Pipeline Therapeutics

[xyz]

I just read that PIPE-505 uses a gamma secretase inhibitor. Isn't that the same thing Audion/Regain has tested? So if Pipeline Therapeutics hasn't got some great reformulation of this drug, this looks not very promising.

 

[FGG]

I just read that PIPE-505 uses a gamma secretase inhibitor. Isn't that the same thing Audion/Regain has tested? So if Pipeline Therapeutics hasn't got some great reformulation of this drug, this looks not very promising.

They do but it seems to have minimal effect on hair cell regrowth and mostly on synapses so my guess is it is a much weaker inhibitor. I did ask them ages ago about support cell depletion and they said for their drug it wasn't a significant amount.

But I would like to know if it is not significant because it does a minimal amount of OHC regeneration and/or if their method produces less support cell depletion. Maybe we can get them on the Tinnitus Talk Podcast at some point and ask them these things.

 

[wwtsai]

Pipeline Therapeutics announced the initiation of their Phase 1 clinical trial for PIPE-307, a drug targeting remyelination. At the bottom of the press release under their About, they state that they're still currently enrolling for their PIPE-505 Phase 1/2 which seems kinda... weird? Based on the last update back in November, the estimated completion date was pushed to this month. If they haven't even finished recruitment then they definitely aren't making that deadline.

Pipeline Therapeutics Initiates Phase 1 Clinical Trial of PIPE-307

 

[Aaron91]

They do but it seems to have minimal effect on hair cell regrowth and mostly on synapses so my guess is it is a much weaker inhibitor. I did ask them ages ago about support cell depletion and they said for their drug it wasn't a significant amount.

But I would like to know if it is not significant because it does a minimal amount of OHC regeneration and/or if their method produces less support cell depletion. Maybe we can get them on the Tinnitus Talk Podcast at some point and ask them these things.

So with FX-322 news imminent, I thought I'd go down the rabbithole a bit with this question in the event Frequency Therapeutics do drop a bombshell on us.

I think most are already aware of this, but PIPE have a transdifferentiation approach when it comes to regenerating hair cells:

To put it simply, FX-322 causes asymmetric division where you end up with a hair cell and a new progenitor, whereas with PIPE the progenitor turns into a hair cell and the progenitor is depleted. Of course people are confused because PIPE are saying they are not seeing much depletion of the support cells, but as @FGG has pointed out this could be down to the fact that they're not getting much regeneration in the first place.

So I did a bit more digging, and found an interesting slide from one of Frequency Therapeutics' presentations. Before I get to that though, here's just a reminder of what Carl LeBel had to say on this:

"What those [transdifferentation] programmes have tried to do is take a progenitor cell and – it's a process we call transdifferentiation – you're taking a supporting cell, a progenitor cell, and turning it into a hair cell, you're making it want to become a hair cell. The problem is, it can't be a fully functional hair cell, because you haven't turned the right genes on. And the other problem is, you have now exhausted now your progenitor pool".​

PIPE use one small molecule, whereas Frequency Therapeutics use two. Again, this is what Carl LeBel had to say:

"The other molecule is a proprietary molecule, and this is a molecule that goes after a specific pathway, and as I said earlier, it's the combination of these two agents that we think is important"​

So far, I don't think I've said anything new, but I just wanted to bring people up to speed in any case. But now, here's the slide from Frequency Therapeutics that I think is interesting and backs up the above:

So what we are looking at here are the differences in cell proliferation when using the two Frequency Therapeutics molecules individually and then together. As I don't have a medical background, the first thing I was interested to know was whether valproic acid (sodium valproate), as a HDAC Inhibitor, is comparable to other notch inhibitor approaches companies such as PIPE have. After a bit of research I came across this paper, which looks at transdifferentiation of pancreatic cells using HDAC. Indeed, it seems to confirm that "HDAC1 regulates retinal neurogenesis by suppressing Wnt and Notch signalling pathways". Obviously we are talking about ears and not eyes here, but I'm assuming the science remains largely relevant.

So one could argue that what we are looking at in the 3rd graph is something close to what one might see when using PIPE's drug, as that too is a notch inhibitor. If anything, it may be even worse in reality, because HDAC seems to also have Wnt inhibition properties as well. What's interesting about the last graph though is that although the red area is more than double, if you look at the x-axis, which indicates the number of green-fluorescent progenitors observed, the numbers are logarithmic. Using Frequency Therapeutics' approach, we are seeing 10x more progenitors compared to single-molecule approaches.

I can only then infer that the reason why there isn't much cell support depletion observed is because there probably isn't much regeneration going on in the first place, at least compared to that observed when using a drug like FX-322.

 

[FGG]

So with FX-322 news imminent, I thought I'd go down the rabbithole a bit with this question in the event Frequency Therapeutics do drop a bombshell on us.

I think most are already aware of this, but PIPE have a transdifferentiation approach when it comes to regenerating hair cells:

View attachment 43636

View attachment 43635

To put it simply, FX-322 causes asymmetric division where you end up with a hair cell and a new progenitor, whereas with PIPE the progenitor turns into a hair cell and the progenitor is depleted. Of course people are confused because PIPE are saying they are not seeing much depletion of the support cells, but as @FGG has pointed out this could be down to the fact that they're not getting much regeneration in the first place.

So I did a bit more digging, and found an interesting slide from one of Frequency Therapeutics' presentations. Before I get to that though, here's just a reminder of what Carl LeBel had to say on this:

"What those [transdifferentation] programmes have tried to do is take a progenitor cell and – it's a process we call transdifferentiation – you're taking a supporting cell, a progenitor cell, and turning it into a hair cell, you're making it want to become a hair cell. The problem is, it can't be a fully functional hair cell, because you haven't turned the right genes on. And the other problem is, you have now exhausted now your progenitor pool".​

PIPE use one small molecule, whereas Frequency Therapeutics use two. Again, this is what Carl LeBel had to say:

"The other molecule is a proprietary molecule, and this is a molecule that goes after a specific pathway, and as I said earlier, it's the combination of these two agents that we think is important"​

So far, I don't think I've said anything new, but I just wanted to bring people up to speed in any case. But now, here's the slide from Frequency Therapeutics that I think is interesting and backs up the above:

View attachment 43638

So what we are looking at here are the differences in cell proliferation when using the two Frequency Therapeutics molecules individually and then together. As I don't have a medical background, the first thing I was interested to know was whether valproic acid (sodium valproate), as a HDAC Inhibitor, is comparable to other notch inhibitor approaches companies such as PIPE have. After a bit of research I came across this paper, which looks at transdifferentiation of pancreatic cells using HDAC. Indeed, it seems to confirm that "HDAC1 regulates retinal neurogenesis by suppressing Wnt and Notch signalling pathways". Obviously we are talking about ears and not eyes here, but I'm assuming the science remains largely relevant.

So one could argue that what we are looking at in the 3rd graph is something close to what one might see when using PIPE's drug, as that too is a notch inhibitor. If anything, it may be even worse in reality, because HDAC seems to also have Wnt inhibition properties as well. What's interesting about the last graph though is that although the red area is more than double, if you look at the x-axis, which indicates the number of green-fluorescent progenitors observed, the numbers are logarithmic. Using Frequency Therapeutics' approach, we are seeing 10x more progenitors compared to single-molecule approaches.

I can only then infer that the reason why there isn't much cell support depletion observed is because there probably isn't much regeneration going on in the first place, at least compared to that observed when using a drug like FX-322.

That's an awesome, well reasoned and well explained deep dig.

Pipeline Therapeutics knew pre-clinically they didn't grow many hair cells but grew synapses well but I think this is obviously preferable when you are dealing with a transducing drug that depletes support cells.

I think what you suggest is part of the picture, however, Audion's drug, if I recall right is also a single molecule (and also a gamma secretase inhibitor like Pipeline Therapeutics' drug) and transduced a non insignificant amount of support cells into "hair cells" as a single molecule.

There has to also be something about the strength of the inhibition, too imo. I.e. maybe a partial or competitive inhibitor doesn't transduce support cells well but stimulates synapse connection.

 

[Tezcatlipoca]

Wow, that's amazing, looks like FREQ may be sitting on the holy grail of regenerative medicine.

 

[Lucifer]

Wow, that's amazing, looks like FREQ may be sitting on the holy grail of regenerative medicine.

FX-322 is going to change the hearing industry for life.

 

[Matchbox]

Pipeline Therapeutics announced the initiation of their Phase 1 clinical trial for PIPE-307, a drug targeting remyelination. At the bottom of the press release under their About, they state that they're still currently enrolling for their PIPE-505 Phase 1/2 which seems kinda... weird? Based on the last update back in November, the estimated completion date was pushed to this month. If they haven't even finished recruitment then they definitely aren't making that deadline.

Pipeline Therapeutics Initiates Phase 1 Clinical Trial of PIPE-307

Auditory nerves at the hair cell base are unmyelinated. This might help MS patients though.

 

[Tezcatlipoca]

Auditory nerves at the hair cell base are unmyelinated. This might help MS patients though.

It is for MS.

 

[Aaron91]

I've had a few more thoughts.

There's been a lot of discussion recently on the FX-322 thread about the pharmacokinetics/dynamics of the two small molecules. Specifically, there has been some concern about VPA, the HDAC inhibitor, being depleted a lot more quickly compared to CHIR99021 in that it does not go as deep into the cochlea, the implication being some regeneration potential goes to waste because Frequency have "undershot" the amount of VPA required. See graph below:

I'm now wondering whether this is actually somewhat intentional.

Given my post yesterday, we can infer that VPA alone, given its inhibition properties, causes progenitors to transduce into hair cells, not divide asymmetrically. The paper I posted yesterday, in paraphrasing another paper, supports this:

"A study recently reported the usefulness of VPA in transdifferentiation of BMSSCs into hepatocytes in vitro."​

So it would appear this is why the two-molecule approach is key, because it is the presence of CHIR99021, the proprietary molecule, that ensures we have asymmetric division and not transdifferentiation, although it's unclear whether the prevention of transdifferentiation is synergetic i.e. either drug alone causes transdifferentiation but together we have asymmetric division.

My point though is this: assuming transdifferentiation is limited to VPA, I'm wondering whether Frequency Therapeutics have purposefully "capped" the amount of VPA relative to CHIR in order to prevent a case transdifferentiation in places where there is not enough CHIR, as an excess of VPA could lead to FX-322 causing unintended support cell depletion.

Thoughts?

Also, do you guys think I should perhaps share this on the Frequency Therapeutics thread?

 

[FGG]

I've had a few more thoughts.

There's been a lot of discussion recently on the FX-322 thread about the pharmacokinetics/dynamics of the two small molecules. Specifically, there has been some concern about VPA, the HDAC inhibitor, being depleted a lot more quickly compared to CHIR99021 in that it does not go as deep into the cochlea, the implication being some regeneration potential goes to waste. See graph below:

View attachment 43662

I'm now wondering whether this is actually somewhat intentional.

Given my post yesterday, we can infer that VPA alone, given its inhibition properties, causes progenitors to transduce into hair cells, not divide asymmetrically. The paper I posted yesterday, in paraphrasing another paper, supports this:

"A study recently reported the usefulness of VPA in transdifferentiation of BMSSCs into hepatocytes in vitro."

So it would appear this is why the two-molecule approach is key, because it is the presence of CHIR99021, the proprietary molecule, that ensures we have asymmetric division and not transdifferentiation, although it's unclear whether the prevention of transdifferentiation is synergetic i.e. either drug alone causes transdifferentiation but together we have asymmetric division.

My point though is this: assuming transdifferentiation is limited to VPA, I'm wondering whether frequency have purposefully "capped" the amount of VPA relative to CHIR in order to prevent a case transdifferentiation in places where there is not enough CHIR, as an excess of VPA could lead to FX-322 causing unintended support cell depletion.

Thoughts?

Also, do you guys think I should perhaps share this on the Frequency Therapeutics thread?

I'm still thinking about this, lol. I have some reading to do. Yeah post it on the Frequency Therapeutics thread...

 

[Diesel]

I've had a few more thoughts.

There's been a lot of discussion recently on the FX-322 thread about the pharmacokinetics/dynamics of the two small molecules. Specifically, there has been some concern about VPA, the HDAC inhibitor, being depleted a lot more quickly compared to CHIR99021 in that it does not go as deep into the cochlea, the implication being some regeneration potential goes to waste because Frequency have "undershot" the amount of VPA required. See graph below:

View attachment 43662

I'm now wondering whether this is actually somewhat intentional.

Given my post yesterday, we can infer that VPA alone, given its inhibition properties, causes progenitors to transduce into hair cells, not divide asymmetrically. The paper I posted yesterday, in paraphrasing another paper, supports this:

"A study recently reported the usefulness of VPA in transdifferentiation of BMSSCs into hepatocytes in vitro."​

So it would appear this is why the two-molecule approach is key, because it is the presence of CHIR99021, the proprietary molecule, that ensures we have asymmetric division and not transdifferentiation, although it's unclear whether the prevention of transdifferentiation is synergetic i.e. either drug alone causes transdifferentiation but together we have asymmetric division.

My point though is this: assuming transdifferentiation is limited to VPA, I'm wondering whether Frequency Therapeutics have purposefully "capped" the amount of VPA relative to CHIR in order to prevent a case transdifferentiation in places where there is not enough CHIR, as an excess of VPA could lead to FX-322 causing unintended support cell depletion.

Thoughts?

Also, do you guys think I should perhaps share this on the Frequency Therapeutics thread?

Interesting thought. If that's the case, then perhaps in additional doses, the VPA would flow past the already OHC/IHC that have asymmetrically divided, and be absorbed by LGR5+ cells deeper in the cochlea. There would likely already be CHIR present. I may re-post this in the Frequency Therapeutics thread...

 

[Aaron91]

Interesting thought. If that's the case, then perhaps in additional doses, the VPA would flow past the already OHC/IHC that have asymmetrically divided, and be absorbed by LGR5+ cells deeper in the cochlea. There would likely already be CHIR present. I may re-post this in the Frequency Therapeutics thread...

Just working on a draft now compiling all posts.

 

[Zugzug]

I've had a few more thoughts.

There's been a lot of discussion recently on the FX-322 thread about the pharmacokinetics/dynamics of the two small molecules. Specifically, there has been some concern about VPA, the HDAC inhibitor, being depleted a lot more quickly compared to CHIR99021 in that it does not go as deep into the cochlea, the implication being some regeneration potential goes to waste because Frequency have "undershot" the amount of VPA required. See graph below:

View attachment 43662

I'm now wondering whether this is actually somewhat intentional.

Given my post yesterday, we can infer that VPA alone, given its inhibition properties, causes progenitors to transduce into hair cells, not divide asymmetrically. The paper I posted yesterday, in paraphrasing another paper, supports this:

"A study recently reported the usefulness of VPA in transdifferentiation of BMSSCs into hepatocytes in vitro."​

So it would appear this is why the two-molecule approach is key, because it is the presence of CHIR99021, the proprietary molecule, that ensures we have asymmetric division and not transdifferentiation, although it's unclear whether the prevention of transdifferentiation is synergetic i.e. either drug alone causes transdifferentiation but together we have asymmetric division.

My point though is this: assuming transdifferentiation is limited to VPA, I'm wondering whether Frequency Therapeutics have purposefully "capped" the amount of VPA relative to CHIR in order to prevent a case transdifferentiation in places where there is not enough CHIR, as an excess of VPA could lead to FX-322 causing unintended support cell depletion.

Thoughts?

Also, do you guys think I should perhaps share this on the Frequency Therapeutics thread?

Extremely nice write up (even going back a couple posts). I've been studying this process to death, but never really took the time to understand the roles of VPA and CHIR -- I just assumed it was out of reach. Whether or not your theory is correct, you explained the science like a high-level ELI5 post. I also now know more about PIPE-505 and even their remyelinating drug program. Good stuff.

As far as your theory, and obviously I'm speculating from a place of total ignorance, I find it unlikely that CHIR has anything to do with HDAC inhibition or transdifferentiation. As even just a layman, it seems like the risk is too high of depleting LGR5+, which would be a major mistake for a company that devised such beautiful synergy.

Something I'm wondering about with those 2 graphs is why the z-axis has different scaling. Is it just a matter of molecular weight differences mostly? I must say, on the math end, it's strange to me that the axes are such perfect numbers like powers of 10. The logarithmic scaling makes sense, but I wonder if they could have produced a more informative VPA graph.

I probably have more thoughts that I will add to the Frequency Therapeutics thread.

 

[FGG]

@Aaron91, did you find anywhere that VPA can transduce cochlear cells in particular? Just because they can affect hepatocytes does not automatically mean the effects would be the same. I'm actually finding studies that show VPA promotes neurite outgrowth, which is possibly why Frequency Therapeutics is using it...

Edit: this paper would help, if I could get the whole thing:

Clonal Expansion of Lgr5-Positive Cells from Mammalian Cochlea and High-Purity Generation of Sensory Hair Cells

 

[dmorg]

Just working on a draft now compiling all posts.

Holy Smokes! Thank you for all of this. Do you ever sleep!?

 

[annV]

Edit: this paper would help, if I could get the whole thing:

Clonal Expansion of Lgr5-Positive Cells from Mammalian Cochlea and High-Purity Generation of Sensory Hair Cells

Hi FGG.

 

[Aaron91]

@Aaron91, did you find anywhere that VPA can transduce cochlear cells in particular? Just because they can affect hepatocytes does not automatically mean the effects would be the same. I'm actually finding studies that show VPA promotes neurite outgrowth, which is possibly why Frequency Therapeutics is using it...

Edit: this paper would help, if I could get the whole thing:

Clonal Expansion of Lgr5-Positive Cells from Mammalian Cochlea and High-Purity Generation of Sensory Hair Cells

Great question. I had the same question and the only example I was able to find was in zebrafish but there could be others as well. Here's the relevant quote from the study though:

"We found that pharmacological inhibition of HDACs using trichostatin A (TSA) or valproic acid (VPA) increased histone acetylation in the regenerated neuromasts following neomycin-induced damage. We also showed that treatment with TSA or VPA decreased the number of supporting cells and regenerated hair cells in response to hair cell damage"​

So to your point it would appear it promotes both neurite growth and hair cell regeneration, but also confirms my theory that VPA does deplete support cells in the process of regeneration. The question I guess now is whether this is applicable to humans. I'm going to keep looking for other papers.

 

[Zugzug]

So to your point it would appear it promotes both neurite growth and hair cell regeneration, but also confirms my theory that VPA does deplete support cells in the process of regeneration. The question I guess now is whether this is applicable to humans. I'm going to keep looking for other papers.

After glancing at the paper, it seems odd they would fail to mention this fact for humans if it was proven to be the case. The introduction paints a picture of human focus, but with zebrafish as something available to show this phenomena. Unless it happened after 2014...

 

[FGG]

Great question. I had the same question and the only example I was able to find was in zebrafish but there could be others as well. Here's the relevant quote from the study though:

"We found that pharmacological inhibition of HDACs using trichostatin A (TSA) or valproic acid (VPA) increased histone acetylation in the regenerated neuromasts following neomycin-induced damage. We also showed that treatment with TSA or VPA decreased the number of supporting cells and regenerated hair cells in response to hair cell damage"​

So to your point it would appear it promotes both neurite growth and hair cell regeneration, but also confirms my theory that VPA does deplete support cells in the process of regeneration. The question I guess now is whether this is applicable to humans. I'm going to keep looking for other papers.

Interestingly, in the paper @annV just linked (you rule as always), it describes one of the functions of VPA as increasing LGR5+ expression so that more support cells can be used as progenitor cells.

The paper also talks about the synergistic effects of notch and wnt co-activation on hair cell regeneration without depleting support cells.

Your paper, @Aaron91 shows VPA on its own depleting support cells. I'm kind of fascinated by the idea that either notch inhibition *or* activation on its own depletes support cells (that's kind of hard for me to wrap my head around).

But maybe you are right. Maybe Frequency Therapeutics designed the drug to "underdose" VPA relative to their proprietary compound.

 

[Aaron91]

After glancing at the paper, it seems odd they would fail to mention this fact for humans if it was proven to be the case. The introduction paints a picture of human focus, but with zebrafish as something available to show this phenomena. Unless it happened after 2014...

Thank you for the kind words further above @Zugzug, you are too kind.

I don't have any answers about the molecular weight thing, that would be a whole other rabbithole lol, but hopefully someone else can chime in.

When you say "they", do you mean Frequency Therapeutics or the team who authored the paper I linked? I must admit it's strange if Frequency Therapeutics know that VPA alone would seem to deplete support cells and not disclose it in any of their communications. Just as an FYI, I understand that the reason zebrafish are often used is because their auditory hair cells are on their skin and so directly and immediately visible. But again, I have no idea how cross-applicable they are to humans.

Interestingly, in the paper @annV just linked (you rule as always), it describes one of the functions of VPA as increasing LGR5+ expression so that more support cells can be used as progenitor cells.

The paper also talks about the synergistic effects of notch and wnt co-activation on hair cell regeneration without depleting support cells.

Your paper, @Aaron91 shows VPA on its own depleting support cells. I'm kind of fascinated by the idea that either notch inhibition *or* activation on its own depletes support cells (that's kind of hard for me to wrap my head around).

But maybe you are right. Maybe Frequency Therapeutics designed the drug to "underdose" VPA relative to their proprietary compound.

I can't get my head around it either, but I'm going to go away and do a bit more reading and see what I can find.

Just to make it clear, I am very much speculating about all this and I know I'm out of my depth in trying to make sense of this stuff. As always I'm just trying to get a conversation going between the brighter minds here.

 

[Tezcatlipoca]

Sorry, even after reading all of this, I can't quite get it. Could you explain it to me like I was a 5-year-old? Please, I really suck with science/medicine stuff.

 

[Aaron91]

Holy Smokes! Thank you for all of this. Do you ever sleep!?

Haha! Not much these days, to be honest. Hyperacusis has turned my life upside down and I'm on a mission to hack this thing or die trying.

 

[FGG]

This article was helpful (I think):

Effects of the histone deacetylase inhibitor valproic acid on Notch signalling in human neuroblastoma cells

It was dealing with VPA's effects on neuroblastoma cells but I think some of the concepts are similar/the same.

"VPA induces loss of proliferative capacity and promotes differentiation"​

I believe that CHIR stimulates proliferation (support cell division) through the wnt pathway but VPA promotes differentiation (through its effects on notch), more below.

I liked this part, btw:

"The mechanisms behind these effects are still not clear"​

Oh good .

"In light of the finding that VPA inhibits HDAC activity, we have studied the effects of the drug on the Notch signalling cascade in NB cells, since this cascade is dependent of HDAC activity and seem to play a role in NB cell differentiation. We here report that VPA induces a more mature phenotype of NB cells along with an increased activity of the Notch signalling cascade."​

It looks like VPA induces the notch pathway but inhibits part of its normal signaling cascade. I.e. it initiates notch but then stops it at a point in the cascade.

How I'm putting this all together:

The notch pathway is responsible for "lateral inhibition", which is basically cell to cell communication telling a neighbor cell not to divide because there is already another cell next to it. My impression (not sure how right I am on this) is that inhibiting this in some way seems vital (and VPA seems to inhibit part of the pathway) so that a support cell (an LGR5+ one) starts dividing again even though it's adjacent to other intact support cells. Without this, notch might prevent the wnt activation from producing new cells.

I could be way off here. Other thoughts?

 

[spacehunter]

So to your point it would appear it promotes both neurite growth and hair cell regeneration, but also confirms my theory that VPA does deplete support cells in the process of regeneration. The question I guess now is whether this is applicable to humans. I'm going to keep looking for other papers.

In the Frequency Therapeutics thread there was a vivid discussion about the VPA concentration dropping much faster than the propriety molecule. If VPA alone really depletes hair cells, maybe the asymmetry in concentration is on purpose to avoid a surplus of VPA depleting the support cells.

 

[Aaron91]

In the Frequency Therapeutics thread there was a vivid discussion about the VPA concentration dropping much faster than the propriety molecule. If VPA alone really depletes hair cells, maybe the asymmetry in concentration is on purpose to avoid a surplus of VPA depleting the support cells.

Indeed, and this was effectively the conclusion I came to in my two rather lengthy posts where I posted the pharmacokinetics models for VPA. I cross-posted them to the Frequency Therapeutics thread yesterday for everyone's benefit.

@FGG, I'm going to need some time to do a bit more reading into this and come back to you. I'm definitely enjoying this debate, so thanks for joining me down this rabbithole, and I hope others will jump in as well.

Whatever the case, it's plausible that even Frequency Therapeutics don't fully understand what's going on here. As many of us know, it wouldn't be the first time a drug has made it to market without fully understanding why or how it works.

All I can say is that it's all incredibly fascinating and I now have some questions for the next podcast I never anticipated on having. Your post on the Frequency Therapeutics thread just now about VPA awakening dormant viruses has also put me on edge a little, but would seem to add credence to my initial thesis that VPA was purposely underdosed, either in the interests of minimising transdifferentiation and/or ensuring safety. I didn't have any concerns previously about AEs, but given the small sample size of Phase 1 I'm now realising that may have been somewhat naive on my part. I'll definitely be looking more closely at AEs in Phase 2 results.

 

[Aaron91]

This article was helpful (I think):

Effects of the histone deacetylase inhibitor valproic acid on Notch signalling in human neuroblastoma cells

It was dealing with VPA's effects on neuroblastoma cells but I think some of the concepts are similar/the same.

"VPA induces loss of proliferative capacity and promotes differentiation"​

I believe that CHIR stimulates proliferation (support cell division) through the wnt pathway but VPA promotes differentiation (through its effects on notch), more below.

I liked this part, btw:

"The mechanisms behind these effects are still not clear"​

Oh good .

"In light of the finding that VPA inhibits HDAC activity, we have studied the effects of the drug on the Notch signalling cascade in NB cells, since this cascade is dependent of HDAC activity and seem to play a role in NB cell differentiation. We here report that VPA induces a more mature phenotype of NB cells along with an increased activity of the Notch signalling cascade."​

It looks like VPA induces the notch pathway but inhibits part of its normal signaling cascade. I.e. it initiates notch but then stops it at a point in the cascade.

How I'm putting this all together:

The notch pathway is responsible for "lateral inhibition", which is basically cell to cell communication telling a neighbor cell not to divide because there is already another cell next to it. My impression (not sure how right I am on this) is that inhibiting this in some way seems vital (and VPA seems to inhibit part of the pathway) so that a support cell (an LGR5+ one) starts dividing again even though it's adjacent to other intact support cells. Without this, notch might prevent the wnt activation from producing new cells.

I could be way off here. Other thoughts?

@FGG I'm still gathering my own thoughts and making notes on all this.

In the meantime, check this paper out, which looks at notch signalling in the intestine. As we already know, the intestine was the initial starting point of Frequency Therapeutics' research:

"More ligand at the cell surface activates Notch signalling in the neighbouring cell which results in reduced ligand production in that cell. This in turn enables the cell with lower Notch activity to increase its ligand production even further, because it receives a weakened inhibitory signal back from its neighbours. The effect of this feedback loop is that any initial difference in Notch activity between them, whether stochastic or genetically controlled, is amplified to drive the neighbouring cells into opposite Notch-level status and hence into different developmental pathways"​

If I'm reading this correctly, am I right in saying that it would appear any cell division induced by inhibiting notch signalling (in this case, through VPA) is self-limiting? Here's the next paragraph from the same paper:

"The fate of the cell also depends on the strength of the Notch signal it receives. The NICD transcriptional program represses genes encoding the Notch ligands (Delta-like, Jagged), so strong Notch activation in the receiving cell reduces its ability to activate its neighbouring cell. Because fate specification is controlled by cell-to-cell signalling between adjacent cells, this process is referred to as "lateral cell fate specification" or "lateral inhibition". At its most basic, lateral inhibition amplifies and stabilises the stochastic initial differences in Notch signalling between two equivalent adjacent cells, rapidly pushing them towards opposite fates".​

So it would appear VPA does have some effect on notch signalling when used with the proprietary molecule, but it would also appear that the very process of inhibiting notch signalling (in the interests of cell proliferation) is self-limiting... which is how nature intended it??? Am I right in saying this, or am I totally off here?

So I now come to your point which, if I'm not mistaken, is that CHIR creates news cells through activating the wnt pathway, but that without notch signalling inhibition (from something like VPA), you have a maintenance of lateral inhibition even in the presence of wnt signalling, or at least partial maintenance of lateral inhibition (in this case, a support cell not dividing because there is already another support cell next to it).

The thing is, we can see from the initial graphs I shared that we do get some cell proliferation when only CHIR is used alone, suggesting then that you simply have progenitors copying themselves. But we can also see that this proliferation tapers off compared to when the two molecules are used together, presumably due to notch signalling, which (I presume) is telling the other progenitors that have not been activated by CHIR (when used alone) to not copy themselves. This would seem very odd, unless of course, as I said, the process is by nature self-limiting.

Until this point, my understanding of this whole process was that FX-322 does the following: a support cell divides in two other cells with opposite fates: one becomes another progenitor, the other becomes a hair cell. But I'm now wondering how this is possible given that one molecule seems to proliferate the same type of cell, while the other seems to transdifferentiate into a different cell entirely? The only explanation I have then is that these molecules clearly behave very differently when used together (perhaps I'm stating the obvious here lol), but it's still a mystery to me as to why.

One of the things I've been reading about today is how HDAC inhibition reduces or stops deacetylation. By preventing deacetylation, it becomes easier for certain transcription factors to bind to DNA, and I'm wondering whether VPA, through its HDAC inhibition properties, actually allows CHIR to have additional gene expression properties that are not seen when CHIR is used alone, because the DNA is too tightly bound to the histone when VPA isn't present. This is all speculation, but say this is true, I'm wondering whether the prevention of deacetylation caused by VPA allows CHIR to induce gene expression relevant to asymmetric division. This would be consistent with what Carl LeBel has said in that, when using the transdifferentiation approach alone, you're not turning on the right genes, suggesting then that CHIR is needed to turn on the right genes, but clearly CHIR alone doesn't do the job. I'm still developing this thesis but any feedback in the meantime would be greatly appreciated.

 

[FGG]

In the meantime, check this paper out, which looks at notch signalling in the intestine. As we already know, the intestine was the initial starting point of Frequency Therapeutics' research:

"More ligand at the cell surface activates Notch signalling in the neighbouring cell which results in reduced ligand production in that cell. This in turn enables the cell with lower Notch activity to increase its ligand production even further, because it receives a weakened inhibitory signal back from its neighbours. The effect
of this feedback loop is that any initial difference in Notch activity between them, whether stochastic or genetically controlled, is amplified to drive the neighbouring cells into opposite Notch-level status and hence into different developmental pathways"​

If I'm reading this correctly, am I right in saying that it would appear any cell division induced by inhibiting notch signalling (in this case, through VPA) is self-limiting? Here's the next paragraph from the same paper:

"The fate of the cell also depends on the strength of the Notch signal it receives. The NICD transcriptional program represses genes encoding the Notch ligands (Delta-like, Jagged), so strong Notch activation in the receiving cell reduces its ability to activate its neighbouring cell. Because fate specification is controlled by cell-to-cell signalling between adjacent cells, this process is referred to as "lateral cell fate specification" or "lateral inhibition". At its most basic, lateral inhibition amplifies and stabilises the stochastic initial differences in Notch signalling between two equivalent adjacent cells, rapidly pushing them towards opposite fates".​

So it would appear VPA does have some effect on notch signaling when used with the proprietary molecule, but it would also appear that the very process of inhibiting notch signaling (in the interests of cell proliferation) is self-limiting... which is how nature intended it??? Am I right in saying this, or am I totally off here?

I'm a mostly visual and analogy based thinker (to the point where if people could view my thoughts, they'd think I was on a constant acid trip, lol), so I'm not always good at explaining thoughts in a more concrete way so bear with me, but the way I have sorted in my mind is that notch seems to act as a directional signal. I.e. "this way is crowded, divide the other way" that is very important in tissue development and embryology for the right shape and structure. It's why the intestines won't "over proliferate" for instance when regenerating. Your link also brought up the concept of "lateral cell fate specification" which seems to suggest a role in limiting the amount of cells that go down a particular fate pathway (which makes sense here as one daughter cell turns into another progenitor cell and one turns into a hair cell).

VPA actually stimulates notch but also inhibits part of the cascade, so it's a bit complicated but to me, it seems to "allow" cell proliferation and transdifferentiation and not "cause" it. That's why it would stimulate transdifferentiation in an already regenerative type tissue but I don't believe it would in the cochlea.

The thing is, we can see from the initial graphs I shared that we do get some cell proliferation when only CHIR is used alone, suggesting then that you simply have progenitors copying themselves. But we can also see that this proliferation tapers off compared to when the two molecules are used together, presumably due to notch signalling, which (I presume) is telling the other progenitors that have not been activated by CHIR (when used alone) to not copy themselves. This would seem very odd, unless of course, as I said, the process is by nature self-limiting.

I don't think you are getting very much proliferation at all when just CHIR is used. If you look at the CHIR graph vs just culture media alone, there really isn't much difference (and no real difference with just VPA alone, which I don't believe is because VPA is transdifferentiating all of the cells perfectly, I think VPA doesn't have that effect on cells who have inactivated their regenerative pathways). I think the cells are likely primed to regenerated by need to receive the appropriate signals from the notch pathways in order to, simply explained, be directed to fate and direction and "go to where they are needed".

One of the things I've been reading about today is how HDAC inhibition reduces or stops deacetylation. By preventing deacetylation, it becomes easier for certain transcription factors to bind to DNA, and I'm wondering whether VPA, through its HDAC inhibition properties, actually allows CHIR to have additional gene expression properties that are not seen when CHIR is used alone, because the DNA is too tightly bound to the histone when VPA isn't present. This is all speculation, but say this is true, I'm wondering whether the prevention of deacetylation caused by VPA allows CHIR to induce gene expression relevant to asymmetric division. This would be consistent with what Carl LeBel has said in that, when using the transdifferentiation approach alone, you're not turning on the right genes, suggesting then that CHIR is needed to turn on the right genes, but clearly CHIR alone doesn't do the job. I'm still developing this thesis but any feedback in the meantime would be greatly appreciated.

I think that's plausibly part of it. It most likely adds to this process (and certainly doesn't impair it).

I really love reading your thoughts on this. There really isn't much "debate" other than I don't think VPA transdifferentiating on its own when present without CHIR is an issue, personally. If Frequency Therapeutics made a deliberate underdosing of VPA decision, I don't think it was for that reason. Luckily, they did say they'd like to be back for a follow up Tinnitus Talk Podcast at some point so there will be lots of questions like this .

 

[Aaron91]

I'm a mostly visual and analogy based thinker (to the point where if people could view my thoughts, they'd think I was on a constant acid trip, lol), so I'm not always good at explaining thoughts in a more concrete way so bear with me, but the way I have sorted in my mind is that notch seems to act as a directional signal. I.e. "this way is crowded, divide the other way" that is very important in tissue development and embryology for the right shape and structure. It's why the intestines won't "over proliferate" for instance when regenerating. Your link also brought up the concept of "lateral cell fate specification" which seems to suggest a role in limiting the amount of cells that go down a particular fate pathway (which makes sense here as one daughter cell turns into another progenitor cell and one turns into a hair cell).

VPA actually stimulates notch but also inhibits part of the cascade, so it's a bit complicated but to me, it seems to "allow" cell proliferation and transdifferentiation and not "cause" it. That's why it would stimulate transdifferentiation in an already regenerative type tissue but I don't believe it would in the cochlea.

I don't think you are getting very much proliferation at all when just CHIR is used. If you look at the CHIR graph vs just culture media alone, there really isn't much difference (and no real difference with just VPA alone, which I don't believe is because VPA is transdifferentiating all of the cells perfectly, I think VPA doesn't have that effect on cells who have inactivated their regenerative pathways). I think the cells are likely primed to regenerated by need to receive the appropriate signals from the notch pathways in order to, simply explained, be directed to fate and direction and "go to where they are needed".

I think that's plausibly part of it. It most likely adds to this process (and certainly doesn't impair it).

I really love reading your thoughts on this. There really isn't much "debate" other than I don't think VPA transdifferentiating on its own when present without CHIR is an issue, personally. If Frequency Therapeutics made a deliberate underdosing of VPA decision, I don't think it was for that reason. Luckily, they did say they'd like to be back for a follow up Tinnitus Talk Podcast at some point so there will be lots of questions like this .

Thank you for the kind words, and of course the feeling is always mutual.

I'm actually mentally exhausted by how much I've read today trying to get my head round all this lol. In short, for reasons I've just given on the FX-322 thread, I think you're right, although I still think there's a good chance Frequency Therapeutics undershot VPA intentionally, either for some safety reasons you've alluded to or some other reason we remain unaware of.

In the meantime, I'm going to look into HDAC inhibition more and its affects on deacetylation.

 

[RichieTheKid]

I'm quite interested in this. Pipeline Therapeutics is very promising.

Anybody aware of the current status of this? Also I thought this was strictly synapse repair, as I see some posts above suggesting hair cell regeneration?