Pharmacology Mixing a chemical with 1) Saline or 2) purified water: can it dramatically alter the nature of the chemical?

[JohnBoy2000]

The chemical in question is "melanotan 2" peptide.

1) Basically last year I mixed it with Saline and it was all good, no problems.

As I understand, as melanotan 2 is acetate based, so mixing it with saline can precipitate sodium-acetate (which leaves a slight off-white residue on the vial) - which can weaken the effect, but it still works.

2) this year I mixed it with purified water (bac water, water + 0.9% benzyl alcohol), no residue on the vial and seems to dilute much more readily - and got a range of different side effects I hadn't experienced previously.

.........

But some were conventional of melanotan 2, such as facial flushing etc - perhaps to be expected as, mixing with purified water = no sodium acetate reaction = a stronger peptide.

.........

Question being, is it possible mixing using 1) saline or 2) purified water, can dramatically alter the composition of a peptide/chemical, such to precipitate or elicit the variation in side effect profile I experienced?

Is the variation in reaction I experience, attributable primarily to simply having a stronger version of the peptide when mixed with 2) purified water?

Thoughts?

 

[Skorpio]

What do you mean it is acetate based? Are you referring to the acetamide? It seems unlikely to be deacetylated without a significant driving force (as acetate is already in the ionic form, but the acetamide isn't).

Usually proteins are stabilized in phisiologic amounts of salts, however, being a cyclic peptide, melanotan II will likely be pretty stable in most mild conditions.

 

[JohnBoy2000]

What do you mean it is acetate based? Are you referring to the acetamide? It seems unlikely to be deacetylated without a significant driving force (as acetate is already in the ionic form, but the acetamide isn't).

Usually proteins are stabilized in phisiologic amounts of salts, however, being a cyclic peptide, melanotan II will likely be pretty stable in most mild conditions.

I guess these are the same, except with an acetate molecule in the first picture?

Each being the chemical structure of mt2.

Based on the potential for interaction with saline, you wouldn't expect any real changes in the peptide?

Organic chemistry is definitely not my strong point.

......

I have as above noted, seen a white precipitate in the mt2 vial when reconstituted with saline;

This doesn't appear with purified water.

I would assume this is a reaction of some sort?

 

[JohnBoy2000]

Usually proteins are stabilized in phisiologic amounts of salts, however, being a cyclic peptide, melanotan II will likely be pretty stable in most mild conditions.

Just to be completely clear here, saline would be consider "mild conditions"?

You wouldn't expect any kind of reaction between it and this peptide?

 

[Skorpio]

Just to be completely clear here, saline would be consider "mild conditions"?

You wouldn't expect any kind of reaction between it and this peptide?

Phisiologic salt concentrations ie saline will stabilize it relative to plain water.

Saline is extremely mild to biomolecules.

 

[JohnBoy2000]

Phisiologic salt concentrations ie saline will stabilize it relative to plain water.

Saline is extremely mild to biomolecules.

Stabilize it relative to plain water (purified water).

Presumably purified water wouldn't therefore conversely, destabilize it (...... a little even..... would it?).

Overall the suggestion is that, in either saline or purified water - neither would be likely to alter melanotan 2, at all really?

I found effects from both to be basically the same in either diluent.

Just curious how last year I tolerated it fine, this year not so much (vials were from the same box, so exactly the same batch).

Perhaps just a developed intolerance?

 

[JohnBoy2000]

I read also that apparently "acetate" is added to "melanotan 2" to:

"extends the halflife, or the duration effectiveness of the amino chain".

....... no idea how that would work.

 

[sekio]

Plain water and saline are functionally identical. The 0.9% sodium chloride will not change anything. In fact, using bacteriostatic water with an additional 0.9% benzyl alcohol would not harm it either.

Generally speaking,, peptides are stable in water solutions, it's the presence of either strong acids or bases, or certain proteolytic enzymes, that you need to worry.

I consider it more important to keep it refrigerated, and sterile. Again, bacteriostatic water would be a smart idea if you are injecting.

> Just curious how last year I tolerated it fine, this year not so much (vials were from the same box, so exactly the same batch).
How have they been stored? Did they have dry powder or solution in them?

>I read also that apparently "acetate" is added to "melanotan 2" to: "extends the halflife, or the duration effectiveness of the amino chain".
Which is nonsense. The acetate is simply the counterion used to form a water soluble salt. Sort of like how you have certain drug hydrochlorides.
Anyway, the acetate is not chemically bound to the drug (like e.g. heroin) so it will have no effect on half life.
In fact, most melanotan II seems to be sold as the acetate salt.

 

[JohnBoy2000]

Plain water and saline are functionally identical. The 0.9% sodium chloride will not change anything. In fact, using bacteriostatic water with an additional 0.9% benzyl alcohol would not harm it either.

Generally speaking,, peptides are stable in water solutions, it's the presence of either strong acids or bases, or certain proteolytic enzymes, that you need to worry.

I consider it more important to keep it refrigerated, and sterile. Again, bacteriostatic water would be a smart idea if you are injecting.

> Just curious how last year I tolerated it fine, this year not so much (vials were from the same box, so exactly the same batch).
How have they been stored? Did they have dry powder or solution in them?

>I read also that apparently "acetate" is added to "melanotan 2" to: "extends the halflife, or the duration effectiveness of the amino chain".
Which is nonsense. The acetate is simply the counterion used to form a water soluble salt. Sort of like how you have certain drug hydrochlorides.
Anyway, the acetate is not chemically bound to the drug (like e.g. heroin) so it will have no effect on half life.
In fact, most melanotan II seems to be sold as the acetate salt.

Wow, great explanation.

As to storage, I stored them un-reconstituted (as powder) in my freezer, as I do all peptides.

Took out a vial, reconstituted as normal etc., nothing different except purified water used in place of saline.

I should mention though, I do seem to have developed intolerances, or hypersensitivities, to other chemicals also.
I used to use this high vitamin shampoo, as an example.

Or tretinoin, certain topical creams - even though they're topical, they legitimately affect my mood.

I use them = mood fluctuation.
Don't use them = no mood fluctuation.

......

Last year however I used mt2 heavily, no problem.

This season, lots of symptoms.

So it may be intolerance, but your explanation reassures me it's unlikely to have been any changes caused by the different diluents.

 

[JohnBoy2000]

One thing to mention however:

I do my own botox.

That's always reconstituted with saline.

But one time I added too much benzyl alcohol by mistake (3% instead of 1%).

When I injected the botox - it had no effect.

Now botox is almost certainly a very different beast to melanotan 2.

But to me that's an example of how the alcohol could have denatured the botox?

........

In the case of mt2 however, I definitely used the normal amounts of benzyl alcohol consistently when reconstituting; even if there was excess used, I don't know if it would affect the mt2 molecule, but it does seem to neutralize botox.

.........

Additionally, when I used the mt2 last year (no symptoms) versus this year (symptoms) - it worked equally well on both occasions in terms of the tan it gave.
It wasn't weaker last year by any means.

i.e. unlike the botox with too much benzyl alcohol - which didn't work at all.

 

[JohnBoy2000]

Last thing I thought could be a possibility?

Is it possible the benzyl alcohol went bad, and somehow that precipitated the symptoms?

I added 1% this year (in making the nasal spray).
I may have added 1.5% last year, cause I used to add a little more as a precaution (again, the mt2 worked very well last year, no change in potency), but this year went with just 1%.

I bought the benzyl alcohol at the start of 2019, and stored it sealed tightly, and kept in a fridge all that time.

I assume it's unlikely that that would have played a factor?

 

[sekio]

too much benzyl alcohol can cause protien aggregation and not denaturaton but the same idea holds: it will inactivate certain protein drugs in excess (>1%)
0.1-0.3% is probably fine, up to 0.9% is regularly used with many drugs, but more is a no-no

> I bought the benzyl alcohol at the start of 2019, and stored it sealed tightly, and kept in a fridge all that time.

It won't go bad at all under those circumstances.

> Additionally, when I used the mt2 last year (no symptoms) versus this year (symptoms) - it worked equally well on both occasions in terms of the tan it gave.
> It wasn't weaker last year by any means.
Well, there's your smoking gun. Negative effects are how your body is reacting to melanotan, it obviously hasn't broken down or been chemically altered if it still has its melanocyte-stimulating effects.

 

[JohnBoy2000]

too much benzyl alcohol can cause protien aggregation and not denaturaton but the same idea holds: it will inactivate certain protein drugs in excess (>1%)
0.1-0.3% is probably fine, up to 0.9% is regularly used with many drugs, but more is a no-no

> I bought the benzyl alcohol at the start of 2019, and stored it sealed tightly, and kept in a fridge all that time.

It won't go bad at all under those circumstances.

> Additionally, when I used the mt2 last year (no symptoms) versus this year (symptoms) - it worked equally well on both occasions in terms of the tan it gave.
> It wasn't weaker last year by any means.
Well, there's your smoking gun. Negative effects are how your body is reacting to melanotan, it obviously hasn't broken down or been chemically altered if it still has its melanocyte-stimulating effects.

Makes sense.

I just find interesting how the excess benzyl alcohol aggregated/weakened the botox.

But didn't have that effect on the melanotan 2 molecule?

(though to be fair, the mistaken percentage used with botox was 3% BA, versus 1.5% used with the mt2)

Perhaps mt2 is just sturdier?

No idea if it's possible to assess that.

Botox:

Molar mass = 149323.05 g·mol−1

Melanotan 2:

Molar mass = 1,024.18 g/mol

 

[JohnBoy2000]

https://www.sciencedirect.com/science/article/abs/pii/S0022354915301908

Sci-Hub | Role of Benzyl Alcohol in the Unfolding and Aggregation of Interferon α-2a. Journal of Pharmaceutical Sciences, 104(2), 407–415 | 10.1002/jps.24105

sci-hub.se

Benzyl alcohol (BA) is the most widely used antimicrobial preservative in multidose protein formulations, and has been shown to cause protein aggregation.

A peptide, as I understand, is distinct from a protein in that it has a shorter length.

For mt2, info I could pull up on google being:

MT2 is just 6 amino acids in length compared to MT1 at 13

M2870-75 Melanotan II (MT-II, MSHa) CAS · Amino Acid Sequence: Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH2

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www.jle.com

Melanotan II, consisting of 7 amino acids

Botox:

The complete sequence of botulinum neurotoxin type A and comparison with other clostridial neurotoxins.

The seven serologically different botulinum neurotoxins are highly potent protein toxins that inhibit neurotransmitter release from peripheral choline…

www.sciencedirect.com

1,296 amino acid residues

We have established the complete sequence of botulinum neurotoxin type A (BoNT/A; 1,296 amino acid residues, Mr = 149,425) and a partial sequence of botulinum neurotoxin type E (273 amino acid residues) as deduced from the corresponding nucleotide sequences of the chromosomally located structural genes.

.......

It's possible the more amino acids, longer the protein chain = more susceptible to aggragation;

Therefore one would expect more weakening of botox, than mt2, in the presence of benzyl alcohol?

Logic check out?

 

[sekio]

As I posted on your Reddit thread, first off, that is not the structure of Botox but rather an organophosphate pesticide called Phoxim.

The 3D "cartoon" drawing showing the actual structure is depicted above from two views. With such a large molecule, we don't bother to draw every atom of every amino acid and instead show the general structure.

Unfortunately, giving the structure, either the amino acid sequence or the shape of the folded protein, doesn't really allow predictions for how it will behave based upon "back of the napkin" calculations, in reality you will either have to conduct computationally expensive molecular dynamics simulations or measure it experimentally (as linked in the Sciencedirect article).

It is the specific tertiary structure (and the distribution of charges and H-bond donor/acceptor sites) that allows interaction of the protein with its receptor and anything that disrupts that shape (denaturants, aggregation, proteolysis) will render it inactive.

And generally speaking, as you suspect, larger proteins are more prone to aggregation than smaller proteins, and almost anything from increased salinity, to alcohols, to denaturing agents can result in loss of activity.

 

[JohnBoy2000]

As I posted on your Reddit thread, first off, that is not the structure of Botox but rather an organophosphate pesticide called Phoxim.

The 3D "cartoon" drawing showing the actual structure is depicted above from two views. With such a large molecule, we don't bother to draw every atom of every amino acid and instead show the general structure.

Okay, I understand. Your replies have been excellent, I appreciate it.

I'm just trying to think - experimentally:

I used

Melanotan 2 with

1) 1.5% benzyl alcohol last year - it worked. I don't know if the BA compromised it's strength, but it did work very well.

2) 1% benzyl alcohol this year - again, it worked well. I'm assuming there was no or less compromise of strength.

So on both occasions with BA, it worked, but I'm left questioning if the strength was compromised with scenario 1).

.......

With scenario 1) = there was no symptom emergence (last year). And I used it very heavily.

Scenario 2) = symptom emergence - so was this attributable to a more potent version of MT2, given the reduced percentage of BA added? And I used it comparatively sparingly (about half the dose of 1) ).

Or, as I initially postulated - just my body responding differently....

And then wondering, would a dose increase overcompensate for the lesser amount of strength?

i.e. even if it was weaker in scenario 1), should the higher dose used not have precipitated symptoms, if it was in fact the mt2 responsible for causing them, and not a tolerance emergence in my biology?
(this may again be very difficult to comment on)

........

In both cases 1) and 2), last and this year - the tanning effect was pronounced and strong;

So the higher BA concentration definitely didn't neutralize the MT2 completely last year.

If there was a stronger effect from the mt2 this year via the lower BA percentage added - could that extra "kick" have caused symptom emergence?

Seems unlikely, but I'm open to suggestion or any thoughts.....?

 

[JohnBoy2000]

Where as with

Botox

1) used with 1% to 1.5% benzyl alcohol (I can't actually remember which) - totally worked, but again I'm unsure if there was a strength or potency compromise.

2) used mistakenly with 3% benzyl alcohol = totally bunk, neutralized it entirely.

.......

Again we're assuming the probability is, Benzyl alcohol would weaken/aggregate botox, more readily than it would MT2.

But for comparative purposes between two different amino acid chains (mt2 versus botox), that was the outcome.

 

[Serhat]

Melanotan II is a synthetic peptide analog of the naturally occurring hormone α-melanocyte-stimulating hormone (α-MSH). It is primarily known for its ability to increase the production of melanin in the skin, leading to tanning without exposure to UV radiation. Additionally, it has other effects that might lead to side effects in users, such as appetite suppression, facial flushing, and increased libido.

To address your questions:

1. Reconstitution with Saline: If you're using normal saline (0.9% NaCl in water), it's possible that the acetate form of the peptide could react with the sodium ions to form sodium acetate. This could indeed precipitate out, leading to the residue you observed. However, sodium acetate is fairly soluble in water, so a significant amount of precipitation would be surprising unless you had a very high concentration of Melanotan II.
2. Reconstitution with Purified Water + 0.9% Benzyl Alcohol: This solution lacks the sodium ions present in saline, so there wouldn't be the same potential for sodium acetate formation. Benzyl alcohol is typically added as a bacteriostatic agent to prevent bacterial growth in the solution.

The difference in side effects you experienced could be attributed to a few factors:

• Concentration: If there was significant precipitation of the peptide when mixed with saline, the effective concentration of the peptide in solution might have been lower. When reconstituted in the purified water + benzyl alcohol solution, you might have had a more concentrated solution of the active peptide, leading to stronger effects.
• Purity: It's possible that the melanotan II you used this year was of a different purity or had a different profile of impurities than the batch you used last year. Impurities or related peptides could cause different side effects.
• Individual Variation: The body's response to substances can vary based on numerous factors, including diet, other medications or supplements being taken, stress levels, and general health. It's possible that some of the variation in side effects was due to changes in your own body rather than the peptide itself.
• Stability: Peptides can degrade over time, especially if exposed to light, heat, or oxygen. The solvent (saline vs. purified water + benzyl alcohol) might influence the stability of the peptide, leading to differences in the amount of intact, active peptide over time.

In summary, it is possible that the choice of reconstitution solution could influence the effectiveness and side effect profile of the peptide. However, it's also important to consider other factors, such as the purity of the peptide, individual variation, and the stability of the peptide in the chosen solution.

 

[JohnBoy2000]

Just to mention

Sci-Hub | Role of Benzyl Alcohol in the Unfolding and Aggregation of Interferon α-2a. Journal of Pharmaceutical Sciences, 104(2), 407–415 | 10.1002/jps.24105

sci-hub.se

Having sci-hubbed that paper, from page 4 on - seems to demonstrate that relative to that peptide being examined, Interferon -2 alpha - the charts demonstrate a weakening or increased aggregation relative to to the percentage BA used.

 

[Serhat]

Just to mention

Sci-Hub | Role of Benzyl Alcohol in the Unfolding and Aggregation of Interferon α-2a. Journal of Pharmaceutical Sciences, 104(2), 407–415 | 10.1002/jps.24105

sci-hub.se

Having sci-hubbed that paper, from page 4 on - seems to demonstrate that relative to that peptide being examined, Interferon -2 alpha - the charts demonstrate a weakening or increased aggregation relative to to the percentage BA used.

If the paper you're referring to indicates that there's an increase in aggregation of Interferon α-2a with increasing concentrations of Benzyl Alcohol (BA), then it means that BA affects the stability and solubility of this protein. This is a significant observation, especially if Interferon α-2a is being considered for therapeutic applications where BA might be present as an excipient or solvent.

Here's a brief explanation of the possible implications:

1. Stability and Solubility: Proteins like Interferon α-2a have specific three-dimensional structures that are crucial for their activity. The unfolding or aggregation of these proteins often means they've lost their native structure, and as a result, they might lose their activity. The presence of substances that induce this aggregation can compromise the efficiency of the protein in therapeutic applications.
2. Therapeutic Implications: If Interferon α-2a is being used as a drug, then the formulation's stability is crucial. If BA is present and causes aggregation, then the drug might lose its effectiveness. The rate and extent of aggregation can also affect the drug's pharmacokinetics and bio-distribution.
3. Formulation Changes: If indeed BA causes aggregation of Interferon α-2a, then pharmaceutical scientists might need to reconsider using BA in formulations containing this protein or find ways to mitigate its effects.
4. Mechanism of Aggregation: It would also be essential to understand the molecular mechanisms behind the observed aggregation. Does BA interact directly with Interferon α-2a? Does it change the solvent properties leading to aggregation? Or are there other indirect effects?