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BNT162b2
Module 2.6.4. Pharmacokinetics Written Summary
CONFIDENTIAL
Page 1MODULE 2.6.4. PHARMA COKINETICS WRITTEN S UMMARY
This document contains confidential information belonging to BioNTech /Pfizer . Except as may be
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Module 2.6.4. Pharmacokinetics Written Summary
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Page 2TABLE OF CONTENTS
LIST OF ABBREVIATION S AND DEFINITION OF TERMS ................................ .............. 3
2.6.4. PHARMACOKINET ICS WRI TTEN SUMMARY ................................ ........................ 4
2.6.4.1. Brief Summary ................................ ................................ ................................ ....4
2.6.4.2. Methods of Analy sis................................ ................................ ............................ 4
2.6.4. 3. Absorption................................ ................................ ................................ ........... 4
2.6.4.4. Distribution ................................ ................................ ................................ .......... 5
2.6.4.5. Metabolism................................ ................................ ................................ .......... 7
2.6.4.6. Excretion ................................ ................................ ................................ ............. 9
2.6.4.7. Pharmacokinetic Drug Interactions ................................ ................................ .....9
2.6.4.8. Discussion and Conclusions................................ ................................ ................ 9
2.6.4.9. References ................................ ................................ ................................ ......... 10
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Page 3LIST OF ABBREVIATION S AND DEFINITION OF TERMS
ADME Absorption, distribution, metabolism, excretion
ALC-0159 Proprietary PEG -lipid included as an excipient in the L NP formulation
used in BNT162b2
ALC-0315 Proprietary amino -lipid included as an excipient in the L NP formulation
used in BNT162b2
[3H]-CHE Radiolabeled [Cholesteryl -1,2-3H(N)] -Cholestery l Hexadecy l Ether
DSPC 1,2-distearo yl-sn-glycero-3-phosphocholine
GLP Good Laboratory Practice
H Human (in metabolite scheme)
IM Intramuscular(ly )
IV Intravenous(l y)
LNP Lipid -nanoparticle
Luc Luciferase (from firefl y Pyractomena lucifera )
Mk Monkey (in metabolite scheme)
Mo Mouse (in metabolite scheme)
modRNA Nucleoside -modified mRNA
mRNA Messenger RNA
PEG Polyethylene gl ycol
PK Pharmacokinetics
R Rat (in metabolite scheme)
RNA Ribonucleic acid
S9 Supernatant fraction obtained from liver homogenate b y centrifuging at
9000 g
WHO World Health Organization
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Page 42.6.4.PHARMACOKINETICS WRI TTEN SUMMARY
2.6.4.1.Brief Summary
TheADME profile of BNT162b2 (BioNTech code number BNT162, Pfizer code number
PF-07302048 ) included evaluation of the PK and metabolism of two novel lipid excipients
(ALC-0315 and ALC -0159) in the LNP and potential biodistribution using luciferase
expression as a surrogate reporter or a radiolabeled lipid marker .The PK study showed the
LNP distributes from the blood to the liver, ~1% of ALC- 0315 and ~ 50% of AL C-0159 were
excreted unchanged in fece s, and there was no detectable excretion of unchange d ALC -0315
and AL C-0159 in the urine .
In a mouse biodistribution study , protein expression was demonstrated at the site of injection
and to a lesser extent in the liver after BALB/c mice received an IM injection of modRNA
encoding luciferase in a nLNP for mulation ,with the identical lipid composition as
BNT162b2 .Luciferase expression was identified at the injection site at 6 hours after injection
and was not detected after 9days.Liver expression was also present at 6 hours after injection
and was no longer detected by 48hours after injection. A quantitative biodistribution study
was also carried out in Wistar Han rats using a radiolabeled lipid marker and a luciferase
modRNA in the same LNP formulation asBNT162b2. Following IM administration, the
greatest mean concentration remained at the injection site , while up to 18% of the
administered dose was found in the liver.
The metabolism of AL C-0315 (aminolipid) and AL C-0159 (PEG- lipid) wasevaluated in
vitro using blood, liver microsomes, S9 fractions, and hepatocytes from mice , rats, monkey s,
and humans. The in vivo metabolism was examined in rat plasma, urine, feces, and liver
samples collected during the PK study .In vitro and in vivo studies indicated ALC-0315 and
ALC-0159 are metabolized slowly by hydrolytic metabolism of the ester and amide
functionalities, respectively , across the species evaluated.
2.6.4.2.Methods of Analysis
No methods of anal ysis have been validated to support GL P TK studies of components of
BNT162b2 ; however, a qualified L C/MS method was developed to support quantitation of
the two novel LNP excipients for the non- GLP IV PK study in
rats(PF-07302048_06Jul20_072424). Methods for immunogenicit y and efficacy studies are
described in Section 2.6.2.12 .
2.6.4.3.Absorption
An intravenous rat PK st udy was performed using L NPs containing surrogate luciferase
RNA, with the identical lipid composition as BNT162b2, to explore the disposition of
ALC-0159 and ALC -0315 ( Table 2.6.4 -1, Study PF-07302048_06Jul20_072424; Tabulated
Summary 2.6.5.3 ). The distribution of the L NP from the blood to the liver was rapid and
essentially complete by 24h, with <1% of the maximum obse rved plasma concentrations
remaining (Figure 2.6.4-1 ).The liver appears to be the major site of drug uptake from the
blood.
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Page 5Table2.6.4-1.PKof ALC-0315 and ALC -0159 in Wistar Han Rats After IV
Administration of LNPs Containing Surrogate Luciferase RNA at 1 mg/kg
Analyte Dose
of Analyte (mg/kg)Gender /N t½
(h)AUCinf
(µg•h/mL)AUClast
(µg•h/mL)Estimated fraction
of dose distributed
to liver (%)a
ALC -0315 15.3 Male/3b139 1030 1020 60
ALC -0159 1.96 Male/3b72.7 99.2 98.6 20
a.Calculated as highest mean amount in the liver (µg)/total mean dose (µg) of ALC -0315 or ALC -0159.
b. 3 animals per timepoint; non -serial sampling.
Figure 2.6.4 -1.Plasma and Liver Concentrations of ALC -0315 and ALC- 0159 in Wistar
Han Rats After IV Administration of LNP sContaining Surrogate
Luciferase RNA at 1 mg/kg
No absorption studies were conducted for BNT162b2, as the administration route is I M.
Pharmacokinetic studies have not been conducted with BNT162b2 and are generall y not
considered necessary to support the development and licensure of vaccine products for
infectious diseases ( WHO, 2005 ; WHO, 2014).
2.6.4.4.Distribution
In an in vivo study in BALB/c mice ( Study R-20-0072 ; Tabulated Summary 2.6.5.5A), the
biodistribution of BNT162b2 was assessed using luciferase as a surrogate marker protein .
RNA encoding luciferase was formulated like BNT162b2, with the identical lipid
composition, andmice received IM injections of 1 µg each in the right and left hind leg (for a
total of 2 µg) of LNP -formulated modRNA encoding luciferase. Luciferase protein
expression was detected at different timepoints, by measuring the in vivo bioluminescence
(Figure 2.6.4 -2) after injection of luciferin substrate, at the site of injection and to a lesser
extent in the liver. Distribution to the liver is likely mediated b y LNPs entering the blood
stream. The repeat -dose toxicity stud iesin rats showed no evidence of liver injury (Section
2.6.6.3 ). The luciferase expression at the injection site, the tissue with the highest
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Page 6bioluminescence , dropped to background levels after 9 day s.As detailed in Section 2.6.4.3 ,
following s ystemic (IV) administration the liver appear sto be the major organ into which the
LNPs distribute, this is consistent with the observations made following IM administration .
Figure 2.6.4 -2. Bioluminescence Emission in BALB/c Mice after IM Injection of an LNP
Formulation of modRNA Encoding Luciferase
These qualitative data are supported b y a biodistribution study (Study 185350 ;Tabulated
Summary 2.6.5.5B ) carried out with LNPswith a comparable lipid composition as
BNT162b2 but with a luciferase mRNA and a [3H]-CHE lipid radiolabel. Following IM
administration to male and female Wistar Han rats at a dose of 50 µg (1.29 mg total lipid),
the greatest mean concentration was found remaining in the injection site at each time point
in both sexes. Outside the injection site, the highest levels of radioactivity were observed in
plasma at 1-4 hours post -dose. Over 48 hours, the radiolabel distributed mainly to the liver,
adrenal glands, spleen and ovaries, with maxim um concentrations observed at 8 -48 hours
post-dose. Total recovery of radioactivity (% of injected dose) outside the injection site was
greatest in the liver (up to 18%) and was much less in the spleen (≤1.0%), adrenal glands
(≤0.1 1%) and ovaries ( ≤0.095 %). The mean concentrations and tissue distribution pattern
were broadl y similar between sexes.
The biodistribution of the expression of the antigen encoded b y the RNA component of
BNT162b2 is expected to be dependent on the LNP distribution. Therefore, results of these
biodistribution studies should be representative for BNT162b2 , as the LNP -formulated
luciferase -encoding modRNA had the same lipid composition.
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Page 72.6.4.5.Metabolism
Metabolism studies were conducted to evaluate ALC -0315 (aminolipid) and AL C-0159
(PEG-lipid). These novel lipids
were evaluated for in vitro metabolic stability in CD-1/ICR
mouse, W istar Han and/or Sprague Dawley
rat, cynomolgus monkey , and human liver
microsomes, S9 fractions, and hepatocy tes. ALC-0315 and AL C-0159 were stable (> 82%
remaining) over 120 min in liver microsomes and S9 fractions and over 240 min in
hepatocytes in all species and test systems
(Studies 01049-20008 , 01049-2 0009,
01049-20010 , 01049-2 0020, 01049-20021 , and 01049-20022; Tabulated Summaries
2.6.5.10A and 2.6.5.10B) .
The metabolism of ALC-0315 and ALC-0159 was further evaluated (Study
PF-07302048_05Aug20_043725; Tabulated Summaries 2.6.5.9, 2.6.5.10C, and 2.6.5.10D)
in vitro using blood, liver S9 fractions, and hepatocytes from CD-1 mice, Wistar Han rats,
cynomolgus monkey s, and humans and in vivo using the rat plasma, urine, feces, and liver
from the PK study (Section 2.6.4.3 ). This study determined ALC -0315 and AL C-0159 are
metabolized slowly and undergo hy drolytic metabolism of the ester and amide
functionalities, respectively . This hy drolytic metabolism was observed across the species
evaluated, as shown in Figure 2.6.4-3 and Figure 2.6.4-4 .
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Page 8Figure 2.6.4 -3. Proposed Biotransformation P athway of ALC -0315 in Various Species
Metabolism of AL C-0315 occurs via two sequential ester h ydrol ysis reactions, first y ielding
the monoester metabolite ( m/z528) followed b y the doubly deesterified metabolite ( m/z290).
Subsequent metabolism of the do ubly deesterified metabolite resulted in a glucuronide
metabolite ( m/z466), which was onl y observed in urine from the rat PK study . The acid
product of both h ydrol ysis reactions of AL C-0315, 6-hexyldecanoic acid ( m/z255), was also
identified.
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Page 9Figure 2. 6.4-4. Proposed Biotransformation Pathway of ALC -0159 in Various Species
The primary route of metabolism identified for ALC -0159 involves amide bond hy drolysis
yielding N,N-ditetradecy lamine (m/z410). This metabolite was identified in mouse and rat
blood, as well as hepatocy tes and liver S9 from mouse, rat, monkey ,and human. No
metabolites of AL C-0159 were identified from in vivo samples.
The other two lipids in the LNP are naturall y occurring (cholesterol and DSPC) and will be
metabolized and excreted like other endogenous lipids. As the protein encoded by the mRNA
in BNT162b2 is expected to be proteoly tically degraded and RNA is degraded by cellular
RNases and subjected to nucleic acid metabolism, no RNA or protein metabolism or
excretion studies will be conducted.
2.6.4.6.Excretion
In the rat PK stud y (Section 2.6.4.3 ), there was no detectable excreti on ALC -0315 and
ALC-0159 in urine after IV administration of LNP scontaining surrogate luciferase RNA at 1
mg/kg. The percent excreted unchanged in feces was ~1% for AL C-0315 and ~ 50% for
ALC-0159. Metabolites of AL C-0315 were detected in the urine of rats (Figure 2.6.4-3 ). No
excretion studies have been conducted with BNT162b2 for the reasons described in Section
2.6.4.5 .
2.6.4.7.Pharmacokinetic Drug Interactions
No PK drug interaction studies have been conducted with BNT162b2 .
2.6.4.8.Discussion and Conclusions
In the rat PK stud y, concentrations of ALC -0159 dropped approximately 8000 -and
>250 -fold in plasma and liver, respectively , during this 2 -week study . For AL C-0315, the
elimination of the molecule from plasma and liver wasslower, but concentrations fell
approximately 7000 -and 4 -fold in two weeks for plasma and liver, respectivel y. Overall, the
apparent terminal t½in plasma and liver were similar in both tissues and were 2- 3 and 6 -8
days for ALC -0159 and AL C-0315, respectively . The apparent terminal t ½in plasma likely
represents the re -distribution of the respective lipids from the tissues into which they have
distributed as the LNP back to plasma where they are eliminated.
Overall, it appears that 50% of ALC -0159 was eliminated unchanged in feces. M etabolism
playeda role in the elimination of AL C-0315 ,as little to no unchange d material was detected
in either urine orfeces. Investigations of urine, feces and plasma from the rat PK study
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Page 10identified a series of ester cleavage products of ALC -0315 ;this likely represents the primary
clearance mechanism acting on this molecule, although no quantitative data is available to
confirm this hy pothesis. In vitro, AL C-0159 was metabolized slowly by hydroly tic
metabolism of the amide functionalit y.
The potential biodistribution of BNT162b2 was assessed using luciferase expression as a
surrogate reporter. Protein expression was demonstrated at the site of injection and to a lesser
extent , and more transiently ,in the liver after BALB/c mice received an IM injection of RNA
encoding luciferase in anLNP formulation like BNT162b2 .Luciferase expression was
identified at the injection site at 6 hours after injection and was not detected by 9days.
Expression in the liver was also present at 6 hours after injection and was not detected by
48hours after injection. These findings are supported by a quantitative biodistribution study
in Wistar Han rats. After IM administration of a radiolabeled lipid marker and a luciferase
modRNA in the same LNP formulation as BNT162b2 to rats, the percent of administered
dose was greatest at the injection site. Outside of the injection site, total recovery of
radioactivity was highest in the liver and much lower in the spleen, with very little recovery
in the adrenal glands and ovaries.
2.6.4.9.References
1. World Health Organization. Annex 1. Guidelines on the nonclinical evaluation of
vaccines. In: WHO Technical Report Series No. 927, Geneva, Switzerland. World
Health Organization; 2005:31-63.
2. World Health Organization. Annex 2. Guidelines on the nonclinical evaluation o f
vaccine adjuvants and adjuvanted vaccines. In: WHO Technical Report Series No. 987,
Geneva, Switzerland. World Health Organization 2014:59-100.
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