Nanoparticles are currently being investigated in a number of human clinical trials. As information on how nanoparticles function in humans is difficult to obtain, animal studies that can be correlative to human behavior are needed to provide guidance for human clinical trials. Here, we report correlative studies on animals and humans for CRLX101, a 20- to 30-nm-diameter, multifunctional, polymeric nanoparticle containing camptothecin (CPT). CRLX101 is currently in phase 2 clinical trials, and human data from several of the clinical investigations are compared with results from multispecies animal studies. The pharmacokinetics of polymer-conjugated CPT (indicative of the CRLX101 nanoparticles) in mice, rats, dogs, and humans reveal that the area under the curve scales linearly with milligrams of CPT per square meter for all species. Plasma concentrations of unconjugated CPT released from CRLX101 in animals and humans are consistent with each other after accounting for differences in serum albumin binding of CPT. Urinary excretion of polymer-conjugated CPT occurs primarily within the initial 24 h after dosing in animals and humans. The urinary excretion dynamics of polymer-conjugated and unconjugated CPT appear similar between animals and humans. CRLX101 accumulates into solid tumors and releases CPT over a period of several days to give inhibition of its target in animal xenograft models of cancer and in the tumors of humans. Taken in total, the evidence provided from animal models on the CRLX101 mechanism of action suggests that the behavior of CRLX101 in animals is translatable to humans

Case, Roy I.

Chao, Joseph

Cole, Roderic O.

Davis, Mark E.

Eliasof, Scott

Han, Han

Hwang, Jungyeon

Lazarus, Douglas

Lin, James

Peters, Christian G.

Schluep, Thomas

Wiley, Devin T.

Yen, Yun

Zuckerman, Jonathan E.

Caltech Authors - Main

Supplementary Information 
 
Supplementary Materials and Methods: 
 
Synthesis of CRLX101 for rat PK study 
 CRLX101 was synthesized with different drug loading and polymer molecular weight for the rat 
PK experiment (Table 1) using methods described elsewhere (18). 
Cryo-TEM Imaging 
Samples of CRLX-101 (3 ul of a 2.5 mg/ml solution) were vitrified on R2-2 Quantifoil™ grids 
in liquid ethane using a Vitrobot™ (FEI Company, Hillsboro) under the conditions: 2 second 
blot, 1 second drain time, offset -4.5mm, 100% chamber humidity, 22.5 oC chamber temperature.  
Images were collected on a Tecnai (FEI Company, Hillsboro) 120keV transmission electron 
microscope equipped with a Gatan 2k×2k UltraScan CCD camera (Gatan, Pleasanton, CA).  At 
least 500 nanoparticles were sized using the “measure” function on ImageJ. 
CRLX101 In Vitro Release Kinetics 
Release of CPT from CRLX101 was conducted at 0.32 mg CPT/mL in BALB/c mice plasma, 
human plasma, and PBS at pH 7.4 in the presence and absence of 100 units/mL of 
butyrylcholinesterase and or 3 mg/mL LDL. CRLX101 was mixed into the relevant pre-warmed 
media and placed back into humidified incubator at 37 oC with 5% CO2. Samples were taken out 
at predetermined time points and immediately frozen at -80 oC until time for analysis. Fraction of 
free and bound CPT was determined as previously described (14).   
Plasma and Urine Pharmacokinetics (rat, dog, human)  
Rats were dosed at 2.59 mg/kg (Fig. 2) or 3 mg/kg CRLX101 (CPT equivalent) and beagle dogs 
were dosed at 0.58 m/kg CRLX101 (CPT equivalent) by intravenous injection. Patients from the 
Phase 1 and 2a clinical trials were dosed as described previously (7). Blood was collected at 
various time points and plasma was separated by centrifugation and frozen until sample analysis. 
Plasma was thawed and analyzed by HPLC for free, conjugated and total CPT, as described 
previously (14). PK curves were analyzed using WinNonLin (Pharsight Inc.). Patient urine from 
the Phase 1 and 2a clinical trials was analyzed for conjugated and released CPT using methods 
described elsewhere (7). Briefly, polymer was collected from the urine of patients and the CPT 
removed by treatment with NaOH (procedure similar to that used for blood and tissue PK). 
Mouse Tumor PK 
Tumor samples were homogenized in 1:1, DMSO:water, 0.1% formic acid. The homogenates 
were centrifuged and the supernatant collected for processing. Samples were treated with 2N 
NaOH to release all CPT from the polymer. Proteins were precipitated by the addition of 
acetonitrile. Samples were diluted with aqueous ammonium formate and assayed for CPT by 
HPLC/MS.  
Measurement of Mouse Tumor Topoisomerase-1 
8x106 NCI-H1299 tumor cells in 100 µL RPMI-1640 were implanted in the mammary fat pad of 
6-7 week old female homozygous NCR nude mice (Taconic Farms, Germantown, NY) and were 
dosed intravenously once with 6 mg/kg CRLX101 when the average tumor volume reached 471 
±182  mm3. At the time points indicated, mice were sacrificed, tumors were harvested and  
homogenized for immunoblotting. Immunoblotting was performed using anti-topoisomerase 1 
(1:1000, Abcam, Cambridge, MA) and anti-actin (1:2500, Santa Cruz Biologicals, Santa Cruz, 
MA) antibodies.  IRDye conjugated anti-mouse and anti-rabbit secondary antibodies (1:10000, 
LI-COR) were visualized on the Odyssey CLx infrared imaging system, and Western blots were 
quantified using Image Studio software (LI-COR Biosciences) and presented as the mean of 
three independent mice.   
Tumor Penetration 
5x106 NCI-H2122 tumor cells in 100 µL RPMI-1640 were implanted in the mammary fat pad of 
6-7 week old female homozygous NCR nude mice (Taconic Farms, Germantown, NY) and were 
dosed intravenously once with 10 mg/kg CRLX101 when the average tumor volume reached 677 
± 85 mm3. At the time points indicated, mice were sacrificed and the tumors were embedded in 
OCT and frozen. In order to positively identify CPT in these tissues, the entire tissue was 
scanned under epifluorescence that facilitated rapid screening of large areas of tissue for signal 
with CPT specific features, e.g., correct color and punctate signal as seen in the mouse xenograft 
images). Human gastric adenocarcinoma sample was obtained from a patient enrolled on a 
CRLX101 pilot trial and consented in accordance with City of Hope IRB guidelines (City of 
Hope IRB protocol #11276). The patient underwent endoscopic biopsies 48 hours post cycle 1, 
day 1 infusion of CRLX101.  The biopsies were immediately frozen in OCT media on dry ice, 
and transferred to the City of Hope Translational Research Laboratory prior to processing. 
Sections of mouse xenografts and the human tumor biopsies were imaged on a Zeiss LSM 510 
inverted confocal scanning microscope (Zeiss PlanApochormat×63/1.4 oil objective). The 
excitation wavelength for camptothecin was 710 nm (two-photon laser) and 488 nm for Sytox 
Green and Alexafluor488 labeled anti-rabbit secondary antibody for CD31 detection (CD31 
antibody, BD biosciences). Their corresponding emission filters were 390-465 nm, 565-615 nm, 
and LP 510 respectively.  
 
 
 
 
 
Figure S1. Schematic diagram of how CRLX101 is synthesized and formulated into vials. 
 
 
 
 
  
 
 
Lot	   Parent	  Polymer	  MW	  
(kDa)	  
CPT	  Loading	  	  
(wt%)	  
Particle	  Diameter	  	  
(nm)	  
A	   64	   11.4	   20	  (22)a	  
B	   65	   10.0	   20	  (25)a	  
C	   65	   11.3	   18	  (18)a	  	  
D	   63	   10.7	   21	  
E	   66	   8.6	   23	  
aValue in parenthesis is from the size measured from cryo-TEM images.  
Zeta potential measured in 10 mM phosphate buffer is ca. -5 mV. 
 
Table S1. Properties of clinical lots of CRLX101. Nanoparticle sizes were measured using 
DLS (Brookhaven Instrument) or cryo-TEM (in parenthesis). Zeta potentials were measured on a 
Zeta Pals Instrument.  
 
 
 
 
 
Figure S2. Representative cryo-TEM of CRLX101 (lot A). 
 
Figure S3. Cryo-TEM nanoparticle size distribution for lot A. 
 
 
Figure S4. Cryo-TEM nanoparticle size distribution for lot C. 
 
Figure S5. DLS nanoparticle size distribution for lot C. 
 
 
 
 
 
Dose 
Level 
(mg/m2) 
Conjugated CPT Unconjugated CPT 
Cmax 
(µg/L) 
T1/2 
(h) 
AUCinf 
(h*mg/L) 
CL 
(mL/h) 
Vss  
(L) 
Cmax 
(µg/L) 
Tmax 
(h) 
T1/2 
 (h) 
AUCinf 
(h*mg/L) 
6 3580 ( 457) 
28.9 
( 5.0) 
116 
(22) 
99 
(22) 
3.3 
(0.6) 
116 
(61) 
17.7 
(10.3) 
48.4 
(16.1) 
13.2 
(7.0) 
12 5620 ( 872) 
30.2 
(5.2) 
188 
(38) 
132 
(24) 
4.6 
(1.1) 
203 
(63) 
19.3 
(7.5) 
50.1 
(30.6) 
16.6 
(3.1) 
15 7190 (1800) 
31.5 
(1.5) 
335 
(122) 
94 
(41) 
2.3 
(1.3) 
268 
(93) 
24.5 
(1.1) 
43.3 
(7.9) 
25.4 
(8.5) 
18 8770 (1830) 
33.5 
(6.9) 
291 
(71) 
103 
(45) 
3.5 
(1.6) 
351 
(146) 
22.8 
(6.5) 
41.9 
(10.6) 
31.5 
(10.9) 
 
Table S2. Human PK parameters as a function of dose level in the Phase 1a clinical trial.  
Numbers are represented as mean and standard deviation (in parentheses). Six patients were 
evaluated at each dose level. CRLX101 lots A and E were used in this study. 
 
 
 
 Dose # N 
Conjugated CPT Unconjugated CPT 
Cmax 
(µg/L) 
t1/2 
(h) 
AUCinf 
(h*mg/L) 
CL 
(mL/h) 
Vss  
(L) 
Cmax 
(µg/L) 
tmax 
(h) 
Cycle 1 
Day 1 6 
7333	  
(1777)	  
27.6	  
(2.)	  
270	  
(53)	  
105.7	  
(19.1)	  
2.8	  
(0.5)	  
363	  
(292)	  
24.9	  
(.2)	  
Cycle 6 
Day 1 6 
7188	  
(1795)	  
27.5	  
(3.8)	  
335	  
(122)	  
94	  
(41)	  
2.3	  
(1.3)	  
302	  
(90)	  
22.1	  
(7.3)	  
 
Table S3. Human PK parameters as a function of dose from the Phase 2a clinical trial.   
All patients were dosed at 15 mg/m2 every 14 days. Numbers are represented as mean and 
standard deviation (in parentheses). There were no statistically significant differences between 
doses, as determined using unpaired, two-tailed t-test (non-equal variances). CRLX101 lots A, B 
and C were used in this study. 
 
 
 
 
 
 
 
Figure S6. Human PK did not change between the first dose (Cycle 1, Day 1) and the 
eleventh dose (Cycle 6, Day 1). Shown are (A) exposure (AUC) and (B) clearance. Lines 
connect individual patients. CRLX101 lots A, B and C were used in this study. 
Text 
Modeling of the PK Dynamics 
 
 In order to model the dynamics of the various forms of CPT in the blood, we developed a 
mathematical model from the following scheme that is used to represent the dynamics in the 
blood of animals and humans: 
 
 
 
CPT = Camptothecin that is not in the nanoparticle or bound to albumin (moles) 
CPT:NP = Camptothecin that is in the nanoparticles (moles) 
CPT:Alb = Albumin bound camptothecin (moles) 
CPT,bladder = Camptothecin excreted by kidneys into the urine (moles) 
 
The rate equations for the various forms of CPT can be written as a series of ordinary differential 
equations: 
 
 
 
with initial conditions of (the initial amount of CPT in the nanoparticles is representative of the 
values used in our animal and human studies): 
 
 
CPT:NP CPT CPT:Alb
CPT,bladder
kdeg
k2
k-2
kclear
These equations describe the nanoparticle release of CPT, the urinary clearance of non-bound 
CPT and the binding of CPT to albumin. In order to have estimates of the rates of CPT release 
and non-bound CPT clearance, we calculated rate constants from the experimental half-lives of 
the nanoparticles and CPT, respectively:  
 
 
 
 
CPT is assumed to quickly reach equilibrium with CPT:Alb and so constants k2 and k-2 can be 
constrained by: 
 
 
 
The amount of albumin in humans is 3.3 X 10-3 moles. Since this value is well above the amount 
of CPT administered, the fraction of albumin that is bound with CPT at any given time is small. 
Thus, for the simulations, we let the value for unbound albumin be constant. We solved the set of 
governing differential equations using Matlab with ode solver ode15s. The values for k2 and Keq 
are not known and were chosen in order to mimic the human data. The results give k2 = 6 x 103 
hr-1 mole-1  and Keq = 3 x 103 mole-1 and the dynamics are shown in Fig. S7.  
 
The same governing differential equations, initial conditions, and rate constants apply to SN-38, 
except for its binding strength to human albumin, described by Keq, that is reported to be 50 
times lower than CPT (1). Decreasing Keq to 60 mole-1 the blood profiles for SN-38 are 
illustrated in Fig. S8. 
 
References: 
 
1.   Y. Kurono, M. Miyajima, K. Ikeda, Interaction of Camptothecin Derivatives with Human 
Plasma Proteins in Vitro.  Yagaku Zasshi 113, 167-175 (1993). 
2.   Z. Mi, T. G Burke, Marked Interspecies Variations Concerning the Interactions of 
Camptothecin with Serum Albumins: A Frequency-Domain Fluorescence Spectroscopic Study. 
Biochemistry 33, 12540-12545 (1994). 
 
 
 
Figure S7. Simulated CPT profiles. 
 
 
Figure S8. Simulated SN-38 profiles. 
 
 
 
 
 
 
Formulation	  
Physical	  Properties	   Conjugated	  CPT	   Unconjugated	  CPT	  
Parent	  
Polymer	  
MW	  
(kDa)	  
Loading	  	  
(weight	  
%)	  
Particle	  
Size	  
(nm)	  
Cmax	  
(ug/mL)	  
T1/2	  
(h)	  
AUC	  
(hr*ug/mL)	  
Cmax	  
(ug/mL)	  
AUC	  
(hr*ug/mL)	  
CRLX101	   64	   11.4	   20	   74.5	  (13.9)	  
18.9	  
(2.4)	  
874	  
(29)	  
1.27	  
(0.78)	  
5.69	  
(1.38)	  
LMW-­‐LL	   47.5	   5.4	   23	   84.1	  (10.8)	  
18.1	  
(3.7)	  
768	  
(36)	  
1.53	  
(0.39)	  
6.98	  
(1.53)	  
LMW-­‐HL	   47.5	   10.8	   17	   85.2	  (2.8)	  
23.9	  
(1.6)	  
931	  
(25)	  
1.05	  
(0.21)	  
4.923	  
(0.70)	  
HMW-­‐LL	   86.6	   6.4	   31	   93.2	  (4.8)	  
18.0	  
(3.4)	  
1200	  
(78)	  
1.57	  
(0.16)	  
8.95	  
(0.61)	  
HMW-­‐HL	   86.6	   9.9	   31	   78.4	  (5.2)	  
21.2	  
(3.3)	  
1069	  
(61)	  
0.88	  
(0.18)	  
5.00	  
(0.79)	  
 
Table S4. Rat PK parameters as a function of formulation, where polymer molecular 
weight and drug loading were varied.   
 
 
 
Lot N 
Conjugated CPT Unconjugated CPT 
Cmax 
(µg/L) 
T1/2 
(h) 
AUCinf 
(h*mg/L) 
CL 
(mL/h) 
Vss 
(L) 
Cmax 
(µg/L) 
Tmax 
(h) 
T1/2 
(h) 
AUCinf 
(h*mg/L) 
A 24 8818	  (1300)	  
28.3	  
(3.7)	  
298	  
(67)	  
97	  
(30)	  
2.8	  
(1.1)	  
297	  
(80)	  
25.6	  
(8.6)	  
46.2	  
(11.5)	  
31.9	  
(11.5)	  
B 18 7694	  (1238)	  
28.5	  
(1.9)	  
312	  
(48)	  
88	  
(22)	  
2.3	  
(0.6)	  
305	  
(217)	  
21.3	  
(7.9)	  
42.5	  
(11.)	  
23.4	  
(4.3)	  
 
Table S5. Human PK parameters from patients receiving doses from two different lots in 
the Phase 2a clinical trial.  Numbers are represented as mean and standard deviation (in 
parentheses). N= the number of patients. 
 
 
 
Figure S9. No consistent changes in patient urine excretion were observed when individual 
patients were dosed with two different lots of CRLX101. Data points represent total 
conjugated (A,C) and unconjugated (B,D) CPT measured in urine, normalized to the dose 
delivered to patients that were dosed with two different lots of CRLX101 at different times 
during their treatment course. Data points represent mean and error bars represent standard 
deviation. Lines connect individual patients.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure S10.  Fluorescence from CPT component of CRLX101.  (A) Fluorescence spectrum of 
camptothecin. Exmax = 370 nm and  Emmax = 435nm. (B) Confocal micrograph of a section of 
NCI-H2122 Kras mutant NSCLC xenografts 24hrs after dosing with CRLX101. Sections with 
anti-rabbit secondary only, demonstrating no non-specific signal in the 488nm channel. (C) 
Confocal micrograph of a section of NCI-H2122 Kras mutant NSCLC xenografts 24hrs after 
dosing with vehicle control only (no CPT).  Autofluorescence was observed in 370nm channel 
used to image CRLX101.   
 
 
 
 
 
 
 
Figure S11. Inhibition of Topoisomerase-1 by CRLX101. NCI-H1299 NSCLC tumor-bearing 
mice were injected IV with CRLX101 at 6 mg/kg and topoisomerase-1 expression was measured 
in tumors at the time points indicated after treatment via Western blots. These data were used to 
generate the plot shown in Fig. 5D. 
 
 
 
Figure S12. Inhibition of HIF-1alpha by CRLX101. NCI-H1299 NSCLC tumor-bearing mice 
were injected IV with CRLX101 at 6 mg/kg and HIF-1alpha expression was measured in tumors 
72 hours after treatment via Western blots.  
 
 
 
  
Vehicle 
1 Day 
3 Days 
5 Days 
7 Days 
9 Days 
11 Days 
13 Days 
15 Days 
17 Days 
Loading 
Control 
Vehicle CRLX101 


Correlating preclinical animal studies and human clinical trials of a multifunctional, polymeric nanoparticle

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Correlating preclinical animal studies and human clinical trials of a multifunctional, polymeric nanoparticle

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