cause of death in Americans 65 years and older ([3]). 

Approximately 1 million people in the U.S. suffer from PD, and 60,000 new cases are diagnosed each year ([4]).  PD is more common in people age 60 and older ([5]).  MS is an illness diagnosed in over 350,000 persons in the United States today ([6]).  Ninety percent of MS patients diagnosed are between the ages of 16 and 60; but MS can make its first appearance in early childhood or after age 60.  There were roughly 2 million cases of traumatic brain injury in 1990 in the U.S, including over 50,000 deaths ([7]).  Between 20 and 30% of the injuries that occur each year are severe enough to result in lifelong disability.

NeuroTriptin

NeuroTriptin is a proprietary agent of Pharmagenesis.  It is a prodrug of PG490, a natural product supplied by Pharmagenesis.  Pharmagenesis activity in the area of neuroprotection will focus on the AD and PD indications.  In vitro assays to be conducted include PC12 cells incubated with glutamate or beta-amyloid, activation of microglia, and inflammatory effects upon primary neuronal cultures.  The transgenic mouse models expressing mutant or truncated human beta-amyloid will initially be employed to generate enabling pharmacology data with NeuroTriptin relevant to AD.  Unilateral transection of the Medial Forebrain Bundle (MFB) of rats will serve as the basis for an animal model of PD.  This model produces a precisely defined lesion of dopaminergic afferents resulting in degenerative changes in a subpopulation of dopaminergic cell bodies in the SNpc with subsequent degeneration of the striatal dopaminergic nerve terminals.

a)    Neuroprotection

Glutamate induces necrosis and apoptosis in the PC12 pheochromocytoma cell line, which is responsive to Nerve Growth Factor and has been used as a cellular model of AD.  PG490 inhibits glutamate-induced cell death, ROS formation and the decrease of mitochondrial membrane potential.  Glutamate is thought to be instrumental in neuronal death in several neurological diseases.  The protection from neuronal death induced by glutamate suggests that NeuroTriptin would be useful in treating neurodegenerative disease such as AD.

Cerebral amyloid angiopathy due to beta-amyloid is one of the specific pathological features of AD, and PC12 cells are extremely sensitive to induction of neurotoxicity by mutant beta-amyloid protein aggregates.  PC12 cells treated with 50 mM beta-amyloid for 48 hours exhibit increased intracellular accumulation of Ca(2+) and undergo apoptotic death.  PG490 at 10 pM markedly inhibits apoptosis induction by beta-amyloid and the increase in intracellular Ca(2+).  PG490 protects PC12 cells from the deleterious and pro-apoptotic effects of treatment with beta-amyloid.  This protection from the neurotoxic effects of beta-amyloid suggests that NeuroTriptin would be useful in treating AD.

PG490 exerts a powerful inhibitory influence over microglia.  Pretreatment with PG490 dose-dependently reduces the lipopolysaccharide (LPS)-induced NO accumulation and TNF-a and IL-1b release from LPS-activated microglia.  PG490 reduces LPS-stimulated mRNA expression of  these inflammatory cytokines.  These results demonstrate that PG490 can inhibit inflammatory responses of microglia to inflammatory stimulation via a mechanism involving the inhibition of the synthesis and release of inflammatory factors.  Microglia inflammatory responses are mediated via the NF-kB pathway.  Treating AD with NeuroTriptin may also benefit by reducing inflammation through PG490 suppression of NF-kB activation.

Tripchlorolide spares substantia nigra (SN) neurons in rats with MFB  transection, a widely used animal model of PD.  This PG490 prodrug preserves SN neurons and dendritic processes of domaminergic neurons and reduces dopamine depletion in these rats.  Activated microglia may play an active role in the degeneration of dopaminergic neurons, as massively activated microglia are found throughout the ventral mesencephalon in this model, particularly in the most medial part of the SN.  Tripchlorolide inhibits the inflammatory and neurotoxic activity of microglia, and shows potent in vivo activity in this model of SN domaminergic neuronal loss, suggesting that NeuroTriptin may be useful in the treatment of PD.

LPS induces the degeneration of dopaminergic neurons that mimics the process of inflammatory dopaminergic degeneration.  The density of microglial cells is higher in the SN compared to other mid-brain areas, the dopaminergic system is most susceptible to inflammation-mediated damage, and microglial cells are implicated in this process.  In primary mesencephalic neuron/glia mixed culture, PG490 blocks the activation of microglia and reduces the production of TNF-a and NO induced by LPS.  PG490 protects against the reduction in dopamine uptake, attenuates the loss of dopaminergic neurons and ameliorates the reduction in dendrite number and truncation of axons caused by LPS.  PG490 treatment without LPS appears to produce more dopaminergic neurons with longer neurites, indicating neurotrophic activity.  These results are relevant to PD, and further illustrate the potential neuroprotective potential of NeuroTriptin. 

Neurotrophic factors enhance the survival of CNS and peripheral neurons under insult, and enhance the outgrowth of neurites after nerve injury. Immunosuppressive agents like cyclosporin and FK506 possess neurotrophic properties.  PG490 and NeuroTriptin are immunosuppressive, and PG490 has neuroprotective activity in vitro. Tripchlorolide promotes growth of long axon-like processes in primary cultures of rat mesencephalic neurons, and stimulates mRNA production for brain derived neurotrophic factor.  This nonproprietary PG490 prodrug was more potent than FK506 in this neurotrophic activity.  NeuroTriptin may be useful in treating acute neurological insults as well as more chronic neurodegenerative diseases, because of antiinflammatory and neuroprotective activity,  as well as neurotrophic properties.

 b)    Clinical Development of NeuroTriptin

The strategy for the development of NeuroTriptin for AD and PD will be an intravenous formulation, with the possibility of an oral formulation for less intense, long-term administration subsequently.  Pharmagenesis will seek a partner for later stages of development of NeuroTriptin in AD and PD.  

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