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Pharmacology Sciences

[Sciences/Pharmacokinetics] Do nano-particles of the Pfizer COVID-19 vaccine cross the blood-brain barrier and infect your brain with mRNA (or will fritz your gonads)?

1. Introduction
[EDIT: Updated the article on 07/05/2021 to reflect some updates on my analysis]

I have recently seen some claims I considered moot resurfacing on social media: first that COVID-19 vaccines render women infertile; second that mRNA vaccines cross the blood-brain barrier and therefore lead to neurological diseases.
These claims have been rebutted by various science communicators including Edward from Deplatform Disease and myself on Skeptical Raptor few months ago, as the Pfizer and Moderna vaccines were rolling out in the US.

Thing is, with anti-vaxxers, claims are never completely dead and keep rising up like some zombies straight out of a Walking Dead episode.

This time, it seems to be materialized through this screenshot, that appear to spread virally on social media over the weekend, especially in various iterations of that screenshot, with a yellow highlight in a table with the following tissue: “ovaries” and total lipid concentrations as only information.

Screenshot depicting estimated aminolipid contents in rats following injection of the Pfizer COVID-19 nanoparticle formulation (source: Facebook).

2. What is the screenshot coming from?

As always, getting back to the source of a document is essential to put this information back into the context. This screenshot appeared to be coming from a leaked document (if I have to judge on the “Pfizer – Confidential” footers) that I was able to find the source. Unfortunately the document is in Japanese but I can speculate this document likely came from an application packet submitted to the Japanese equivalent of the FDA to seek authorization of sale of the vaccine on the Japanese market. 3. What is the document about?
It seems the document provides us with some pharmacokinetics data on the mRNA vaccine done in rats (Wistar Han strain, both males and females) to assess the pharmacokinetics of the nanoparticles inside these rodents to assess the pharmacokinetics of both the lipid nanoparticles and the mRNA (using the luciferase as reporter of mRNA transcription, I will explain it later).
For the majority of the experiments, we have the following situation been used (according to Table 1):

Nanoparticles were used using two aminolipids (ALC-0315 and ALC-1059) at concentrations of 15.3 and 1.96mg/kg respectively. mRNA was encapsulated in these nanoparticles at 1mg/kg (to give you an idea, the actual dose of mRNA in a Pfizer shot is 30ug or 0.03mg from patients ranging of 12 years and older)

Table 1 provides us with some pertinent PK parameters including the half-life (time to eliminate 50% of a drug), the AUC (to compare the relative bioavailability, distribution and calculate the clearance of a drug) and finally the Kanji translated by Google Translate (sorry but that poor Gaijin is illiterate to Japanese despite decades of anime) as “Distribution ratio to the liver“, with 60% of ALC-0315 found in the liver, 20% of ALC-0159 respectively. The number of animals also appear to be N=3/group (male, female as groups).

We have therefore extensive data on the aminolipids metabolism and the metabolites obtained both in vivo (plasma samples mostly), in vitro (using liver microsomes homogenates, a classic in PK/PD studies); distribution of LNPs in tissues and organs using a non-metabolized radio-tracer ([3H]-08-A01-C0 which I quote the document “[3H]-08-A01-C0 = An aqueous dispersion of LNPs, including ALC-0315, ALC-0159,distearoylphosphatidylcholine, cholesterol, mRNA encoding luciferase and trace amounts of radiolabeled [Cholesteryl-1,2-3H(N)]-Cholesteryl Hexadecyl Ether, a non-exchangeable, non-metabolizable lipid marker used to monitor the disposition of the LNPs“, which was given at a dose of 50ug in animals) and finally bio-luminescence assays in which it consisted of injecting 2ug of RNA encapsulated in the LNP formulation in the hind-limbs of rats (we can assume these were adult rats, therefore a weight of 200-250g is not unheard of), followed by live imaging of the animals to track the luciferase activity (following injection of coelenterazine, the conversion of this substrate by luciferase results in bio-luminescence at close proximity which can be detected through a special camera, as Figure 2).

4. What the data is telling us?


The first thing I would tell is that the person behind the yellow highlight not only have absolutely no idea of what to look for in Table 3 but also went into a cherry-picking expedition to use numbers in scaring people with numbers. That person is providing us with amount of the radiolabeled tracer detected in the tissue (e.g. ug/g tissue), with the approximation of total lipids amount in tissue. This assumes that the nanoparticles made it through the tissue complete, but we cannot exclude that we are maybe measuring only the 08-A01-C0 compound accumulation.
In practice, we usually focus our attention on the percentage of injected dose (% ID) when it comes to appreciate the distribution and the delivery of a drug into an organ/tissue. In some fields, like the BBB, such value is usually not sufficient, and we further correct these values to sort the amount that diffused across the blood-brain barrier (BBB) against the amount that is retained in the cerebral vasculature by the time of euthanasia.
Therefore, we have to put our attention on the right-half of the table. I have plotted these values into a plotting software (Graphpad Prism 9) to have a graphical representation.

What we can see is that the LNPs reach a Cmax value of 52.9% of the ID by 1 hour following IM injection and see a biphasic phase of distribution and elimination (which I suspect the drug would follow a 2-model compartment). Liver is the organ with the highest uptake (we know that 60% of the LNPs are uptaken by the liver) with a Cmax of about 18% of the injected dose by 8 hours. This is expected as liver has a formidable blood flow compared to other organ (Q=1500mL/min). Spleen (very important lymphoid organs) comes in as a good second with a Cmax~1%ID by 8 hours. Kidneys in the other hand sees a much lower uptake despite being an organ with a decent blood flow (GFR=~120mL/min) with a Cmax`0.2%ID, suggesting these LNPs maybe eliminated mostly via hepatic clearance route (including metabolism).

[EDIT: I have performed an area-under-the curve analysis, just for the fun of it. We are lacking data, so we will use for informational purpose. The use of the AUC trapezoidal method can allow to guesstimate how much of that radiotracer accumulated in the tissue/organs over the 48 hours period.
If we look at the AUC values of these from 0 to 48 hours, about 57% of the injected dose is found in the liver, 3% in the spleen, 0.25% in the kidneys, 0.17-0.18% in the gonads and finally 0.04% of the injected dose is found in the brain). ]

What about ovaries? Well we are in the same ballpark than kidneys and indeed nothing really much about (0.1%ID after 48 hours). Interestingly, the author hyper focused on female gonads and occulted to show that male gonads (testes) were getting the same %ID (0.07%). I don’t think it was an accident from the author, just a sign of a deliberate attempt to manipulate the narrative by spinning the numbers.
And last, brain, my favorite organ. The amount entering the brain is maybe the lowest of our organ of interest as we measured a meager 0.02% ID there. Keep in mind, we have to be careful on this number as we may have an overestimation here. In the field, when you do brain perfusion and you are about to collect your last plasma timepoint before sacrificing the animal, you have to be sure to perform a “flushing” of your cerebral blood vessels with PBS, to remove any residual blood volume that can contain your drug. Unless you can correct for the vascular volume (which is not as simple), you have to perform this procedure as we did in a paper I collaborated on. Failure to do so can can lead to overestimation of your brain uptake. Until I have evidence of such flushing occurred, we can hypothesize that the investigators sacrificed their animals at the time points, extracted and weighted all organs and proceeded with the radioactive counts. Therefore, that 0.02% ID should be considered as a grand maximum, likely overestimating the real concentration.

Taken together, we can see that aside of the liver and spleen, the uptake of the radiolabeled tracer (and by extension nanoparticles) remains very low in gonads and in the brain, with amounts of 0.1% and 0.01% respectively at 48 hours.

The second set of data we have to look at is the bio-luminescence data (see Page 5). The lab injected 1ug of mRNA in each hind leg, totaling 2ug mRNA in each rat. Considering an average weight of 200g per rat, we can approximate a dose of 10ug/kg for the luciferase assay. As a control (to remove the background noise), control animals were injected with saline buffer. The average bio-luminescence signals were given, and I personally added 10% of this average as an estimated standard deviation to have an error margin, which a value commonly accepted in biological sciences (10% variation around average is considered pretty good data variability).
[Added: The bio-luminescence is also set to a mininum of 10E6 AU, which is important for the rest of the analysis.]

We can see that the luciferase activity at the injection site (which we can refer as our reference tissue) is significantly high within hours of injection (2 hours being the first reported timepoint) and decreases over time. [Added: What is important to note is how does the %ID actually compares to the bioluminescence. The common sense would be the more of the lipids are biodistributing in the tissue, the more mRNA (and therefore luciferease activity) we should detect, no? Well it is more complicated than this. Let’s plot the %ID in the tissue versus the bio-luminescence.

As you can see, an increase of lipid tracer in the tissue does not correlate with an increase in mRNA activity (as seen by Luc activity). It can be meaning two different things:
* The accumulation of the radiotracer present in the LNPs does accumulate in the tissue because of its non-metabolization and therefore may overestimate the half-life of the LNPs.
* Lets assume the LNPs found a way in the tissues, does not mean they made it safely with their cargo. They may accumulate as residues, or may come as empty shells with little or no mRNA left.]


We can assume that the luciferase expression at the injection site last for up to 10 days before being no different of background noise (we also have to be careful to not extrapolate as-is for the spike S protein, as the mRNA and protein kinetics of luciferase enzyme may greatly differ from the recombinant spike protein). However, the risk of off-target effect and having the mRNA expressed outside the injection site seems to be quite dim. Luciferase activity in the liver (which apparently uptake 60% of the injected dose) is down to background level by 48 hours post-injection. [Added: If we look at the profile, we can guess there is some metabolism in the liver that makes the clearance of LNPs and/or mRNA faster than the muscle tissue. From the data of the muscle bio-luminescence, we can see the decay of the bio-luminescence follows a first-order kinetics and puts with a half-life of ~0.75 days].
Ovaries luciferase activity was basically in the range of the saline group (and would be barely detected over noise, if we refer to the expected min. The penetration of the dye emission wavelength should be enough to be caught by the camera, even through solid tissue. If we don’t see any luminescence, it is likely because it is below or same intensity than background in saline) and brain luciferase activity in the brain was basically noise from the beginning to start (remember we have no access on the standard deviation but the numbers being that close from saline suggest we are scrapping background noise).
In conclusion, the risk of having the mRNA expression outside the injection is very unlikely and meaningless when it comes to biological activity.

5. The perils of dismissing the dose and the allometric scale in assessing the risk
So, we have evidence that the LNPs are pretty safe by barely accumulating in gonads and in the brain, that the mRNA activity is mostly not being found to have off-target, but what about the dose and how does it correlate to clinical situation?
This is where important concept of doses and allometric scale have to be introduced.
First, the dose used for the PK study. It was 1mg/kg of mRNA given in rats. As a comparison, the regular dose of the Pfizer vaccine is 30 ug (0.03mg) given to any patient of 12 years and older.  An average 12-years old girl would be 40 kgs per the CDC chart (rounded up to the lower value and for the ease of calculation). This would indicate a dose of 0.00075mg/kg. That’s already a difference of 1333-fold between what we gave to these rats and what we gave to humans, but there is more!
We also have to account to the allometric factor, because rats are not small human. [EDIT: For adjusting to the allometric scale, we will use this calculator ]. The allometric scale tells us that 1mg/kg dose in rats results in a human-equivalent dose (HED) of 68mg/kg if your patient is a 70-kgs adult; 45mg/kg if you are a 40-kgs teenager (~12 year old girl falling in the 50th percentile of the CDC growth chart).

Therefore, we have to multiply it by 45 (40-kgs patient) or 68 (70-kgs), which means if we want to transpose the PK findings as done in the rats, we would need to inject about 60’000 doses of the Pfizer vaccine in ONE girl (91’000 doses if you are a 70-kgs adult). That’s about half one-fourth of all doses distributed to Amarillo until now given to only ONE person [EDIT: One 12-year old teenage girl that is in the 50th percentile], ALL AT ONCE! You see where we going? The very extreme implausibility of the claims that COVID19 vaccines affect ovaries and the brain.
To finish it up, we can also look at the actual mRNA and luciferase.
We know that 8microg/kg was sufficient to see some liver activity, but no activity in gonads and brain. How does it translate to humans? First, lets apply the allometric scale (68x). We would need 544microg/kg for the HED, and translated to a 12-years old girl that would be 21760microg of mRNA delivered, which is about 725 doses of Pfizer given in ONE person at once! You can see that since we cannot detect notable activity if I give 725 doses at once, chance are I will not detect any activity when given a single dose or even two doses of Pfizer.

6. Concluding remarks

In conclusion, we can take the following messages:
– This is a document leaked on the PK of nanoparticles as found in the Pfizer vaccine, showing animal studies have been done before or during the clinical trials and we have the documentation.
– It helps clarify an ambiguous statement made by Pfizer in their summary submitted to the European Medicine Agency a couple of weeks ago about the distribution of the mRNA vaccine.
– The studies were done in a very conservative fashion at doses exceptionally high and impossible to reach in humans
– At such doses, it was shown that aside from the liver and spleen, the distribution of LNPs was minimal in gonads and the brain.
– The amount of mRNA required to be present in the tissue to appreciate an off-target effect is ridiculously low and impossible to achieve in real life and was transient in the liver.
– When accounting for the clinical dose and the allometric scale, this study shows that the Pfizer vaccine is very safe with a very low incidence of the off-target effect. To achieve the same result in humans, it would take a ridiculously high amount and a sheer incompetent healthcare practice to have the probability of having any issues of off-target effect occur in humans.

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Junk Sciences Junk Sciences Sciences Uncategorized

[Sciences/Junk Sciences] Reconsideration of the immunotherapeutic pediatric safe dose levels of aluminum (Lyons-Weiler and Ricketson, J Trace Elem Med Biol 2018)

This is a post I wanted to write about a long time ago but for some reasons, I have been putting on the back burner for many different reasons.
You know what can be the most irritating to read? Papers from anti-science in general. You see, if the data was sound, the experimental setup was robust then I would consider their arguments are valid and sound. The problem with the anti-science papers I have been reading so far (anti-vaccines, anti-GMOs) are most of the time written by scientists that are lacking the expertise and credentials (in terms of publication record) to discuss on a topic, are based on speculation (the experimental data to support their hypothesis is at best paper-thin), the experimental data are most of the time missing the rigor and paradigm needed to make an objective outcome and often cherry-pick the literature.
Under normal conditions, such papers would not even pass a normal peer-review filter and would have been rejected outright. Yet, such papers found their way in very low impact factor journals or in predatory journals (that will publish any garbage study, as long as there is a valid payment method).

1. Who are the authors?
This is the case of this manuscript written by James Lyons-Weiler and Robert Ricketson. In this study, they claim that the current immunization schedule is dangerous, blaming on the extraordinary amount of aluminum and using questionable and speculative pharmacokinetics to support their claims (of course, there is no experimental data to support their claims, only speculation). A tenet in scientific publication is to assess how credible the authors are in the field, this can be judged by the authors affiliation and publication records. James Lyons-Weiler has  (according to his LinkedIn profile) a PhD in Ecology, Evolution and Conservation Biology and currently affiliated to the “Instittute for Pure and Applied Knowledge”. This is not a scientific institute as the Salk Institute, but rather an frontstore for some quackery posing as a “scientific institute”. The second author, Robert Ricketson, is no better. Indeed, he is even worse. Apparently Dr. Ricketson has a history of medical malpractice as a spine surgeon, and has been implicated in a medical malpractice lawsuit in 2001 for inserting a screwdriver in a patient spine. At the publication date, Ricketson affiliation is another “scientific institute” named “Hale O’mana’o Research” in Edmond, OK. A quick verification on his LinkedIn profile suggest that these two Ricketson are the same Ricketson. To summarize, we have two authors with ZERO expertise in pharmacokinetics (including one doctor that got fined over $5 millions for medical malpractice), working in institutes with questionable scientific credientials but established anti-vaccine stance, under the disguise of “vaccine safety” (here and here), published in a journal in which a notorious anti-vaccine scientist is sitting in the editorial board. Is it surprising? For me, it is not. Just a classical MO for anti-vaccine scientist.

2. What the paper is about?
You can find the paper here, since it is behind paywall I cannot legally share the information, so I would request the reader to corroborate my claims by getting the full-text. In this study, the authors consider the safety studies done in animals are not correct and underestimate the toxicity of aluminum because they are based on animal body weight. And thats where the trouble start. The authors solely consider the amount of aluminum injected into animals and patients SOLELY based on the body weight.
They ignore the administration route, they ignore the existing literature and even questions the outcomes and recommendation of the World Health Organization as mentioned as “We found two important errors in the provenance and derivation of provisional aluminum intake levels from World Health Organization (WHO; Supplementary Material) which, unfortunately, led to overestimation of safe exposure levels.” That’s a bold statement by the beginning, coming from two non-experts in pharmacokinetics and toxicokinetics.
So how do they ended up using such claim? By using a derived version of the Clarke’s equation:

Child dose (mg) = Adult Dose (mg) * (child bodyweight (lbs)/adult bodyweight (lbs))

The Clarke’s equation is a common equation used for therapeutic dosing, as we commonly refer to administer doses as x mg/kg. Knowing the patient weight, you can easily calculate the dose administered.  This formula is great…….if you already know the target concentration (or the average plasma concentration) you aim to target. This is usually supported by empirical data and further confirmed by lab tests (you can dose the drug in the patient plasma and assess if such amount falls within the therapeutical window). But this equation tells you nothing about the pharmacokinetics of the drug, or about the bioavailability of the drug, or differences in the administration routes.
It only tells you one thing “How many miligrams of X should I administer to obtain a plasma concentration of X falling into therapeutical range?” That’s it. You assume a dosing regimen (mg/kg), you know your patient weight (in kgs) and thus you can obtain the dose needed (loading dose or maintenance dose).  However, the authors manipulated the equation to be able to transpose the minimal risk level from adults to children as the following:

CED (mg/kg)= HED(adult) mg/kg x [BW(child) (kg)/BW(adult) (kg)]

The rest of the paper is SOLELY based on speculation, no experimental data to support the claim (we rather have a post hoc ergo fallacy unfolding). If the authors wanted to make their claims valid, they would provide experimental data (in forms of blood sampling) for 2 months babies before immunization (baseline control) and 6-24 hours after immunization to show that Al levels in plasma are significantly altered by the immunization. But they never show that data.
Indeed, what they show is a blatant misuse of the data and recommendation of the FDA and a serious miscalculation that a 12th grader would not even do.
They compared the dietary MRL as “JECFA provisional tolerable daily intake from dietary and additive exposures of 140 μg/kg/day and current provisional tolerable daily intake of 290 μg/kg/day per day both before and after the safety factor of 10 is applied (Fig. 3).
We end up in the classical cases of “apples versus oranges” and trying to make the claim they are the same. Which they are not. Yes, both are extravascular routes and follow similar fate. But in the same time, we have to compare the physics-chemical aspects and the bioavailability of Al in both routes. One is administered by oral route, the other by intramuscular route. In both cases, the bioavailability falls within the same range, with the oral showing about 0.3% and the IM from 0.6% (based on Flarend et al., Vaccine 1997) and 0.9% (Yokel and McNamara estimate, Pharm Tax 2001).

What the authors show us is basically a graph that assume the WHOLE Al injected in 100% bioavaialable at once, exceeding the MRL adjusted to pediatrics) as seen in Figure 4:
Lyons_Weiler_2018_Fig4

There is one thing to consider: The ATSDR. The ATSDR considers the MRL of 1mg/kd/day of ingested aluminum (that is about 100x lower than the NOAEL and adjusted to the bioavailability, as described here: https://www.atsdr.cdc.gov/toxprofiles/tp22-c8.pdf). If we assume a bioavailability of 0.3%, then we expect that out of 1mg/kg/day ingested, we can estimate that about 3microg (or 0.003mg)/kg/day would contribute in the total burden in the Al plasma level. This graph would be correct if 100% of the aluminum injected ended up in the systemic circulation at ONCE and spiked Al levels significantly high. But thats not the case, and the authors blatantly ignored this critical information, coming from previous studies. In order to compare these two items, you have to compare and estimate how much of each of these routes will contribute in the total Al plasma/blood levels.
You cannot just plot the total amount injected (adjusted per kg) and assume it is representing the same variable than estimated plasma levels from the MRL. Now, let consider that the Al injected is available at the rate of 1% a day, the graph will look more like that.

Lyons_Weiler_2018_Fig4_Adjusted

You see, we have a complete different scenario. If we consider that the aluminum is slowly released into the body at a rate of 1% per day we are now being way under the MRL and within safe levels. Again, we consider the MRL of 1mg (1000microg)/kg/day. If we consider a 0.3% bioavailability and difference in 5th and 95th percentiles weight (grey bars), we conclude that the daily burden of Al via dietary route should not exceed a value ranging from 13.20-18.72microg/day. Our values matches the MRL from Lyons-Weiler. In other words, our assumption is correct. If we consider an average weight of 5.35kg (50th percentile) at 2 months and 1mg as a cumulative dose of the immunization occurring the same day (conservative estimate), the amount delivered that day would be 0.187mg or (187microgram). Considering a bioavailability of 1% per day via IM, we have about 1.87microgram of burden from the vaccine added each day to a maximum MRL of 16.05microgram/day for the 50th percentile (weight 50th percentile=5.35kgs). Thats about 11.7% aluminum to be removed from the daily MRL, but within negligible range to have a statistical significance (you need at least 30% to consider it as statically meaningful).  This of course has to be confirmed by studies assessing plasma levels of Al after injections but there are already a literature out there with such data avaialable and reported here and here. Both studies concluded no changes in total Al plasma levels in regard of the vaccination status, including 6-24 hours after immunization.

3. Concluding remarks

Anti-science know how to bangs for their bucks, by sensationalizing claims knowing that the lay person will not or be capable to verify their claim. Most of the times, such claims come from persons that are legitimate scientists in their field, but completely speak out of their expertise domain. This is a common trope we see when people cite Linus Pauling, Otto Warburg or Luc Montagnier. Each of them have done remarkable discoveries in their field, got their Nobel Prizes but once they speak outside their expertise have proven to be wrong or have seen their claims manipulated by quack-peddlers. Lets take Linus Pauling that has been incremental in modern chemistry by describing the chemical bonds, but later claimed cancer(s) can be cured with Vitamin C. Coincidentaly, he died of prostate cancer in 1994.
Same applies in this paper. We have two authors with ZERO knowledge of pharmacokinetics, yet they have given themselves the role to demonize aluminum at all cost, bending and occulting facts to fit their narrative and their conclusion. This paper is the evidence that they are not serious about “vaccine safety”. They are staunch anti-vaccines, and they will use their status of scientists to vilify it at all costs, even if it means reaching outside their expertise, make extraordinary claims without extraordinary evidences (they did not have evidence for this study) and get published in a journal that will favor their claims and obviously lacked the rigor in the review.
Negating the neurotoxic effects of aluminum is not a correct statement either. Aluminum is neurotoxic, but as anything in toxicology it is all about the dose. And one parameter that is critical to assess the safety of aluminum is its plasma level. This safety level is driven by how much aluminum access the systemic circulation (from IV parenteral nutrition bags or from extravascular routes such as vaccines or dietary exposure). What matter at the end is the Al plasma levels and the FDA set a limit on that daily exposure (5 micrograms/kg/day via IV route). This is a problem encountered by patients suffering from non-functional kidneys (95% of aluminum is cleared via renal route) and from patient continuously fed via IV route (total parenteral nutrition).
Both Lyons-Weiler and Ricektson failed to applied basic concepts of pharmacokinetics, ignored the differences between vascular and extravascular routes and willfully used a calculation method that is not appropriated for this purpose. I would even what is worse is that none of their claims is supported by hard data, making their claims even more questionable.
Unfortunately, such “junk paper” felt through the cracks of peer-review and has been used repeatedly used by anti-vaxxers as supporting evidence. As Andrew Wakefield has his second paper removed after 16 years, how long it will take to remove that paper?
I dont know but the damage is done, and until “vaccine safety” scientists come with robust and foul-proof studies published in highly respected journals, they will be considered by me and others as junk scientists, keeping on feeding the literature with their garbage studies that should have been wiped out by a rigorous peer-review process.