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Mar 6, 2022Liked by Modern Discontent

Wasn’t there an earlier Japanese study finding similar concentrations of LNPs in those organs? As soon as I saw that the ovaries were collecting that stuff the shot was a big NOPE for my 12-year-old daughter. It still horrifies me that just about all her friends got the shots, no questions asked by their parents. Well, that all healthy kids got it, really, but that’s another rant 😜

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Mar 7, 2022Liked by Modern Discontent

It's the same study, it's just that the results of the study were passed to different authorities in advance of authorization and the Japanese were the first to release it.

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I should mention it's hard to discern what the ramifications are here. Remember that this study specifically may only be examining where the 3H-CHE is ending up specifically since the other LNP components and the mRNA were not radiolabeled, so we can only speculate that LNPs are reaching ovaries, although it would be more nuanced to state that the 3H-CHE are.

The biggest issue here is why this was not addressed or examined further by the researchers. Also, why was this essentially brushed off within the abstract and conclusions? The main takeaway a non-discerning reader may get is that the LNPs stay within the injection site, and yet clearly that data is not available based on the Female rats. Paired with the high expression in ovaries compared to testes and one is left wondering why the inconsistent data was never addressed.

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Mar 7, 2022Liked by Modern Discontent

"I tend to overlook the materials and methods sections"

Haha, that's where I usually head to first. I don't wait until the results appear suspicious, I assume all study results are suspicious to begin with! And yet ironically, I find the authors' rationale here to be compelling. Accurate sampling for non-injection-site organs was easier than for the injection site, and so the wild variations in injection-site values are just darts that missed the bullseye and should not necessarily condemn the non-injection-site results, which are a lot more consistent for most organs except the hearts (Table 3). In a sense, there was no point attempting to quantify the injection site to begin with. The variations are extreme even at .25 hours (appendix). It's a lot harder to sample "area in the leg that was hit by a needle" than "bladder" since one can "move" and the other can't.

A really great walk-through (I especially got value from the examination of whether distribution can be measured with fidelity by this type of experiment to begin with), and thanks again for the kind link. Not only should this have prompted rescinding the EUA application, it's sickening to think that the results here didn't even wrap until Phase II/III was well underway.

Obviously, your cholesterol-transport mechanism, if correct, would still potentially be competing with capillary fenestration for determination of uptake, and this could account for both glandular and liver-targeting. See figure 5 of https://www.nature.com/articles/nrd4278 (which was cited by Pfizer in their FDA application!).

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It definitely is harder to isolate regional injections than harvesting organs. My biggest issue with that argument was that the injection site results were used to validate the idea that most of the radioactivity occurred within the injection site and therefore a lot of the LNPs stayed around the injection site. If they can't collect the samples properly as was that the case with the female rats it doesn't make me feel confident that they can make such a claim. Remember that the tagline has been that "it stays within the injection site" and based on how the injection site results never addressed the sex differences due to poor sampling for female rats I can't help but feel like they intentionally skipped over that.

The heart is really spotty. Quantitatively we can't tell if the inconsistency may be due to individual differences, in which case this would be concerning within the context of myocarditis. However, there's no way of knowing the significance. I did a brief examination of the male hearts just to see if maybe there were higher rates of distribution there. At the same time the heart may need even just a tiny bit to cause bad things to happen.

I've only seen radiolabeled studies within the context of drug design and ADME (absorption, distribution, metabolism, and excretion) so I used a similar approach with this study and was curious if any of the radiolabeled cholesteryl alkyl ether may have been metabolized. Fortunately the manufacturer states that it shouldn't be active so we may need to take their word on that. It's an interesting study, but perspective-wise even though I stated that this is showing LNP distribution it really is measuring distribution of the 3H-CHE and we are assuming that 3H-CHE movement is the same as LNP movement. I think if they used an active luciferase enzyme or spike protein where cell lysis may occur the results would have been far different.

Wow, Brian! I feel like every time I'm looking into a paper you provide me with something else that perks the ears up! There's quite a few interesting segments in that review. Also, that broadness of that figure is quite scary especially if mRNA can be quite broad in its target cells, which of course was seen as a plus.

There's also quite a few concerning paragraphs:

"The current data do not indicate that there is any induction of immunogenicity against IVT mRNA itself. However, mounting evidence suggests that patients with systemic lupus erythematosus and other autoimmune diseases can develop anti-self RNA autoantibodies that have a role in the induction and progression of autoimmunity187. Thus, under certain circumstances, such as long-term repetitive systemic application of mRNAs, anti-RNA antibodies may potentially form and mediate immune pathology. One might consider screening mRNA sequences to avoid conformations that are prone to inducing mRNA-specific antibodies188. Clinical monitoring of autoimmune phenomena and laboratory tests for antinuclear antibodies are therefore advised."

"It is also conceivable that the expression of a foreign protein together with the pro-inflammatory effects mediated by the mRNA backbone may result in immunopathology on the tissue level. For immunotherapeutic approaches, this may be of minor relevance as antigen-presenting cells are the target cells of mRNA delivery and these are short-lived once they have transitioned into the mature state. If, however, other organs such as the liver, kidney, lungs or myocardium are targeted, this risk needs to be addressed. Various applications that are being pursued use the liver as the target organ, as it has been shown (at least for various siRNA delivery platforms) that nucleic-acid-based drugs are routed to the liver by default, and therefore liver targeting can be accomplished without further optimizing delivery195. As hepatic toxicity may be life threatening, particular caution is warranted and liver enzymes such as transaminases need to be measured. However, given the unique immunological properties of this organ, it may even be de-risking to use the liver as a depot organ for protein expression of the first-generation of mRNA-based therapeutics for protein replacement, as its capacity to induce antigen-specific tolerance may counteract immunogenecity196."

That's a lot of food for thought!

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Mar 7, 2022Liked by Modern Discontent

"Food for thought," haha. I see Brain > Endothelium > Paracrine activity of protein expression and put a mental checkmark under "This 'tech' is a sham and will never work."

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Thanks, very interesting article!

If the LNP delivers its payload, does it necessarily stay with that cell or is it likely to detach and continue traveling? IOW do we expect a strong relationship between presence of LNP and delivery and presumably expression of the mRNA?

I also seem to recall reading that these LNPs are only metabolized in the liver due to their unusual composition.

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The assumption here is that it's high hydrophobicity/lipophilicity would prevent it from travelling afterwards. However, we also have to deal with the assumption that cells that express the spike protein would be designated to cell death. Afterwards, the questions of where the LNPs go would be an issue but at that point it would not be so much of a concern considering that most of these LNPs are either things endogenous to our bodies or things that are readily metabolized. It would be more of a concern about which cells they target for entry.

The pairing of this study with the luciferase study is important because the luciferase study at least allows for examination of the downstream protein expression. In that study liver cells expressed luciferase. The results of that study paired with this study would suggest that the LNPs are both travelling to the liver and are being uptaken by liver cells to express luciferase. The assumption here is that spike protein may be expressed by the liver as well.

https://moderndiscontent.substack.com/p/preliminary-examination-of-biontech?s=w

https://moderndiscontent.substack.com/p/clarifying-yesterdays-lipid-nanoparticle?s=w

The limitations of this study is that it only shows where the 3H-CHE is appearing, not the LNPs as a whole or if the mRNA is being translated to produce protein (the mRNA used here was an inert luciferase mRNA so no proteins were expressed).

Most exogenous substances are metabolized by the liver. As an aside, it's the reason why the liver is one of the main areas of concern with drug studies (the kidneys as well) because the liver metabolizes so many of what we take in. Sometimes, the exogenous substance may produce a toxic metabolite which ends up being hepatotoxic.

But speaking specifically of the LNPs, like I said the prior study with luciferase activity suggest that the mRNA is being taken up by liver cells in order to show luciferase activity, which implies more is going on here than just metabolism of the LNPs.

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Thank you for your response!

Would the luciferase expression likely cause the cells to be killed? How long does that take (does it require the immune system to identify the foreign chemical first in order to flag the cells?)

I ask because I notice the trend in the concentrations seemed to rise and then fall for some organs, and just keep rising for others. If the LNPs stick until the cell is killed, then I guess the concentration would only go down if the cell was killed and the detritus is metabolized and excreted. But by the same token, I would expect this to happen in all the organs.

If LNPs adhere to organs but do not transfect the cells, then those LNPs could stay there indefinitely, as there might be no reason for them to ever be metabolized.

It looks like my belief about metabolism only in the liver seems incorrect as they appear to show that metabolism also occurred in blood and plasma.

Re 3H vs. the whole LNP, they seem to suggest that they screened for whole LNPs in urine and feces (not sure how they did this) vs. just looking for the radio tags.

On another note, I noticed that they did not do a reprotox analysis for "V8" (the EUA version?), only for "V9" (the unobtainable "approved" version)? Perhaps a minor point but not a good thing (section 2.4.4.6 of the overview).

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That question is harder to assess. The luciferase study was not powered to examine that. It did indicate a possible adaptive T-cell immune response against luciferase, although the actual ramifications of that response are difficult to discern. Unlike the spike protein luciferase expression is sequestered within the cell so the immune response is likely to be different than spike proteins which are likely to be transporter to the outer membrane of the cell and expose to the extracellular fluid.

So the organs have to be analyzed from the perspective of distribution and that the actual amount of radioactivity is finite. Therefore, the idea should be that as percentages decrease in certain areas it should increase in others. More simply put, as the percentage decreases within the injection site the assumption would be that the percentage should increase elsewhere. However, it's important to keep in mind that the radioactive half life of tritium should be thought about although the level of decay is likely to be negligible over the course of two days. You also have to keep in mind that different cohorts of rats were sacrificed and tissues collected at each time point meaning that high variability between cohorts should be considered as well.

The LNPs adhering is interesting. Unfortunately there's no evidence to support or refute that notion as none of these studies were designed to measure that. Usually what you would do with these studies is to collect the material through feces or urine and measure how much of the original compound was collected (this doesn't take into account any metabolites that may be released through gas exchange and breathed out), however considering that the 3H-CHE that was used is not metabolically active the researchers could have had a cohort that they collected waste material from and examined at what point a large portion of the 3H-CHE was collected. That would at least rebut the idea that the LNPs may adhere indefinitely.

The feces and urine looked like they just measured for waste (the tables measure in grams). I think this may have been done to examine for changes in eating behaviors or possible changes in kidney or GI issues? I'm not quite sure as they did not explain too much in that regard.

As for the vaccine used that's interesting. I did a brief overview but I was looking up stuff on long COVID and was going to write about that a bit. I may return and look at that overview. Do you know the difference between the two? I haven't looked too closely into those slight variations.

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Re. excretion of the LNPs and metabolic products - there is a reference to some analysis in the nonclinical overview document https://phmpt.org/wp-content/uploads/2022/03/125742_S1_M2_24_nonclinical-overview.pdf .

"An intravenous rat PK study, using LNPs with the identical lipid composition as BNT162b2, demonstrated that ALC-0315 and ALC-0159 distribute from the plasma to the liver. While there was no detectable excretion of either lipid in the urine, the percent of dose excreted unchanged in feces was ~1% for ALC-0315 and ~50% for ALC-0159. " So this seems to imply that they did analyze the excretions and were able to identify whole un-metabolized LNPs, but it doesn't say whether they did a radiological survey to find the total amount of tags disposed into the excretions. However looking at the tables of distribution at the 2 day mark, they only account for 43% or so of the radiological tags, and it must have gone somewhere, so presumably excreted?

Re. the difference between the EUA and FDA version, it's not really clear. I recall reading something enumerating the differences, I thought in one of the application docs, but after spending an hour looking I can't find it again. The non-clinical overview doc does make reference to it here: "The nonclinical safety evaluation included 2 variants of BNT162b2: V8 and V9. BNT162b2 (V9), the candidate submitted for licensure, differs from BNT162b2 (V8) only in the presence of optimized codons to improve antigen expression, but the amino acid sequences of the encoded antigens are identical. Two GLP repeat-dose toxicity studies for BNT162b2 (V8) and BNT162b2 (V9), one study for each variant, have been completed. In both studies, the nonclinical toxicology findings were similar between BNT162b2 (V9) and BNT162b2 (V8). BNT162b2 (V9) was assessed for development and reproductive toxicity in rats."

I seem to also remember that the formulation changed out some of the other ingredients, but I haven't been able to find that document again. There's no reason to think any of these differences are significant, but they are differences nonetheless.

In terms of my original question, I guess it's not clear. The evidence still shows an increasing concentration in the ovaries at 48 hours, which is not explained and a longer test would be indicated. Whether that LNP concentration implies transfection, or just presence of "spent" LNPs isn't proven one way or the other, but could reasonably be assumed to be implying transfection... which might not be good for fertility. I'd really like to see this experiment done: https://igorchudov.substack.com/p/steve-kirsch-germ-line-mutation-allegations?s=r

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Mar 7, 2022Liked by Modern Discontent

Thank you for taking us through the study! 👍🏽💕

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Of course! Hopefully it was translated well and people understood many of the underlying implications.

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I am wondering what your take on this study is now, more than a year later. Now that we have several autopsy reports finding damage to the endothelial lining. Studies that show that the only thing needed to trigger syncytia formation in ACE2 presenting tissue is the spike protein, the fact that capillary beds are made solely of endothelial cells. Overall I would think that the approval of the new bivalent boosters is right up the alley of bad studies like the study #185350.

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"These results helped to validate the use of these vaccines in millions of people, and if Pfizer and BioNTech could not bother to follow proper lab procedures or to redo studies how do we trust that these vaccines are safe and effective without any proper studies?"

What does this imply about the review or regulatory process that there wasn't a directive to redo this study? Is this a deviation from normal standards?

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