Electronic Supplemental Material
Morphine and Metabolites in Young Children: a Population Pharmacokinetic Model
Authors: Nieves Velez de Mendizabal*1,2, Ricardo Jimenez-Mendez*3, 4, 5, Erin Cooke6, Carolyne J.
Montgomery6, Joy Dawes6, Michael J. Rieder7, Katarina Aleksa8, Gideon Koren8, Carlos O. JacoboCabral9, Rodrigo Gonzalez-Ramirez9, Gilberto Castañeda-Hernandez9 and Bruce C. Carleton3,4,5
Affiliations:
1
Division of Clinical Pharmacology, Department of Medicine, Indiana University School of Medicine,
Indianapolis
2
Indiana Clinical and Translational Sciences Institute (CTSI), Indianapolis, IN, USA
3
Division of Translational Therapeutics, Department of Paediatrics, Faculty of Medicine, University of
British Columbia, Vancouver, Canada
4
Pharmaceutical Outcomes Programme, BC Children’s Hospital, Vancouver, Canada
5
Child & Family Research Institute, Vancouver, Canada
6
Pediatric Anesthesia Research Team, University of British Columbia and Department of Pediatric
Anesthesia, BC Children's Hospital, Vancouver, Canada
7
Department of Physiology and Pharmacology, Western University, London, Canada
8
The Motherisk Program, Division of Clinical Pharmacology/Toxicology, The Hospital for Sick Children
Toronto, Canada
9
Departamento de Farmacología, Centro de Investigación y de Estudios Avanzados del Instituto
Politécnico Nacional, México, DF.
Corresponding author:
Dr. Bruce C. Carleton
Child and Family Research Institute
950 West 28th Avenue, Rm. A3-207
Vancouver, BC. V5Z 4H4, Canada
e-mail: bcarleton@popi.ubc.ca
* Nieves Velez de Mendizabal and Ricardo Jimenez-Mendez contributed equally to this work.
Running Title: PK model for morphine and its metabolites
Keywords: Morphine, M3G, M6G, NONMEM, children
Abbreviations: M3G, morphine-3-glucoronide; M6G, morphine-6-glucoronide; IV, intravenous; ISV,
inter-subject variability; CWRES, conditional weighted residuals; NPDE, normalized prediction
distribution errors; RSE, relative standard error; CV, coefficient of variation; PK, pharmacokinetics
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Online Resource 1: Bio-analytical assay
After induction of anaesthesia, a second IV cannula was inserted. Blood samples were drawn at 30, 60,
90, 120, 180 and 240 minutes. Blood samples were collected in heparinized tube and refrigerated.
Samples were centrifuged at 3000 bpm and stored at -80 °C until plasma concentration of morphine,
M6G and M3G were measured using a validated high performance liquid chromatography essay.
Sample preparation summary: Morphine and morphine glucuronides (morphine-6-glucuronide (M-6-G)
and morphine-3-glucuronide (M-3-G)) were extracted from plasma by solid-phase extraction on C18
cartridges at pH 9.3. Method from Leis HJ et al., (2002)
LC-MS/MS summary
Analysis was performed on an ABSciex QTRAP5500 (ABSciex: Framingham, Massachusetts, USA) and
Agilent 1290 LC system (Agilent Technologies: Santa Clara, California, USA).
Chromatography ran at a flow rate of 800 µL/min on a Kinetex HILIC column 4.6x50mm, 2.6 µm (Agilent)
with a gradient starting at 5% A (90/10 Water/Acetonitrile 5 mM ammonium Formate pH 3.2) and 95% B
(10/90 Water/Acetonitrile 5 mM ammonium Formate pH 3.2) ramping to 100% A at 5 minutes. This
method allowed for baseline chromatographic resolution of the 2 glucuronide isomers.
The mass spectrometer was operated in positive ESI mode with a source temperature of 650°C and an IS
Voltage setting of 5000. Precursor to product ion mass transitions were established by standard
infusions. Data was acquired by MRM mode (Multiple Reaction Monitoring) with mass transitions as
follows:
286→ 165 m/z for Morphine, 289 → 165 m/z for Morphine-d3 (RT 3.0 min)
462 → 286 m/z for Morph-6-Gluc, 465 → 289 for Morph-6-Gluc-d3 (RT 4.85 min)
462 → 286 m/z for Morph-3-Gluc, 465 → 289 for Morph-3-Gluc-d3 (RT. 5.05 min)
Data analysis and peak integration was performed using Analyst 1.5.2 software from ABSciex. Sample
concentrations were calculated by plotting peak area ratios (Analyte/Internal Standard) against
calibration curves of extracted matrix spiked standards.
Quality control follow-up



Sample concentrations were calculated by plotting peak area ratios (Analyte/Internal Standard)
against calibration curves of extracted matrix spiked standards
Linearity was verified by presenting regression line coefficients (r2). The Acceptance Criteria of
the r2 of the regression line was > 0.99
Quality controls of extracted matrix spiked standards were run along with the unknown
samples, and their concentrations were calculated by plotting peak area ratios (Analyte/Internal
Standard) against calibration curves of extracted matrix spiked standards. Percent recovery was
>80% and <120%.
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Online Resource 2: Ordinary Differential Equations ODEs of the selected PK model for
morphine and its metabolites M3G and M6G
dA1
  A1 (t)  k a
dt
dA 2
CL EX
k
 A1 (t)  k a  A 2 (t) 
 A 2 (t)  m
dt
VC
VC
dA 3 CL 2M3G
CL M3G

 A 5 (t) 
 A 3 (t)
dt
VC
VM3
CL M6G
dA 4 CL 2M6G

 A 5 (t) 
 A 4 (t)
dt
VC
VM6
dA 5
CL 2M3G
CL 2M6G
k
 A 2 (t)  m 
 A 5 (t)  A 5 (t)
dt
VC
VC
VC
where A1(t), A2(t), A3(t), A4(t) and A5(t) represent depot, morphine central, metabolite M3G
central, metabolite M6G central and delay transit compartment respectively; ka first order rate
constant of absorption; VC/F is apparent volume of distribution of the central compartment; kmf is the
first order rate constant for the metabolism transit compartment, CL2M3G and CL2M6G are the formation
rates for M3G and M6G respectively and CLM3G and CLM6G are the elimination rates. The metabolite
volumes of distribution (V3M, V6M) were parameterized as VC/F.
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Online Resource 3: NONMEM model
$SUBROUTINES ADVAN13 TOL=9 ;TIME (MINUTES)
$MODEL
COMP(DEPOT)
COMP(PARENT) ; 2 CENTRAL MORFINA
COMP(M3G)
; 3 METABOLITO M-3-G
COMP(M6G)
; 4 METABOLITO M-6-G
COMP(METAB) ; 5 LIVER METABOLISM
$PK
KA = THETA(1)*EXP(ETA(1))
; KA (min-1)
VC = THETA(2)*EXP(ETA(2))
; V CENTRAL MORPHINE (L)
CLEX = THETA(3)*EXP(ETA(3))
; CLEX MORPHINE-ELIMINATION (L/min)
F1 = THETA(4)*EXP(ETA(4))
; Bioavailability
CL2M3G = THETA(5)*EXP(ETA(5)) ; FORMATION DE M-3-G
CLM3G = THETA(6)*EXP(ETA(6)) ; ELIMINATION DE M-3-G
CL2M6G=THETA(7)*EXP(ETA(7)) ; FORMATION DE M-6-G
CLM6G=THETA(8)*EXP(ETA(8)) ; ELIMINATION DE M-6-G
KMF=THETA(9)*EXP(ETA(9))
; METABOLISM (DELAY)
VM3=VC
; V MORPHINE-3-GLUCURONIDO
VM6=VC
; V MORPHINE-6-GLUCURONIDO
;-------- scales -------S2=VC/1000
; AMT= mg, DV= ng/ml
S3=VM3G/1000 ; AMT= mg, DV= ng/ml
S4=VM6G/1000 ; AMT= mg, DV= ng/ml
$DES
DADT(1)=-KA*A(1)
DADT(2)=KA*A(1)-(CLEX/VC)*A(2)-(KMF/VC)*A(2)
;MORPHINE
DADT(3)=(CL2M3G/VC)*A(5)-(CLM3G/VM3)*A(3)
;M-3-G
DADT(4)=(CL2M6G/VC)*A(5)-(CLM6G/VM6)*A(4)
;M-6-G
DADT(5)=(KMF/VC)*A(2) -(CL2M3G/VC)*A(5) - (CL2M6G/VC)*A(5)
…
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Online Resource 4: Individual and population predictions versus observations
Fig. S1 Individual and population predictions versus observations. Dots stand for observations, solid
lines for the model individual predictions and dashed red lines for population predictions. Grey color
represents morphine, blue M3G and green M6G concentrations. Results are stratified by dose: (A) 0.1
mg/kg; (B) 0.2 mg/kg and (C) 0.3 mg/kg.
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