Drugs Interactions with LNPs (so to speak)

Drugs and other agents affecting endosomal escape of the mRNA and/or endocytosis

1. Introduction

I think one aspect (among others) that is missing about understanding the mechanisms of action of vaccines is what exactly is happening with the LNPs.

THERE IS NO CORRELATION BETWEEN THE AMOUNT OF mRNA AND SPIKE PROTEIN QUANTITY

THERE IS NO CORRELATION BETWEEN THE AMOUNT OF mRNA AND ANTIBODY PRODUCTION

the reason is because only about 2-5% of all the LNPs injected into the body end up making spike protein and that is because the bottleneck of the jab is the process of endosomal escape as explained in my previous post’

Transfection and Endosomal Escape

THIS MEANS 95% OF THE ‘DOSE’ OF THE JABS IS GOING TO WASTE

So it is not only is there a targeting problem with the LNPs, there is the endosomal escape problem. This is why there is work on

1. saRNA (less number of LNPs/RNA with same amount of protein production)¹

2. addition of cytokines (you can give less LNPs and get the same amount of protein production)

3. research on the LNP structure activity relationship²³

4. new ionizable LNPs, new LNP formulations, non-LNP particles, polymers etc (too many to mention)

It is all to get the product to be more efficient.

And that also means, research on drugs or other substances that helps with endosomal escape, which I hope to review in this post.

(PS and what happens to that 95% of the LNPs that is not utilized for making spike protein? Huh? Why is no one talking about this?)

2. BUT FIRST, the Role of Endolysosomes in SARS-CoV2 Infection

On thing you learn quickly is how these jabs mimic infection with the coronavirus itself. Which gives more meaning to the fact that we are dealing with synthetic biology and these jabs are a corollary to an infection itself.

This paper⁴ gives a fascinating description on how endosomal uptake of the virus occurs.

Here the authors discuss how the virus itself uses the endosomes and which drugs which could help fight the virus by de-acidifying the endosome, for example.

This is how the virus infects you and replicates.

Endolysosome-mediated therapeutic strategies against SARS-CoV-2: SARS-CoV-2 enters cells following interactions between viral spike proteins and cell surface ACE2 receptors. Once endocytosed, spike proteins in endosomes are primed in late endosomes/lysosomes by cathepsin enzymes (B/L); this enhances virus entry. Post-fusion, virus is either released from or degraded in endolysosomes. SARS-CoV-2 once released from endolysosomes, enters the cytosol where it produces a replication complex to generate viral genomic and sub-genomic RNA. Following replication, viral structural proteins get inserted into the ER and move to the ERGIS (endoplasmic reticulum–Golgi intermediate compartment) secretory pathway for virus assembly. Following assembly, virions are transported to vesicles and released from cells by exocytosis.

Based on these mechanisms, the authors suggest the following repurposed drugs/nutraceuticals which may help with infection with the virus.

1. de-acidification of endolysosomes and blocking the priming of spike proteins by deactivating serine proteases.

   1. spermine/spermidine

   2. vitamin D

   3. baicalein (from Chinese skullcap)

   4. trehalose

   5. ketones (or fasting!)

   6. 17beta-estradiol

2. blocking clathrin-mediated endocytosis.

   1. chlorpromazine (and other antipsychotics?)

   2. camostat

3. NPC1 (Neiman-Pick 1) inhibitors could effectively inhibit the virus infection by de-acidifying endolysosomes and blocking the trafficking of cholesterol.

   1. imipramine

   2. posoconazole, itraconazole

4. Anti-TPC (two-pore channel) inhibitors examples also helps with de-acidifying the endosomes

   1. tetrandine (Stephania tetrandia from

   2. naringenin (a flavone from citrus)

TPCs and NPC1 have both been implicated in coronavirus infectivity.

3. What improves Endosomal Escape (since endosomal escape is the Rate Limiting Step) for the JABS?

Since endosomal escape is a huge problem, a whole lot of money is being spent on trying to increase the endosomal escape problem, which hopefully would decrease the amount of LNPs (and toxicity) and the amount of mRNA (and resulting protein toxicity).

What are the options?

1. pH Responsive Fusogenic LNPs (ie ALC-0315 and SM-102 and the like)

   • currently, that is what is being used with the ionizable lipids, helper lipids and cholesterol (remember the PEG sloughs off and is not needed for transfection per se

   • lots of work trying to improve the ratio, type of lipids, yadayada

2. Biomimetic LNPs coated with endosomal vescicle membrane proteins⁵

   1. exploits NATURAL endosomal escape pathways

3. Adding small organic molecules which affect the endosome as co-delivery

   1. ADDING A DRUG to the LNP

4. Phototriggered/Thermosensitive LNPs⁶

   1. can you imagine????

So for this post I am going to talk about ADDING a small organic molecule, like a drug to the LNPs to improve endosomal escape. Does everyone see where this is going? Does that mean there are drugs which increased the release of mRNA from the endosomes? Would that make adverse events WORSE? or paradoxically BETTER?

4. DRUGS WHICH HELP IN ENDOSOMAL ESCAPE OF LNPS

A. TRICYCLIC CATIONIC AMPIPHILIC DRUGS⁷

Here, we describe how well-known tricyclic cationic amphiphilic drugs (TCADs) can be repurposed as both structural and functional components of lipid-based NPs for mRNA formulation, further referred to as CADosomes.

What is a tricyclic cationic amphiphilic drug? These drugs have the following features

1. Tricyclic Structure

• The molecule contains three fused rings, typically aromatic.

2. Cationic Nature

• At physiological pH (~7.4), these drugs become positively charged (cationic), usually due to a basic amine group that is protonated.

3. Amphiphilic Properties

• Amphiphilic means the molecule has both:

  • Hydrophobic (lipophilic) parts: the tricyclic ring system is fat-soluble.

  • Hydrophilic (charged) parts: the cationic amine is water-soluble.

What does THAT sound like? A little bit like ionizable lipids, doesnt it?

So what drugs are TCADs?

Tricyclic antidepressants

First generation antipsychotics (chlorpromazine)

First generation antihistamines

Fluoxetine

These researchers made a TCAD LNP using nortriptylline, a tricyclic antidepressant. Looks like THIS, and WITHOUT the ionizable lipids. Worked ok in rabbits, specifically for the cornea.

Do any of the previous drugs sound familiar? The previous article suggested a tricyclic antidepressant like imipramine (very closely related to nortriptyline) could help with SARS-CoV2 infection. Acidifying of the lysosomes degrades the virus, but also helps with endosomal escape….

2. CATIONIC AMPIPHILIC DRUGS (the previous TCAD as just a subtype because they have a tricyclic structure).

CADs (Cationic Amphiphilic Drugs) is a broad physicochemical classification that includes many drugs not limited to tricyclics. These drugs share structural and functional features that cause them to accumulate in acidic organelles (like lysosomes), potentially disrupt membranes, and sometimes cause phospholipidosis (a lysosomal storage-like side effect).

And what is the poster child drug for CADs? CHLOROQUINE AND HYDROXYCHLOROQUINE

These researchers⁸ are looking at adding the following drugs to improve endosomal escape and have the following caveats. (table with the assistance of ChatGPT)

So what does adding these drugs to the LNPs do?

1. Enhance endosomal escape by accumulating in lysosomes, then they protonate and cause membrane destabilization, resulting in increased transfection efficiency

2. They can add to the proton sponge effect of the ionizable lipids

3. They can degrade lysosomal pathways to recycling endosomes or the cytosol

4. But they can accumulate in lysosomes causing lipid buildup. This results in toxicity and immunogenicity.

BUT ISN’T THAT WHAT THE LNPS DO ALL ON THEIR OWN?

I would say that these drugs might help with a single dose of an RNA/LNP particle but really can’t be used on an ongoing basis.

HOWEVER, if accumulation in lysosomes is occuring with the mRNA shots, would a dose or two of these drugs help with endosomal escape and thus clearing of the mRNA and lipids (which are likely degraded)?

OR, did patients on these drugs regularly, have WORSE outcomes and AEs with the mRNA jabs? Or paradoxically better?

Oh, and BTW, these CAD drugs are also known for

1. QT prolongation

2. CNS effects

3. Drug-drug interactions mostly 3A4 and 2C9 (REMEMBER that from my previous post?)

So not exactly benign products you can add to the lipids and use as a vaccine. At least in the world I used to know.

However, OTHER CAD have been used including

1. the antihistamine ebastine as an intracellular siRNA delivery enhancer⁹

2. The alpha-adrenergic antagonist prazosin¹⁰ promotes cytosolic siRNA delivery from lysosomal compartments

4. LEUKOTRIENE D4 RECEPTOR ANTAGONISTS (MONTELUKAST [Singulair], ZAFIRLUKAST [Accolade])

This was one of my most fascinating finds. Leukotriene receptor antagonists have been looked at with regards to endosomal escape since 2017!! So the manufacturers KNEW that endosomal escape was a problem and that these drugs might INCREASE mRNA in the cytosol.

OK look at THIS!!¹¹

The top row is just the LNPs and the bottom row is with the addition of MK-571 s a small-molecule inhibitor best known as a selective antagonist of the MRP1 (Multidrug Resistance-associated Protein 1) transporter. It was originally developed by Merck as a leukotriene D₄ receptor antagonist (similar to montelukast). Big difference.

So does any commercially available leukotriene inhibitors do the same?

Hmmm it may be concentration dependent but pranlukast (Onon) and zafirlukast (Accolate) might be clinically relevant.

Strange I haven’t found anything since. Let me know in the comments if there are any other studies on using LTRAs with LNPs.

WHAT ARE THE IMPLICATIONS?

5. PROTON PUMP INHIBITORS (Esomeprazole)

OK so maybe chloroquine or hydroxychloroquine is not really safe enough because it could add to the lipidosis problem and LTRAs are ineffectual so what about PROTON PUMP INHIBITORS? They’re safe, aren’t they?

Ok so this is brand new¹². Now esomeprazole is an “adjuvant”

As a potential solution for improving mRNA delivery, we propose the use of esomeprazole (ESO), a type of PPI, as an adjuvant to enhance its delivery efficiency. ….. In this regard, our study aimed to improve mRNA antigen delivery by utilizing ESO as an adjuvant.

So in this study they made LNPs with esomeprazole and added it to SM-102 (Moderna) or DLin-MC3-DMA (which is in Onpattro) to see the effects of esomeprazole in reducing endo−/lysosomal membrane integrity (increasing endosomal escape). The esomeprazole-containing LNP formulation successfully induced robust antigen-specific humoral and cellular immune responses. This was for a melanoma mouse model.

SO DOES TAKING A PPI (proton pump inhibitor like Nexium and the like) at the time one received the vaccine increased the risk of adverse events? If there was increased endosomal escape and more mRNA and more spike protein produced? Or no difference?

6. Why Would We Want to Increase Endosomal Escape?

This is another great review paper on current strategies for endosomal escape. The problem is, it is due to the LNP mechanism of action on the endosomes/lysomes. More endosomal escape usually means more inflammation.

Hypothetically, a pure lipid membrane fusion mechanism would not expose the interior of endosomes and thus not trigger a Gal-8 recruitment. Recent findings investigating LNPs [81] correlate galectin recruitment with the formation of large, irreversible membrane lesions, ultimately triggering inflammatory responses.

These findings on galectin are in this preprint from 2024.¹³ Given its findings, it is surprising that it has not been published. They find

Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation.

LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models.

Because of the complexity of the endosomal system and differences between cell types, several questions remain.

1. why are the hepatocytes the most permissive for cytosolic delivery than any other tissue

2. is maximal endosomal escape the best option?

3. Or release at a lower more continuous level?

4. Which strategy is most productive and biocompatible? It sure isn’t the current LNPs

5. Is fast and early escape better than a slow continuous release?

This is a great review article which really describes mRNA technology and the LNPs. Highly recommended. Here is a figure on what is being done to improve endosomal escape.¹⁴

NP3.47 is a small-molecule inhibitor of Niemann-Pick type C1 (NPC1), a protein involved in endosomal cholesterol transport. Inhibiting NPC1 with NP3.47 has been shown to enhance the intracellular retention of lipid nanoparticle (LNP)-encapsulated small interfering RNA (siRNA), thereby increasing the opportunity for endosomal escape and improving gene silencing efficacy

So the manufacturers need to ADD an inhibitor to enhance endosomal escape resulting in 4 FOLD INCREASE IN gene silencing using LNPs.¹⁵

Well, that says it all, doesn’t it.

SUMMARY AND IMPLICATIONS

1. Small molecule drugs have been used to improve endosomal escape in mice models. Cationic Ampiphilic Drugs (CAD) have been known to do this for several years. (tricyclic antidepressants, HCQ and some antipsychotics…..I note these may have had activity vs the virus as well).

2. The LNPs should be considered a CAD on its own, and have its toxicological profile as such delineated.

3. Possible adverse effects should have been studied to determine if being on these drugs, or any others, posed a risk when taking the mRNA/LNP vaccines

4. Other drugs such at LTRA like montelukast and the rest, appear to have activity vs the virus, especially in combination with an antihistamine. Could this combination increase risk in patients who received the vaccine?

5. Even common drugs like PPIs could be implicated

6. Paradoxically, could these drugs be USED to manage some of the LNP toxicities? Like lysosomal-storage like effects? Assuming the mRNAs are more or less degraded and cannot be translated into the spike protein, could these drugs help in the endosomal/lysosomal cycling pathway? And relieve some of the lysosomal storage effects that may be occurring?

7. Could some of the nutraceuticals mentioned by Khan et al¹⁶ or other drugs regarding de-acidifying make things worse? or better?

8. Retrospective pharmacovigilance studies should be performed to determine if certain drugs may have posed an increased risk to patients. This should be followed up with long term in vivo and in vitro experiments.

9. And what are the implications of 95% of the dose being excess drug because only a small proportion of the dose actually does what it is supposed to do? Yeah.

Please let me know what you think in the comments. And as always pray the rosary

Chemical reactivity of lipid membranes

1. https://pmc.ncbi.nlm.nih.gov/articles/PMC10974399/

2. https://www.biorxiv.org/content/10.1101/2024.09.01.610730v1

3. https://pubs.rsc.org/en/content/articlelanding/2025/nr/d5nr00433k

4. https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2020.595888/full

5. https://www.oaepublish.com/articles/evcna.2024.19

6. https://pmc.ncbi.nlm.nih.gov/articles/PMC12082745/

7. https://www.sciencedirect.com/science/article/abs/pii/S0168365922005144

8. https://www.sciencedirect.com/science/article/abs/pii/S1367593124001078

9. https://www.sciencedirect.com/science/article/abs/pii/S0378517323007688

10. https://www.sciencedirect.com/science/article/abs/pii/S0168365923006648

11. https://pmc.ncbi.nlm.nih.gov/articles/PMC5623340/pdf/nihms902546.pdf

12. https://www.sciencedirect.com/science/article/abs/pii/S0168365925003761?via%3Dihub#preview-section-references

13. https://www.biorxiv.org/content/10.1101/2024.04.16.589801v1

14. https://jbiomedsci.biomedcentral.com/articles/10.1186/s12929-024-01080-z

15. https://pubmed.ncbi.nlm.nih.gov/27633442/

16. https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2020.595888/full