Reconstituting agent for Cagrilintide

[hexagonal]

Respectfully, I feel like you (and others) are conflating issues that have nothing to do with each other and creating heightened risks where they simply don't exist.

Whether or not Cagrilinitide is safe for long term use is completely separate from the issue of fibril formation. There may be 0 practical risk of fibril formation happening, but it could be proven unsafe for other reasons. In that regard we should treat it just like any other unapproved drug.

There is a huge difference between "pH you need to formulate a specific compound" and reconstitution pH. It is very normal that compounds only form at a certain pH, temperature, pressure, concentration, and are then perfectly stable after. Pretty much all of the inorganic substances we interact with on a daily basis operate this way.

It's possible that the Cagrilinitide we buy from grey sources experiences more "stress", but it doesn't matter if that stress isn't comparable to the stress needed for fibril formation - 41 hrs, 98°, in a centrifuge.

No, it's not separate from the issue of fibril formation. But you are correct that even if fibril formation isn't an issue, it could still be unsafe for other reasons.

But we know that fibril formation is a health risk that NN considers important enough to have selected for formulations that minimize them.

The NN patent also specifies that Cagrilintide should be stored at that PH until injection time when it is mixed with Semaglutide in their patent. They mention that acetic acid is used to keep the pH stable post formulation, and that the concentration of buffer must keep it at 4.0 in the medical device. They're explicit all over the patent about it needing to be right around that pH all the way until delivery, even though it means having to store it separately in the device to the semaglutide. It does not seem like it's just a matter of needing to be at that pH only during the original formulation.

The centrifuge experiment is meant just to speed up the process - it's quite possible that 2 weeks sitting in a warehouse and then being transported by <truck/train/plane> before being lyophilized ultimately has the same result - and lyophilization won't do anything to the fibrils besides also turning them to powder, and then they'll reconstitute just like the peptide. It's like how they'll "age" whisky by putting the barrels on a boat - 6 months there produces similar results to sitting in a barrel house for a decade or so.

NN might be doing all of this out of an overabundance of caution and it's all fine regardless, but I'm a pretty firm believer that unless we really know that the fibrils are a nonissue that confidently telling people it's a nonissue is a mistake.

For example, this is all I would feel comfortable saying about the potential safety of cagri:

"We do not know what pH the original Cagrilintide raw material was formulated at and what the conditions were prior to lyophilization. Novo Nordisk believes that cagrilintide must be kept stable at around 3.5 - 4.5 pH, favoring 4.0, due to concerns around fibril formation. There are potential concerns around health risks from fibrils. We know that under relatively extreme conditions that cagrilintide at a 7.5 pH will form fibrils. We do not know how closely those conditions match the process for what we have available for purchase or how transferable that information is to other conditions. You can use acetic acid to get the pH for your cagrilintide down to 4 after the fact. You can use a filter to potentially remove any fibrils that have aggregated together, but singular fibrils that did not aggregate or ones that did not aggregate to larger than the filter's size will not be caught. There are of course also the general risks of unknown long-term side effects even unrelated to fibril formation. Choose whether these concern you enough to skip cagrilintide or not based on your own risk tolerance."

 

[noteablequotable]

The centrifuge experiment is meant just to speed up the process - it's quite possible that 2 weeks sitting in a warehouse and then being transported by <truck/train/plane> before being lyophilized ultimately has the same result

That's not what the paper says at all:

"Propensity toward formation of fibrils upon exposure to mechanical stress was assessed"

"Mechanical stress was applied using a Fluoroskan Ascent FL fluorescence plate reader (Bie & Berntsen A/S, Roedovre, Denmark) at 37 °C incubation, 960 rpm, 1 mm amplitude"

The NN patent also specifies that Cagrilintide should be stored at that PH until injection time when it is mixed with Semaglutide in their patent. They mention that acetic acid is used to keep the pH stable post formulation, and that the concentration of buffer must keep it at 4.0 in the medical device. They're explicit all over the patent about it needing to be right around that pH all the way until delivery, even though it means having to store it separately in the device to the semaglutide. It does not seem like it's just a matter of needing to be at that pH only during the original formulation.

Here's how I read the paper that I think we're both discussing, but hasn't been explicitly quoted so far:

The first time pH is mentioned it is because attempting to formulate their first compound at a ph of 7 resulted in deamination - it wasn't stable enough.

"We found that formulation at pH 7 caused chemical instability including deamidation of asparagine residues, as reported for pramlintide, (17,18) and this could not be solved by formulations or minor pH adjustments. In order not to deviate too much from h-amylin, formulation at low pH seemed necessary."

It was much more stable at a pH of 4, but that did not stop fibril formation.

"Formulation at pH 4 was preferred for chemical stability based on published pramlintide data, as described above. (18) However, when 1 was taken into further preclinical development and high doses were given subcutaneously to rats, there were quite severe injection site reactions with signs of necrosis. A precipitate was observed at the injection site that appeared to have a fibrillar structure resembling classical h-amylin fibrils when studied with electron microscopy."

This appeared to be the result of fibril formation at higher a pH when solubility was low

"The hypothesis emerged that compound 1 in low doses binds to albumin at the injection site and is kept soluble by albumin despite low solubility at pH 7.4 in albumin-free aqueous solutions. At high doses, there might not be enough albumin at the injection site, and compound 1 precipitates and forms fibrils as pH rises."

They did not solve this by tinkering with the pH, they tossed this product (compound 1), and started looking for something that had better solubility and lower propensity to form fibrils in the pH range that offered the most stability.

"As fibrils of h-amylin are known to be cytotoxic, (31) the new design strategy therefore focused at identifying compounds with better solubility in the pH range 4.0–7.4 and with low tendency to form fibrils."

The explicit purpose of the centrifuge test was to evaluate tendency to form fibrils.

"Two assays were introduced to accommodate the selection. First, a simple solubility assay (Table 4) in the pH range from formulation pH (4.0) to physiological (7.4) was used... Second a fibrillation assay used in insulin research was introduced to filter out compounds with a high propensity to fibrillate (Table 5). It was based on Thioflavin T fluorescence of fibrils after exposure to mechanical stress over 2 days.

They created over 800 compounds while trying to get this right. Only four resisted fibril formation in the assay (mechanical centrifuge test) for more than 35 hours and showed significant potency.

"Among the more than 800 amylin-based peptides made..."

"Only nine compounds had a lag-time longer than 35 h, and of those, only four had attractive in vivo properties—high potency and long duration of action (8, 22, 23, and 24)."

They ultimately went with #23 because they expected it to perform better when in humans. Compound #23 is Cagrilinitide.


"In the final choice between 22 and 23 (differing at position 17; Figure 2), the species difference in vitro indicated that 23 might have the largest potency difference in favor of the human receptor compared to 22."

 

[hexagonal]

That's not what the paper says at all:

"Propensity toward formation of fibrils upon exposure to mechanical stress was assessed"

"Mechanical stress was applied using a Fluoroskan Ascent FL fluorescence plate reader (Bie & Berntsen A/S, Roedovre, Denmark) at 37 °C incubation, 960 rpm, 1 mm amplitude"

I'm not sure I understand what you think the purpose of the test was, then. Purely to see what happens if consumers happen to centrifuge it at high heat before they use it? It's obviously intended to be a proxy for accelerating the degradation that they believe can happen over time - only testing for 45h after creation would be useless for any sort of commercial use case - no one is taking it within that timeframe.

Here's how I read the paper that I think we're both discussing, but hasn't been explicitly quoted so far:

The first time pH is mentioned it is because attempting to formulate their first compound at a ph of 7 resulted in deamination - it wasn't stable enough.

"We found that formulation at pH 7 caused chemical instability including deamidation of asparagine residues, as reported for pramlintide, (17,18) and this could not be solved by formulations or minor pH adjustments. In order not to deviate too much from h-amylin, formulation at low pH seemed necessary."

It was much more stable at a pH of 4, but that did not stop fibril formation.

"Formulation at pH 4 was preferred for chemical stability based on published pramlintide data, as described above. (18) However, when 1 was taken into further preclinical development and high doses were given subcutaneously to rats, there were quite severe injection site reactions with signs of necrosis. A precipitate was observed at the injection site that appeared to have a fibrillar structure resembling classical h-amylin fibrils when studied with electron microscopy."

This appeared to be the result of fibril formation at higher a pH when solubility was low

"The hypothesis emerged that compound 1 in low doses binds to albumin at the injection site and is kept soluble by albumin despite low solubility at pH 7.4 in albumin-free aqueous solutions. At high doses, there might not be enough albumin at the injection site, and compound 1 precipitates and forms fibrils as pH rises."

They did not solve this by tinkering with the pH, they tossed this product (compound 1), and started looking for something that had better solubility and lower propensity to form fibrils in the pH range that offered the most stability.

"As fibrils of h-amylin are known to be cytotoxic, (31) the new design strategy therefore focused at identifying compounds with better solubility in the pH range 4.0–7.4 and with low tendency to form fibrils."

The explicit purpose of the centrifuge test was to evaluate tendency to form fibrils.

"Two assays were introduced to accommodate the selection. First, a simple solubility assay (Table 4) in the pH range from formulation pH (4.0) to physiological (7.4) was used... Second a fibrillation assay used in insulin research was introduced to filter out compounds with a high propensity to fibrillate (Table 5). It was based on Thioflavin T fluorescence of fibrils after exposure to mechanical stress over 2 days.

They created over 800 compounds while trying to get this right. Only four resisted fibril formation in the assay (mechanical centrifuge test) for more than 35 hours and showed significant potency.

"Among the more than 800 amylin-based peptides made..."

"Only nine compounds had a lag-time longer than 35 h, and of those, only four had attractive in vivo properties—high potency and long duration of action (8, 22, 23, and 24)."

They ultimately went with #23 because they expected it to perform better when in humans. Compound #23 is Cagrilinitide.


"In the final choice between 22 and 23 (differing at position 17; Figure 2), the species difference in vitro indicated that 23 might have the largest potency difference in favor of the human receptor compared to 22."

But if you look at Table 5, #23 was still found to create fibrils.

"The strategy here was to select compounds with as low propensity for amyloid fibril formation as possible, that is, significantly longer lag-time than 1 and preferable no amyloid fibril formation at all within the assay time frame of 45 h"

The lag time for 23 at a pH of 4.0 meets that 45 h bar, but it does not at the 7.5 pH and the recovery % also drops significantly compared to 4.0


The patent is even more interesting than the study, though. They found even at a pH of 4 that it was buffer dependent on if fibrils formed, via testing at ambient temperature and 100 end-over-end inversions of syringes per day. Look at the section starting around Table 2-1. This is a significantly less extreme test than the heated centrifuge, but we still got fibrils. Glutamate significantly outperformed the other buffering agents - even at 4.0 some of the buffering agents formed fibrils quite fast, particularly benzoate.

We have no idea what the pH or buffering agents are for these when purchased from China.


Again, I'm not saying that I know this stuff is dangerous - maybe they're using glutamate and keeping the pH at 4 and not throwing it around all over the place. If so, sweet! But, maybe they're not bothering to drop the pH to 4 and are instead keeping it closer to 7.5, and maybe they're using acetate for the buffering agent, and maybe it's getting tossed around all over the place after formulation. We just don't know! And that's why I'd advocate against saying fibrils are a nonissue, because I don't think we have the information available to us to be confident in that.

 

[penewbie]

Having read the patent, there is absolutely no way NN would design a whole new pen with TWO different chambers of differing pH in it if they thought CagriSema could co-exist in the same pH.

Now the question remains, is this because long term storage of liquid peptide solution in sub-optimal pH harms the peptide, or even short term storage does the same.

If it's only long term storage, we don't need to be concerned, no one here keeps it longer than 6 months anyway, and even that is a serious stretch.

However if even short term storage is a problem, well it spooks me enough to not try this peptide in pH 7 until we have better evidence.

 

[Lullabytreehugger]

How do i drop my reconstitution of cagri with bac water down to ph4?

 

[Christine S]

You don't need to worry about fibrils unless you plan to also hear up your Cagri and put it in a centrifuge

Войдите на Facebook

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[Christine S]

Войдите на Facebook

Войдите на Facebook, чтобы общаться с друзьями, родственниками и знакомыми.

www.facebook.com

Novo has admitted to amylin fibrils.

 

[Lullabytreehugger]

Novo has admitted to amylin fibrils.

Ohh goodness so what shall we do? I have cagri in my freezer but still haven’t reconstituted it …

 

[Christine S]

Im assuming reconstitute and use asap or not at all.

 

[GimmeABreak]

Having read the patent, there is absolutely no way NN would design a whole new pen with TWO different chambers of differing pH in it if they thought CagriSema could co-exist in the same pH.

Now the question remains, is this because long term storage of liquid peptide solution in sub-optimal pH harms the peptide, or even short term storage does the same.

If it's only long term storage, we don't need to be concerned, no one here keeps it longer than 6 months anyway, and even that is a serious stretch.

However if even short term storage is a problem, well it spooks me enough to not try this peptide in pH 7 until we have better evidence.

Since BAC water will raise the PH, could you reconstitute a sacrificial vial with sterile water and check if the PH is in the four range as a test of the manufacturer's process. If you are hellbent on researching this peptide would it also make sense to buy the smallest mg vials and reconstitute new vials as often as possible?

 

[noteablequotable]

Novo has admitted to amylin fibrils.

Ohh goodness so what shall we do? I have cagri in my freezer but still haven’t reconstituted it …

This is literally the paper I was posting direct quotes from above....did you read through any of my previous posts at all?

 

[TooBigtoFail]

Here is my contribution on the pH:
Pramlintide put Novo on the track for Cagrilintide, very stable at pH4.
Read this paper "Kinetics of pramlintide degradation in aqueous solution as a function of temperature and pH"
https://pmc.ncbi.nlm.nih.gov/articles/PMC2784819/

It provides info on Pram pH@4.0, temperature and stability of a peptide improved on, to create Cagri. Now read this article on Cmpd C, a new peptide better that Cagri. See how bad Cagri does at pH7.4 on a standard thermal test to evaluate stability (this is used to figure out its survivability during poor transport conditions) .

"789-P: Discovery of a Novel, Long-Acting Amylin Receptor Agonist for Body Weight Control"
https://diabetesjournals.org/diabet...789-P-Discovery-of-a-Novel-Long-Acting-Amylin

My plan is to target a ph4.0 for my Cagri recon, to retain a decent potency for 28 days or more. I am days away to get all my supply in, for my first recon of Cagri and can't wait to correct my Tirz stall with Cagri.

Based on a few tests, I need to add between 2cc to 6cc of AA@0.6% to get down to pH4.0, unless QSC 's lyophilized vials have the correct buffer already mixed in. One simple option is that if I blindly add 2cc of AA on an already buffered QSC vial, and get a pH3.5, Cagri patent say it is fine at pH3.5. It may string (and I will have to add bac to my next syringe before I poke) but will have to do, until I find a way to accurately measure recon pH. I really do not want to open and waste a Cagri recon just to use my electronic test probe.

 

[exploitedworkerbee]

unless QSC 's lyophilized vials have the correct buffer already mixed in. One simple option is that if I blindly add 2cc of AA on an already buffered QSC vial, and get a pH3.5

I don’t know about the most recent stuff but I checked ph on my qsc cagri purchased about 4 months ago and it was around 7. So, no buffer.

 

[FreeSpectator8]

Ohh goodness so what shall we do? I have cagri in my freezer but still haven’t reconstituted it …

Novo has admitted to amylin fibrils.

The only thing that whole long meaningless facebook post says is that in manufacturing the drug needs to be at PH 4 and no higher. It has nothing to do with storage after formation.

 

[FreeSpectator8]

This is literally the paper I was posting direct quotes from above....did you read through any of my previous posts at all?

No they clearly didn't. And they clearly don't understand the research they are quoting and are spreading misinformation and fearmongering.

 

[hexagonal]

The only thing that whole long meaningless facebook post says is that in manufacturing the drug needs to be at PH 4 and no higher. It has nothing to do with storage after formation.

They are testing for fibrils on the manufactured material in it's post-buffered state in both the patent and in the paper quoted. They even assess it over multiple weeks in the patent. This is not just during the manufacturing process, this is in it's post-buffer, post-constitution state.

There are reasonable arguments about whether or not any of this matters (and indeed, NN is testing a single-chamber pen for cagrisema now), but you are incorrect in this statement.

 

[JohnnyGobbs]

I tested my nexa cagri with Amazon bac, ph was approx 6. I then added acetic acid and got it down to approx 4.5-4.0.

 

[FreeSpectator8]

I tested my nexa cagri with Amazon bac, ph was approx 6. I then added acetic acid and got it down to approx 4.5-4.0.

Can you share roughly the amount of each liquid you used? Also if you dont mind linking the products you used?

 

[janedoe]

Can you share roughly the amount of each liquid you used? Also if you dont mind linking the products you used?

I'm curious about this for Cagri as well. My plan is to start with BAC to test ph, then lightly add AA if needed. However, it would be interesting to see others recon trials if they have to adjust PH for Cagri and the amount consensus of each. I also contemplated using Hospira BAC w/ AA mix, but that may make ph more difficult to tweak, if tweaking is needed.

 

[FreeSpectator8]

They are testing for fibrils on the manufactured material in it's post-buffered state in both the patent and in the paper quoted. They even assess it over multiple weeks in the patent. This is not just during the manufacturing process, this is in it's post-buffer, post-constitution state.

There are reasonable arguments about whether or not any of this matters (and indeed, NN is testing a single-chamber pen for cagrisema now), but you are incorrect in this statement.

First of all, I was not talking to you. I was directly responding to a user who claimed that NN had admitted that the current Cagri compound will create fibrils.

That however is not what that hogwash of a facebook post says, it only talks about the chemicals that were tried in the process of developing Cagri.

Now addressing you, you seem to be completely misunderstanding the entire point of the research paper you keep referencing. The research was *not* conducted in such a way as to find out if chemical 23 (Cagrilintide) is dangerous, it is the culmination of all the different research they did before deciding that Cagrilintide is the safest and most effective choice for human use.

That means Novo went and paid probably millions of dollars trying to develop this drug, and they decided that Cagrilintide was the safest and best choice to move forward with for testing, OK? That means after spending all that money, they decided to move foward with this drug for trials, knowing that if they get approved and it later ends up being unsafe they will be paying out the ass in lawsuits.

Now, does that mean we dont have to buffer cagri? I cannot say, but the solubility of 23 (Cagri) is fully soluble and stable all the way up to a PH of 8. Additionally, compound 1 still fibrilated in high dosages despite being in a 4 PH solution, they theorize that is because compound 1 was not soluble at higher PH and after injection the chemical is mixed with the human bodies fluids which are by and large a very constant 7.4-ish PH, no amount of buffering will change this, and you dont want it to because if you did it would fuck you up. It is theorized at low doses compound 1 was kept soluble by albumin, a chemical in human serum fluid, so in higher dosages, as the compound binds and uses up all local albumin, it allows the chemical to fibrilate, and the fibrils do not necessarily redisolve once the albumin returns to the area because the compound is not soluble above PH 5.

What this very clearly shows is that the solubility of the chemical is leaps and bounds more important than the PH of the fluid. YES in storage a ph of 4 is probably better, but when the chemical gets in your blood it is going to be in a 7.4ph solution until it is fully removed from your body.

Cagrilintide (Compound 23) solves this issue by being soluble up to a ph of 8.

It is in my opinion that with the information blatantly available, Cagri is most likely perfectly safe in the short term after reconstitution whether you buffer it to PH 4 or not. As you have said they are already mixing the to in CagriSema.

As far as your concerns about their testing showing that high heat and wear and tear on the chemical can cause fibrils, I would just remind you that these medications are stored in a form that is not reconstituted, and once reconstituted are nowhere near those conditions.

 

[FreeSpectator8]

I'm curious about this for Cagri as well. My plan is to start with BAC to test ph, then lightly add AA if needed. However, it would be interesting to see others recon trials if they have to adjust PH for Cagri and the amount consensus of each. I also contemplated using Hospira BAC w/ AA mix, but that may make ph more difficult to tweak, if tweaking is needed.

One thing to keep in mind is your body is PH7.4... so really worrying about the PH of the recon really only matters for the short term storage while you use the vial.

Research wise and scientifically, there was only fibrilation of cagri with high temperature and physical stress, which cagri will not (or atleast shouldnt lol) experience sitting in your fridge.

 

[FreeSpectator8]

The thing i would be much more concerned about is if the Chinese labs are manufacturing the peptides at 4 PH before lyophilization. which we have no way of knowing really.

 

[janedoe]

One thing to keep in mind is your body is PH7.4... so really worrying about the PH of the recon really only matters for the short term storage while you use the vial.

Research wise and scientifically, there was only fibrilation of cagri with high temperature and physical stress, which cagri will not (or atleast shouldnt lol) experience sitting in your fridge.

I should have clarified... the duration of the shelf stability once reconned is actually my focus of the proper PH. I do know I don't plan on shaking it around like a martini though.

 

[FreeSpectator8]

I should have clarified... the duration of the shelf stability once reconned is actually my focus of the proper PH. I do know I don't plan on shaking it around like a martini though.

Someone probably (or a group finance kind of deal) need to get a few bottles of cagri, recon some with BAC and some with AA to a PH of 4 and let both sit in a fridge for like 6 months or a year and then have them tested against some unrecon'd bottles of the same batch.

 

[janedoe]

Someone probably (or a group finance kind of deal) need to get a few bottles of cagri, recon some with BAC and some with AA to a PH of 4 and let both sit in a fridge for like 6 months or a year and then have them tested against some unrecon'd bottles of the same batch.

Though in my trials, I would/do not use anything reconned more than 6-8 weeks, ( Cagri no more than 1 month), your idea would certainly be an interesting research project.

 

[49metal]

I have posted this info elsewhere, but I think it could be partly useful to answer OP: I also wanted to target a lower pH for reconstituted cagri, so I used Hospira Bacteriostatic 0.9% Sodium Chloride. Specifically, the monograph reads "Each milliliter (mL) contains sodium chloride 9 mg and 0.9% (9 mg/mL) benzyl alcohol added as a bacteriostatic preservative. May contain hydrochloric acid for pH adjustment . . . . The pH is 5.0." When I reconstituted cagri with this, I got a solution with a pH of about 5.0 to 5.25. I tested the refrigerated solution's pH over the course for a few weeks and it stayed in that range and the stuff kept working on RS. I'm presently on hiatus from cagri due to a case of sluggish oligomerphobia, but I have an acetic acid vial on hand to someday attempt to lower such a cagri solution to 4.0. This is just my narrow experience and I have precious little clue what I'm up to, so take it for what it is worth.

[Presently, I take no position on how any of this relates to cagri oligomerpyleing, fibriliferating, or manifesting an intermediate Cascade-like sheeting action on the way to slimming your brain, pancreas, or waistband.]

 

[JohnnyGobbs]

Need to hear @hexagonal rebuttal to freespectators points. Hex talking points about cagri is what got my layman brain concerned in the first place to tweak the PH lol

 

[JohnnyGobbs]

I have posted this info elsewhere, but I think it could be partly useful to answer OP: I also wanted to target a lower pH for reconstituted cagri, so I used Hospira Bacteriostatic 0.9% Sodium Chloride. Specifically, the monograph reads "Each milliliter (mL) contains sodium chloride 9 mg and 0.9% (9 mg/mL) benzyl alcohol added as a bacteriostatic preservative. May contain hydrochloric acid for pH adjustment . . . . The pH is 5.0." When I reconstituted cagri with this, I got a solution with a pH of about 5.0 to 5.25. I tested the refrigerated solution's pH over the course for a few weeks and it stayed in that range and the stuff kept working on RS. I'm presently on hiatus from cagri due to a case of sluggish oligomerphobia, but I have an acetic acid vial on hand to someday attempt to lower such a cagri solution to 4.0. This is just my narrow experience and I have precious little clue what I'm up to, so take it for what it is worth.

[Presently, I take no position on how any of this relates to cagri oligomerpyleing, fibriliferating, or manifesting an intermediate Cascade-like sheeting action on the way to slimming your brain, pancreas, or waistband.]

Yea this is basically my scenario too. Took 5mg vial of cagri, 1ml of bac (amazon) that was about 5.5-6 PH. Then Added .4ml of AA (Amazon) and it dropped to 4-4.5 PH. I stopped at 4-4.5 and Pinned and it worked fine (Another .1ml-.2ml of AA would prob get it into the 3’s). I’m also a total amateur and scared of fibrils and oligomers lol.

 

[TooBigtoFail]

I'm curious about this for Cagri as well. My plan is to start with BAC to test ph, then lightly add AA if needed. However, it would be interesting to see others recon trials if they have to adjust PH for Cagri and the amount consensus of each. I also contemplated using Hospira BAC w/ AA mix, but that may make ph more difficult to tweak, if tweaking is needed.

I buy StatLab bac 2ml vials (actually has ~3.1-3.5ml) on Amazon. I had one vial half empty (1.6ml) so I added AA 0.6% (from Quality Research Chemical 10 ml on Amazon). After adding 1.4ml of AA, I got pH4.5. No sting, so for next Cagri recon I will do 75% AA to get ~pH4.0. Looks like pure AA should be pH2.8 @ 0.6%. (0.1Mole). My AA was about pH3.5. Was it mixed with water? not really 0.6% ? Is AA alone a good enough peptide preservative for 30 days? Works for pickling ! USP standard allows a lot of variance for the pH of bac water: Hospira bac pH5.7 can be between pH4.5 to 7.0. The mannitol filler is pH6.3. Conclusion: need to test the pH of the bac/AA, and also the final recon pH.

 

[JohnnyGobbs]

I buy StatLab bac 2ml vials (actually has ~3.1-3.5ml) on Amazon. I had one vial half empty (1.6ml) so I added AA 0.6% (from Quality Research Chemical 10 ml on Amazon). After adding 1.4ml of AA, I got pH4.5. No sting, so for next Cagri recon I will do 75% AA to get ~pH4.0. Looks like pure AA should be pH2.8 @ 0.6%. (0.1Mole). My AA was about pH3.5. Was it mixed with water? not really 0.6% ? Is AA alone a good enough peptide preservative for 30 days? Works for pickling ! USP standard allows a lot of variance for the pH of bac water: Hospira bac pH5.7 can be between pH4.5 to 7.0. The mannitol filler is pH6.3. Conclusion: need to test the pH of the bac/AA, and also the final recon pH.

This basically. I also used stat labs and quality research brands. Great minds… 🍻

 

[FreeSpectator8]

I got lambda water BAC water, and Quality Research Chemicals AA, we will see how it goes. Bought PH test strips to see if they can work instead of having to buy a 50+ dollar PH tester.

 

[JohnnyGobbs]

I got lambda water BAC water, and Quality Research Chemicals AA, we will see how it goes. Bought PH test strips to see if they can work instead of having to buy a 50+ dollar PH tester.

Yea I used the strips. They worked fine.

 

[janedoe]

I buy StatLab bac 2ml vials (actually has ~3.1-3.5ml) on Amazon. I had one vial half empty (1.6ml) so I added AA 0.6% (from Quality Research Chemical 10 ml on Amazon). After adding 1.4ml of AA, I got pH4.5. No sting, so for next Cagri recon I will do 75% AA to get ~pH4.0. Looks like pure AA should be pH2.8 @ 0.6%. (0.1Mole). My AA was about pH3.5. Was it mixed with water? not really 0.6% ? Is AA alone a good enough peptide preservative for 30 days? Works for pickling ! USP standard allows a lot of variance for the pH of bac water: Hospira bac pH5.7 can be between pH4.5 to 7.0. The mannitol filler is pH6.3. Conclusion: need to test the pH of the bac/AA, and also the final recon pH.

As I am more concerned with stability after recon, I read this a couple times to absorb this..... it makes sense. Thanks for adding your experience.

 

[hexagonal]

First of all, I was not talking to you. I was directly responding to a user who claimed that NN had admitted that the current Cagri compound will create fibrils.

That however is not what that hogwash of a facebook post says, it only talks about the chemicals that were tried in the process of developing Cagri.

Now addressing you, you seem to be completely misunderstanding the entire point of the research paper you keep referencing. The research was *not* conducted in such a way as to find out if chemical 23 (Cagrilintide) is dangerous, it is the culmination of all the different research they did before deciding that Cagrilintide is the safest and most effective choice for human use.

That means Novo went and paid probably millions of dollars trying to develop this drug, and they decided that Cagrilintide was the safest and best choice to move forward with for testing, OK? That means after spending all that money, they decided to move foward with this drug for trials, knowing that if they get approved and it later ends up being unsafe they will be paying out the ass in lawsuits.

NN is explicit that fibrils do form in cagrilintide, in both that research paper and in the patent. I have quoted those sections extensively across these threads. In the research paper, yes, they use a test that puts it in relatively extreme conditions vs. what is going to be seen after it is reconstituted by most people here, but we don't know what it looks like on the manufacturing side, how roughly it is treated there, how it is stored, etc. The patent filing uses a significantly less torturous test, but we still see fibril formation in the longer term with that test as well.

Part of the testing in the paper is to determine if fibrils are formed. I have never claimed this makes it dangerous - I have explicitly said I do not understand enough about the science to know if these fibrils truly are dangerous. What I will say is that in the testing conditions in the paper and the patent filing, they do find that cagrilintide produces fibrils.

Now, does that mean we dont have to buffer cagri? I cannot say, but the solubility of 23 (Cagri) is fully soluble and stable all the way up to a PH of 8. Additionally, compound 1 still fibrilated in high dosages despite being in a 4 PH solution, they theorize that is because compound 1 was not soluble at higher PH and after injection the chemical is mixed with the human bodies fluids which are by and large a very constant 7.4-ish PH, no amount of buffering will change this, and you dont want it to because if you did it would fuck you up. It is theorized at low doses compound 1 was kept soluble by albumin, a chemical in human serum fluid, so in higher dosages, as the compound binds and uses up all local albumin, it allows the chemical to fibrilate, and the fibrils do not necessarily redisolve once the albumin returns to the area because the compound is not soluble above PH 5.

What this very clearly shows is that the solubility of the chemical is leaps and bounds more important than the PH of the fluid. YES in storage a ph of 4 is probably better, but when the chemical gets in your blood it is going to be in a 7.4ph solution until it is fully removed from your body.

Cagrilintide (Compound 23) solves this issue by being soluble up to a ph of 8.

It is in my opinion that with the information blatantly available, Cagri is most likely perfectly safe in the short term after reconstitution whether you buffer it to PH 4 or not. As you have said they are already mixing the to in CagriSema.

As far as your concerns about their testing showing that high heat and wear and tear on the chemical can cause fibrils, I would just remind you that these medications are stored in a form that is not reconstituted, and once reconstituted are nowhere near those conditions.

You're writing a whole lot that doesn't actually say anything one way or other about the concern. No one is arguing that the PH remains low in your body.

No one has produced details on what PH is being used for the single chamber pen - we seem to be assuming it is a higher PH, and I wouldn't be surprised if that is the case, but they might also be going with a lower pH for the entire solution, unless someone has evidence that semaglutide is harmed at the lower pH.

Again, the patent involves a significantly more gentle test, at temperatures below 100F and just doing 100 end-over-end inversions of the vial on the liquid form of cagrilintide + buffering agent each day.

I've also been quite explicit my primary point is we have no idea what is happening with the manufacturing before the lyophilization. How long was it in the pre-lyophilized state? What pH was it manufactured at and stored at in between? What were the conditions during that time period? If I thought that just reconstituting it at a lower pH solved all ills, I would be using cagrilintide myself - I'm certainly confident in my ability to lower the pH of a solution. All I've said about it in reconstituted form is that if I had decided to press on and use it regardless of the rest, I would filter it and reconstitute at a lower pH to do what I could to prevent further fibril formation.

 

[hexagonal]

Need to hear @hexagonal rebuttal to freespectators points. Hex talking points about cagri is what got my layman brain concerned in the first place to tweak the PH lol

No one should be taking anything I say as anything beyond my own layperson interpretation of the paper and patent filing, lol. I'm certainly no expert.

 

[exploitedworkerbee]

No one should be taking anything I say as anything beyond my own layperson interpretation of the paper and patent filing, lol. I'm certainly no expert.

Appreciate your humble and thoughtful approach to all this. I think one of the reasons these conversation get ugly is that team fibril has several vocal members (leaders?) who are… ummmm… less measured in their tone and less lucid in their language. Your interpretation was helpful.

 

[hexagonal]

Appreciate your humble and thoughtful approach to all this. I think one of the reasons these conversation get ugly is that team fibril has several vocal members (leaders?) who are… ummmm… less measured in their tone and less lucid in their language. Your interpretation was helpful.

Thanks!

My biggest worry in engaging on all of this is people assuming I'm some sort of professional and taking my opinion as anything more than a nerd reading papers and interpreting them to the best of his ability.

 

[TooBigtoFail]

Great discussions. Google AI results say it may be over the course of 10-20 years of accumulation before plaque forms. Specific conditions. Is there enough material in Cagri 2.5mg weekly, taken for 6 months to be a concern? These are all relatively young GPL medication. In a study, to create fibrils, they had to keep the temperature near 37celcius for days and then use a 0.22um filter to count them(fibrils measure 10nm) Am I to conclude that using a .22um filter would remove them? Should I be filtering my grey market peps anyway as I expect to stay on maintenance for 1 or 2 years after I reach my goal? A survey on filtering?

 

[hexagonal]

Great discussions. Google AI results say it may be over the course of 10-20 years of accumulation before plaque forms. Specific conditions. Is there enough material in Cagri 2.5mg weekly, taken for 6 months to be a concern? These are all relatively young GPL medication. In a study, to create fibrils, they had to keep the temperature near 37celcius for days and then use a 0.22um filter to count them(fibrils measure 10nm) Am I to conclude that using a .22um filter would remove them? Should I be filtering my grey market peps anyway as I expect to stay on maintenance for 1 or 2 years after I reach my goal? A survey on filtering?

.22um is 220nm, so individual fibrils would still pass through.

My understanding is fibrils do tend to clump so you could potentially filter out any large enough clumps, which is why I would personally filter if I were to decide to use cagri.

 

[JohnnyGobbs]

No one should be taking anything I say as anything beyond my own layperson interpretation of the paper and patent filing, lol. I'm certainly no expert.

I know but your rebuttals and skepticism are pretty solid. Debate is healthy.

 

[JohnnyGobbs]

Great discussions. Google AI results say it may be over the course of 10-20 years of accumulation before plaque forms. Specific conditions. Is there enough material in Cagri 2.5mg weekly, taken for 6 months to be a concern? These are all relatively young GPL medication. In a study, to create fibrils, they had to keep the temperature near 37celcius for days and then use a 0.22um filter to count them(fibrils measure 10nm) Am I to conclude that using a .22um filter would remove them? Should I be filtering my grey market peps anyway as I expect to stay on maintenance for 1 or 2 years after I reach my goal? A survey on filtering?

I haven’t taken the leap to use a syringe filter yet only the lower PH. Haven’t learned the whole syringe filter process. Does anyone have a link of which one to buy?

 

[FreeSpectator8]

NN is explicit that fibrils do form in cagrilintide, in both that research paper and in the patent. I have quoted those sections extensively across these threads. In the research paper, yes, they use a test that puts it in relatively extreme conditions vs. what is going to be seen after it is reconstituted by most people here, but we don't know what it looks like on the manufacturing side, how roughly it is treated there, how it is stored, etc. The patent filing uses a significantly less torturous test, but we still see fibril formation in the longer term with that test as well.

Part of the testing in the paper is to determine if fibrils are formed. I have never claimed this makes it dangerous - I have explicitly said I do not understand enough about the science to know if these fibrils truly are dangerous. What I will say is that in the testing conditions in the paper and the patent filing, they do find that cagrilintide produces fibrils.



You're writing a whole lot that doesn't actually say anything one way or other about the concern. No one is arguing that the PH remains low in your body.

No one has produced details on what PH is being used for the single chamber pen - we seem to be assuming it is a higher PH, and I wouldn't be surprised if that is the case, but they might also be going with a lower pH for the entire solution, unless someone has evidence that semaglutide is harmed at the lower pH.

Again, the patent involves a significantly more gentle test, at temperatures below 100F and just doing 100 end-over-end inversions of the vial on the liquid form of cagrilintide + buffering agent each day.

I've also been quite explicit my primary point is we have no idea what is happening with the manufacturing before the lyophilization. How long was it in the pre-lyophilized state? What pH was it manufactured at and stored at in between? What were the conditions during that time period? If I thought that just reconstituting it at a lower pH solved all ills, I would be using cagrilintide myself - I'm certainly confident in my ability to lower the pH of a solution. All I've said about it in reconstituted form is that if I had decided to press on and use it regardless of the rest, I would filter it and reconstitute at a lower pH to do what I could to prevent further fibril formation.

From the paper on Oral Semaglutide: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2019/213051Orig1s000ChemR.pdf

"Semaglutide tablets are co-formulated with 300 mg of salcaprozate sodium (SNAC, a
permeation enhancer). The isoelectric point of the semaglutide is 5.4. The peptide has a
low solubility at pH range 2-6. Semaglutide is considered a BCS class 4 molecule (low
permeability and low solubility). It is hypothesized that SNAC facilitates the oral
absorption of semaglutide in stomach either by transiently increasing the transcellular
permeability in gastric epithelium or through buffering action on the local environment
near the site of action to provide a high pH and thereby protecting the semaglutide from
degradation."

Additionally:

A Review on the Efficacy and Safety of Oral Semaglutide - PMC

There are numerous treatment options currently available for patients with type 2 diabetes mellitus; however, a multitude of patients continue to have inadequately controlled glycemic levels with their current antihyperglycemic regimen. Furthermore, ...

pmc.ncbi.nlm.nih.gov

"The effect of pepsin on oral semaglutide stability was most profound at low pH, with oral semaglutide being most labile toward pepsin at pH 2.6 (t½ = 16 min)" Its clear that Semaglutide is unstable at low PH and will degrade quicker, making storing it in a low PH solution not ideal.

So there you go as far as your question about how we can say they likely aren't lowering the PH of the CagriSema mixture. Especially since the custom designed Cagri to be soluble and long term stable in a pH up to 8.

Again, no, part of the paper was not to find if fibrils form in cagri this is already a-given, as the chemical type that is the basis of Cagri will fibrillate. The whole point of the experiment was to find the best chemical composition that resisted fibrillation the longest and best while providing the best effect and any other sought after qualities. These tests would be used to simulate the maximum amount of degradation the product could possibly experience in its good til shelf life.

It is a false equivalence to say that because Cagri CAN fibrillate, then it DOES fibrillate. This is simply not true.

NN is not going to open itself up to liability of long term health effects caused by there drug because they figured it "probably wouldnt be a problem" they have a duty to test to the fullest extent and guarantee individuals safety. Not to mention the class action lawsuits that could and would happen if it became obvious they had shipped a product that can and will degrade and harm you during its shelf life.

As far as manufacturing concerns, there is only one way to create the specific formulation that is cagrilinitide, that's kind of how chemicals work. Any sort of impurities will come up in a purity test from janoshik, and if the chemical is wrong it wont match the sample that janoshik or any other testing party uses.

 

[penewbie]

As far as manufacturing concerns, there is only one way to create the specific formulation that is cagrilinitide, that's kind of how chemicals work.

There were lots of kinda dodgy things you said in that comment.

But this specific sentence is so egregiously wrong it actually made me laugh.

 

[hexagonal]

From the paper on Oral Semaglutide: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2019/213051Orig1s000ChemR.pdf

"Semaglutide tablets are co-formulated with 300 mg of salcaprozate sodium (SNAC, a
permeation enhancer). The isoelectric point of the semaglutide is 5.4. The peptide has a
low solubility at pH range 2-6. Semaglutide is considered a BCS class 4 molecule (low
permeability and low solubility). It is hypothesized that SNAC facilitates the oral
absorption of semaglutide in stomach either by transiently increasing the transcellular
permeability in gastric epithelium or through buffering action on the local environment
near the site of action to provide a high pH and thereby protecting the semaglutide from
degradation."

Additionally:

A Review on the Efficacy and Safety of Oral Semaglutide - PMC

There are numerous treatment options currently available for patients with type 2 diabetes mellitus; however, a multitude of patients continue to have inadequately controlled glycemic levels with their current antihyperglycemic regimen. Furthermore, ...

pmc.ncbi.nlm.nih.gov

"The effect of pepsin on oral semaglutide stability was most profound at low pH, with oral semaglutide being most labile toward pepsin at pH 2.6 (t½ = 16 min)" Its clear that Semaglutide is unstable at low PH and will degrade quicker, making storing it in a low PH solution not ideal.

So there you go as far as your question about how we can say they likely aren't lowering the PH of the CagriSema mixture.

I don't know how applicable the oral semaglutide formulation is, but I'll accept it as being 1:1 for the sake of argument.

Especially since the custom designed Cagri to be soluble and long term stable in a pH up to 8.

Except that they explicitly say that Cagri does not meet their design thresholds at the 7.5 pH, much less 8. If it did, why would they have bothered developing the dual chamber pen and keeping it at a lower pH to begin with? They obviously had a reason at the time, considering the additional cost and complexity incurred.

Again, no, part of the paper was not to find if fibrils form in cagri this is already a-given, as the chemical type that is the basis of Cagri will fibrillate. The whole point of the experiment was to find the best chemical composition that resisted fibrillation the longest and best while providing the best effect and any other sought after qualities. These tests would be used to simulate the maximum amount of degradation the product could possibly experience in its good til shelf life.

You seem to be arguing semantics here. Yes, of course they knew fibrils could form. I believe from context it is obvious that the discussion is about the timelines and conditions for fibril formulation.

It is a false equivalence to say that because Cagri CAN fibrillate, then it DOES fibrillate. This is simply not true.

NN is not going to open itself up to liability of long term health effects caused by there drug because they figured it "probably wouldnt be a problem" they have a duty to test to the fullest extent and guarantee individuals safety. Not to mention the class action lawsuits that could and would happen if it became obvious they had shipped a product that can and will degrade and harm you during its shelf life.

If we're going to be arguing semantics, in your previous portion of your text, you explicitly said "cagri will fibrillate."

The other paragraph is the whole crux of the conversation and what set this all off to begin with: NN, for most of the time cagrilintide has existed, was focusing their efforts on storing it at a low pH, with all of the research papers and patent filing showing a higher tendency to fibrillate at higher pH.

As far as manufacturing concerns, there is only one way to create the specific formulation that is cagrilinitide, that's kind of how chemicals work. Any sort of impurities will come up in a purity test from janoshik, and if the chemical is wrong it wont match the sample that janoshik or any other testing party uses.

What are you even talking about? Nothing I discussed in my manufacturing portion would mean using a different formulation. It was explicitly about pH, temperature, amount of abuse it suffers, etc. If it sits around on shelves in a hot warehouse for a month and then takes two weeks in a hot trailer as it moves from the raw manufacturer to the finisher, vibrating along the roads, that environment is suddenly getting closer to the sort of stress seen in the the research paper and patent filings where they found fibrils forming.

Also, we already know we often get different forms of these peptides from China and the compounding pharmacies - we get them in their salt forms, which is part of EL and NN's whole argument about them being potentially unsafe.

Nothing is 100% on purity tests - we don't get a report saying that the .7% impurities are fibrils and oligomers or whatever. And other substances won't show up on the purity test - you certainly don't see the excipient in there, etc. It's just the purity of the peptide itself, despite most people thinking it is a vouchsafe for the rest of the contents in the vial.

 

[FreeSpectator8]

There were lots of kinda dodgy things you said in that comment.

But this specific sentence is so egregiously wrong it actually made me laugh.

Ok, sure go ahead and explain how you plan to get the compound known as cagrilintide, without creating the exact same chain of compounds, as seen here: