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PF-07302048 Biotransformation
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Page 1of 34INVESTIGAT ION OF THE BIOTRANSF ORMATION OF ALC- 0159 AND ALC -0315
IN VITRO AND IN VIVO IN RATS
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Page 2of 34LIST OF AB BREVIATIONS
Abbreviation Term
ALC-0159 P roprietary PEG-lipid included as an excipient in the L NP formulation used
in the COVID-19mRNA vaccine
ALC-0315 P roprietary amino-lipid included as an excipient in the LNP f ormulation
used in theCOVID-19 mRNA vaccine
COVID-19 Coronavirus disease 2019
DMSO Dimethyl sulfoxide
LNP Lipid-nanoparticles
MeCN Acetonitrile
modRNA Nucleoside -modified mRNA
mRNA Messenger RNA
NAD+ Nicotinamide adenine dinucl eotide (oxidized form)
NADH Nicotinamide adenine dinucl eotide (reduced form)
NADP+ Nicotinamide adenine dinucl eotide phosphate (oxidized form)
NADPH Nicotinamide adenine dinucl eotide phosphate ( reduced form)
PAPS 3'-Phosphoadenosine -5'-phosphosulfate
PEG Polyethylene glycol
S9 Supernatant obtained from liver homogenate by centrifuging a t 9000g
UDPGA Uridine diphosphate glucuroni c acid
UHPLC Ultra high-performance liquid chromatography
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Page 3of 341.ABSTRACT
The metabolism of the novel excipients, ALC-0159 and AL C-0315,wasexamined invitro
using blood, liver S9 fractions and hepatocy tes, all from mouse, rat, monkey and human .
Thein vivometabolism was exami ned in rat plasma, urine, feces, and l iverfrom a rat
pharmacokinetics stud y where a luciferase -encoding mod RNAformulated i n LNPwith an
identical lipid composition as PF -07302048 ( COVID-19 mRNA Vaccine ; BioNTech code
number BNT162) wasadministered intravenous lyat a 1 mg/kgdose.
The primary route of metabolism identified for ALC-0159involves am ide bond hy drolysis
yielding N,N-ditetradecylamine(m/z410). This metabolit ewas identifie d inmouse and rat
blood,as well as hepatocy tesandliver S9 from mouse, rat, monkey and human. No
metabolites of ALC-0159 were identified from in vivosamples.
Metabolism of AL C-0315 occurs via two se quentialester hydrolysis reactions, first yielding
themonoester metabolite (m/z528)followed bythe doubly deesterified met abolite (m/z290).
The monoester metabolite was observed in vitroin ratblood,monkey S9fraction,and invivo
inratplasmaandrat liver. The doubly deesterified metabolite was observed in vitroin
mouseand rat blood; monkeyliver S9 fract ion;and invivoinratplasma, urine, fecesand
liver. Subsequent metabolism of the doubly deesterified metabolite r esultedin a glucuronide
metabolite (m/z466)which was obse rved inurine only from the rat p harmacokinetics study .
Additionally, 6-hexyldecanoic acid(m/z255), the acid product of both ester hydrolysis
reactions of AL C-0315,was identified invitroinmouse andratblood;mouse, rat, monkey
andhuman hepatocy tes; mouse, rat andhuman liver S9 fractions ; andin vivoinrat plasma.
Based on nonquantitative ion current data for parent depletion and metabolite formation,
metabolism for both ALC -0159 and AL C-0315 appears to occur relativel y slowly across
most species in vit roand in vivo. Overall, it can be concluded that bot h ALC-0159 and
ALC-0315 are metabolized by hydrolytic metabolism of the amide or ester functionalities,
respectivel y, and this hy drolytic metabolism is obse rved across the species evaluated.
2.OBJECTIVES
The objective of this study was to provide a prelimi nary qualitative a ssessment of the
biotransformation of the novel excipients ALC-0159 and AL C-0315in blood, liver S9
fractions and hepato cytesfrom mouse, ra t, monkey andhumanas well as in plasma, urine,
feces and liver samples from a rat pharmacokinetics study.
ALC-0159(n = 40-51)
n = 45, major com ponent
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Page 4of 34
ALC-0315
3.MATERIALS AND METHOD S
3.1.Materials
ALC-0159 (2 -[(polyethyleneglycol]-2000]-N,N-ditetradecylacetamide, Lot#GALC0159-
10), ALC-0315 ((4-hydroxybutyl)azanediyl)bis(hexane -6,1-diyl) bis(2-hexyldecanoate), Lot#
GALC0315-11), and Carboxy-MPEG2 (methoxy polyethylene glycol 2000 acetic acid , Lot#
792354-01-011) were obta ined from Ava ntiPolar Lipids, Inc. NAD+, reducedNADH,
reduced N ADPH, NADP+,alamethicin, adipic acid, diethylene glycol, triethylene glycol,
tetraethyleneglycol, myristic acid, tetradec ylamine, 6-hexyldecanoic acid, 4-aminobut yric
acid, and 6- aminohexa noic acid wereobtained from Millipore-Sigma (St. Louis, MO).
N,N-Ditetradecylaminewas obtained fr om Ambeed(ArlingtonHeights,IL). All other
reagents were the highest gr ade commerciall y available.
Blood from mouse(female,CD-1), rat(male, Wistar Han), monkey(male, cynomolgus), and
human(one male and one fe male)was obtained fromin-houseuntreated animals and from
human donorsnot taking any medications. Potassium EDTA (K2EDTA)was used as the
anticoagulant for all species. In allspecies except rat, t he blood used for thein vitro
assessmentswasa pool of 2 or more animals or donors. Mouse(male, CD-1, B ioIVT, lot
YKA), rat (male, Wistar Han, BioIVT, lot DTO), monkey (male, cynomolgus, BioIVT, lot
DNB), and hum an (mixed gender, BioIVT, lot SPB) hepatocytes were used in the invitro
assessments. Mouseliver S9 fraction(Xenotech,female, CD -1, lot# 0310217, 20 mg/mL
protein), rat liverS9 fraction (BD Gentest, male Wistar Han, lot#58237, 20 mg/mL protein),
monkeyliverS9 (Xenotech, male, cynomolgus, lot# 0210398, 20 mg/mL)andhuman liver
S9(Celsis,LotABT,20mg/mLprotein)were utilized for the invitroassessments.
3.2.Blood
Mouse, rat, monke y and humanbloodwere spiked with ALC-0159and ALC-0315 stock
solutions (1 mM, each dissolvedin DMSO )to give a final concentration of 10M. A
solvent control was alsoincluded where DMSO was adde din place of test compound. Aft er
addition of test compoundor DMSO, blood samples were maintained at 37 °C.Aliquots
(500 L) were removed at 0, 0.5, 1, 2, 4, 6, and 24hand quenched with 6 volumes of i ce-
cold MeCN. Samples were subseque ntly centrifuged at 1860 x gfor 5minutes. The s amples
were then transferred to clean 15 mL glass tubesand evaporated to dry ness using a Genevac
evaporative centrifuge. To expedite analy ses, onlythe0 and 24 h samples were reconstitut ed
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Page 5of 34in 100L of1%MeCN in wate rand analyzedas described below . The remaining sampl es
were stored at -40 °C.
3.3.Hepatocytes
Mouse, rat, monkey and human hepatocyte incubatio ns (0.75x 106cells/mL), were
conducted at a final concentration of ALC-0159and ALC-0315 of 10Musing 1mM
stocks, each dissolvedin DMSO. A solvent control was also included where DMSO was
added in place of test compound . After addition of testcompound or DMSO,sampleswere
maintained in an incubator at 37 °C, 95% humidity, and 5% c arbon dioxide. Aliquots (500
L) were removed at 0 , 0.5, 1, 2,and 3h and a 250 Lat 4 hand quenched with 6 vol umes
of ice-cold MeCN. Samples were subsequentl y centrifuged at 1860x gfor 5 minutes. The
samples werethen transferr ed to clean 15mL glass tubes and evaporated to dry ness using a
Genevac ev aporative centrifu ge. To expedite anal yses, onlythe 0 and4 h samples were
analyzed. The 0 h sample s werereconstituted in 100Lof1% MeCN in water and 4 h
samplesin 50Lof1% MeCN in water and analyzedas described bel ow. Theremaining
samples were stored at - 40 °C.
3.4.Liver S9 Fractions
Liver S9 fractions from mou se, rat, monkey , and human suspended in 0.1 M phosphate
buffer (pH 7.4)containing3.3 mM magnesium chloridewere preincubated with a lamethicin
(9 g/mL) for 15 mi n onice. Incubations were started by addition of a mixture of test
compound dissolved in DMSO and buf fer, test compounddissolved in DM SO, buffer, and
cofactor mix A (1 mM NADPH, 1mM NADH, 0.5 mM PAPS, and2.5 mM UDPGA), or test
compound , buffer and cofactor mix B(1 mM NADP+, 1 mM NAD+, 0.5 mM PAP S, and 2.5
mM UDPGA),bringing theincubation to a total volume of1 mL witha final protein
concentration of 1 mg/ mLand a final concentration ofALC-0159 or ALC-0315 of 10 M.
Incubation mixtures were warmed to 37 °C,and aliquots (150 L) were rem oved at 0, 0.5, 1,
2, 4, 6, and 24 h and quenched b yaddition to MeCN (400 L). Samples were subsequentl y
centrifuged at 3000 rpm (1860 x g) for5minutes. The samples wer e then transferred to
clean1mL glassdolphin-nosedtubesand evaporated t o dryness using a Genevac
evaporative centrifuge. To expedite analy ses, onlythe0 and 24 h samples were re constituted
in 100L of1%MeCN in w ater andanalyzedas described be low. The remaining samples
were stored at -40 °C.
3.5.Rat Pharmacokinetics Study Samples
Plasma, urine, feces and liver samples wereobtained from a 14-dayrat pharma cokinetics
study(StudyPF-07302048_06Jul20_07242 41)where aluciferase -encoding mod RNA
formulated in LNPwith an identical l ipid composition as PF -07302048 ( COVID-19mRNA
Vaccine; BioNTech code number BNT162) was administered intravenously at a1 mg/kg
mod RNA dose to male, Wistar Han rats . At this mod RNA dose ,the dose of AL C-0159 was
1.96mg/kg and of AL C-0315 was 15.3mg/kg. While add itional time pointsamples were
obtainedof pharmac okinetic anal ysis,for metabolite identificat ion studies, pl asmaand livers
from three rats per time pointat the following timepointswere used : pre-dose, 0.1, 24, 96,
192, and 336 h post-dose. Fecal and urine samples from three rats per time point frompre-
dose, 0-24, 24-48, 72- 96, 168-192, and 312- 336 h post-dose were used.
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Page 6of 343.5.1. Plasma S ample Preparati on
Plasma(50 µL)from each ratper time point was pooled to generate pools for the pre -dose,
0.1, 24, 96, 192, and 336 h time points. P roteins were precipi tatedwith 4volumesof ice-
cold MeCN, centrifuged, and t he supernatant blown to dryness. Residues were reco nstituted
in 100L of 1% MeCN in waterandanalyzedas described be low.
3.5.2. UrineSample Preparatio n
Urinesamples(100 µL from each rat )for the pre-dose, 0-24, 24-48, 72- 96, 168-192, and
312-336 h timepointswere combinedto generate a sample pool for each of the se time
points.Pooled urine samples were cent rifugedat 17000 x gfor 10minutes. Supernatants
weretransferred to anal yses tubes and analyzed wi thout further ma nipulationandanalyzedas
described below.
3.5.3.FecesSamplePreparation
Feces samplesfor each rat fecal sample from the pre-dose,0-24, 24-48, 72-96, 168-192,and
312-336 htime points were diluted 1:9(w/v) with homogenization solution (6 0:40 isopropy l
alcohol/water) and homogenized with aMini-Beadbeater-96 (BioSpec Products)u sing 2 mm
zirconia beads and a2 minutehomogenization time. Homogenized samples ( 300 µL) for the
three ratsamplesper time point were pooled . Proteins were precipitated with 4volumesof
ice-cold MeCN, centrifuged, and thesupernatant blown to dryness. Residues were
reconstitutedin 100L of 1% MeCN in waterandanalyzedas described below.
3.5.4.LiverSample Preparation
Liversamplesfor each ratfrom the pre-dose, 0.1, 24, 96, 192, and 336 h time points were
diluted1:4(w/v)with homogenization solution ( 60:40 isopropy lalcohol/water)and
homogenized with a Mini-Beadbeater-96 (BioSpec Products)using 2 mmzirconia beadsand
a 2 minutehomogeniza tion time. Homogenized samples (2 00 µL) for thethree ratsamples
per time point were pooled. Proteins were prec ipitatedwith 4 volumes of ice-cold MeCN,
centrifuged, and t he supernatant blown to dryness. Residue s were reconstitutedin100L of
1% MeCN in waterandanalyzedas described be low.
3.6. UHPLC-MS/MS Analysis
3.6.1. UHPLC-MS/MSSample Analysis of ALC-0159
Reconstituted samples were anal yzedusing the sameUHPLC method bu t with separate
MS/MSanalyses inpositive ion and negative ion electrospraymodes using a Thermo
OrbitrapElite mass spectrometer. Xcalibursoftware ver sion3.0.63 was used to contro l the
UHPLC-MSsystem. Injections of 2 µ L were made by a CTC PAL autosampler .Fullscan
data werecollected at 15,000 resol ution. The UHPLCsystem consisted of an Accel a
quaternary solvent delivery pump(Thermo Electr on Corporation). AnAcquityUPLC C8
100 Å column was used (2.1 x 1 00mm, 1.7µm) with a flow rate of0.4 mL/min heated to45
°Cin aHotSleeve column heater (Analytical Sales and Servic es). Mobile phase A was 10
mM ammonium a cetate buffer (pH 4.5)and mobile phase B wasMeCN.
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Page 7of 34Time, min%A %BFlow Rate
(L/min)
0.0 1000 400
2.5 1000 400
5.0 4060 400
23.0 595 400
26.0 595 400
26.1 1000 400
30.0 1000 400
3.6.2. UHPLC-MS/MSSample Analysis of ALC-0315
Reconstituted samples w ere analyzed using the same UHPLC method but with separa te
MS/MSanalyses inpositive ion and negative ion electrospray modesusing a Therm o
OrbitrapElitemassspectrometer. Xcalibursoftware version 3.0.63 was used to control the
UHPLC-MS system. Injections of 5µL were madeby a CTC PALautosampler. Full scan
data were collected at 15,000 resol ution. The UHPLCsystem consisted of an Accela
quaternary solvent delivery pump(ThermoElectron Corporation ). AnAcquityUPLC C18
100 Å column was use d (2.1 x 150 mm, 1.7 µm) with a flow rate of 0.3mL/min heated to 45
°Cin a HotSleeve column heater (Analytical Sales and Servic es).Mobile phase A w as 0.1
% formicacidinwaterand mobilephase BwasMeCN.
Time, min%A %BFlow Rate
(L/min)
0.0 1000 300
2.5 1000 300
5.0 9010 300
10.0 5050 300
17.5 595 300
21.5 595 300
21.6 1000 300
25.0 1000 300
4.RESULTS & DISCUSSION
As shown in Figure 9.1, the primary route of metabolism identified for ALC-0159involves
amide bond hydrolysis yielding N,N-ditetradecylamine(m/z410). This metabol ite was
identified inmouse and rat blood as wellas hepatocytesandliver S9 from mouse, rat,
monkey and human. Theoretical metabolites were arrived at via examination of the excipient
molecules and consideration of commonly observed biotransformations ( hydroxylation,
N-dealkylation, hydrolysis, glucuronidation, sulf ation, oxidation and combinations thereof) .
Summaries of the masses of theoretical and observed metabolites of ALC-0159 for blood,
hepatocytes, liver S9 fractions, and rat pharmacokinetics s amplesare presented in Tables 8.1,
8.2, 8.3, and 8.4, res pectively. Representative e xample chromatograms from in vitro
incubationsof ALC-0159 withmouse hepatocytes, human hepatocytes, and in vivosamples
from a rat pharmaco kinetics study are presented in Figures 9.3, 9.4, and 9.5, respe ctively. No
metabolites of ALC-0159 were identified frominvivosamples.
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Page 8of 34Metabolism of ALC-0315 occurs via two se quentialester hydrolysis reactions, first yielding
themonoester metabolite (m/z528)followedbythe doubly deesterified metaboli te (m/z290)
as shown in Figure 9.2. The monoester m etabolite was observed in vitro in ratblood,
monkey S9fraction,and invivoinratplasmaand rat liver. Thedoubly deesterified
metabolite was observed in vitroin mouseand rat blood; monkey liver S9 fraction; and in
vivoinratplasma, urine, fecesandliver.Subsequent metabolism of the doubly desterified
metabolite r esultedina glucuronide metabolite ( m/z466)which was observed in urine only
from the rat pharm acokinetics study . Additionally, 6-hexyldecanoic acid(m/z255), the acid
product of both hydrolysis reactions of AL C-0315,was identifiedinvitroinmouse and rat
blood;mouse, rat , monkeyand human he patocytes; mouse, rat andhuman liv erS9 fractions ;
andin vivoinrat plasma . Summarie s of the masses of theoretical and o bserved metaboli tes
of ALC-0315for blood, hepatocy tes, liver S9 fr actions, and rat pharmacokinet icssamplesare
presented i n Tables8.5,8.6, 8.7,and 8.8, respectively.Representative example
chromatogra ms fromin vitroincubations of ALC-03159 withmonkey liver S9 fraction ,
humanhepatocytes, and in vivosamplesfrom a rat pharmacokinetics s tudy are presented in
Figures 9.6, 9.7, and 9.8, respectivel y.
Based on nonquan titative ion current data for parent depletion and metaboli te formation,
metabolism for both ALC-0159 and AL C-0315 appears t o occur relatively slowlyacross
most species invitroandin vivo. Overall, it can b e concluded that both ALC -0159 and
ALC-0315 are metabolized by hydrolytic metabolism of the amide or este r functionalities,
respectivel y, and this hydrolytic metabolism i s observed across thespecies evaluat ed.
4.1.Mass Spectral Anal ysisof ALC-0159
Mass spectrometricanalyses of ALC -0159 indicate tha t itis a mixture of var ying
polyethylene glycol (PEG) len gths ranging between approximately 40-51 ethylene glycol
units. Addit ionally,the mass spectrum (Figure9.9) indicates that thi s mixture of compounds
also exists in +2, +3,and +4charge sta teswith the +4 charge state being the mos t abundant.
Deconvolution o fthe mostabundant ion in the +4 cha rge state(m/z629.6939, tR= 19.1
minutes)is consistent with a t riply ammoniated , protonated species. For simplicity of
analyses and description, PEG-containing metabolites of AL C-0159,where standards are not
available,were searched for based on modif icationsof the most abundant and intense parent
mass (m/z629.6939).
4.2.Mass Spectral An alysisof ALC-0159 m/z410metabolite
Anm/z410 metabolite of A LC-0159 hada retention time of approxima tely 16.9minutes
witha protonated mol ecular ion of m/z410.4715.It wasobservedin mouse and rat blo od,as
well as hepatocytesandliver S9 from mouse , rat, monkey and human.The product ion
spectrumfor m/z410 possessed a single fragment io n of m/z214 whichcorresponds to loss
ofone of the 14 -carbon aliphatic ch ains. Both the observed retention time and frag mentation
patternfor m/z410 match thos e obtained f rom N,N-ditetradecy lamine (Figure9.10)
4.3.Mass Spectr al Analysisof ALC-0315
ALC-0315was identified at a retention ti me of 20.0 minutesand m/z766.7254. Product ion
spectrumfragment io nsat m/z748, 694 and 510 wer e observed in as shown in Fi gure9.11.
The m/z748fragment is consistent with a water lossfrom butylalcohol substitue nt. The m/z
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Page 9of 34694fragment cor responds to loss of the buty lalcohol substituent. The m/z272 fragment i s
consistent with lossof one of the 6-hexyldecanoic acid moiet iesalong with H+.
4.4.Mass Spectral A nalysisof ALC-0315m/z528 Metabolite
Ametabolite of ALC-0315 witha retention ti me of 15.9 min utesand m/z528.4975.This
metabolite was observed in rat blood, monkey liver S9fraction, rat plasma and rat liver
samples. Product ion spectrum fragment ionsat m/z510, 456, 272and 218were observed as
shown in Figure 9.12.The m/z510 fragment is consistent wit h a waterloss from one ofthe
twoalkyl alcohol substit uents. The m/z456 fragment corresponds to loss of the buty lalcohol
substituent. The m/z272 fragment i s consistent with loss of the 6-he xyldecanoic acid moiet y
alongwith H+.
4.5.Mass Spectral A nalysisofALC-0315m/z290 Metabolite
Ametabolite of ALC-0315 was observe dat 8.0minutes with m/z290.2688. This metabolite
was observed in mouse and rat blood, monkey liver S9 fraction, and pla sma, urine, feces and
liver from the rat phar macokinetics study . The product ion spectrumdisplays fragment ions
of m/z272 (lossof water) and m/z218 (loss of buty lalcohol subst ituent) (Figure 9.13).
4.6. Mass Spectral Anal ysisof ALC-0315 m/z466 Metabolite
The m/z466 metabolite of AL C-0315 was observed at 7.9 minuteswith m/z466.3006 only in
rat urine. The product ion spectra of shows a single fragment with m/z290(Figure 9.14). A
neutral loss of 176 Da for thi s metabolite is consistent with a glucuronide conjugate to one of
the three alcohol moieties of the doubly deesterifiedmetabolite , m/z290.
4.7. Mass Spectral Anal ysisof ALC-0315 m/z255 Metabolite
An m/z255metabolite of AL C-0315was observed at approximat ely 19.7 min with m/z
255.2324 in mouse plasm a. This metabolite was observed in mouse and ratblood;mouse,
rat, monkeyandhuman hep atocytes; mouse, rat andhuman live rS9 fractions ; andin vivoin
rat plasma. This metabolitematches b y bothretention time and exact mass with a synthetic
standard of 6 -hexyldecanoic acid . However , product ion spectra coul d not beobtained for
either the metabolite or the 6-hexyldecanoic acidstandard.
5. CONCLUSIONS
The metabolism of the novel excipients, ALC-0159 and AL C-0315, wereexamined invitro
using blood, liver S9 fractions and hepatocy tes, all frommouse, rat, monkey and human.The
in vivometabolism was examin ed in rat plasma, urine, feces, a nd liver from a rat
pharmacokinetics stud ywhere aluciferase -encoding mod RNAformulated in anLNP with
an identic al lipid composition as PF -07302048 wasadministered intravenou sly at a1 mg/kg
dose.
The primary route of metabolism identified for ALC-0159involves am ide bond hy drolysis
yielding N,N-ditetradecylamine(m/z410). This metabolit ewas identifie d inmouse an d rat
blood, as well as hepatocy tesandliver S9 from mouse , rat, monkey and human. No
metabolites of ALC-0159 were ide ntified fromin vivosamples.
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Page 10of 34Metabolism of AL C-0315 occurs v ia two sequentialester hydrolysis reactions, first yielding
themonoester metabolite ( m/z528)followed bythedoubly deesterified metabolite (m/z290).
The monoester metabolite was observed in vitroinratblood,monkey S9fraction,and invivo
inratplasmaand rat liver. Thedoubly deesterifiedmetabolite was observedin vitroin
mouseand rat blood; monkey liver S9 fraction; andinvivoinratplasma, urine, fecesand
liver. Subsequent m etabolism of the doubly deesterifiedmetabolite r esultedin a glucuronide
metabolite (m/z466)which was observed inurine only fromtherat pharmacokinetics stu dy.
Additionally, 6-hexyldecanoicacid(m/z255), the acid product of both hydrolysis reactions
ofALC-0315,was identified invitroinmouse and ratblood;mouse, rat, monkeyand human
hepatocytes; mouse, rat andhuman liver S9 fractions; and in vivoinrat plasma .
Based on nonquantita tive ioncurrent data for parent depletion and metabolite formation,
metabolism for both ALC -0159 and AL C-0315 appears to oc cur relatively slowly across
most species in vitroand in vivo. Overall,it can be concluded that both ALC -0159 and
ALC-0315 are me tabolizedby hydrolytic metabolism of the amide or ester f unctionalities,
respectivel y, and this hy drolytic metabolism is ob served across the species evaluated.
6. ARCHIVING
Data presented in this report can be found in the f ollowing locations:
Laboratory Notebooks /Root/PDM/Groton/
VBN#00710777/COVID _Excipient/200715_COVID_
Novel_Excipients_HHEP
/Root/PDM/Groton/
VBN#00710777/COVID_Excipient/200728_COVID_
Novel_Excipie nts_Blood_Stability
/Root/PDM/Groton/
VBN#007107 77/COVID _Excipient/200728_COVID_
Novel_Excipients_S9
/Root/PDM/Groton/
VBN#00710777/COVID_Excipient/200820_CO VID_
excipient_rat_PK_met_ID
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(b) (6)
(b) (6)
(b) (6)
(b) (6)
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Page 11of 34Laboratory Notebooks /Root/PDM/Groton/
VBN#00710777/COVID _Excipient/200715_COVID_
Novel_Excipients_HHEP
/Root/PDM/Groton/
VBN#00710777/COVID_Excipient/200728_COVID_
Novel_Excipie nts_Blood_Stability
/Root/PDM/Groton/
VBN#007107 77/COVID _Excipient/200728_COVID_
Novel_Excipients_S9
/Root/PDM/Groton/
VBN#00710777/COVID_Excipient/200820_CO VID_
excipient rat PK met ID
Analytical Archive ReferenceOpen Lab:
Enterprise/Content/ Target Archive /COVID vaccine
excipients/Biotransformation/
200730_COVID_Excipient_HEP
Enterprise/Content/TargetArchive/ COVID vaccine
excipients/ Biotransformation/
200730_COVID_Excipients_L S9
Enterprise/Content/Target Archive /COVIDvaccine
excipients/ Biotransformation/
200731_COVID_Ex cipient_Blood
Enterprise/Content/ Target Archive /COVID v accine
excipients/Biotransformation/
200822 COVID Excipient Rat PK
7.REFEREN CES
1. PF-07302048_06Jul20_072424_A single dose pharmacokinetics stu dy of ALC-0315 and
ALC-0159 following intravenous bolus injecti on of PF-07302048 Nanoparticle Formulation
in Wistar Han Rats. 01 Sept 2020.
090177e194e3ef10\Approved\Approved On: 11-Sep-2020 18:13 (GMT)
(b) (6)
(b) (6)
(b) (6)
(b) (6)
FDA-CBER-2021-5683-0709412
PF-07302048 Biotransformation
PF-07302048_05Aug20_ 043725
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Page 12of 348.SUPPORTIVE TABLES
8.1.In VitroAssessment of ALC-0159Metabolites in Mouse, Rat , Monkey and Human
Blood
m/z Biotransformation t R, minBlood
Mouse RatMonkeyHuman
107.0703bO-Demethylation,
O-dealkylation1.2cND ND ND ND
151.0965bO-Demethylation,
O-dealkylation3.1cNDND ND ND
195.1227bO-Demethylation,
O-dealkylation4.8cND ND ND ND
214.2529b Hydrolysis,
N-dealkylation7.3cND ND ND ND
227.2017aN-Dealkylation, oxidation9.1cNDNDND ND
410.4720bHydrolysis (amine) 16.9c++ND ND
531.5849bN,N-Didealkylation NDNDND ND ND
580.6396bN-Dealkylation ND NDND ND ND
629.6853bO-Demethylation,
oxidationND NDND ND ND
633.6931bHydroxylation ND NDND ND ND
637.1880bω-Hydroxylation,
oxidationND ND ND ND ND
708.7721bHydrolysis (acid) 5.8cNDND ND ND
a.Negative ion mode
b. Positive ion mode
c.Determined us ing standard
ND–Not Detected, + = metabolite identified.
090177e194e3ef10\Approved\Approved On: 11-Sep-2020 18:13 (GMT)
FDA-CBER-2021-5683-0709413
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Page 13of 348.2.In VitroAssessment ofALC-0159 Metabolites in Mouse, Rat , Monkey and Human
Hepatocyt es
m/z Biotransformation tR, minHepatocytes
Mouse RatMonkeyHuman
107.0703bO-Demethylation,
O-dealkylation1.2cND ND ND ND
151.0965bO-Demethylation,
O-dealkylation3.1cNDND ND ND
195.1227bO-Demethylation,
O-dealkylation4.8cND ND ND ND
214.2529b Hydrolysis,
N-dealkylation7.3cND ND ND ND
227.2017aN-Dealkylation, oxidation 9.1cNDND ND ND
410.4720bHydrolysis (amine) 16.9c++ + +
531.5849bN,N-Didealkylation ND NDND ND ND
580.6396bN-Dealkylation ND NDND ND ND
629.6853bO-Demethylation,
oxidationND ND ND ND ND
633.6931bHydroxylation NDNDND ND ND
637.1880bω-Hydroxylation,
oxidationND ND ND ND ND
708.7721bHydrolysis (acid) 5.8cNDND ND ND
a.Negative ion mode
b. Positive ion mode
c.Determined using standard
ND–Not Detected, + = metabolite identified .
090177e194e3ef10\Approved\Approved On: 11-Sep-2020 18:13 (GMT)
FDA-CBER-2021-5683-0709414
PF-07302048 Biotransformation
PF-07302048_05Aug20_ 043725
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Page 14of 348.3.In VitroAssessment ofALC-0159Metabolites in Mouse, Rat , Monkey and Human
Liver S9Fractions
m/z Biotransformation tR, minLiver S9 Fr actions
MouseRatMonkeyHuman
107.0703bO-Demethylation,
O-dealkylation1.2cND ND ND ND
151.0965bO-Demethylation,
O-dealkylation3.1cND ND ND ND
195.1227bO-Demethylation,
O-dealkylation4.8cNDND ND ND
214.2529b Hydrolysis,
N-dealkylation7.3cND ND ND ND
227.2017aN-Dealkylation, oxidation9.1cNDND ND ND
410.4720bHydrolysis (amine) 16.9c++ + +
531.5849bN,N-Didealkylation ND NDND ND ND
580.6396bN-Dealkylation ND NDND ND ND
629.6853bO-Demethylation,
oxidationND ND ND ND ND
633.6931bHydroxylation ND NDND ND ND
637.1880bω-Hydroxylation,
oxidationND ND ND ND ND
708.7721bHydrolysis (acid) 5.8cNDND ND ND
a.Negative ion mode
b. Positive ion mode
c.Determined us ing standard
ND–Not Detected, + = metabolite identified .
090177e194e3ef10\Approved\Approved On: 11-Sep-2020 18:13 (GMT)
FDA-CBER-2021-5683-0709415
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Page 15of 348.4.Assessment of Meta bolites of ALC-0159in Plasma, Urine, Feces, and Liver from a
Rat Pharma cokinetics Study.
m/z Biotransformation tR, minRatIn Vivo
PlasmaUrineFeces Liver
107.0703bO-Demethylation,
O-dealkylation1.2cND ND ND ND
151.0965bO-Demethylation,
O-dealkylation3.1cND ND ND ND
195.1227bO-Demethylation,
O-dealkylation4.8cND ND ND ND
214.2529b Hydrolysis,
N-dealkylation7.3cND ND ND ND
227.2017aN-Dealkylation, oxidation 9.1cND ND ND ND
410.4720bHydrolysis (amine) 16.9cND ND ND ND
531.5849bN,N-Didealkylation NDND ND ND ND
580.6396bN-Dealkylation ND ND ND ND ND
629.6853bO-Demethylation,
oxidationND ND ND ND ND
633.6931bHydroxylation ND ND ND ND ND
637.1880bω-Hydroxylation,
oxidationND ND ND ND ND
708.7721bHydrolysis (acid) 5.8cND ND ND ND
a.Negative ion mode
b. Positive ion mode
c.Determined us ing standard
ND–Not Detected, + = metabolite identified .
090177e194e3ef10\Approved\Approved On: 11-Sep-2020 18:13 (GMT)
FDA-CBER-2021-5683-0709416
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Page 16of 348.5.In VitroAssessmen t ofALC-0315Metabolites in Mouse, Rat, Monkeyand Human
Blood
m/z Biotransformation tR, minBlood
Mouse RatMonkey Human
102.0561aN-Dealkylation, oxidation ND NDND ND ND
104.0706bN-Dealkylation, oxidation 1.2cNDND ND ND
130.0874aN-Dealkylation, oxidation ND NDND ND ND
132.1019bN-Dealkylation, oxidation 1.9cNDND ND ND
145.0506aN-Dealkylation,
hydrolysis, oxidation7.7cND ND ND ND
255.2330aHydrolysis(acid) 19.7c++ND ND
271.2279aHydrolysis, hydroxylationND NDND ND ND
290.2690bBis-hydrolysis(amine)8.1 ++ND ND
431.2650a Hydrolysis,
glucuronidationND ND ND ND ND
464.2865aBis-hydrolysis(amine),
glucuronidationND ND ND ND ND
466.3011bBis-hydrolysis(amine),
glucuronidation7.9ND ND ND ND
528.4986bHydrolysis(amine)15.9ND +ND ND
704.5307bHydrolysis(amine ),
glucuronidationND ND ND ND ND
778.6930aOxidation to acid ND NDND ND ND
780.7076bOxidation to acid ND NDND ND ND
782.7232bHydroxylation ND NDND ND ND
844.6706aSulfation ND NDND ND ND
846.6851bSulfation ND NDND ND ND
940.7458aGlucuronidation ND NDND ND ND
942.7604bGlucuronidation ND NDND ND ND
a.Negative ion mode
b. Positive ion mode
c.Determine d usingstandard
ND–Not Detected, + = metabolite identified .
090177e194e3ef10\Approved\Approved On: 11-Sep-2020 18:13 (GMT)
FDA-CBER-2021-5683-0709417
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Page 17of 348.6.In VitroAssessment of ALC-0315Metabolites in Mouse, Rat, Monkey and Human
Hepatocyt es
m/z Biotransformation tR, minHepatocytes
Mouse RatMonkey Human
102.0561aN-Dealkylation, oxidation ND NDND ND ND
104.0706bN-Dealkylation, oxidation 1.2cNDND ND ND
130.0874aN-Dealkylation,oxidation ND NDND ND ND
132.1019bN-Dealkylation, oxidation 1.9cNDND ND ND
145.0506aN-Dealkylation,
hydrolysis, oxidation7.7cND ND ND ND
255.2330aHydrolysis(acid) 19.7c++ + +
271.2279aHydrolysis, hydroxylationND NDND ND ND
290.2690bBis-hydrolysis(amine)8.1NDND ND ND
431.2650a Hydrolysis,
glucuronidationND ND ND ND ND
464.2865aBis-hydrolysis(amine),
glucuronidationND ND ND ND ND
466.3011bBis-hydrolysis(amine),
glucuronidation7.9ND ND ND ND
528.4986bHydrolysis(amine) 15.9NDND ND ND
704.5307bHydrolysis(amine),
glucuronidatio nND ND ND ND ND
778.6930aOxidation to aci d NDNDND ND ND
780.7076bOxidation to acid ND NDND ND ND
782.7232bHydroxylation NDNDND ND ND
844.6706aSulfation ND NDND ND ND
846.6851bSulfation ND NDND ND ND
940.7458aGlucuronidatio n ND NDND ND ND
942.7604bGlucuronidation ND NDND ND ND
a.Negative ion mode
b. Positive ion mode
c.Determined us ing standard
ND–Not Detected, + = metabolite identified .
090177e194e3ef10\Approved\Approved On: 11-Sep-2020 18:13 (GMT)
FDA-CBER-2021-5683-0709418
PF-07302048 Biotransformation
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Page 18of 348.7.In VitroAssessment of ALC-0315Metabolites in Mouse, Rat , Monkey and Human
Liver S9Fractions
m/z Biotransformation t R, minLiver S9 Fractions
Mouse RatMonkey Human
102.0561aN-Dealkylation, oxidation ND NDND ND ND
104.0706bN-Dealkylation, oxidation 1.2cNDND ND ND
130.0874aN-Dealkylation, oxidation ND NDNDND ND
132.1019bN-Dealkylation, oxi dation 1.9cNDND ND ND
145.0506aN-Dealkylation,
hydrolysis, oxidation7.7cND ND ND ND
255.2330aHydrolysis(acid) 19.7c++ND +
271.2279aHydrolysis, hydroxylationND NDND ND ND
290.2690bBis-hydrolysis(amine)8.1NDND + ND
431.2650a Hydrolysis,
glucuronidationND NDND ND ND
464.2865aBis-hydrolysis(amine),
glucuronidationND ND ND ND ND
466.3011bBis-hydrolysis(amine),
glucuronidation7.9ND ND ND ND
528.4986bHydrolysis(amine) 15.9NDND + ND
704.5307bHydrolysis(amine),
glucuronidati onND ND ND ND ND
778.6930aOxidation to acid ND NDND ND ND
780.7076bOxidation to acid ND NDND ND ND
782.7232bHydroxylation ND NDND ND ND
844.6706aSulfation ND NDND ND ND
846.6851bSulfation ND NDND ND ND
940.7458aGlucuronidation ND NDND ND ND
942.7604bGlucuronidation ND NDND ND ND
a.Negative ion mode
b. Positive ion mode
c.Determined us ing standard
ND–Not Detected, + = metabolite identified .
090177e194e3ef10\Approved\Approved On: 11-Sep-2020 18:13 (GMT)
FDA-CBER-2021-5683-0709419
PF-07302048 Biotransformation
PF-07302048_05Aug20_ 043725
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Page 19of 348.8.Assessment of Meta bolites of ALC-0315in Plasma, Urine, Fe ces, and Liver from a
Rat Pharmacokinetics Study.
m/z Biotransf ormation tR, minRat
PlasmaUrineFeces Liver
102.0561aN-Dealkylation, oxidation ND ND ND ND ND
104.0706bN-Dealkylation, oxidation 1.2cND ND ND ND
130.0874aN-Dealkylation, oxidation ND ND ND ND ND
132.1019bN-Dealkylation, oxidation1.9cND ND ND ND
145.0506aN-Dealkylation,
hydrolysis, oxidation7.7cND ND ND ND
255.2330aHydrolysis(acid) 19.7c+ND ND ND
271.2279aHydrolysis, hydroxylationND ND ND ND ND
290.2690bBis-hydrolysis(amine)8.1 + + + +
431.2650a Hydrolysis,
glucuronidationND ND ND ND ND
464.2865aBis-hydrolysis(amine),
glucuronidationND ND ND ND ND
466.3011bBis-hydrolysis(amine),
glucuronidation7.9ND +ND ND
528.4986bHydrolysis(amine) 15.9+ND ND +
704.5307bHydrolysis(amine),
glucuronidationND ND ND ND ND
778.6930aOxidation to acid ND NDND ND ND
780.7076bOxidation to acid ND ND ND ND ND
782.7232bHydroxylation ND ND ND ND ND
844.6706aSulfation ND ND ND ND ND
846.6851bSulfation ND ND ND ND ND
940.7458aGlucuronidation ND ND ND ND ND
942.7604bGlucuronidation ND ND ND ND ND
a.Negative ion mode
b. Positive ion mode
c.Determined us ing standard
ND–Not Detected, + = metabolite identified .
090177e194e3ef10\Approved\Approved On: 11-Sep-2020 18:13 (GMT)
FDA-CBER-2021-5683-0709420
PF-07302048 Biotransformation
PF-07302048_05Aug20_ 043725
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Page 20of 349. SUPPORTIVE FIGURES
9.1.Proposed Biotransformation Pathway of ALC-0159in Mouse (Mo ), Rat (R),
Monkey (Mk) and Human ( H)
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Page 21of 349.2.Proposed Biotransfor mation Pathway of ALC-0315inMouse (Mo ), Rat (R),
Monkey (Mk) and Human ( H)
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PF-07302048 Biotransformation
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Page 34of 3410. CONTRIB UTING SCIENTISTS
The following scientists were involved in the conduct of this study , and are responsible for
the scientific content of this research r eport.
Contributing ADME Scien tist
11.APPROVAL
The author and approver are responsible for the accurate representation of the data in this
research report.
ReportAuthor
Pharmacokine tics, Dynamics and Metabolism, Pfizer, Groton, CT, USA
.
Report Appr over
Pharmacokinetics, Dynamics and Metabol ism, Pfizer, Groton, CT,USA
090177e194e3ef10\Approved\Approved On: 11-Sep-2020 18:13 (GMT)
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