1. What is EMT and MET?

Cancer metastasis is a complex process, central to which are Epithelial-to-Mesenchymal Transition (EMT) and Mesenchymal-to-Epithelial Transition (MET). The EMT is a process by which epithelial cells lose their cell polarity and cell-to-cell adhesion and gain migratory and invasive properties. MET occurs when circulating cancer cells settle in a new environment and revert to an epithelial state to establish secondary tumors. This transition is essential for the colonization phase of metastasis, allowing cancer cells to grow and form new tumors in distant organs.

2. What IPA-3?
It is a highly selective inhibitor of group-1 P21 activated kinases (PAKs).

3. What are P21 activated kinases (PAKs)?
PAKs are a family of intracellular serine-threonine kinases in 6 different isoforms, namely PAK1, PAK2, PAK3, PAK4, PAK5, and PAK6.

4. What are the different classes of PAKs? Why are they categorized in separate groups?
PAKs are categorized into two groups: Group-1 PAKs that include PAK1, PAK2, and PAK3 and Group-2 PAKs that have PAK4, PAK5, and PAK6. Group-1 and Group-2 PAKs are categorized based on their mechanism of action and their potential cellular function.

5. What is the mechanism of action of Group-I and Group-II PAKs?
Group-1 PAKs are activated downstream of two small GTPases, namely Rac and CDC42, which are necessary to form two cellular processes called lamellipodia and filopodia, respectively. Lamellipodia are the membrane protrusions from a cell.  Filopodia are spike-like proteins that make new contacts with the substrate (Thus, both these cellular structures are formed due to actin cytoskeletal remodeling modulated via the activation of Group-1 PAKs by Rac and CDC42 in lamellipodia and filopodia, respectively.
Group-2 PAKs are NOT activated by either Rac or CDC42. As of today, the actual mechanisms leading to their activation are not fully understood. Group-2 PAKs are known to regulate the cell cycle, cell proliferation, and survival.

6. Why is targeting Group-1 PAKs an attractive strategy to treat patients with metastatic cancers?
The regulation of actin cytoskeleton formation for lamellipodia and filopodia, and cell migration are regulated by Group-1 PAKs, primarily the most ubiquitously expressed PAK isoform, PAK1. Several cellular and preclinical studies performed by the MetasTx founders and others in the late 2000s revealed that PAK1 is essential for cancer cell invasion and metastasis. Hence, the idea was born that suppressing the ability of the cancer cells to migrate and invade adjacent tissues could prevent and/or treat metastatic cancers.
Studies by the MetasTx founders revealed the integral role of PAK1 in the regulation of ‘epithelial-to-mesenchymal-transition” (EMT) and Mesenchymal-to-Epithelial Transition (MET), processes necessary for the early cancer cells to switch to the metastatic ones. IPA-3 was able to inhibit prostate cancer cell EMT and suppress metastasis to other organs. In addition, IPA-3 was also found to stop the growth of metastatic prostate cancer cells in the bone and thus to prevent cancer-induced bone remodeling and loss of tissue.

7. It has been over ten years since PAK1 was identified to regulate cancer metastasis. What caused a delay in its therapeutic development?
Before using a cellular molecule for human therapy, it is essential to have a well-characterized pharmacological inhibitor. Until 2008, no compounds were identified to target the enzymatic activity of any PAKs specifically. However, in 2008, a group of scientists identified IPA-3 (Inhibitor Targeting Pak1 activation-3) among a screen of 33000 compounds to have the ability to suppress PAK1 activity.
Since 2008, several compounds were identified as PAK inhibitors, which include G‑5555 (in 2015), FL172 (in 2008), PF‑3758309 (in 2010), PRT062607 (in 2012), LCH-7749944 (in 2012), FRAX486 (in 2014), FRAX597 (in 2014), FRAX1036 (in 2015), AZ13705339 (in 2016), and PF‑3758309 (in 2016). Later, G-5555, PF‑3758309, AZ13705339, and FL172 were identified to have off-target effects. Also, PF‑3758309 and LCH-7749944 were identified to be predominantly PAK4 (Group-II) inhibitors.
A clinical trial on PF‑03758309 for advanced solid tumors was terminated, due to the undesirable pharmacokinetic characteristics, such as unfavorable levels of the drug in the plasma and the lack of a dose-response association.
Because of the reasons above, the development of therapeutics to target PAKs for cancer metastasis was delayed.

8. When there are several compounds available today, why is IPA-3 a preferred drug?
As mentioned above, most PAK inhibitors identified so far have either off-target effects with the potential to develop serious side effects in patients. These compounds target the ATP binding domain that is highly conserved in a large group of serine-threonine kinases.
IPA-3, among these, is unique because of its allosteric nature in suppressing Group-I PAK activity. Furthermore, it does not target the ATP-binding domain of any kinase. Because of these properties, IPA-3 is highly selective in suppressing PAK1 activity in various cell types with minimal effects on other cellular functions, and hence fewer side-effects are anticipated.

9. Is IPA-3 ready for clinical trials?
All the studies performed to date used a research-grade IPA-3. Prior to its application in humans, GLP and GMP batches of IPA-3 must be manufactured.  Some pre-clinical studies must be repeated with GLP IPA-3 prior to starting clinical trials.

10. What do we know about the characteristics of IPA-3?
There are some concerns regarding the stability of IPA-3 in plasma. This is because of the presence of a disulfide bond in it, which is critical for the inhibition of PAK1. Once in plasma, IPA-3 quickly becomes inactive because of the disulfide bond breakage, thus limiting its therapeutic benefits.

11. Is there a method to increase the stability of IPA-3 in plasma?
Yes. The stability can be improved by encapsulating IPA-3 in a nano-liposomal formulation.

12. What is liposome encapsulated IPA-3?
The founders of MetasTx have developed a formulation of sterically stabilized liposomal encapsulated IPA-3 (SSL-IPA3). Today, the formulation is known as “MTX-101”. The stability of MTX-101 and its efficacy in inhibiting metastatic prostate cancer cells in vitro and in vivo has been tested. MTX-101 treatment demonstrated superior efficacy to inhibit prostate tumor growth and metastasis in various mouse models of prostate cancer.

13. What are the advantages of MTX-101 over free IPA-3?
There are several benefits of using MTX-101 for metastatic cancer therapy over free IPA-3. These include:

• increased stability in the plasma by preventing the break down of IPA-3
• improved half-life by increasing the stability of IPA-3
• reduced toxicity as the ecapsulation of IPA-3 in to liposome takes advantage of the leakiness of the tumor blood vessels to allow targeted distribution to the tumor, as opposed to non-cancer cells.
• reduced chances of any potential side-effects by ehanced targeting to the tumor
• increased efficacy as more IPA-3 is delivered to the tumor

The most important among all the advantages is the reduced duration of drug administration. Whereas daily administration of free IPA-3 for 21 days was necessary to suppress prostate tumor growth and metastasis in preclinical models, the same efficacy was achieved with just two times per week administration of MTX-101.

14. What other therapies to treat cancer are there?

• The mainstay for treatment of prostate cancer that has not spread to other parts of the body include radiation, surgery or small molecule inhibitors of the androgen receptor, typically called hormone therapy.
  • These stop your body from producing testosterone, a male-centric hormone that causes prostate cancer cells to grow.
    • Leuprolide
    • Gosereline
    • Triptoreline
    • Many others
  • Another class of medication does not stop testosterone from being produced, but block it’s ability to to reach cancer cells. These include:
    • Bicalutamide
    • Flutamide
    • Nilutamide
  • There are other small molecules that do not focus on the production or function of testosterone. These include:
    • Abiraterone
    • Ketoconazole
    • Estrogen
    • Some steroid drugs
• For prostate cancer that has spread to other parts of the body the primary classes are:
  • Chemotherapy
  • Immunotherapy
  • Bone building medications
  • Radioactive drugs

15. What are advantages of IPA-3?
IPA-3 isn’t a hormone therapy and doesn’t target the androgen receptor. This means it has the advantage that the cancer cell cannot develop androgen reistance. A primary mechanism by which prostate cancer cells evade the cancer killing effect of small molecules.
IPA-3 also targets a protein whose expression is increased in prostate cancer cells. This adds to the specficiity and selectivity of the molecules.  This is different from other chemotherapy treaments that target microtubules or DNA.
The above advantages suggest that IPA-3 may be less likely to induce drug resistance and less likekly to induce the deleterious effects of chemotherapy (like hair loss, bone loss or naseua).

16. Is MTX-101 toxic to cells?
Compared to the toxicities and side effects (off-target effects) caused by other cancer therapies currently in use, the toxicity by MTX-101 administration is expected to be extremely low. This is because of its high specificity to PAK1 inhibition, minimal off-target effects, increased stability, and reduced frequency of drug administration because of the improved pharmacokinetic properties. Compared to the currently used taxanes such as Docetaxel (Taxotere) and Cabazitaxel (Jevtana), both inhibit microtubules and cell proliferation, preclinical studies on MTX-101 suggests much lower toxicity and side-effects as compared to the current standard of care.

17. Why/how your PAK inhibitor promises better safety than other PAK inhibitors that have been developed and studied?
As the investor has pointed out, we are aware of the several PAK inhibitors available in the market. Many of them are of less clinical interest due to the lack of efficacy, and specificity, having off-target effects, and/or serious side effects. Many that have been used in clinical trials are met with side effects and a lack of specificity issues. Hence, allosteric inhibitors, that do not target the ATP-binding domain, are preferred for higher efficacy, fewer off-target effects, and better safety profile.

As of today, primarily two compounds have been identified as allosteric inhibitors of group-I PAKs (PAK1, PAK2, and PAK3). These are ‘NVS-PAK1’ and ‘IPA3’, out of which, the former only inhibits PAK1, and the latter inhibits all 3 group-I PAKs. Since cancers are known to upregulate the expression and/or activity of all the 3 isoforms of group-I PAKs, IPA-3 is preferred over NVS-PAK1 and all other currently available PAK inhibitors.

IPA3 is the active ingredient of MTX-101, encapsulated in a liposomal ‘shell’. This formulation not only improves the safety profile of IPA3 but also increases its efficacy (our patented findings on the novel composition of matter). Apart from this, as Dr. Cummings has pointed out, the liposomal encapsulation of IPA3 has the following added advantage:
“The primary reason for the increased safety of our PAK inhibitor is it is in a nanoparticle that is targeted to the tumor site. Tumors have leaky vasculature or holes in their blood vessels. Normal tissue doesn’t have these leaky vessels. It’s kind of like a run-down house that has leaky pipes, but a well-kept house doesn’t.

Putting our drug into nanoparticles allows it to bypass the liver and other tissue, but accumulate selectively in the tumor site, again because of their leaky vessels. The nanoparticles are able to do this because of their size. They are too large to get through blood vessels into the healthy tissue, but tumor tissue has larger holes in their blood vessels through which the nanoparticle can enter. This means more drug goes to the tumor, but less goes to healthy tissue. This decreases the toxicity of the inhibitor to non-cancer cells.

Bibliography:

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2. Somanath PR, Vijai J, Kichina JV, Byzova T, Kandel ES. The role of PAK-1 in activation of MAP kinase cascade and oncogenic transformation by Akt. Oncogene. 2009 Jun 25;28(25):2365-9.
3. Kichina JV, Goc A, Al-Husein B, Somanath PR, Kandel ES. PAK1 as a therapeutic target. Expert Opin Ther Targets. 2010 Jul;14(7):703-25.
4. Goc A, Al-Azayzih A, Abdalla M, Al-Husein B, Kavuri S, Lee J, Moses K, Somanath PR. P21 activated kinase-1 (Pak1) promotes prostate tumor growth and microinvasion via inhibition of transforming growth factor β expression and enhanced matrix metalloproteinase 9 secretion. J Biol Chem. 2013 Feb 1;288(5):3025-35.
5. Al-Azayzih A, Gao F, Somanath PR. P21 activated kinase-1 mediates transforming growth factor β1-induced prostate cancer cell epithelial to mesenchymal transition. Biochim Biophys Acta. 2015 May;1853(5):1229-39.
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7. Najahi-Missaoui W, Quach ND, Jenkins A, Dabke I, Somanath PR, Cummings BS. Effect of P21-activated kinase 1 (PAK-1) inhibition on cancer cell growth, migration, and invasion. Pharmacol Res Perspect. 2019 Sep 6;7(5):e00518.
8. Verma A, Artham S, Alwhaibi A, Adil MS, Cummings BS, Somanath PR. PAK1 inhibitor IPA-3 mitigates metastatic prostate cancer-induced bone remodeling. Biochem Pharmacol. 2020 Jul;177:113943.
9. Verma A, Najahi-Missaoui W, Cummings BS, Somanath PR. Sterically stabilized liposomes targeting P21 (RAC1) activated kinase-1 and secreted phospholipase A₂ suppress prostate cancer growth and metastasis. Oncol Lett. 2020 Nov;20(5):179.
10. Najahi-Missaoui W, Quach ND, Somanath PR, Cummings BS. Liposomes Targeting P21 Activated Kinase-1 (PAK-1) and Selective for Secretory Phospholipase A₂ (sPLA₂) Decrease Cell Viability and Induce Apoptosis in Metastatic Triple-Negative Breast Cancer Cells. Int J Mol Sci. 2020 Dec 10;21(24):9396.

Studies on PAKs by other investigators

1. Deacon SW, Beeser A, Fukui JA, Rennefahrt UE, Myers C, Chernoff J, Peterson JR. An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chem Biol. 2008 Apr;15(4):322-31.