[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):
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.