A85 Japanese Pfizer bio distribution study April 2021.

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SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.4 Summary of pharmacokinetic study

TABLE OF CONTENTS

LIST OF TABLES ..................................................... ................................................................. ........................................ 1

LIST OF FIGURES ..................................................... ................................................................. ...................................... 1

Terms and abbreviations used in this section ..................................... ................................................................. ............................ 2

Summary ...................................................... ................................................................. ................................................................. .... 3
Analytical method ...................................................... ................................................................. ................................................................. .....Four
Absorption ............................................ ................................................................. ................................................................. ........Four
Distribution ..................................................... ................................................................. ................................................................. ........Five
Metabolism ..................................................... ................................................................. ................................................................. ........ 7
Excretion ..................................................... ................................................................. ................................................................. ........ 9
Pharmacokinetic drug interactions ..................................... ................................................................. ........................ 9
Other pharmacokinetic studies ..................................................... ................................................................. .............................. 9
Discussions and conclusions .................................................. ................................................................. ........................................ 9
Chart ...................................................... ................................................................. ................................................................. ....Ten

References ................................................................ ................................................................. ................................................................. ..Ten

LIST OF TABLES

Table 1 Luciferase RNA-encapsulated LNP intravenously to Wistar Han rats at a dose of 1 mg RNA / kg

Pharmacokinetics of ALC-0315 and ALC-0159 when administered orally ..................... ........Four

LIST OF FIGURES

Figure 1 Luciferase RNA-encapsulated LNP venous in Wistar Han rats at a dose of 1 mg RNA / kg

Plasma and liver concentrations of ALC-0315 and ALC-0159 when administered orally ..................... 5

Figure 2 In vivo in BALB / c mice intramuscularly administered with luciferase RNA-encapsulated LNP

Luminous ..................................... ................................................................. ....................................... 6

Figure 3 Estimated in vivo metabolic pathway of ALC-0315 in various animal species .................................. ............................ 8 Figure 4 Estimated in vivo metabolic pathway of ALC-0159 in various animal species .................................. ............................ 9

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2.6.4 Summary of pharmacokinetic study

Terms and abbreviations used in this section

Term / AbbreviationNot abbreviated expressions or definitions

ALC-0159 PEG lipid added to this drug

^ALC-0315Amino lipid added to this drug

[ 3 H]-CHE Radiolabeled [cholesteryl-1,2- 3 H (N)] - cholesteryl hexadecyl Ether: radiolabeled [cholesteryl

Lil-1, 2-3 H (N)] Hexadecyl ether

DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine: 1,2-distearoyl-sn-glycero-3-phosphocholine

Rin

GLP Good Laboratory Practice: Criteria for conducting non-clinical studies on drug safety LNP Lipid-nanoparticle: Lipid nanoparticle

modRNA Nucleoside-modified mRNA: Modified nucleoside mRNA mRNA Messenger RNA: Messenger RNA m / z m / z (m over z): Obtained by dividing the mass of an ion by the unified atomic mass unit (= Dalton).

The dimensionless quantity obtained by dividing the obtained dimensionless quantity by the absolute value of the number of charges of the ion.

PEG Polyethylene glycol: Polyethylene glycol

PK Pharmacokinetics: Pharmacokinetics

RNA Ribonucleic acid: Ribonucleic acid

S9 Supernatant fraction obtained from liver homogenate by centrifuging at 9000 g: liver homogenate

Supernatant fraction centrifuged at 9000 g

WHO World Health Organization: World Health Organization

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2.6.4 Summary of pharmacokinetic study

1. Summary

BNT162b2 (BioNTech code number: BNT162, Pfizer code number: PF-07302048) is a severe acute call.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike glycoprotein (S protein) full length

It is a modified nucleoside mRNA (modRNA) that encodes against SARS-CoV-2 infection.

Development is underway as the essence of the mRNA vaccine. When formulating BNT162b2, there are two

Functional lipids ALC-0315 (aminolipid) and ALC-0159 (PEG lipid) and two structural lipids

By mixing with DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and cholesterol

Lipid nanoparticles (LNP) that encapsulate BNT162b2 are formed (hereinafter, "BNT162b2-encapsulated LNP").

ALC-0315 and ALC-0315 contained in LNP to evaluate the nonclinical pharmacokinetics of BNT162b2 encapsulated LNP

In vivo and in vitro studies assessing absorption (PK), metabolism and excretion of ALC-0159 and BNT162b2

Biodistribution studies using luciferase or radiolabeled lipids as an alternative reporter for Was carried out.

Based on the fact that the development of vaccines aimed at preventing infectious diseases does not require evaluation of systemic exposure.

(WHO, 2005; Non-clinical study guidelines for infectious disease preventive vaccines) 1 , 2 , BNT162b2 Encapsulated LNP muscle

No internal PK study was performed. In addition, two other types of lipids (choleste) contained in this drug

Rolls and DSPCs) are naturally occurring lipids that are thought to be metabolized and excreted in the same way as endogenous lipids.

available. In addition, BNT162b2 is degraded by ribonucleases in the cells that have taken it up, resulting in nucleic acid charges.

Apologize, the S protein from BNT162b2 is expected to undergo proteolysis. From the above,

It was considered unnecessary to evaluate the metabolism and excretion of these components again.

LNP (Luciferase) encapsulating RNA encoding luciferase as an alternative reporter for BNT162b2

Lase RNA is encapsulated in an LNP having the same lipid composition as the BNT162b2-encapsulated LNP:

In a PK study in which ZeRNA-encapsulated LNP ") was intravenously administered to Wistar Han rats, plasma, urine, feces and

Liver samples were collected over time and the concentrations of ALC-0315 and ALC-0159 in each sample were measured. The conclusion

As a result, ALC-0315 and ALC-0159 were shown to be rapidly distributed from the blood to the liver. Also,

About 1% and about 50% of the doses of ALC-0315 and ALC-0159 are excreted in feces as unchanged drug, respectively.

All of them were below the detection limit in urine.

In the biodistribution test, luciferase RNA-encapsulated LNP was intramuscularly administered to BALB / c mice. That

As a result, the expression of luciferase was observed at the administration site, and the expression level was lower than that in the liver.

Was also recognized. Expression at the administration site of luciferase was observed from 6 hours after administration, and 9 days after administration.

Disappeared. Expression in the liver was also observed 6 hours after administration and disappeared by 48 hours after administration. Also, Intramuscular administration of radiolabeled LNP containing luciferase RNA to rats to quantify biodistribution

Upon evaluation, the radioactivity concentration was the highest at the administration site. Liver is highest except at the administration site It was good (up to 18% of the dose).

Metabolism of ALC-0315 and ALC-0159 in CD-1 / ICR mice, Wistar Han or Sprague Dawley rats,

In vitro using cynomolgus monkey or human blood, liver microsomes, liver S9 fraction and hepatocytes evaluated. In addition, plasma, urine, feces and liver samples collected in the above rat intravenous administration PK test were used.

We also examined in vivo metabolism. From these in vitro and in vivo studies, ALC-0315 and ALC-0159 was added to ester and amide bonds in all animal species tested.

The solution showed that it was slowly metabolized.

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From the above nonclinical pharmacokinetic evaluation, it was shown that LNP that reached the circulating blood is distributed in the liver.

In addition, metabolism and fecal excretion may be involved in the disappearance of ALC-0315 and ALC-0159, respectively.

It was suggested.

Analytical method

Report number: PF-07302048_06 _072424

Intravenous administration of rats without GLP PK test (M2.6.4.3), ALC-0315, which is a constituent lipid of LNP, and

ALC-0159 We have developed an LC / MS method with appropriate performance for quantifying the concentration. That is, 20 µL

Plasma, liver homogenate (homogenates are prepared using sections collected from three parts of the liver, and they are used.

Dilute with a blank matrix as appropriate), urine and fecal homogenate (as appropriate, bran)

Dilute with kumatrix) Divide each sample with acetonitrile containing an internal standard substance (PEG-2000) After protein, it was centrifuged and the supernatant was subjected to LC-MS / MS measurement.

Absorption

Report number: PF-07302048_06 _072424 , Summary table: 2.6.5.3

Male luciferase RNA-encapsulated LNP to study the pharmacokinetics of ALC-0315 and ALC-0159

A single intravenous dose of 1 mg RNA / kg was administered to Wistar Han rats over time (pre-dose, post-dose 0.1, 0.25,

Sparse plasma and liver 0.5, 1, 3, 6 and 24 hours and 2, 4, 8 and 14 days after dosing)

Sampling was performed (3 animals / time point). ALC-0315 and ALC-0159 in plasma and liver

The concentration was measured and the PK parameters were calculated (Table 1). ALC-0315 and ALC-0159 in the blood are thrown

It was promptly distributed to the liver by 24 hours after administration. In addition, the plasma concentration 24 hours after administration is the highest in plasma.

It was less than 1% of the concentration (Figure 1 ). The apparent terminal phase elimination half-life (t½) is in plasma and liver At the same level, ALC-0315 took 6 to 8 days and ALC-0159 took 2 to 3 days. From the results of this test, the liver is in the blood It was suggested that it is one of the major organizations that take up ALC-0315 and ALC-0159 from.

Results of examination of urinary and fecal concentrations of ALC-0315 and ALC-0159 conducted in this study Is M2.6.4.Described in Section 6 .

Table 1 Intravenous injection of luciferase RNA- encapsulated LNP into Wistar Han rats at a dose of 1 mg RNA / kg

Pharmacokinetics of ALC-0315 and ALC-0159 when given
Analytical material Dosage of analyte Gender / N ^t½^{( h )}AUC ^inf (mg / kg) (μg • h / mL)	AUC last ( Μg • h / mL )	To the liver Distribution ratio (%) a
ALC-0315 15.3 Male / 3 b 139 1030	1020	60
ALC-0159 1.96 Male / 3 b 72.7 99.2	98.6	20

Calculated as [maximum liver distribution (μg)] / [dose (μg)]. b. 3 animals at each time point. Sparse sampling.

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2.6.4 Summary of pharmacokinetic study

Figure 1 Intravenous injection of luciferase RNA- encapsulated LNP into Wistar Han rats at a dose of 1 mg RNA / kg

Plasma and liver concentrations of ALC-0315 and ALC-0159 when given

ALC-0315 in plasma ALC-0315 In the liver

ALC-0159 In the liver ALC-0159 in plasma

l) )

/ m / gg g μ μ

μ μ

concentration( concentration(

Elapsed time (h) Elapsed time (h)

4. Distribution

Report number: R- -0072 , 185350, Summary table: 2.6.5.5A, 2.6.5.5B

Female BALB / c mice (3 mice) were administered luciferase RNA-encapsulated LNP to emit luciferase luminescence.

The biodistribution of BNT162b2 was examined as an alternative marker. That is, luciferase RNA inclusion

LNP was intramuscularly administered to the left and right hind limbs of mice at a dose of 1 μg RNA (2 μg RNA in total). After that, Le

Intraperitoneal administration of luciferin, a luminescent substrate, 5 minutes before detection of cipherase luminescence, isoflurane hemp

Intoxication, in vivo luminescence 6 and 24 hours after administration using Xenogen IVIS Spectrum and 2,

By measuring on days 3, 6 and 9, the expression of luciferase protein in the same individual was estimated over time.

Evaluated the transfer. As a result, expression of luciferase at the administration site was observed from 6 hours after administration, and it was administered.

It disappeared 9 days after giving. Expression in the liver was also observed 6 hours after administration and disappeared by 48 hours after administration.

It was. Regarding the distribution to the liver, a part of locally administered luciferase RNA-encapsulated LNP reaches the circulating blood, and the liver

It was thought to indicate that it was taken up by the viscera. M2.6.4.Lucife in rats, as detailed in Section 3.

When intravenously administered with Lase RNA-encapsulated LNP, the liver is the major ALC-0315 and ALC-0159.

It has been suggested that it is a distributed organ, which is the finding of the results of this study, which was intramuscularly administered to mice.

It was a match. Toxicity findings indicating liver damage were observed in the rat repeated-dose toxicity test.

Not available ( M2.6.6.3 ).

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2.6.4 Summary of pharmacokinetic study

Figure 2 In vivo luminescence in BALB / c mice intramuscularly administered with luciferase RNA- encapsulated LNP

Buffer solution Luciferase RNA-encapsulated LNP

Male and female Wistar Han rats labeled with [ 3 H] -cholesteryl hexadecyl ether ([ 3 H] -CHE) LNP

Luciferase RNA-encapsulated LNP using luciferase RNA was intramuscularly administered at a dose of 50 µg RNA, and 15 minutes after administration.

Blood, plasma and tissue were collected from 3 males and 3 females at 1, 2, 4, 8, 24 and 48 hours each.

The biodistribution of LNP is evaluated by measuring the radioactivity concentration by the liquid scintillation counting method.

Worth it. In both males and females, the radioactivity concentration was highest at the administration site at all measurement points. The radioactivity concentration in plasma was the highest 1 to 4 hours after administration. Also, mainly the liver, spleen, adrenal glands and Distribution to the ovaries was observed, and the highest radioactivity concentration in these tissues was 8 to 48 after administration.

It was time. The total radioactivity recovery rate for doses other than the administration site is the highest in the liver (up to 18%). Significantly lower in the spleen (1.0% or less), adrenal gland (0.11% or less) and ovary (0.095% or less) compared to the liver won. In addition, the average concentration of radioactivity and the tissue distribution pattern were generally similar between males and females.

The in vivo expression distribution of the antigen encoded by BNT162b2 is considered to depend on the LNP distribution. For this test

Is the lipid composition of the luciferase RNA-encapsulated LNP the same as that of the submitted preparation of BNT162b2?

Therefore, the results of this test are considered to indicate the distribution of BNT162b2-encapsulated LNP.

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2.6.4 Summary of pharmacokinetic study

5. Metabolism

Report number: 01049-008 , 01049-009 , 01049- 010 , 01049- 020 , 01049-021 , 01049-022 ,

PF-07302048_05 _043725 , Summary table: 2.6.5.10A , 2.6.5.10B , 2.6.5.10C , 2.6.5.10D

CD-1 / ICR mouse, Wistar Han or Sprague Dawley rat, cynomolgus monkey and human liver mi

In vitro metabolic stabilization of ALC-0315 and ALC-0159 using crosome, liver S9 fraction and hepatocytes

Gender was evaluated. Liver microsomes or liver S9 fractions of each animal species with ALC-0315 or ALC-0159 (120)

Incubate) or add to hepatocytes (240 minutes incubation) and incubate

The proportion of unchanged drug after vation was measured. As a result, which of ALC-0315 and ALC-0159

It was also metabolically stable in animal species and test systems, with the final proportion of unchanged drug being over 82%.

Furthermore, the metabolic pathways of ALC-0315 and ALC-0159 were evaluated in vitro and in vivo. this

In these studies, CD-1 mouse, Wistar Han rat, cynomolgus monkey and human blood, liver S9 fractions

And hepatocytes were used to evaluate metabolism in vitro. In addition, plasma, urine, and feces collected in the rat PK test.

And liver samples were used to evaluate metabolism in vivo (M2.6.4.Item 3 ). From the test results, ALC-0315 And ALC-0159 are both slowly metabolized, with hydrolysis of ester and amide bonds, respectively.

It was revealed that it was metabolized by.Hydrolytic metabolism shown in Figures 3 and 4 Was found in all the animal species evaluated.

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2.6.4 Summary of pharmacokinetic study

Figure 3 Estimated in vivo metabolic pathway of ALC-0315 in various animal species

In blood (Mo, R)

In hepatocytes (Mo, R, Mk, H)

Liver S9 (Mo, R, H)

Plasma (R)

In blood (Mo, R)

Liver S9 (Mk)

Plasma (R)

Liver (R)

In blood (Mo, R)

In hepatocytes (Mo, R, Mk, H)

Liver S9 (Mo, R, H)

Plasma (R)

In blood (Mo, R)

Liver S9 (Mk) Plasma (R)

Urinary (R)

Feces (R)

Liver (R)

Glucuronide

Urinary (R)

H: human, Mk: monkey, Mo: mouse, R: rat

ALC-0315 is metabolized by undergoing ester hydrolysis twice in a row. These two hydrolysiss

First produces a monoester metabolite ( m / z 528) and then a double deesterified metabolite ( m / z 290).

Will be done. This double deesterified metabolite is further metabolized to the glucuronide conjugate ( m / z 466).

However, this glucuronic acid conjugate was detected only in urine in the rat PK test. Also, two hydrolysiss It was also confirmed that all of the acidic products of were 6-hexyldecanoic acid ( m / z 255).

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2.6.4 Summary of pharmacokinetic study

Figure 4 Estimated in vivo metabolic pathway of ALC-0159 in various animal species

In blood (Mo, R)

In hepatocytes (Mo, R, Mk, H)

In liver S9 (Mo, R, Mk, H)

N, N-ditetradecylamine

m / z 410

H: human, Mk: monkey, Mo: mouse, R: rat

In ALC-0159, N , N -ditetradecylamine ( m / z 410) is produced by hydrolysis of the amide bond.

The pathway was the main metabolic pathway. This metabolite is found in mouse and rat blood as well as in mouse and rat. It was detected in monkey and human hepatocytes and liver S9 fractions. Metabolites of ALC-0159 from in vivo samples Not confirmed.

6. Excretion

PK study of intravenous luciferase RNA-encapsulated LNP in rats at a dose of 1 mg RNA / kg

(M2.6.4.The concentrations of ALC-0315 and ALC-0159 in urine and feces collected over time were measured in (3).

Neither ALC-0315 nor ALC-0159 unchanged form was detected in urine. On the other hand, in the feces

Unaltered forms of ALC-0315 and ALC-0159 were detected, at a rate of approximately 1% per dose, respectively.

It was about 50%. Also,Figure 3 As shown in, a metabolite of ALC-0315 was detected in urine.

7. Pharmacokinetic drug interactions

No pharmacokinetic drug interaction studies have been conducted with this vaccine.

8. Other pharmacokinetic studies

No other pharmacokinetic studies of this vaccine have been conducted.

9. Discussion and conclusion

Plasma and liver ALC-0315 levels were highest in rat PK studies by 2 weeks post-dose

It is reduced to about 1/7000 and about 1/4, respectively, and the ALC-0159 concentration is about 1/8000, respectively.

And reduced to about 1/250. t½ is comparable in plasma and liver, ALC-0315 is 6-8 days,

ALC-0159 was 2-3 days. The plasma t½ value is that each lipid is distributed in the tissue as LNP.

After that, it is considered to indicate that it was redistributed in plasma during the disappearance process.

Little unchanged form of ALC-0315 was detected in either urine or feces, but in the rat PK study

Monoester metabolites, double deesterified metabolites and 6-hexy from fecal and plasma samples collected in

Ludecanoic acid was detected in urine, and a glucuronic acid conjugate, a double deesterified metabolite, was detected in urine. This metabolism

The process is thought to be the major disappearance mechanism of ALC-0315, but quantitative data have been obtained to test this hypothesis. Absent. On the other hand, about 50% of the dose of ALC-0159 was excreted in feces as unchanged drug. In vitro metabolism experiment In, it was slowly metabolized by hydrolysis of the amide bond.

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2.6.4 Summary of pharmacokinetic study

Since the in vivo expression distribution of the antigen encoded by BNT162b2 is considered to depend on the LNP distribution,

Intramuscularly administered luciferase RNA-encapsulated LNP to BALB / c mice as an alternative reporter protein

The biodistribution was examined. As a result, expression of luciferase was observed at the administration site, and more than that.

Although the expression level was low, it was also observed in the liver. Expression at the administration site of luciferase is post-administration

It was observed from 6 hours and disappeared 9 days after administration. Expression in the liver was observed from 6 hours after administration, and it was administered.

It disappeared by 48 hours after giving. Locally administered luciferase RNA-encapsulated LNP circulates in the liver

It was considered to indicate that it reached the ring blood and was taken up by the liver. Also, Luciferer on rats

When the radioactivity-labeled body of ZeRNA-encapsulated LNP was intramuscularly administered, the radioactivity concentration was the highest at the administration site.

Indicated. Other than the site of administration, it was highest in the liver, followed by the spleen, adrenal glands and ovaries.

Total radioactivity recovery for doses in these tissues was significantly lower than in the liver. This result is

This was consistent with the expression of luciferase in the liver in the mouse biodistribution test. In addition, it should be noted.

No toxic findings indicating liver damage were found in the rat repeated-dose toxicity test ( M2.6.6.3 ).

From the above nonclinical pharmacokinetic evaluation, it was shown that LNP that reached the circulating blood is distributed in the liver.

In addition, metabolism and fecal excretion may be involved in the disappearance of ALC-0315 and ALC-0159, respectively.

It was suggested.

10. Chart

Charts are shown in the text and in the summary table.

References

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.
Non-clinical study guidelines for infectious disease preventive vaccines (No. 0527 from Yaksik Examination) No. 1, May 27, 2010)

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SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048) 2.6.5 Pharmacokinetic study summary table
2.6.5.1. PHARMACOKINETICS OVERVIEW						Test Article: BNT162b2
Type of Study Single Dose Pharmacokinetics	Test System	Test item	Method of Administration	Testing Facility		Report Number
Single Dose Pharmacokinetics and Excretion in Urine and Feces of ALC-0159 and ALC-0315 Distribution	Rat (Wistar Han)	modRNA encoding luciferase formulated in LNP comparable to BNT162b2	IV bolus	Pfizer Inc a		PF-07302048_06	_072424
In Vivo Distribution	Mice BALB / c	modRNA encoding luciferase formulated in LNP comparable to BNT162b2	IM Injection		b b	R- -0072
In Vivo Distribution Metabolism In Vitro and In Vivo Metabolism	Rat (Wistar Han)	modRNA encoding luciferase formulated in LNP comparable to BNT162b2 with trace amounts of [ 3 H] -CHE as nondiffusible label	IM Injection		c	185350
In Vitro Metabolic Stability of ALC-0315 in Liver Microsomes	Mouse (CD-1 / ICR), rat (Sprague Dawley and Wistar Han), monkey (Cynomolgus), and human liver microsomes	ALC-0315	In vitro	d		01049- 008
In Vitro Metabolic Stability	Mouse (CD-1 / ICR), rat	ALC-0315	In vitro			01049-009

of ALC-0315 in Liver S9 (Sprague Dawley),

monkey (Cynomolgus), d and human S9 liver fractions

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2.6.5 Pharmacokinetic study summary table

2.6.5.1. PHARMACOKINETICS OVERVIEW

Type of Study	Test System	Test item	Method of Administration
In Vitro Metabolic Stability of ALC-0315 in Hepatocytes	Mouse (CD-1 / ICR), rat (Sprague Dawley and Wistar Han), monkey (Cynomolgus), and human hepatocytes	ALC-0315	In vitro
In Vitro Metabolic Stability of ALC-0159 in Liver Microsomes	Mouse (CD-1 / ICR), rat (Sprague Dawley and Wistar Han), monkey (Cynomolgus), and human liver microsomes	ALC-0159	In vitro
In Vitro Metabolic Stability of ALC-0159 in Liver S9	Mouse (CD-1 / ICR), rat (Sprague Dawley), monkey (Cynomolgus), and human S9 fractions	ALC-0159	In vitro
In Vitro Metabolic Stability of ALC-0159 in Hepatocytes	Mouse (CD-1 / ICR), rat (Sprague Dawley and Wistar Han), monkey (Cynomolgus), and human hepatocytes	ALC-0159	In vitro
Biotransformation of ALC-0159 and ALC-0315 In Vitro and In Vivo in Rats	In vitro: CD-1 mouse, Wistar Han rat, cynomolgus monkey, and human blood, liver S9 fractions and hepatocytes In vivo: male Wistar Han rats	ALC-0315 and ALC-0159	In vitro or IV (in vivo in rats)

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2.6.5 Pharmacokinetic study summary table

2.6.5.1. PHARMACOKINETICS OVERVIEW

Type of Study Test System Test item Method of

Administration

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Test Article: BNT162b2

Testing Facility Report Number

01049- 010

01049- 020

01049-021

01049- 022

Pfizer Inc e PF-07302048_05 _043725

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Test Article: BNT162b2

Testing Facility Report Number

La Jolla, California.
, Germany.
, UK.
, China.
Groton, Connecticut.

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2.6.5 Pharmacokinetic study summary table

2.6.5.3. PHARMACOKINETICS: Test Article: modRNA encoding luciferase in LNP

PHARMACOKINETICS AFTER A SINGLE DOSE Report Number: PF-07302048_06 _072424

Species (Strain) Rat (Wistar Han)

Sex / Number of Animals Male / 3 animals per timepoint a

Feeding Condition Fasted Method of Administration IV

Dose modRNA (mg / kg) 1

Dose ALC-0159 (mg / kg) 1.96

Dose ALC-0315 (mg / kg) 15.3

Sample Matrix Plasma, liver, urine and feces

Sampling Time Points (h post dose): Predose, 0.1, 0.25, 0.5, 1, 3, 6, 24, 48, 96, 192, 336

Analyte ALC-0315 ALC-0159

PK Parameters: Mean b Mean b

AUC inf (µg • h / mL) c 1030 99.2

AUC last (µg • h / mL) 1020 98.6

Initial t ½ (h) d 1.62 1.74

Terminal elimination t ½ (h) e 139 72.7

Estimated fraction of dose distributed to liver (%) f 59.5 20.3

Dose in Urine (%) NC g NC g

Dose in Feces (%) h 1.05 47.2

ALC-0159 = 2-[(polyethylene glycol)-2000]-N, N-ditetradecylacetamide), a proprietary polyethylene glycol-lipid included as an preferably in the LNP formulation used in BNT162b2; ALC-0315 = (4-hydroxybutyl) azanediyl) bis (hexane-6,1-diyl) bis (2-hexyldecanoate), a proprietary aminolipid included as an preferably in the LNP formulation used in BNT162b2; AUC inf = Area under the plasma drug concentration-time curve from 0 to infinite time; AUC last = Area under the plasma drug concentration-time curve from 0 to the last quantifiable time point; BLQ = Below the limit of quantitation; LNP = Lipid nanoparticle; modRNA = Nucleoside modified messenger RNA; PK = Pharmacokinetics; t ½ = Half-life. a. Non-serial sampling, 36 animals total.

Only mean PK parameters are reported due to non-serial sampling.
Calculated using the terminal log-linear phase (determined using 48, 96, 192, and 336 h for regression calculation).
ln (2) / initial elimination rate constant (determined using 1, 3, and 6 h for regression calculation).
ln (2) / terminal elimination rate constant (determined using 48, 96, 192, and 336 h for regression calculation).
Calculated as follows: highest mean amount in the liver (µg) / total mean dose (µg) of ALC-0315 or ALC-0159.g. Not calculated due to BLQ data.

h. Fecal excretion, calculated as: (mean µg of analyte in feces / mean µg of analyte administered) × 100

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2.6.5 Pharmacokinetic study summary table

2.6.5.5A. PHARMACOKINETICS: ORGAN Test Article: modRNA encoding luciferase in LNP

DISTRIBUTION Report Number: R- -0072

Species (Strain): Mice (BALB / c)

Sex / Number of Animals: Female / 3 per group

Feeding Condition: Fed ad libitum

Vehicle / Formulation: Phosphate-buffered saline

Method of Administration: Intramuscular injection

Dose (mg / kg): 1 µg / hidden leg in gastrocnemius muscle (2 µg total)

Number of Doses: 1

Detection: Bioluminescence measurement

Sampling Time (hour): 6, 24, 48, 72 hours; 6 and 9 days post-injection

Time point Total Mean Bioluminescence signal (photons / second) Mean Bioluminescence signal in

the liver (photons / second)

Buffer control modRNA Luciferase in LNP modRNA Luciferase in LNP

6 hours 1.28 × 10 5 1.26 × 10 9 4.94 × 10 7

24 hours 2.28 × 10 5 7.31 × 10 8 2.4 × 10 6

48 hours 1.40 × 10 5 2.10 × 10 8 Below detection a

72 hours 1.33 × 10 5 7.87 × 10 7 Below detection a

6 days 1.62 × 10 5 2.92 × 10 6 Below detection a

9 days 7.66 × 10 4 5.09 × 10 5 Below detection a

LNP = Lipid nanoparticle; modRNA = Nucleoside modified messenger RNA. a. At or below the background level of the buffer control.

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2.6.5 Pharmacokinetic study summary table

2.6.5.5B. PHARMACOKINETICS: ORGAN Test Article: [ ₃ H]-Labelled LNP-mRNA formulation containing

DISTRIBUTION CONTINUED ALC-0315 and ALC-0159

Report Number: 185350

Species (Strain): Rat (Wistar Han)

Sex / Number of Animals: Male and female / 3 animals / sex / timepoint (21 animals / sex total for the 50 µg dose)

Feeding Condition: Fed ad libitum

Method of Administration: Intramuscular injection

Dose: 50 µg [ 3 H] -08-A01-C0 (lot # NC-0552-1)

Number of Doses: 1

Detection: Radioactivity quantitation using liquid scintillation counting

Sampling Time (hour): 0.25, 1, 2, 4, 8, 24, and 48 hours post-injection

Sample

Adipose tissue

Adrenal glands

Bladder

Bone (femur)

Bone marrow (femur) Brain

Eyes

Heart

Injection site

Kidneys

Large intestine

Liver

Lung

Mean total lipid concentration (µg lipid equivalent / g (or mL))

48 h

0.181

18.2

0.365

0.687

3.77

0.068

0.112

0.546

165

0.425

1.34

24.3

1.09

0.25 h

0.001

0.000

0.007

0.000

0.018

19.9

0.050

0.008

0.602

0.052

% of administered dose (males and females combined)

24 h

0.066

0.002

0.010

0.002

0.027

29.1

0.054

0.692

15.4

0.101

48 h

0.106

0.002

0.009

0.003

0.030

24.6

0.057

0.762

16.2

0.101

0.25 h

0.057

0.271

0.041

0.091

0.479

0.045

0.010

0.282

128

0.391

0.013

0.737

0.492

(males and females combined)

24 h

0.084

13.8

0.247

0.342

2.49

0.069

0.091

0.451

195

0.426

1.10

19.2

1.04

1 h

0.007

0.001

0.013

0.001

0.056

52.6

0.124

0.025

2.87

0.101

2 h

0.010

0.001

0.020

0.001

0.084

31.6

0.211

0.065

7.33

0.178

4 h

0.015

0.001

0.016

0.002

0.060

28.4

0.109

0.192

11.9

0.169

8 h

0.035

0.001

0.011

0.002

0.042

21.9

0.075

0.405

18.1

0.122

1 h

0.100

1.48

0.130

0.195

0.960

0.100

0.035 1.03

394

1.16

0.048

4.63

1.21

2 h

0.126

2.72

0.146

0.266

1.24

0.138

0.052 1.40

311

2.05

0.093

11.0

1.83

4 h

0.128

2.89

0.167

0.276

1.24

0.115

0.067

0.987

338

0.924

0.287

16.5

1.50

8 h

0.093

6.80

0.148

0.340

1.84

0.073

0.059

0.790

213

0.590

0.649

26.5

1.15

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2.6.5 Pharmacokinetic study summary table

2.6.5.5B. PHARMACOKINETICS: ORGAN Test Article: [ ₃ H]-Labelled LNP-mRNA formulation containing

DISTRIBUTION CONTINUED ALC-0315 and ALC-0159

Report Number: 185350

Sample Total Lipid concentration (µg lipid equivalent / g [or mL]) % of Administered Dose (males and females combined)

(males and females combined)

0.25 h 1 h 2 h 4 h 8 h 24 h 48 h 0.25 h 1 h 2 h 4 h 8 h 24 h 48 h

Lymph node 0.064 0.189 0.290 0.408 0.534 0.554 0.727 - - - - - - -

(mandibular)

Lymph node 0.050 0.146 0.530 0.489 0.689 0.985 1.37 - - - - - - -

(mesenteric)

Muscle 0.021 0.061 0.084 0.103 0.096 0.095 0.192 - - - - - - -

Ovaries 0.104 1.34 1.64 2.34 3.09 5.24 12.3 0.001 0.009 0.008 0.016 0.025 0.037 0.095

(females)

Pancreas 0.081 0.207 0.414 0.380 0.294 0.358 0.599 0.003 0.007 0.014 0.015 0.015 0.011 0.019

Pituitary gland 0.339 0.645 0.868 0.854 0.405 0.478 0.694 0.000 0.001 0.001 0.001 0.000 0.000 0.001

Prostate 0.061 0.091 0.128 0.157 0.150 0.183 0.170 0.001 0.001 0.002 0.003 0.003 0.004 0.003

(males)

Salivary 0.084 0.193 0.255 0.220 0.135 0.170 0.264 0.003 0.007 0.008 0.008 0.005 0.006 0.009 glands

Skin 0.013 0.208 0.159 0.145 0.119 0.157 0.253 - - - - - - -

Small intestine 0.030 0.221 0.476 0.879 1.28 1.30 1.47 0.024 0.130 0.319 0.543 0.776 0.906 0.835

Spinal cord 0.043 0.097 0.169 0.250 0.106 0.085 0.112 0.001 0.002 0.002 0.003 0.001 0.001 0.001

Spleen 0.334 2.47 7.73 10.3 22.1 20.1 23.4 0.013 0.093 0.325 0.385 0.982 0.821 1.03

Stomach 0.017 0.065 0.115 0.144 0.268 0.152 0.215 0.006 0.019 0.034 0.030 0.040 0.037 0.039

Testes (males) 0.031 0.042 0.079 0.129 0.146 0.304 0.320 0.007 0.010 0.017 0.030 0.034 0.074 0.074

Thymus 0.088 0.243 0.340 0.335 0.196 0.207 0.331 0.004 0.007 0.010 0.012 0.008 0.007 0.008

Thyroid 0.155 0.536 0.842 0.851 0.544 0.578 1.00 0.000 0.001 0.001 0.001 0.001 0.001 0.001

Uterus 0.043 0.203 0.305 0.140 0.287 0.289 0.456 0.002 0.011 0.015 0.008 0.016 0.018 0.022

(females)

Whole blood 1.97 4.37 5.40 3.05 1.31 0.909 0.420 - - - - - - -

Plasma 3.97 8.13 8.90 6.50 2.36 1.78 0.805 - - - - - - -

Blood: Plasma 0.815 0.515 0.550 0.510 0.555 0.530 0.540 - - - - - - -

ratio a

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2.6.5 Pharmacokinetic study summary table

2.6.5.5B. PHARMACOKINETICS: ORGAN Test Article: [ ₃ H]-Labelled LNP-mRNA formulation containing

DISTRIBUTION CONTINUED ALC-0315 and ALC-0159

Report Number: 185350

-= Not applicable, partial tissue taken; [ 3 H] -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 nonexchangeable, nonmetabolizable lipid marker used to monitor the disposition of the LNPs; ALC-0159 = 2-[(polyethylene glycol)-2000]-N, N--ditetradecylacetamide), a proprietary polyethylene glycol-lipid included as an preferably in the LNP formulation used in BNT162b2; ALC-0315 = (4--hydroxybutyl) azanediyl) bis (hexane-6,1diyl) bis (2-hexyldecanoate), a proprietary aminolipid included as an preferably in the LNP formulation used in BNT162b2; LNP = Lipid nanoparticle; mRNA = messenger RNA.

The mean male and female blood: plasma values were first calculated separately and this value represents the mean of the two values.

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Page 19 SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048) 2.6.5 Pharmacokinetic study summary table				Masking location: Adjusting
2.6.5.9. PHARMACOKINETICS: METABOLISM IN VIVO,				Test Article: modRNA encoding luciferase in LNP
RAT				Report Number: PF-07302048_05				_043725
Species (Strain):				Rat (Wistar Han)
Sex / Number of animals				Male / 36 animals total for plasma and liver, 3 animals for urine and feces
Method of Administration:				Intravenous
Dose (mg / kg):				1
Test System:				Plasma, Urine, Feces, Liver
Analysis Method:				Ultrahigh performance liquid chromatography / mass spectrometry
Biotransformation		m / z		Metabolites of ALC-0315 Detected
				Plasma Urine Feces				Liver
N- dealkylation, oxidation		102.0561 a		ND ND ND				ND
N- Dealkylation, oxidation		104.0706 b		ND ND ND				ND
N- dealkylation, oxidation		130.0874 a		ND ND ND				ND
N- Dealkylation, oxidation		132.1019 b		ND ND ND				ND
N- dealkylation, hydrolysis, oxidation		145.0506 a		ND ND ND				ND
Hydrolysis (acid)		255.2330 a		+ ND ND				ND
Hydrolysis, hydroxylation		271.2279 a		ND ND ND				ND
Bis-hydrolysis (amine) Hydrolysis, glucuronidation Bis-hydrolysis (amine), glucuronidation Bis-hydrolysis (amine), glucuronidation Hydrolysis (amine) Hydrolysis (amine), Glucuronidation Oxidation to acid Oxidation to acid Hydroxylation Sulfation Sulfation Glucuronidation Glucuronidation Note: Both theoretical and observed metabolites are included.	290.2690 b 431.2650 a 464.2865 a 466.3011 b 528.4986 b 704.5307 b 778.6930 a 780.7076 b 782.7232 b 844.6706 a 846.6851 b 940.7458 a 942.7604 b		+ ND ND ND + ND ND ND ND ND ND ND ND		+ ND ND + ND ND ND ND ND ND ND ND ND	+ ND ND ND ND ND ND ND ND ND ND ND ND	+ ND ND ND + ND ND ND ND ND ND ND ND
m / z = mass to charge ratio; ND = Not detected; + = minor metabolite as assessed by ultraviolet detection. a. Negative ion mode. b. Positive ion mode. PFIZER CONFIDENTIAL Page 9

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2.6.5 Pharmacokinetic study summary table

Test Article: ALC-0315

2.6.5.10A. PHARMACOKINETICS: METABOLISM IN VITRO

Report Numbers: 01049- 008

01049-009

01049- 010

Type of Study: Stability of ALC-0315 In Vitro

Study System: Liver Microsomes + NADPH S9 Fraction + NADPH, UDPGA, and Hepatocytes

alamethicin

ALC-0315 1 µM 1 µM 1 µM

Concentration:

Duration of 120 min 120 min 240 min

Incubation (min):

Analysis Method: Ultra-high performance liquid chromatography-tandem mass spectrometry

Incubation time Percent ALC-0315 remaining

(min) Liver Microsomes Liver S9 Fraction Hepatocytes

Mouse Rat Rat Monkey Human Mouse Rat (SD) Monkey Human Mouse Rat Rat Monkey Human

(CD- (SD) (WH) (Cyno) (CD- (Cyno) (CD- (SD) (WH) (Cyno)

1 / ICR) 1 / ICR) 1 / ICR)

0 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00

15 98.77 94.39 96.34 97.96 100.24 97.69 98.85 99.57 95.99 - - - - -

30 97.78 96.26 97.32 96.18 99.76 97.22 99.62 96.96 97.32 101.15 97.75 102.70 96.36 100.72

60 100.49 99.73 98.54 100.00 101.45 98.61 99.62 99.13 94.98 100.77 98.50 102.32 97.82 101.44

90 97.78 98.66 94.15 97.96 100.48 98.15 98.85 98.70 98.33 101.92 99.25 103.09 100.0 100.36

120 96.54 95.99 93.66 97.71 98.31 96.76 98.46 99.57 99.33 98.85 97.38 99.61 96.36 100.72

180 - - - - - - - - - 101.15 98.88 103.47 95.64 98.92

240 - - - - - - - - - 99.62 101.12 100.00 93.82 99.64 t ½ (min) > 120 > 120 > 120 > 120 > 120 > 120 > 120 > 120 > 120 > 240 > 240 > 240 > 240 > 240

-= Data not available; ALC-0315 = (4-hydroxybutyl) azanediyl) bis (hexane-6,1-diyl) bis (2-hexyldecanoate), a proprietary aminolipid included as an preferably in the lipid nanoparticle formulation used in BNT162b2; Cyno = Cynomolgus; NADPH = Reduced form of nicotinamide adenine dinucleotide phosphate; NC = not calculated; SD = Sprague Dawley; t ½ = half-life; WH = Wistar-Han; UDPGA = uridine-diphosphate-glucuronic acid trisodium salt.

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2.6.5 Pharmacokinetic study summary table

Test Article: ALC-0159

2.6.5.10B. PHARMACOKINETICS: METABOLISM IN VITRO

Report Numbers: 01049- 020

							01049-021 01049- 022
Type of Study:			Stability of ALC-0159 In Vitro
Study System:		Liver Microsomes + NADPH	S9 Fraction + NADPH, UDPGA, and alamethicin			Hepatocytes
ALC-0159 Concentration:		1 µM	1 µM			1 µM
Duration of Incubation (min):		120 min	120 min			240 min
Analysis Method:			Ultra-high performance liquid chromatography-tandem mass spectrometry
Incubation time (min)		Liver Microsomes	Percent ALC-0159 remaining Liver S9 Fraction			Hepatocytes
	Mouse (CD1 / ICR)	Rat Rat Monkey Human Mouse Rat (SD) Monkey Human (SD) (WH) (Cyno) (CD-1 / ICR) (Cyno)		Mouse (CD1 / ICR)	Rat (SD)	Rat (WH)	Monkey (Cyno)	Human

CONTINUED

0 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00

15 82.27 101.24 112.11 100.83 99.59 98.93 84.38 91.30 106.73 - - - - -

30 86.40 93.78 102.69 85.12 92.28 91.10 90.87 97.96 107.60 100.85 93.37 113.04 90.23 106.34

60 85.54 98.34 105.38 86.36 95.53 102.85 97.97 105.56 104.97 94.92 91.81 105.07 92.93 101.58

90 85.41 95.44 100.90 94.63 97.97 90.75 93.51 108.33 109.36 94.28 90.25 112.80 94.59 92.67

120 95.87 97.10 108.97 93.39 93.09 106.76 92.70 105.74 119.59 87.08 89.47 104.11 97.51 96.04

180 - - - - - - - - - 94.92 93.96 102.90 89.81 93.66

240 - - - - - - - - - 102.75 94.93 98.79 92.93 102.57 t ½ (min) > 120 > 120 > 120 > 120 > 120 > 120 > 120 > 120 > 120 > 240 > 240 > 240 > 240 > 240

-= Data not available; ALC-0159 = 2-[(polyethylene glycol)-2000]-N, N-ditetradecylacetamide), a proprietary polyethylene glycol-lipid included as an preferably in the lipid nanoparticle formulation used in BNT162b2; Cyno = Cynomolgus; NADPH = Reduced form of nicotinamide adenine dinucleotide phosphate; NC = not calculated; SD = Sprague Dawley; WH = Wistar-Han; UDPGA = uridine-diphosphate-glucuronic acid trisodium salt.

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2.6.5 Pharmacokinetic study summary table

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2.6.5.10C. PHARMACOKINETICS: METABOLISM

Test Article: ALC-0315

Report Number: PF-07302048_05 _043725

IN VITRO CONTINUED

Type of study

Metabolism of ALC-0315 In Vitro

Study system

Blood

Hepatocytes

Liver S9 Fraction

ALC-0315 concentration

10 µM

Duration of incubation

24 h

4 h

24 h

Analysis Method:

Ultrahigh performance liquid chromatography / mass spectrometry

Biotransformation

m / z

Blood

Hepatocytes

Liver S9 Fraction

Mouse

Rat Monkey Human Mouse

Rat

Monkey Human Mouse

Rat

Monkey Human

N- dealkylation, oxidation

102.0561 a

ND ND

N- Dealkylation, oxidation

104.0706 b

ND ND

N- dealkylation, oxidation

130.0874 a

ND ND

N- Dealkylation, oxidation

132.1019 b

ND ND

N- dealkylation, hydrolysis, oxidation

145.0506 a

ND ND

Hydrolysis (acid)

255.2330 a

ND ND

Hydrolysis, hydroxylation

271.2279 a

ND ND

Bis-hydrolysis (amine)

290.2690 b

ND ND

Hydrolysis, glucuronidation

431.2650 a

ND ND

Bis-hydrolysis (amine), glucuronidation

464.2865 a

ND ND

Bis-hydrolysis (amine), glucuronidation

466.3011 b

ND ND

Hydrolysis (amine)

528.4986 b

ND ND

Hydrolysis (amine), glucuronidation

704.5307 b

ND ND

Oxidation to acid

778.6930 a

ND ND

Oxidation to acid

780.7076 b

ND ND

Hydroxylation 782.7232 b Sulfation 844.6706 a

Sulfation 846.6851 b

Glucuronidation 940.7458 a Glucuronidation 942.7604 b

Note: Both theoretical and observed metabolites are included.

m / z = mass to charge ratio; ND = Not detected; + = metabolite present. a. Negative ion mode.

b. Positive ion mode.

ND ND ND

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SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.5 Pharmacokinetic study summary table

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2.6.5.10D. PHARMACOKINETICS: METABOLISM

Blood

10 µM

24 h

Blood

Test Article: ALC-0159

Report Number: PF-07302048_05 _043725

IN VITRO CONTINUED

Type of study

Study system

ALC-0159 concentration

Duration of incubation Analysis Method:

Biotransformation

O- Demethylation, O- dealkylation

Hydrolysis, N -Dealkylation

N- Dealkylation, oxidation

Hydrolysis (amine)

N , N -Didealkylation

Dealkylation
Demethylation, oxidation

Hydroxylation

ω-Hydroxylation, Oxidation

Hydrolysis (acid)

m / z

107.0703 b

151.0965 b

195.1227 b

214.2529 b

227.2017 a

410.4720 b

531.5849 b

580.6396 b

629.6853 b

633.6931 b

637.1880 b

708.7721 b

Mouse

Metabolism of ALC-0159 In Vitro Hepatocytes

10 µM

4 h

Liver S9 Fraction

10 µM

24 h

Liver S9 Fraction

Ultrahigh performance liquid chromatography / mass spectrometry

Hepatocytes

Rat Monkey Human Mouse

Rat

Monkey Human Mouse

Rat

Monkey Human

ND ND

Note: Both theoretical and observed metabolites are included.

m / z = mass to charge ratio; ND = Not detected; + = metabolite present. a. Negative ion mode.

b. Positive ion mode.

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Global Prophecies

A85 Japanese Pfizer bio distribution study April 2021.

LIST OF TABLES

LIST OF FIGURES

Terms and abbreviations used in this section

1. Summary

4. Distribution

5. Metabolism

6. Excretion

7. Pharmacokinetic drug interactions

8. Other pharmacokinetic studies