Magnetic resonance imaging with an iron oxide nanoparticle

Magnetic resonance imaging with an iron oxide nanoparticle demonstrates preclinically the feasibility of predicting
intratumoral uptake and activity of MM-398, a nanoliposomal irinotecan (nal-IRI)
Ashish Kalra1, Joseph Spernyak2, Jaeyeon Kim1, Walid Kamoun1 , Arnold Sengooba1, Stephan Klinz1, Nancy Paz1, Jason Cain1,
Ninfa Straubinger3, Yang Qu3, Sheryl Trueman3, Eliel Bayever1, Ulrik Nielsen1, Daryl Drummond1, Jonathan Fitzgerald1, Robert M. Straubinger3
1 Merrimack Pharmaceuticals, Cambridge, MA; 2 Roswell Park Cancer Institute, Buffalo, NY; 3 University at Buffalo, SUNY, Buffalo, NY
CES activity in tumor
CES activity in blood
Irinotecan 40 mg/kg
2
nal-IRI permeability
SN-38 permeability
CPT-11 permeability
10
nal-IRI 40 mg/kg
0
24
48
72
96 120 144 168
-0.5
0
0.5
60
72
1
1
23
sP
C
C. Quantifying tumor uptake of
labeled (DiI5) liposomes
D
A
AM
B
1
80
27
A
T2 Rate (1/s)
A549
A2780
iron concentration
( g/ml)
M
B2
31
As
PC
1
27
80
A
SK
ES
1
M
DA
48
M
Liposome
-1
Ferumoxytol (Prussian blue)
1
Sensitivity
Macrophages (F4/80)
A549
A2780
D. Tumor irinotecan (CPT-11) and SN-38 concentrations correlate with the
intratumor concentrations of FMX
Macrophage Content
Experimental validation
4X
Nuclei
DiI5-Liposome
Bright field
Pseudocolor
4X
B. Correlating FMX uptake to liposome uptake in 18254 (pancreatic PDX) and HT-29 (colon) xenograft
Ferumoxytol positive area
Tumor activation
1. nal-IRI is a 100nm nanoliposomal formulation of
irinotecan that displays higher plasma and tumor
exposure compared to free irinotecan in preclinical
models
2. Sensitivity analysis highlights two parameters, (1) tumor
drug uptake: “tumor deposition” and (2) ability of tumor
carboxylesterase enzyme to activate pro-drug irinotecan
to SN-38: “tumor activation” as critical factors driving nalIRI in vivo activity
3. Experimental data in a panel of xenograft models confirm
the critical role of tumor drug deposition and activation.
4. Ferumoxytol (FMX) is a 30nm iron-oxide, superparamagnetic nanoparticle, known to be taken up by
macrophages
5. We are currently evaluating the feasibility of correlating
FMX-based MRI acquisition to estimate tumor drug
delivery in a pilot clinical study (NCT01770353)
SK
ES
A5
49
Tumor SN-38 (ng/g)
Tumor SN38 (ng/g)
A
54
9
Tumor CPT-11 (ng/g)
Plasma CPT-11 (ng/ml)
Plasma SN-38 (ng/ml)
nal-IRI clearance rate in plasma
SN-38 clearance rate in plasma
Time (hr)
36
B. IHC analysis to measure intratumor FMX
uptake (Prussian blue staining)
Ferumoxytol co-localizes with labeled liposomes and TAMs
CPT-11 clearance rate in plasma
10
24
Time (hr)
A. IHC analysis to determine the intratumor distribution of liposomes, ferumoxytol and
tumor associated macrophages in a pancreatic PDX tumor
1
irinotecan payload
12
Tumor CPT-11 at 72h
(ng/g)
Internal
aqueous
space
Liposome release rate in tumor
Liposome release rate in blood
deposition
0
Tumor SN-38 at 72h
(ng/g)
100110nm
Tumor SN-38 (nM)
Lipid
Membran
e
10
*
Sensitivity analysis
activation
A549
20
0
Figure 1. (A) HT-29 colon tumor bearing NOD SCID mice
were injected (i.v.) with FMX followed (24h later) with nalIRI (20 mg/kg). (B) Tumor bearing NOD SCID mice were
injected with FMX (20 mg/kg) followed (24h later) with nalIRI (10 mg/kg). Plasma and tumor samples were analyzed
by HPLC to determine CPT-11 and SN-38 levels. (C) HT-29
colon tumor bearing NOD SCID mice were treated with
FMX (20 mg/kg) followed by injection of DiI5-liposomes
4
3
A2780
HT29
30
C. Ex vivo FACS analysis to determine impact of FMX administration on liposome
uptake by tumor cells (EPCAM+) and macrophages (CD11b+)
10
10
40
Tumor CPT-11 at 72h
(ng/g)
Tumor PK
A. Iron concentration was estimated from MRI acquisition (T2 relaxation rates)
across different xenograft models
Kidney
Tumor CPT-11 (ng/g)
tumor
plasma
Fe Signal
nal-IRI
B. nal-IRI uptake in a panel of
xenografts following FMX
A. Effect of FMX administration on nal-IRI PK
nal-IRI and ferumoxytol (FMX) background
PEG-DSPE
Tumor models with high ferumoxytol concentrations showed
correlation with high intratumor irinotecan pro-drug levels
nal-IRI PK and intratumor distribution remains unaltered by
ferumoxytol
Sustained intratumoral delivery of cytotoxic agents is a major challenge for effective cancer treatment, and motivated
the development of MM-398, nanoliposomal irinotecan (nal-IRI) that has been shown to have an extended plasma
half-life and greater tumor deposition than free irinotecan in preclinical models. We have previously shown through a
systems pharmacology approach that tumor deposition of nal-IRI and the subsequent carboxylesterase conversion of
irinotecan to the active metabolite, SN-38, are determinants for nal-IRI activity in vivo.
Ferumoxytol (FMX) is a 30nm iron-oxide, super-paramagnetic nanoparticle, known to be taken up by macrophages (as
is nal-IRI), and exhibits magnetic resonance imaging properties. Since the size of a nanoparticle affects the rate of
transcapillary transport significantly, we hypothesized that nal-IRI tumor biodistribution may be predicted by FMXbased MRI (Fe-MRI).
Biodistribution and imaging studies were performed in mice bearing cell-line derived (A2780, HT-29, A549) and
patient-derived (pancreatic adenocarcinoma) tumor xenografts. The protocol consisted of a baseline MRI scan, an i.v.
injection of FMX (20 mg/kg), followed by an i.v. injection of fluorescently labeled nal-IRI (10 mg/kg) 24hr later. Mice
were sacrificed 24hr and 72hr after nal-IRI injection, and irinotecan and SN-38 concentrations were determined in
plasma, tumor, and tissues by HPLC analysis.
The presence of FMX did not interfere with nal-IRI PK or biodistribution. Cellular distribution of liposomes within
tumors was also not affected by FMX at up to 50 mg/kg as measured by flow cytometry. Furthermore,
immunohistochemistry showed that both liposomes and FMX were co-localized with tumor-associated macrophages.
The drug metabolite measurements from tissue samples showed that the xenograft tumor models display wide ranges
of nal-IRI deposition capacity (irinotecan concentrations at 24hr: 2,104-20,096 ng/g). A2780 tumors displayed the
highest concentration of both iron (3.92 mg/ml) and irinotecan (9,466 ng/g) at 72hr after nal-IRI injection, whereas
A549 tumors displayed the lowest levels of both iron (0.23 mg/ml ) and irinotecan (436 ng/g). We observed a
correlation between the tumor Fe-MRI signal and intratumoral levels of irinotecan 72hr after nal-IRI injection (R2=0.9,
p<0.001). Furthermore, in vivo activity studies confirmed that xenograft models with higher intratumoral levels of
irinotecan and SN-38 at 72hr showed greater tumor growth inhibition.
In summary, preclinical studies demonstrate the potential of utilizing Fe-MRI as a potential diagnostic tool to identify
patients with higher tumor permeability. Based on encouraging preclinical data, a pilot study in patients with
advanced solid tumors with extensive Fe-MRI scanning and paired tumor biopsies (NCT # 01770353) is being
conducted.
iron concentration
( g/ml)
Abstract
Abstract #2065
Tumor deposition
Ferumoxytol
269
254
Liposome uptake
(Dil5 positive area)
Di5-Liposome
Figure 2. Pancreatic patient-derived
xenograft models 18254 and 18269
injected with FMX (20 mg/kg) followed
by DiI5-labeled liposomes. (A) IHC
analysis of 18254 model shows colocalization of liposomes, FMX and
macrophages. (B) Correlating liposome
uptake to FMX levels within tumors
(left panel). Comparing co-localization
of FMX and DiI5 liposomes in HT-29
xenograft (right panel).
Figure 3. (A) CB17 tumor bearing mice were injected with FMX (20 mg/kg). Contrast enhanced MRI was performed
using 4.7T CSI magnet incorporating Bruker AVANCE electronics and the ParaVision 3.0.2 imaging. Iron concentrations
were extrapolated from the T2 relaxation rates using a phantom FMX standard curve. A2780 tumors displayed higher
FMX (B) and DiI5 liposome (C) uptake compared to A549 tumors. (D) FMX-MRI confirms high FMX uptake in A2780
tumors which correlates with irinotecan (CPT-11) and SN-38 tumor levels.
Summary
• In preclinical studies ferumoxytol is co-localized with liposomes as well as tumor associated
macrophages within the tumor microenvironment and does not alter the pharmacokinetic properties
of nal-IRI.
• Contrast-enhanced MRI using ferumoxytol shows correlation between the FMX MRI signal and tumor
drug uptake particularly in tumors with high liposomal drug delivery.
• A clinical study to assess the feasibility of ferumoxytol as tumor imaging agent prior to investigational
nal-IRI treatment is ongoing (NCT01770353).

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