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

[sekio]

Interferon is a lot bigger than melanotan though: The mature protein is made of 165 amino acids, one less than the other human IFNα subtypes.

Instead of theorizing, which we can do all day and accomplish nothing, I went and hit the books, and what do you know: this question has already been answered. A comprehensive review on Botox says:
"All the above products are in powder form and need to be reconstituted prior to application. There is debate with regard to reconstitution with normal saline with no preservatives and preserved (0.9% benzyl alcohol) saline. The manufacturer’s suggested diluent is normal saline with no preservatives. Several authors have reported equivalent clinical effectiveness with BoNT-A diluted with preserved saline. (sekio edit: i.e. with 0.9% benzyl alcohol) However, there is a debate on whether the preservatives in the saline deactivate the toxin partially.[ref1][ref2] Alam et al, in a double-blind, randomized controlled trial, reported that preserved saline containing benzyl alcohol used in reconstitution of [Botox] did not affect its potency or clinical outcome and made the injections less painful.[ref] This was confirmed by two other studies.[ref1][ref2]"

So, unless you use ridiculous amounts of benzyl alcohol, Botox is not effected by the 0.9% BA typically used. Likewise, I would not expect other peptides to react with 0.9% BA or less, Melanotan II included.

If you observed an equal tanning effect on both batches of Melanotan then it obviously is working equally well, and I would put money on the symptoms experienced being either psychogenic or due to continued usage (MT-II crosses the blood brain barrier and produces effects in the brain and spinal cord [ref])

 

[Serhat]

Interferon is a lot bigger than melanotan though: The mature protein is made of 165 amino acids, one less than the other human IFNα subtypes.

A comprehensive review on Botox says:
"All the above products are in powder form and need to be reconstituted prior to application. There is debate with regard to reconstitution with normal saline with no preservatives and preserved (0.9% benzyl alcohol) saline. The manufacturer’s suggested diluent is normal saline with no preservatives. Several authors have reported equivalent clinical effectiveness with BoNT-A diluted with preserved saline. However, there is a debate on whether the preservatives in the saline deactivate the toxin partially.[ref1][ref2] Alam et al, in a double-blind, randomized controlled trial, reported that preserved saline containing benzyl alcohol used in reconstitution of ONA did not affect its potency or clinical outcome and made the injections less painful.[ref] This was confirmed by two other studies.[ref1][ref2]"

So, unless you use ridiculous amounts of benzyl alcohol, Botox is not effected by the 0.9% BA typically used.

Your observations underscore the importance of understanding that different proteins and peptides can have different sensitivities and interactions with various excipients or additives, including benzyl alcohol (BA).

Here's a breakdown:

1. Size and Complexity: As you mentioned, Interferon α (with 165 amino acids) is indeed larger than melanotan. The size, shape, and specific amino acid sequence of a protein can significantly influence its stability and interactions with other molecules. For example, a large protein like Interferon may have more domains or regions that can be destabilized by certain agents compared to smaller peptides.
2. Botox (Botulinum toxin type A): Botox is a complex protein toxin. The reference you provided indicates that there's debate over the effect of BA on Botox. However, as noted, several studies have found that the typical concentration of 0.9% BA used in saline does not adversely affect the potency or clinical outcomes of Botox. This highlights that not all proteins or peptides are affected the same way by BA.
3. Specificity of Interactions: Just because one protein (like Botox) is unaffected by BA doesn't mean another protein (like Interferon α) will have the same resilience. The interactions can be very specific. The effects of BA might be based on particular regions of the protein that could interact with or be destabilized by BA.
4. Context: The concentration and context in which BA is used matter. A higher concentration of BA could have more pronounced effects. Moreover, the formulation's overall composition, including pH, ionic strength, and presence of other stabilizing agents, can also influence the protein's stability.

It's essential to appreciate that formulation science, particularly with biologics like proteins, is a nuanced field. Small changes can lead to significant differences in stability, efficacy, and safety. When considering the use of any additive or excipient with a protein or peptide, empirical studies, like the ones you referenced for Botox, are crucial for understanding their potential impacts.

 

[JohnBoy2000]

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?

It does seem to be the case certainly.

Comparing with melanotan 2:

Melanotan-II

www.raybiotech.com

The molecular formula of Melanotan-II is C50H69N15O9. It has the amino acid sequence of Ac-Nle-cyclo [Asp-His-D-Phe-Arg-Trp-Lys]-NH2 and a molecular weight of 1024.2 Dalton.


IFN alpha 2a

https://www.genscript.com/protein/Z00358-Interferon_Alpha_2a_IFN_Alpha_2a_Human.html#:~:text=Human%20Interferon%20alpha%202a%20(IFN,molecular%20mass%20of%2019%2C241%20Da.

Human Interferon alpha 2a (IFN alpha 2a) produced in E. coli is a single, non-glycosylated, polypeptide chain containing 165 amino acids and having a molecular mass of 19,241 Da.

........

MT2 = 1 kDA molar mass, 6 amino acids in length.

IFN alpha 2a = 19 kDA molar mass, 165 amino acids in length.

Am I correct there?

I'm just speculating, based on this, whether mt2 would be less prone to aggregation than IFN alpha 2a?

 

[JohnBoy2000]

Interferon is a lot bigger than melanotan though: The mature protein is made of 165 amino acids, one less than the other human IFNα subtypes.

Instead of theorizing, which we can do all day and accomplish nothing, I went and hit the books, and what do you know: this question has already been answered. A comprehensive review on Botox says:
"All the above products are in powder form and need to be reconstituted prior to application. There is debate with regard to reconstitution with normal saline with no preservatives and preserved (0.9% benzyl alcohol) saline. The manufacturer’s suggested diluent is normal saline with no preservatives. Several authors have reported equivalent clinical effectiveness with BoNT-A diluted with preserved saline. (sekio edit: i.e. with o.9% benzyl alcohol) However, there is a debate on whether the preservatives in the saline deactivate the toxin partially.[ref1][ref2] Alam et al, in a double-blind, randomized controlled trial, reported that preserved saline containing benzyl alcohol used in reconstitution of [Botox] did not affect its potency or clinical outcome and made the injections less painful.[ref] This was confirmed by two other studies.[ref1][ref2]"

So, unless you use ridiculous amounts of benzyl alcohol, Botox is not effected by the 0.9% BA typically used. Likewise, I would not expect other peptides to react with 0.9% BA or less, Melanotan II included.

Sounds solid.

It's unfortunate that I went up to 1.5% BA with melanotan 2 (from experience, it still worked - but I couldn't specifically measure it's complete efficacy, naturally).

But drawing comparisons, it would appear it probably didn't effect its efficacy........

.......

However, when you say "ridiculous amounts of BA", I would guess 3% is ridiculous (mistake on my part), cause with botox, that did neutralize it.

 

[Serhat]

It does seem to be the case certainly.

Comparing with melanotan 2:

Melanotan-II

www.raybiotech.com

The molecular formula of Melanotan-II is C50H69N15O9. It has the amino acid sequence of Ac-Nle-cyclo [Asp-His-D-Phe-Arg-Trp-Lys]-NH2 and a molecular weight of 1024.2 Dalton.


IFN alpha 2a

https://www.genscript.com/protein/Z00358-Interferon_Alpha_2a_IFN_Alpha_2a_Human.html#:~:text=Human%20Interferon%20alpha%202a%20(IFN,molecular%20mass%20of%2019%2C241%20Da.

Human Interferon alpha 2a (IFN alpha 2a) produced in E. coli is a single, non-glycosylated, polypeptide chain containing 165 amino acids and having a molecular mass of 19,241 Da.

........

MT2 = 1 kDA molar mass, 6 amino acids in length.

IFN alpha 2a = 19 kDA molar mass, 165 amino acids in length.

Am I correct there?

I'm just speculating, based on this, whether mt2 would be less prone to aggregation than IFN alpha 2a?

Based on the information you've provided:

1. Melanotan II (MT2):
   • Molecular weight: 1024.2 Dalton (or Da)
   • This means it has a molar mass of approximately 1 kDa (since 1 kDa = 1000 Da).
   • Amino acid sequence provided shows 7 amino acids (including the cyclic portion), not 6.
2. Interferon alpha 2a (IFN alpha 2a):
   • Molecular weight: 19,241 Da
   • This translates to a molar mass of approximately 19 kDa.
   • Contains 165 amino acids.

Your interpretation of the molar masses is correct. However, Melanotan II has 7 amino acids in the sequence you provided, not 6.

Regarding aggregation propensity:

In general, larger and more complex proteins (like IFN alpha 2a) can be more prone to aggregation due to the increased number of hydrophobic patches, disulfide bonds, and potential folding intermediates. However, protein aggregation is influenced by numerous factors, such as protein structure, solution conditions (pH, ionic strength), presence of excipients, and processing conditions (like freeze-thawing, shaking, or shear forces).

While it's reasonable to speculate that IFN alpha 2a might be more prone to aggregation than MT2 due to its larger size and complexity, the actual propensity for aggregation would need to be studied experimentally for each protein under specific conditions.

Additionally, even small peptides like MT2 can aggregate, especially due to their hydrophobic interactions or incorrect disulfide bond formations. So, size is one factor, but it's not the only factor that determines aggregation propensity.

 

[JohnBoy2000]

even small peptides like MT2 can aggregate, especially due to their hydrophobic interactions or incorrect disulfide bond formations. So, size is one factor, but it's not the only factor that determines aggregation propensity.

Does MT2 have hydrophobic interactions?

Or incorrect disulfide bond formations?

I'd love to read the link if you have it available? I'm trying to inform myself as much as possible on the specifics of this moleclue.

I agree with the former statement:

While it's reasonable to speculate that IFN alpha 2a might be more prone to aggregation than MT2 due to its larger size and complexity, the actual propensity for aggregation would need to be studied experimentally for each protein under specific conditions.

Unfortunately we can't do that, so based on
- examining the variables,
- comparisons with other molecules, and
- the "clinical outcomes" (mt2 worked with saline and 1.5% BA, albeit I can't say if it was weakened or not - but it worked, i.e. definitely wasn't entirely neutralized),

I guess we're trying to make some kind of determination as to the potential for weakening/degradation/aggregation, under these variable conditions (saline vs pure water, 1% BA versus 1.5% BA).

I very much appreciate your thoughts and insights.

 

[Serhat]

Does MT2 have hydrophobic interactions?

Or incorrect disulfide bond formations?

I'd love to read the link if you have it available? I'm trying to inform myself as much as possible on the specifics of this moleclue.

I agree with the former statement:



Unfortunately we can't do that, so based on
- examining the variables,
- comparisons with other molecules, and
- the "clinical outcomes" (mt2 worked with saline and 1.5% BA, albeit I can't say if it was weakened or not - but it worked, i.e. definitely wasn't entirely neutralized),

I guess we're trying to make some kind of determination as to the potential for weakening/degradation/aggregation, under these variable conditions (saline vs pure water, 1% BA versus 1.5% BA).

I very much appreciate your thoughts and insights.

Given its sequence, MT2 has the potential for various interactions:

1. Hydrophobic Interactions: MT2 does have hydrophobic residues in its sequence, such as phenylalanine (Phe) and tryptophan (Trp). These residues can partake in hydrophobic interactions. When peptides dissolve in water, hydrophobic residues may tend to orient themselves away from the aqueous environment, leading to potential interactions.
2. Disulfide Bonds: The MT2 peptide sequence you've provided previously does not contain cysteine residues. Cysteines are the amino acids responsible for forming disulfide bonds. Therefore, MT2 would not have the capability to form disulfide bonds.

That said, these properties and interactions can influence how MT2 behaves in solution and potentially how it interacts with its target receptors in the body. However, it's worth noting that MT2, given its size and design, has been optimized for specific biological activities and likely has a stable conformation favorable for its function.

I guess we're trying to make some kind of determination as to the potential for weakening/degradation/aggregation, under these variable conditions (saline vs pure water, 1% BA versus 1.5% BA).

Absolutely, I understand your intent. Let's further delve into the considerations and analysis of Melanotan II (MT2) under the mentioned conditions:

1. Stability in Different Solvents:
   • Saline: Saline (sodium chloride in water) primarily affects the ionic strength of the solution. In the case of MT2, it might impact the solubility and possibly the stability of the peptide. However, peptides generally have a balance of hydrophilic (water-loving) and hydrophobic (water-repelling) amino acids, and MT2 is no exception. The presence of salt might not have a dramatic effect on MT2's stability or propensity to aggregate, especially given its size.
   • Pure Water: Without the added ionic strength from salts, MT2 might exhibit a different solubility profile in pure water. But considering MT2's balance of hydrophobic and hydrophilic residues, it's likely still soluble in pure water, and stability might not be dramatically different from saline.
2. Benzyl Alcohol (BA) Effect:
   • 1% vs. 1.5% BA: The concentration change is relatively minor. The primary function of BA in such formulations is its bacteriostatic property, not necessarily to stabilize the peptide. The difference in these concentrations is unlikely to have a substantial effect on MT2's stability. If at all, a higher concentration might impact the peptide's solubility slightly, but not its inherent stability.
3. Potential for Aggregation:
   • MT2's small size and sequence make it less likely to aggregate than larger, more complex proteins. While changes in solvent and BA concentration might shift the equilibrium of dissolved peptide to any aggregated form, the fact that MT2 worked in your application suggests that if any aggregation occurred, it wasn't significant enough to impact its biological activity.
4. Weakening/Degradation:
   • Peptides can degrade over time, especially if exposed to conditions like high temperatures, light, or certain chemicals. BA isn't known to degrade peptides, so the choice between 1% and 1.5% is unlikely to cause degradation. However, always storing the peptide in a cool, dark place will further ensure its stability.

In conclusion, based on the provided conditions and the nature of MT2:

• The difference between using saline and pure water is likely minimal in terms of peptide stability and aggregation.
• The difference between 1% and 1.5% BA probably has minimal (if any) impact on the peptide's stability or propensity for aggregation.
• The observed efficacy of MT2 under these conditions further suggests that any potential degradation, weakening, or aggregation is not significantly impacting its activity.

However, as always, it's essential to mention that these are educated estimations based on the peptide's nature and general principles of peptide chemistry. Direct experimentation would be the gold standard for conclusive evidence.

 

[JohnBoy2000]

Benzyl Alcohol (BA) Effect:

• 1% vs. 1.5% BA: The concentration change is relatively minor. The primary function of BA in such formulations is its bacteriostatic property, not necessarily to stabilize the peptide. The difference in these concentrations is unlikely to have a substantial effect on MT2's stability. If at all, a higher concentration might impact the peptide's solubility slightly, but not its inherent stability.

Given the fact that, as you've acknowledge, the MT2 still worked relative to both - that would suggest our conclusion here is correct, that this small percentage difference probably didn't alter the integrity of the peptide dramatically.

As I think I mentioned in the pm though - 1% versus 3% BA, mixed with botox, definitely DID have a dramatic impact (neutralized it entirely at 3%, worked well at 1%).

And as you mentioned, higher percentages can also cause irritation - which also happened.
Cause when I injected the botox, my forehead swole up and went numb for about a week (expected as BA is an anesthetic agent also).

But then, that was 3%, a hell of a lot more than 1.5%........
Conversely, the botox didn't work at all at 3%, unlike the MT2 at 1.5% which worked perfectly fine.

However, as always, it's essential to mention that these are educated estimations based on the peptide's nature and general principles of peptide chemistry

Absolutely, that's what I'm looking for here, and it's highly appreciated!

 

[JohnBoy2000]

Putting aside the MT2 for moment though:

Curious why 3% BA would in fact neutralize Botox?

When the standard 1% is considered and proven to be fine.

The factors taken into consideration for mt2:
- aggregation
- molecular mass
- amino acid chain length

Given its size/weight, perhaps just more vulnerable to that small difference?

The manufactures do recommend non-preserved diluent/saline, perhaps they were aware than, BA at low doses may not impact the integrity massively, but may somewhat.

But re mt2, smaller etc., less prone to this probably.

.........

The former scienedirect article on benzyl alcohol does also mention in can exert its aggregating effect with time.

Curious whether the controlled study of Botox with preserved water examined its potency at the time of reconstitution - but also after storage also? (i.e. storage/freezing after reconstitution).

@sekio did that study you read mention anything on effects of storing botox reconstituted in preserved water? (versus using it immediately upon reconstitution?)

 

[Serhat]

Putting aside the MT2 for moment though:

Curious why 3% BA would in fact neutralize Botox?

When the standard 1% is considered and proven to be fine.

The factors taken into consideration for mt2:
- aggregation
- molecular mass
- amino acid chain length

Given its size/weight, perhaps just more vulnerable to that small difference?

The manufactures do recommend non-preserved diluent/saline, perhaps they were aware than, BA at low doses may not impact the integrity massively, but may somewhat.

But re mt2, smaller etc., less prone to this probably.

.........

The former scienedirect article on benzyl alcohol does also mention in can exert its aggregating effect with time.

Curious whether the controlled study of Botox with preserved water examined its potency at the time of reconstitution - but also after storage also? (i.e. storage/freezing after reconstitution).

Botox, or botulinum toxin type A, is a highly potent neurotoxin. Its activity can be affected by various factors, including the pH and preservatives in its diluent. When it comes to benzyl alcohol (BA), its concentration in the solution matters, and different substances may have different susceptibilities to BA-induced degradation or aggregation.

Why might 3% BA neutralize Botox when 1% is considered fine?

1. Mechanism of Action: Benzyl alcohol can denature proteins, and at higher concentrations, this effect might be more pronounced. The exact mechanism by which BA could neutralize Botox is not fully understood, but it may involve protein aggregation, disruption of the protein's conformation, or interference with its mechanism of action.
2. pH Alterations: Botox has an optimal pH range for stability. Benzyl alcohol might alter the pH of the solution, and a higher concentration of BA might have a more significant impact. This could affect Botox's potency.
3. Time-Dependent Aggregation: As you pointed out, BA can exert its aggregating effect over time. Even if Botox remains active initially, prolonged exposure to BA, especially at higher concentrations, might lead to degradation or loss of efficacy.

Melanotan II (MT2) considerations:

MT2 is a peptide, and peptides, in general, can be sensitive to environmental conditions like pH, temperature, and exposure to certain chemicals. The factors you mentioned – aggregation, molecular mass, and amino acid chain length – can indeed influence a peptide's stability. Given its distinct structure and properties, MT2's susceptibility to BA might differ from that of Botox.

Regarding the controlled study of Botox with preserved water:

I don't have the specific study you're referring to in front of me, but it would indeed be essential to see if the study considered both immediate and long-term potency after reconstitution and storage. Generally, for a study to be comprehensive and conclusive about the effects of BA on Botox, it should look at potency immediately after reconstitution and after storage (whether refrigerated or frozen) over varying periods.

Finally, while manufacturers recommend non-preserved saline for Botox reconstitution, this recommendation might be out of an abundance of caution. It ensures maximum potency and minimizes potential risks.