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EXECUTIVE SUMMARY:

A Drug for the Prevention & Treatment of Liver Injury and Cirrhosis


1) Our Target Market
The Target Market includes all patients with chronic liver diseases. There are approximately 200 million people with Chronic Hepatitis C infection worldwide; 1 million cirrhosis patients admitted to hospitals in Europe and the USA; 4 million with Hepatitis B or C in the USA; 10 million with non-alcoholic steatohepatitis (NASH) related to obesity and Diabetes type 2 in the USA. These chronic liver diseases have high morbidity and mortality related to many clinical complications, including internal bleeding, confusion and liver cancer.
There is no FDA approved medication for the prevention or treatment of liver cirrhosis.


2) Our Drug:
Our drug is highly effective in preventing the development of liver cirrhosis and inducing the regression of established liver cirrhosis in a physiologically relevant animal model. Thus, our drug would prevent patients with chronic liver diseases to progress into liver cirrhosis as well as to induce the regression of liver cirrhosis. In addition, our drug would improve liver injury and inflammation in patients with chronic liver diseases. These beneficial effects would improve the quality of life, decrease hospitalizations and increase survival among patients with chronic liver diseases.


3) Patients’ Issues, Needs and Wants. How Xfibra Addresses Them:
Patients with chronic liver diseases are afraid of the complications, particularly liver cancer. Patients need an effective and safe medication. They prefer a simple administration, free of pain. Also, the patients, HMOs, Medicare, and VA want an inexpensive medication.

Xfibra has addressed these issues:

i] the drug by inhibiting liver injury and inflammation and by preventing/regressing cirrhosis would markedly decrease the risk for liver cancer;

ii] the drug is highly effective and extremely safe in animal models and human liver cell studies;

iii] our drug would be administered once per week under the skin in a very small volume ( ~ 50 ul); a simple, painless and practical approach; and

iv] the production cost of the PEG-30kDa-peptoid is very low. The GMP synthesis of the drug is easy and inexpensive.


4) Projected Revenues
From the animal efficacy studies and using the FDA conversion Tables for human dosing, it is expected that a dose of 1mg (50 μL) administered weekly should be effective in patients with chronic liver diseases.

For 1 patient: 1 mg/week [~ 50 mg/year]; if 1 mg market price would be hypothetically $ 100/week (a very low estimate when Sovaldi's treatment for Hepatitis C costs $ 7,000/week)
For every 200,000 patients: 200 g/week [~ 10 kg/year]. The predicted yearly revenues would be ~$ 1 billion/ year

NOTE: Given that the Target Market is several folds larger than 200,000 patients and that the Market Price would likely be higher than $ 100/week, the annual revenues are expected to exceed $ 1 billion.

5) What Makes Our Drug Different
The novelties of the Approach are:

i] the unique molecular target is essential for the activation and survival of liver myofibroblasts, which are the cells responsible for the production of the scar tissue in cirrhosis; and

ii] the therapeutic peptoid by effectively and selectively blocking the target, prevents activation of liver myofibroblasts, thereby, inhibiting liver scarring.

This kind of molecular target has never been proposed as a potential therapeutic. This is not to be confused with other molecular targets such as the inhibition of a kinase, while potentially still specific, is more impactful on the molecular landscape of the cell, blocking possibly dozens of downstream phosphorylation events on dozens of factors. Our therapeutic is designed to block a single posttranslational modification on one protein, a single event critical to liver scar tissue production and nonessential to other mechanistic processes.
Therefore, our drug is highly specific, which minimizes any potential off-target toxicity.


The PEG-peptoids in culture systems and in animal models of acute liver injury /cirrhosis underwent a systematic analysis of efficacy and safety in a chronic liver fibrosis model. CCl4 (a human hepatotoxin known to cause liver injury, cirrhosis, and cancer) was administered to mice for up to 16-wk as a classical model of liver cirrhosis. We administered the PEG-peptoid once a week IP (5 μg of peptoid linked to 300 μg PEG-30kDa) starting at week 8 once severe liver cirrhosis was already established. The efficacy of the selected PEG peptoids included quantitative analysis of liver scar tissue.

The PEG-peptoid also decreased the liver injury. The serum ALT (a clinical end-point used routinely in clinical practice and by the FDA in the evaluation of liver injury in drug studies) was markedly induced by the hepatotoxin CCl4 but improved in spite of only a single treatment with the PEG-30kDa-peptoid  8 hr after the injury was established .
The PEG-30kDa-peptoid showed no evidences of toxicity at 100-fold the effective dose in cultured human primary hepatocytes (the main liver cell) and at 100-fold the therapeutic dose in vivo in mice.

Medicinal chemistry optimization performed to date.
Synthesis and analysis of peptoids containing non-naturally occurring amino acids with and without an N-terminal PEG-30kDa and a modified carboxy terminus has been performed. The lead PEG- 30kDa-peptoid easily achieves the required purity by the FDA and it has an outstanding solubility: >125 mg/ml water. This outstanding solubility will allow the FDA required escalation dose in human studies (estimated 0.1mg; 0.5mg; 2 mg of the peptoid; subcutaneous) using volumes of < 100 ul. The PEG-30kDa will allow weekly administration of the drug.

The competitive landscape in liver scar tissue: There is no available medication for the treatment of liver cirrhosis at present. Our original publication reporting the novel mechanisms by which liver scar tissue production can be prevented or reversed received an Editorial in Cell and a Commentary in Science.

In strong support of our innovative approach, the NIH Action Plan for Liver Diseases Progress Review (Chapter 2 Liver Injury, Inflammation, Repair, and Fibrosis) stated:


“C3. Develop mechanism-based drug therapy in fibrotic disease, targeting pro-fibrogenic and fibrosis resolution pathways. Several attractive approaches to prevention or reversal of hepatic fibrosis have been suggested in studies in animal models. Activation of stellate cells ( “myofibroblast”) is a central step in hepatic fibrogenesis, and the pathway to activation has been shown to be mediated, at least in part, by activation of RSK and phosphorylation of C/EPBβ (Buck M and Chojkier M. PLoS ONE 2007; 2: e1372) “ .

As an acknowledgment of its innovation, our therapeutic strategy received the Second Award in the coveted National McGinnis Venture Competition Award for Life Sciences, and the only $1 million NIH-RC-1 Challenge grant for “Medication for Hepatic Fibrosis”; as well as a VA Merit Award, an NIH R-37 Merit Award and three NIH STTR R-41 grants.

High Stability of the Peptoid:

We have already developed a sensitive and specific LC-MS/MS method to identify and quantify the peptoid. In addition, we have determined that the peptoid has excellent stability in human liver microsomes and in human plasma. Further, the peptoid did not inhibit any of the major human CYP-450 metabolizing enzymes. Collectively, these data predicts an excellent stability for the peptoid in Clinical Studies .

Intellectual Property:

The University of California has obtained several patents to facilitate the Research & Development of the dominant negative peptides and peptidomimetics. UCSD has licensed these patents to Xfibra, Inc (a Company founded by the inventors Drs. Martina Buck and Mario Chojkier)


6) Why Is Our Drug Very Likely to Reach the Market ?


• Our drug is safe and efficacious for a disease that has no FDA-approved treatment.
• Exceptional efficacy in several animal models of liver cirrhosis.
• No evidences of toxicity at 100-fold the therapeutic dose.
• Our transgenic mice carrying a protein mimicking the drug are fertile, have a normal life span, are resistant to liver scar tissue formation by liver toxins.
• Our transgenic mice are the first example of an animal carrying the “drug-like” in all tissues without evidences of any toxicity.
• As an Advisor to the FDA (CDER for GI Drugs), Dr. Chojkier has the experience, motivation and ability to successfully execute the Clinical Studies.
• As the main inventor of the Peptoid and as an expert in Molecular Medicine, Dr. Martina Buck has discovered surrogate markers that will dramatically simplify the Clinical Studies endpoints, timelines and costs.


APPENDIX:

REPRESENTATIVE ADDITIONAL EFFICACY AND SAFETY RESULTS


1] Preliminary efficacy and safety of the pre-therapeutic peptoids.

 Neither the parent peptide nor any of the three selected peptoids induced hepatocyte injury to human or mouse cultured primary hepatocytes or to mice in vivo (at 100-fold the therapeutic dose) .


2] Lack of toxicity of the parent peptide: it does not induce apoptosis in cultured normal primary mouse hepatocytes.

The parent peptide was not toxic to normal mouse hepatocytes even at 100-fold the effective dose (1000 ng/mL), suggesting an excellent safety profile.

                                                                                                                                                                   

3] A sensitive and specific LC-MS/MS method to identify and quantify the lead peptoid
We have developed a sensitive and specific LC-MS/MS method to identify and quantify the lead peptoid. LC-MS/MS was able to detect and quantify the precursor and the ionization product with high sensitivity.


4] The lead peptoid is highly stable in human liver microsomes and in human plasma
We have determined that the lead peptoid is stable in mouse and human liver microsomes and in mouse and human plasma ex vivo .


5] Pilot Pharmacokinetic studies in mice

The  PEG-30kDa-peptoid was administered intravenously to mice (7.5 mg/kg of peptoid). Using the LC-MS/MS bioassay we were able to measure significant levels of the lead peptoid as it was released from the PEG-30kDa-peptoid at all time-points assayed (4 hr : 65.8 ng/ml; 8 hr : 79.2 ng/ml; 24 hr : 70.2 ng/ml; and 48 hr : 67.6 ng/ml) .  Therapeutic levels of the peptoid were detected in the livers of these animals . 

As expected, these results indicate the slow release of the peptoid to achieve steady-state concentrations. Similar PK are expected for comparable effective doses in humans, suggesting very feasible PK studies in clinical Phase-1 and Phase-2.


6] The lead peptoid is not immunogenic to human CD-4+ T-cells

Immunogenicity is always a concern during development of any therapeutic peptides. Although the parent peptide was effective and free of any immunogenicity during prolonged treatment of mice, we altered the peptide to decrease any potential immunogenicity in humans. These modifications should minimize the possible immunogenicity of the peptoids in humans. Moreover, binding to MHC class II molecules in CD-4+ T-cells and induction of immunogenicity requires at least a core 9-mer peptide.

Thus, the lead 4-mer peptoid cannot bind to MHC class II molecules and induce immunogenicity through this classic mechanism. Further, we show in this proposal that the lead peptoid is not immunogenic to human CD-4+ T-cells (purified from 50 normal human donors), as determined by the normal production of IFN-gamma; IL-4; IL-6; and IL-12 and by the lack of induction of CD-4+T-cell proliferation. In contrast, IFN-β a known immunogenic protein, markedly stimulated cytokine production and T-cell proliferation .

7] Pilot studies of in vivo efficacy and safety of the lead PEG-30kDa-peptoid in acute liver injury and activation of myofibroblasts.


To induce liver injury CCl4 was administered  to normal mice. Eight hours later, animals received an IP injection of the lead PEG-30kDa-peptoid (5 μg of the peptoid linked to the PEG-30kDa). Animals were sacrificed after 30 hr, at the peak of hepatocyte death as measured by ALT (a clinical end-point used routinely in clinical practice and by the FDA in the evaluation of liver toxicity in human drug studies).

Mice receiving only CCl4 had intense expression of alpha-SMA in the liver, a main indicator of activated liver myofibroblasts, compared with the minor expression of mice receiving both CCl4 and the lead PEG-peptoid.

There was severe acute liver injury in animals receiving CCl4 but mild-to-moderate injury in animals receiving both CCl4 and the lead pre-therapeutic compound.

The lead peptoid blocked the typical liver discoloration and granular appearance of severe liver injury induced by the hepatotoxin CCl4 and the livers of these treated mice were similar to control livers . The histopathology induced by CCl4 was one of severe liver injury and collapse of the architecture as reflected by the standard clinical stains (H&E and Reticulin).

The lead peptoid decreased the acute liver injury . The serum ALT was markedly induced by the hepatotoxin CCl4  but improved in spite of only a single treatment of the lead peptoid, 8 hr after the injury was established .

The albumin mRNA (the major indicator of normal liver-specific gene expression) was decreased  and the IL-6 and TNF-alpha mRNAs (the main indicators of liver inflammation) were increased by  CCl4 from control values. The peptoid markedly improved these values .

8]  High Efficacy of the Lead Therapeutic Peptoid in a Chronic Mouse Model of Liver Fibrosis.

The therapeutic PEG-peptoids underwent a systematic analysis of efficacy and safety in a classical chronic liver fibrosis model . CCl4 (a human hepatotoxin) was administered to mice for 16-wk as a classical model of liver fibrosis .  We administered the three selected therapeutic PEG-peptoids once a week IP (7 μg of peptoid linked to PEG) starting at week 8, once severe liver fibrosis was already established. The efficacy of the three selected PEG peptoids included quantitative analysis of liver fibrosis.

As expected, CCl4 administration to mice for 16-wk induced liver cirrhosis.  We found that the lead PEG-30kDa peptoid (given once per week from week-8, once the liver fibrosis was already severe) decreased  liver fibrosis by > 8-fold. The two alternate PEG-30kDa peptoids also effectively decreased  liver fibrosis (~ 3-fold and ~ 2-fold, respectively).

This mimics the treatment given for several years to a patient with established severe liver fibrosis.

9] Lack of Cardiotoxicity in In Vitro Models.

The therapeutic peptoid did not induce any toxicity either in the hERG Assay (The cardiac potassium channel) or in in Human Cardio-myocytes (derived from human Stem Cells)
 

 


Thus, the early safety and efficacy profile of the peptoid is very high in acute and chronic mouse models of liver injury and cirrhosis and activation of liver myofibroblasts.

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B.  Drug for the Prevention & Treatment of Lung Injury and  Fibrosis

  SIGNIFICANCE:
 

1. The Challenge and Potential Impact: A medication that would decrease or prevent the progression of lung fibrosis would impact the healthcare of patients with Idiopathic Pulmonary Fibrosis (IPF). IPF affects 5 million people worldwide and 200,000 patients in the US. No therapy is known to improve significantly health-related quality of life or survival in patients with IPF and these patients live only 3 to 5 years after diagnosis. Our central hypothesis is that the phosphorylation of C/EBPβ on threonine217 is critical for lung fibrogenesis  since, C/EBPβ expression and Thr217 phosphorylation is essential for survival of activated lung myofibroblasts (LM). Th Founders have shown that inhibition of C/EBPβ-Thr217 phosphorylation with either a single point mutation (Ala217, a dominant negative transgene), or an inhibitory tetra-peptide (a dominant negative mutated phosphoacceptor site) stops the progression of lung fibrosis induced by Bleomycin in mice . Phosphorylation of human C/EBPβ on Thr266 (the human phosphoacceptor exact homologue of the mouse Thr217) was induced in collagen-activated human LMF in culture as well as observed in activated LMF in lungs of patients with IPF but not in control lungs, suggesting that this phosphorylation is relevant in human lung fibrosis. 

The goal of Xfibra is to develop this lead clinical candidate and the associated data packet which will strongly support an IND approval by the FDA for the initiation of clinical target validation (Clinical Phase-1/2A Studies).

 

 

2. The Therapeutic Target and its Potential Clinical Relevance: The human C/EBPβ-Thr266 phosphorylation is  critical for lung fibrogenesis since this phosphorylation is essential for survival of activated human LMF .  Quiescent lung fibroblasts produce negligible amounts of extracellular matrix proteins (ECM), but upon activation, develop a myofibroblast phenotype, proliferate and become the main contributors of ECM (8). There is consensus that this step is critical for the development of lung fibrosis that leads to the decreased of both vital capacity (VC) and diffusion of CO (DLCO). The clinical indication to be targeted for FDA approval is the inhibition of the deterioration of VC and DLCO in IPF. Our original publication reporting the novel mechanisms by which either unphosphorylated mouse C/EBPβ-Thr217 or unphosphorylated human C/EBPβ-Thr266, associates with procaspase 8 inducing its self-activation and stimulating apoptosis of  myofibroblasts upon their activation received an Editorial in Cell and a Commentary in Science.  As an acknowledgment of its potential for innovation, if developed as a therapeutic strategy, the inhibitory peptide received the coveted RC1 NIH Award for the topic Treatment for Tissue Fibrosis and the Second Award ($ 100,000 to the UCSD MBA student who presented the virtual project) in the coveted National McGinnis Venture Competition Award for Life Sciences.

     

    INNOVATION:

The Therapeutic development of novel peptoids (a lead and two alternates) created based on the inhibitory peptide (parent peptide) is proposed in this application. They have never been reported and no medication of this kind has ever been developed for any disease. A blockade of a single phosphorylation site on a specific protein has never been proposed as a potential therapeutic. This is highly innovative. It is not to be confused with the inhibition of a kinase, while potentially still specific, is folds more impactful on the molecular landscape of the cell, blocking possibly dozens of downstream phosphorylation events on dozens of factors. Our therapeutic is designed to block one phosphorylation site on one protein, a single event critical to the survival and proliferation of activated LMF and fibrosis, yet nonessential to other mechanistic processes in the lung.

1.   The Therapeutic Development Rationale:

Activation of LMF is responsible for the development of lung fibrosis in IPF . Phosphorylated C/EBPβ-Thr217 facilitates survival of activated LMF by binding to the inactive procaspase 8 complex and preventing its self-cleavage and activation.  Phosphorylation of C/EBPβ-Thr217 in activated LMF is critical for the progression of lung fibrosis. This was determined by the PIs using classical Bleomycin-induced lung fibrosis models in mice, primary mouse and human LMF in tissue culture and cell-free systems.  Phosphorylation of human C/EBPβ-Thr266 in activated LMF occurs in human lung fibrosis of IPF.  Mice expressing the non-phosphorylatable C/EBPβ-Ala217 transgene are refractory to the induction of LMF activation and proliferation by Bleomycin . The non-phosphorylatable C/EBPβ-Ala217 facilitates the death of activated LMF by binding to the inactive procaspase and inducing its self-cleavage and activation .  The non-phosphorylatable C/EBPβ-Ala217 dominant negative transgene was present within the death receptor complex II, with active caspase 8, and was linked to apoptosis of activated LMF in transgenic mice.  Blocking phosphorylation of C/EBPβ-Thr217 with the C/EBPβ-Ala217 transgene or by C/EBPβ gene knock-out decreases the fibrotic response of the lung .  The decreased fibrotic response of the lung to Bleomycin in C/EBPβ-ko mice  suggests that the critical target of RSK in activated LMF is C/EBPβ-Thr217 rather than other RSK phosphoacceptors on c-Fos, CREB, CBP or other proteins.  The inhibitory parent peptide prevents the phosphorylation of C/EBPβ-Thr217 in LMF activated in culture by a collagen type 1 matrix or in vivo by lung injury. The inhibitory parent peptide blocks active fibrogenesis through this apoptotic mechanism in vitro and in vivo, preventing progression and allowing regression of lung fibrosis .

 

2.  The competitive landscape:  The Development of a medication for lung fibrosis will be addressed with the FDA required assays that are IND-enabling. There is no approved medication that directly inhibits or reverses lung fibrosis at present.  Current treatments.in this area include Esbriet (pirfenidone) and OFEV (nintedanib)  are only partially effective and have significant limitations and very high cost to the patient.

In 2014, FDA provided “Fast-Track” and “Breakthrough Therapy” designations to OFEV and Esbriet. While the drugs appear to inhibit important pathways that help prevent scarring and significantly slow the progression of the disease, neither drug is considered a cure and IPF may still advance after patients use these drugs. Moreover, the pathways inhibited may not be specific, for example, OFEV is a kinase inhibitor and blocks multiple pathways that may be involved in the scarring of lung tissue; however, this also indicates that use of the drug may result in serious adverse effects. As described above, OFEV's use has several safety concern associated with it. The two IPF drugs are also very expensive and affordability to patients would be a very likely hurdle. 

 3. Preliminary Results:

 - The phosphorylation of human C/EBPβ-Thr266 appears to be essential for preventing induction of death cascades in LMF upon their activation . Inhibition of this phosphorylation by the Ala-217 transgene, the parent peptide, or potentially our novel peptoid would result in a dose-response apoptosis curve of activated LMF, and blockade of this fibrotic pathway.

-Prevention of C/EPBβ phosphorylation on human Threonine 266 either indirectly by inhibiting the activation of RSKp90, or directly with the parent peptide, stimulates apoptosis of activated human LMF.  

- Activation of RSK and phosphorylation of human C/EBPβ on Thr266 was induced in collagen-activated human LMF in culture as well as observed in activated LMF in lungs of patients with IPF but not in control lungs, suggesting that this phosphorylation is also relevant in human lung fibrosis.

- The Lead peptoid is Highly Stable in Human Lung Microsomes and Human Plasma Ex Vivo.

- We were able to detect effective therapeutic concentrations of the free peptoid in the lung ( ~ 70 ng/g )  and in the  liver ( ~ 90 ng/g), after being released from the PEG-30kDa in plasma.


 

  

           mchojkier@gmail.com