Development of a Prodrug of Camptothecin for Enhanced Treatment of Glioblastoma Multiforme

Article abstract of DOI:10.1021/acs.molpharmaceut.0c00968

A novel therapeutic approach for glioblastoma multiforme (GBM) therapy has been carried out through in vitro and in vivo testing by using the prodrug camptothecin-20-O-(5-aminolevulinate) (CPT-ALA). The incorporation of ALA to CPT may promote uptake of the cytotoxic molecule by glioblastoma cells where the heme synthesis pathway is active, improving the therapeutic action and reducing the side effects over healthy tissue. The antitumor properties of CPT-ALA have been tested on different GBM cell lines (U87, U251, and C6) as well as in an orthotopic GBM model in rat, where potential toxicity in central nervous system cells was analyzed. In vitro results indicated no significant differences in the cytotoxic effect over the different GBM cell lines for CPT and CPT-ALA, albeit cell mortality induced by CPT over normal cell lines was significantly higher than CPT-ALA. Moreover, intracranial GBM in rat was significantly reduced (30% volume) with 2 weeks of CPT-ALA treatment with no significant side effects or alterations to the well-being of the animals tested. 5-ALA moiety enhances CPT diffusion into tumors due to solubility improvement and its metabolic-based targeting, increasing the CPT cytotoxic effect on malignant cells while reducing CPT diffusion to other proliferative healthy tissue. We demonstrate that CPT-ALA blocks proliferation of GBM cells, reducing the infiltrative capacity of GBM and promoting the success of surgical removal, which improves life expectancy by reducing tumor recurrence.

Full text of DOI:10.1021/acs.molpharmaceut.0c00968

pubs.acs.org/molecularpharmaceutics
Article
Development of a Prodrug of Camptothecin for Enhanced
Treatment of Glioblastoma Multiforme
Elisa Checa-Chavarria, Eva Rivero-Buceta, Miguel Angel Sanchez Martos, Gema Martinez Navarrete,
Cristina Soto-Sánchez, Pablo Botella,* and Eduardo Fernández*
Cite This: Mol. Pharmaceutics 2021, 18, 1558−1572
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sı Supporting Information
ABSTRACT: A novel therapeutic approach for glioblastoma multi-
forme (GBM) therapy has been carried out through in vitro and in vivo
testing by using the prodrug camptothecin-20-O-(5-aminolevulinate)
(
CPT-ALA). The incorporation of ALA to CPT may promote uptake of
the cytotoxic molecule by glioblastoma cells where the heme synthesis
pathway is active, improving the therapeutic action and reducing the
side e�ects over healthy tissue. The antitumor properties of CPT-ALA
have been tested on di�erent GBM cell lines (U87, U251, and C6) as
well as in an orthotopic GBM model in rat, where potential toxicity in
central nervous system cells was analyzed. In vitro results indicated no
signi�cant di�erences in the cytotoxic e�ect over the di�erent GBM cell
lines for CPT and CPT-ALA, albeit cell mortality induced by CPT over
normal cell lines was signi�cantly higher than CPT-ALA. Moreover,
intracranial GBM in rat was signi�cantly reduced (30% volume) with 2 weeks of CPT-ALA treatment with no signi�cant side e�ects
or alterations to the well-being of the animals tested. 5-ALA moiety enhances CPT di�usion into tumors due to solubility
improvement and its metabolic-based targeting, increasing the CPT cytotoxic e�ect on malignant cells while reducing CPT di�usion
to other proliferative healthy tissue. We demonstrate that CPT-ALA blocks proliferation of GBM cells, reducing the in�ltrative
capacity of GBM and promoting the success of surgical removal, which improves life expectancy by reducing tumor recurrence.
KEYWORDS: glioblastoma multiforme, camptothecin, 5-aminolevulinic acid, blood−brain barrier, targeting
1
. INTRODUCTION
theless, even with such aggressive treatment, the risk of
7
recurrence is very high. Consequently, the average survival of
Glioblastoma multiforme (GBM) is the most aggressive and
lethal type of brain tumor, having a median survival of
approximately 15 months. It is considered as level IV glioma by
the World Health Organization. It is the most frequent type of
primary tumor of the central nervous system (CNS), with 2−3
new diagnosis per 100 000 inhabitants every year. It has a
8
,9
patients is barely 15 months after diagnosis.
When
recurrence is present, the common treatment used is
10
1
bevacizumab. The major advantage of temozolamide and
bevacizumab is their capacity to move through the BBB, the
major obstacle for the di�usion of chemotherapeutics agents
1
,2
11
into the CNS.
characteristic genetic pro�le that favors a high level of mitotic
activity and angiogenesis, inhibiting apoptosis and enabling a
3
In this context, camptothecin (CPT) is a natural pentacyclic
alkaloid isolated from the oriental tree Camptotheca accuminata
quick generation of resistance against treatment. It also
preserves the capability to migrate from precursor cells,
astrocytes, in�ltrating into the cortical tissue and leading to
metastasis in other regions of the CNS. Due to all these
characteristics, GBM has the ability to grow and evolve faster
than any other CNS tumor, increasing its malignant
phenotype, poor prediction, and lethality. Moreover, the
protection provided by the blood−brain barrier (BBB)
converts GBM into one of the tumor types more di�cult to
cure and with stronger resistance.
12
by Wall et al. in 1966, which is well-known for its antitumor
13
activity against a wide spectrum of human cancers. CPT
4
,5
stabilizes via a noncovalent link the complex formed by the
topoisomerase I and DNA during replication. As a result, the
enzyme is inhibited and DNA structure is damaged, leading to
apoptosis. Because of the mechanism of actuation of this drug,
6
Received: September 26, 2020
Revised: February 20, 2021
Accepted: February 22, 2021
Published: March 1, 2021
7
Current standard therapies include the surgical removal of as
much tumor tissue as possible, followed by an adjuvant
treatment with radiotherapy and chemotherapy, using
8
temozolomide, also known as the Stupp protocol. Never-
©
2021 American Chemical Society
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tumors with a high index of proliferation like GBM are more
2. MATERIALS AND METHODS
.1. General. All reagents and solvents were purchased
from Sigma-Aldrich except for CPT from ABCR and HPLC
solvents (HPLC grade from Scharlab or LC/MS grade Optima
from Fisher).
14,15
sensitive to CPT and its analogues.
Unfortunately, CPT
2
presents some major pharmacological limitations that preclude
its clinical application, like poor water solubility and rapid
lactone ring hydrolysis at physiological pH, leading to the
16
inactive carboxylate form, and hampered di�usion through
the BBB.
Reversed-phase high performance liquid chromatography
RP-HPLC) analysis was performed on an Agilent 1220
(
In an e�ort to improve CPT solubility and pharmacoki-
netics, di�erent structural derivatives have been developed.
Most derivatives of CPT have been obtained through the
substitution of the quinolone ring. However, to date, only two
of these CPT analogues have been approved for clinical use.
These are irinotecan (CPT-11) and topotecan, which are able
In�nity LC coupled to a �uorescence detector 1260 In�nity
with an analytical column (Mediterranean Sea C18, 5 �m, 100
mm × 21 mm). The products were eluted utilizing a constant
solvent mixture (CH CN/H O−TFA, pH 4.5, 50:50 v/v) at
3
2
0.8 mL/min. NMR spectra were recorded on a Bruker AV300
1
Ultrashield spectrometrer. H NMR spectra were acquired at
17
to cross the BBB, but their intrinsic activity is clearly lower
300 MHz employing pulses of 15 �s and a recycle time of 1 s.
13
1
than the pristine drug. Recent studies have been focused on
di�erent conjugates (ester, amide, carbonate, etc.) at the C20
position. These systems improve CPT delivery and bioavail-
ability and may also introduce alternative administration routes
Data for H spectra are reported as follows: chemical shift,
multiplicity (s = singlet, d = doublet, dd = doublet of doublet, t
=
triplet, dt = doublet of triplet, m = multiplet), and
1
3
integration. To obtain C spectra 9 �s pulses at 75 MHz
were applied with a recycle time of 2 s. Both H and
1
13
18−21
C
for parenteral injection.
Conversely, in order to use CPT
experiments were carried out using tetramethylsilane (TMS) as
chemical shift reference. Quadrupole time-of-�ight (Q-ToF)
mass spectra were recorded on an Acquity UPLC Waters
coupled with Xevo QToF MS with an Acquity UPLC BEH
C18 (1.7 �m, 50 mm × 21 mm) column and using positive
electrospray ionization. The products were eluted utilizing a
against GBM, it is compulsory to improve the di�usion to the
CNS.
In this scenario, our group recently described a CPT
prodrug with 5-aminolevulinic acid (5-ALA): camptothecin-
22
2
0-O-(5-aminolevulinate) (CPT-ALA). The rationale for
using 5-ALA to synthesize the prodrug is based on the
targeting and photodynamic properties of this molecule. First,
linear gradient solvent mixture CH CN (0.01% HCOOH)/
3
−1
H O (0.01% HCOOH) at 0.3 mL min (0−13 min, 80:20;
23
2
5
-ALA is able to go through the BBB, which could favor the
1
3−17 min, 0:100; 17−20 min, 80:20). All data collected in
di�usion to the CNS of small molecules like CPT. Second, 5-
ALA is a metabolic precursor of protoporphyrin IX
centroid mode were acquired using Masslynx software (Waters
Corp.).
2
4,25
(
6
PpIX),
a photoactive compound emitting red light at
Cell lines and primary culture cells were incubated at 37 °C
26
35 nm when excited with 375−440 nm wavelength. In this
context, it has been reported that 5-ALA uptake and PpIX
under a humidi�ed atmosphere of 5% CO . Rat C6
2
glioblastoma cells, human U87 and human U251 glioblastoma
cells, and rat cortex primary culture cells were used to evaluate
CPT-ALA anticancer activity and toxicity at nontumor tissues.
All glioblastoma cell lines were purchased from American Type
Culture Collection (ATCC, VA, USA). Cell lines were seeded
in 96-well culture plates in a �nal culture medium volume of
200 �Lwell, using the following seeding densities; C6 and U87
25 000 cell/mL and U251 50 000 cell/mL. C6 cells also were
seeded in 12-well culture plates in a �nal culture medium
volume of 1 mL/well. All cell lines were maintained using
DMEM (1×) (GIBCO by Life Technologies) supplemented
with 10% fetal bovine serum (FBS-Biowest) and penicillin and
streptomycin (Gibco by Life Technologies) 1:100 (v/v).
Rat cortex primary cells were isolated from Sprague Dawley
rat embryos at day E17-E18. Mechanically dissociated tissue
was added and incubated at 37 °C for 15 min with DMEM and
27
synthesis are greatly increased in GBM, which enhances the
targeting role of 5-ALA. Indeed, 5-ALA is used in clinical
practice to locate the tumor in the brain, which enables precise
surgical removal of GBM by �uorescence guided resection,
maximizing tumor resection and having an important e�ect in
improving patient healthcare. Finally, the endogenous PpIX
accumulation promoted by 5-ALA increases tissue photo-
sensitivity. For this reason, ALA has also been used in
photodynamic therapy to treat several types of non-melanoma
28
2
9
skin cancers,
investigated.
and its e�ect in GBM is also being
3
0
In this work, we present a preclinical study of CPT-ALA
prodrug as a novel therapeutic approach for GBM therapy,
showing its anticancer and cytotoxic properties. The
incorporation of 5-ALA to CPT follows a double strategy:
31
trypsine for chemical dissociation. Both cellular cultures were
originated from cortical cells: isolated astrocytes culture and
cortical cells culture were mostly composed of neurons and
astrocytes. The use of both cultures allowed us to determine
the e�ect of CPT-ALA in precursor cells to GBM and other
CNS cells. Cortical cells obtained with this process were
maintained in di�erent culture mediums with the �nal purpose
to obtain both cortical cell culture and astrocytes cell culture.
�rst, promoting CPT di�usion through the BBB and then
targeting the cytotoxic molecule to GBM cells, which should
improve the therapeutic action, and in parallel, reducing the
side e�ects over healthy tissue. For this reason we tested the
antitumor properties of this prodrug on di�erent GBM cell
lines (U87, U251, and C6) as well as in an orthotopic
glioblastoma model in rat while analyzing the potential toxicity
over healthy tissue and CNS cells. Our results suggest that
CPT-ALA prodrug shows high antitumor activity both in vitro
and in vivo, without producing any neuronal damage. In this
sense, our therapeutic system presents a superior potential for
GBM therapy than CPT analogues already approved for
clinical use.
96-well plate pretreated with poly-D-lysine (PDL, Sigma-
Aldrich) and laminine (Sigma-Aldrich) were used to seed both
cells types. Cortical cells were seeded with density 65 000 cell/
mL and maintained in Neurobasal medium (GIBCO by Life
Technologies) supplemented with 2% FBS, 2% B27 growth
factor (GIBCO by Life Technologies), 0.4% Glutamax
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(
1
6
GIBCO-Invitrogen), and 0.4% penicillin and streptomycin
:100 (v/v). Astrocytes were seeded with a seeding density of
0 000 cel/mL and maintained in the same cortical cell
laser confocal scanning microscopy (LCSM) on a Leica TC-
SP2-AOBS microscope. PpIX �uorescence intensity was
monitored at the maxima for excitation (� = 601 nm) and
ex
medium culture, supplemented with 10% FBS and without
B27.
emission (� = 405 nm). Moreover, a control was also done
with no 5-ALA/CPT-ALA addition
All animal experiments were approved by the Ethic
Committee of Universidad Miguel Herna n� dez (Elche, Spain)
according to the directive 2010/63/EU of the European
Parliament and of the Council and the RD 53/2013 Spanish
regulation. Sprague Dawley and Wistar rats were obtained
from Animal Experimental Service (SEA) of Universidad
Miguel Herna n� dez. Animals were maintained at room
temperature in a 12 h light and 12 h dark cycle and fed ad
libitum. Twenty Sprague Dawley rat embryos at day E17-E18
were used to isolate cell cortex primary culture and astrocytes
culture. Eight female Wistar rats at 3−4 months old, weighing
2.4. Cell Viability Assay. Cell treatment with CPT-ALA or
CPT started 24 h after cell seeding in the case of cancer cell
lines and 14 days in the case of cortical cultures. A 1.5 mM
stock solution with dimethyl sulfoxide (DMSO) was prepared
for every compound, and dilutions were done with cell culture
medium (C6, U87, U251, DMEM; cortical cells and
astrocytes, Neurobasal). Cells were treated with CPT-ALA or
CPT at 0.002−4.8 �g of CPT equiv/mL (CPTeq/mL)
concentration range. Cell culture medium was used as cell
death negative control (no cell death) and a 30% DMSO
solution as a positive control (>99% cell mortality). CPT-ALA
and CPT cytotoxicity e�ect over the cell cycle was studied at
two di�erent incubation times, 24 and 72 h. Cells were
incubated with drugs at 37 °C under a humidi�ed atmosphere
2
00−250 g, were used to create an orthotopic model of GBM.
.2. Synthesis of Camptothecin-20-O-(5-aminolevuli-
nate). First, N-tert-butyloxycarbonyl-5-aminolevulinic acid
2
32
(
Boc-ALA) was synthesized by optimizing a known recipe.
of 5% CO . Cell viability was measured by 3-(4,5-
2
1
2 mL of 5-aminolevulinic acid (1 g, 5.95 mmol) aqueous
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay (Sigma-Aldrich). Following cell incubation
during 24 or 72 h with CPT or CPT-ALA, the plates were
incubated at 37 °C for 3 h with a �nal concentration of 1 mg of
MTT/mL. After incubation, cellular medium with MTT was
removed. Then, formazan crystals were dissolved in 100 �L of
DMSO and 595 nm absorbance plate was measured in an
iMark microplate reader. Absorbance values were normalized
with respect to the negative controls and expressed as a
percentage by using eq 1:
solution was adjusted to pH 8−10 with aqueous sodium
hydroxide (0.1 N). Di-tert-butyl dicarbonate (DTBD, 2.76 g,
1
2.68 mmol) was dissolved in 12 mL of 1,4-dioxane (DOX)
and added to the mixture, which was stirred at room
temperature for 18 h. The excess of DTBD was removed by
washing the mixture with diethyl ether (3 × 100 mL). The
aqueous solution was acidi�ed with a hydrochloric acid
solution (1 N) to pH = 0.5. Ethyl acetate was added (3 ×
1
00 mL) to extract the Boc-ALA, and the solvent was removed
in a rotatory evaporator, obtaining 500 mg (36%).
relative cell viability = OD
test sample
× 100
Boc-ALA (500 mg, 2.163 mmol) was dissolved in 165 mL of
anhydrous dichloromethane (DCM) at room temperature, and
to this solution were added N,N �- diisopropylcarbodiimide
595
OD595 control
(1)
(
(
DIC) (335 �L, 2.16 mmol), 4-(dimethylamino)pyridine
DMAP) (176 mg, 1.44 mmol), and CPT (251 mg, 0.72
At least, six independent experiments were performed for every
drug concentration, and each experiment was carried out in
triplicate. IC50 survival data were calculated by nonlinear
regression sigmoidal dose−response (variable slope) curve-
�ting with Prism 6.0 software (GraphPad).
mmol) at 0 °C. Then, the reaction mixture was stirred at room
temperature for 16 h under argon atmosphere. The resultant
solution was washed with hydrochloric acid 0.1 N, and the
solvent was removed in a rotatory evaporator, collecting 792
mg of camptothecin-20-O-(N-tert-butyloxycarbonyl-5-amino-
2.5. Apoptosis Rate and Cell Cycle Analysis by
Fluorescent-Activated Cell Sorting (FACS). 2.5.1. Apop-
tosis Rate. Apoptosis caused by CPT-ALA, CPT, or a physical
mixture of CPT and 5-ALA (CPT+5-ALA, 1:1 M) was studied
on the C6 cell line seeded in 12-well culture plates. Cells were
treated 1 day after seeding at DIV 1 with every molecule at
0.002, 0.4, and 1.6 �g of CPTeq/mL for 24 h, at 37 °C under a
33
levulinate) (CPT-ALA-Boc, 65%). Afterward, CPT-ALA-Boc
198 mg, 0.35 mmol) was dissolved in 5 mL of tri�uoroacetic
(
acid (TFA/DCM 50:50 v/v) and stirred at room temperature
for 1 h. The solvent was removed under reduced pressure and
the product was recrystallized from a mixture methanol/diethyl
ether (MeOH/DEE 50:50 v/v), obtaining 90 mg of CPT-ALA
humidi�ed atmosphere of 5% CO . Then, cells were washed
2
(
45%).
brie�y with PBS and trypsinized, subsequently stained with
100 �L of binding bu�er containing 5 �L of annexin V-FITC
(�uorescein isothiocyanate) and 5 �L of propidium iodide
(PI), and incubated in darkness for 15 min at room
temperature, according to supplier’s protocol (annexin V
apoptosis detection kit, Biotool). Eventually, to it was added
400 �L of binding bu�er per sample to neutralize the staining.
Samples were kept on ice and analyzed immediately on the
FACSCanto system (BD Biosciences). For every concentration
10 000 events were assessed with medium �ow. Cells with no
staining and treatment were used to delimitate C6 population,
whereas cells with no treatment were used as apoptosis and
necrosis negative control. Annexin V−/PI− were considered as
viable cells. Annexin V+/PI− and annexin V+/PI+ cells were
considered as apoptotic cells and annexin V−/PI+ cells as
necrotic cells. The obtained data were analyzed with BD
The complete characterization of all synthesized compounds
1
13
by H and C NMR spectra and mass spectroscopy is
provided in the Supporting Information. Moreover, stability
tests in DMEM and in human serum were carried out in order
to check CPT-ALA bioavailability in real conditions. This is
described in the Supporting Information.
2
.3. Protoporphyrin IX Synthesis in Glioblastoma
Cells. PpIX synthesis boost by CPT-ALA was tested on the C6
cell line. Cells were seeded in 24-well plates with coverslips at
5
0 000 cells/well and then incubated in cell medium with 3.6
mM CPT-ALA for 4 h. For comparison, control cells were also
incubated in the same conditions with 3.6 mM 5-ALA.
Afterward, cells were washed with PBS (1×, pH 7.34), �xed
with paraformaldehyde 4% for 20 min at room temperature,
and mounted on a slide for further image acquisition using
1
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Figure 1. Synthesis scheme for CPT-ALA.
FACSDiva software. Four independent experiments were
performed for every drug concentration, and each experiment
was carried out in triplicate.
and antibiotic (5 mg/kg enro�oxacin) drugs were adminis-
trated subcutaneously to improve animal welfare.
2.6.2. In Vivo Therapy. Treatment with CPT-ALA started 5
days after tumor inoculation. Animals (n = 5) were
administered 1 mg of CPT-ALA/kg (corresponding to 0.8
mg of CPTeq/kg) twice a week for 2 weeks, consistent with
2
.5.2. Cell Cycle Analysis. To study the action over the cell
cycle of CPT-ALA, CPT, or CPT+5-ALA, the cell ratio was
determined in each cycle phase by �ow cytometry over the C6
cell line. For this sake, cells were seeded in 12 well culture
plates and incubated with every molecule at 0.002, 0.4, and 1.6
36
preliminary work. For this purpose, a solution of 1 mg of
CPT-ALA in DMSO/PBS (1:9 v/v) was prepared and slowly
perfused (1 min) through the tail lateral vein with a catheter-
gauge-24 at days 6, 9, 13, and 16 after tumor inoculation.
Besides, the control group (n = 3) was treated with the mixture
DMSO/PBS (1:3 v/v) in the same conditions. Animal weight
was monitored, and animal welfare was evaluated daily using
�g of CPTeq/mL) for 6 or 24 h at 37 °C under a humidi�ed
atmosphere of 5% CO . Afterward, C6 cells were �xed with 2%
2
paraformaldehyde and incubated 30 min at 37 °C with 2.5 mg/
mL RNase A from bovine pancreas (Sigma-Aldrich) and 2.5
mg/mL PI. Finally, C6 cells were stored in ice and analyzed
with FACSCanto system to determine population cell
percentage in each cycle phase. For every drug concentration
37,38
the Morton and Gri�ths scale and facial expressions.
According to this test, if animal weight loss exceeds 20% with
respect to initial animal weight or if the test score is equal or
exceeds 15 points, the animal must be sacri�ced to avoid
su�ering. If the test score is higher than 10 points, euthanasia
should be considered. Finally, rats were sacri�ced with 40 mg/
kg sodic pentobarbital intraperitoneally injected 3 days after
the last CPT-ALA administration.
1
0 000 events were assessed with low �ow. Cells with no
staining and treatment were used to delimitate C6 population,
whereas cells with no treatment were used as controls to de�ne
C6 normal distribution in the cell cycle phases. Data were
analyzed through BD FACSDiva software. Four independent
experiments were performed for every drug, and each
experiment was carried out in triplicate.
2
.6.3. Hystological Analysis. To evaluate CPT-ALA
biocompatibility, samples of brain, spleen, liver, kidney, heart,
and lung were collected for histological analysis. Tissues were
2
.6. In Vivo Testing. 2.6.1. Tumor Induction. Female
Wistar rats of age 3−4 months were pretreated with 1 mg/kg
of dexamethasone 24 and 1 h before surgery. Prior to
intervention, the analgesia state was induced with a single dose
of (40 mg/kg ketamine)/(10 mg/kg xylazine) cocktail using
intraperitoneal injection. During surgery, anesthesia was
maintained with 3% iso�urane gas mixed with oxygen, and
the animal’s body temperature was controlled with an electric
blanket. The animal’s head was �xed with a stereotaxic frame.
A small craniotomy of approximately 1 mm diameter was
performed in stereotaxic coordinates: 1 mm anterior and 3 mm
�
xed using 4% paraformaldehyde (PFA) during 48 h. Organs
were embedded in para�n and cut with a microtome at 7 �m
thickness slices. Sections were depara�nized and stained with
hematoxylin and eosin. Finally, tissue images were analyzed
with an Olympus AX70 microscope.
2.6.4. Antitumor Activity. To evaluate CPT-ALA drug
antitumor activity, brains were �xed with 4% paraformaldehyde
for 48 h and cut using a cryostat at 20 �m thickness slides.
Next, an immunohistochemistry assay was done to measure
both tumor size and cellular proliferation. Nonspeci�c staining
was blocked for 1 h through 10% bovine serum albumin, and
brain slices were permeabilized with 0.5% Triton-X-100.
Antibodies anti-Ki67 (Ki67-polyclonal antibody rabbit IgG,
Thermo Fisher) and anti-GFAP (glial �brillary acidic protein
antibody mouse IgG, clone GA5, Millipore) were used to
target these proteins at nontumoral astrocytes of brain slices.
Ki67 and GFAP were detected through secondary antibodies
3
4,35
left lateral with respect to the Bregma line.
C6
glioblastoma cells were inoculated into the striatum through
craniotomy in order to generate an intracranial glioblastoma
5
animal model. 10 C6 cells resuspended in 1.5 �L of PBS were
injected with a Hamilton 33-gauge needle to a depth of 4 mm.
During the 3 days that followed surgery, analgesic (0.05 mg/kg
buprenorphine), anti-in�ammatory (0.5 mg/kg meloxicam),
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Figure 2. MTT cell viability assays in GBM cell lines and normal CNS cells after 24 and 72 h incubation. Drug concentration is referred to CPT
equivalent (lower scale). Cell viability data are expressed as the mean ± SEM: (A) cortical cells (n = 10); (B) astrocytes (n = 10); (C) C6 cell line
(n = 6); (D) U87 cell line (n = 7); (E) U252 cell line (n = 7).
(
1:100), respectively, Alexa Fluor 488 conjugated donkey anti-
2.7. Statistical Analysis. Statistical analysis was performed
using arithmetic mean values and error bars of statistical error
mean values (SEM). To determine MTT assay signi�cance, t-
tests and Wilcoxon rank-sum tests were performed. Statistical
analyses of all in vivo experiments, e.g., TUNEL, proliferation
assays, and anticancer activity analysis, were carried out with t-
tests. Conversely, statistical signi�cance of �ow cytometry, e.g.,
rabbit IgG (Thermo Fisher), and Alexa Fluor 555 conjugated
donkey anti-mouse IgG (Thermo Fisher). Cellular nuclei in
the slices were stained with HOECHST 33342 (1:300 v/v).
Seven random images per slice were taken with a Carl Zeiss
Apotome2 microscope to calculate cellular proliferation levels
inside the tumor. Total tumor volume was determined by
applying eq 2 from the measurement of the two perpendicular
2
apoptosis and cell cycle analysis, was evaluated using the �
39
radical distances. Tumor 3D representation was done using
Matlab software (MathWorks).
test. The t-tests and Wilcoxon rank-sum tests were performed
2
with Matlab (MathWorks), while the � test was carried out
using Prism 6.0 software (GraphPad).
Vtumor = 4 r r
�
2
x
z
3
(2)
3. RESULTS
2
.6.5. TUNEL Assay. TUNEL assay was performed in 20 �m
3.1. Synthesis of CPT-ALA. The preparation of CPT-ALA
thickness brain slices to compare apoptosis in brain and tumor
of the animals with and without CPT-ALA treatment. Brain
slices were incubated for 60 min at 37 °C with TUNEL
Roche Molecular Biochemicals). Seven random images per
slice were obtained with a Carl Zeiss Apotome2 microscope to
determinate cellular apoptosis inside the tumor.
is presented in Figure 1. All compounds were characterized by
1
13
H NMR, C NMR, and mass spectrometry (Q-Tof analysis)
(see Supporting Information). The synthesis took place in
three steps. First, 5-ALA was reacted with DTBD to give Boc-
ALA. This precursor was then conjugated with CPT by
esteri�cation of the 20-hydroxyl group. Finally, Boc protection
(
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was released with TFA to give CPT-ALA. The complete
process runs with 30% global yield, CPT being the only
impurity. After a separation step, CPT-ALA was obtained with
purity of >99%.
apoptosis cells (annexin V+/PI−); Q2, later apoptosis cells
(annexin V+/PI+); Q3, healthy cells (annexin V−/PI−); Q4,
necrotic cells (annexin V−/PI+). Figure 3A−D shows how
cellular population distribution changed with regard to CPT-
ALA concentration, whereas Figure 3e represents the
quantitative analysis of these results. A signi�cant increment
of apoptosis took place in treated cells (p < 0.001) when
increasing CPT-ALA concentration (e.g., from 0.002 to 1.6 �g
CPTeq/mL) with regard to nontreated cells. In addition,
necrosis variation as a function of CPT-ALA concentration was
not signi�cant. Consistent with MTT assay results, these data
indicate that CPT-ALA drug provokes apoptosis in C6 cells
after 24 h incubation. Furthermore, for the sake of comparison,
a similar study was carried out with free CPT and the physical
mixture of CPT and 5-ALA (CPT+5-ALA, 1:1 M) over C6
cells, and the results are shown at Figure 3E. Here we observed
that although all CPT systems signi�cantly increase apoptosis
at large dose (e.g., 1.6 �g of CPTeq/mL) after 24 h incubation,
CPT-ALA presents a clear superior activity in comparison to
CPT and CPT+5-ALA.
3.2.4. Cell Cycle Analysis. The analysis of cell cycle
distribution of C6 cells treated with CPT-ALA for 6 and 24
h and stained using PI was carried out by FC. C6 population
was sorted out into three subpopulations: G0/G1 phase (P4),
S phase (P5), and M phase (P6). Figure 4 shows changes in
the distribution of the di�erent cell cycle phases with CPT-
ALA concentration. Cell percentage in G0/G1 phase is
signi�cantly increased after 24 h incubation with CPT-ALA,
whereas cell populations in DNA synthesis (S) and mitosis
(M) phases are strongly reduced in the range 0.4−1.6 �g of
CPTeq/mL (p < 0.001). No signi�cant changes were observed
in cell cycle phase distribution after incubation with CPT-ALA
for 6 h at the same concentration. In addition, FC assays after
PI staining demonstrate that CPT-ALA induced cells to stop in
G0/G1 phase and prevented cell cycle progression. Eventually,
these results show that the number of cells in a proliferative
stage is reduced when the incubation time with CPT prodrug
is increased. For comparison, a similar study was carried out
with free CPT and with the physical mixture of CPT and 5-
ALA (CPT+5-ALA, 1:1 M) on C6 cells, and the results are
shown in Figure 4E. Here we observed that all CPT systems
signi�cantly increase (p < 0.001) cell cycle disruption at phase
G0/G1 after 24 h exposure at a wide range of concentration
(0.4−1.6 �g of CPTeq/mL), resulting in glioblastoma cell
apoptosis.
We also carried out stability tests of CPT-ALA in DMEM
and in commercial human serum (male AB, Sigma-Aldrich). In
cellular medium CPT release was lower than 1% at 24 h (data
not shown). Moreover, in human serum no signi�cant CPT
release was detected in the �rst 4 h and, afterward, a slow free
CPT concentration increase was observed, which reached
about 5% at 24 h (Figure S2). This performance is in line with
the stability in physiological �uids of 20-O-acylcamptothecin
40,41
derivatives claimed by di�erent authors
and con�rms the
stability in human serum of these compounds observed
42
previously by our group.
.2. In Vitro Study. 3.2.1. Protoporphyrin IX Synthesis.
3
CPT-ALA promotes the synthesis of PpIX in glioblastoma cells
in a similar way as 5-ALA. This is known on the basis of the
26
PpIX molecular route, which is overexpressed in GBM.
A
short incubation time (e.g., 2 h) in C6 cells is therefore enough
to achieve successful formation of PpIX in vitro, resulting in
high red �uorescence intensity (� = 405 nm), as presented in
Figure S3.
3
.2.2. Cytotoxicity. Figure 2 shows quantitative results of
cell viability by MTT assay. GBM cell line and primary cortical
and astrocytes cultures are represented after the treatment with
CPT and CPT-ALA for 24 and 72 h. Very little e�ect, if any,
was observed after 24 h incubation, but a signi�cant increase of
cell mortality was observed in all cell types when the treatment
was extended up to 72 h (C6, p < 0.0001; U251, p < 0.0001;
U87, p < 0.0001; astrocytes, p < 0.005; cortical cells, p < 0.05),
which is consistent with CPT activity over cell cycle. According
to some authors 5-ALA has shown some cytotoxicity and
43,44
genotoxicity over lymphocytes and cancer cells,
but we do
not think this may be signi�cant in our case, as the cytotoxic
activity of CPT is several orders above. No signi�cant
di�erences in the cytotoxic e�ect over the di�erent GBM
cell lines (U87, U251 and C6) were observed for CPT and
CPT-ALA. However, cell mortality induced by CPT over
normal cell lines is signi�cantly higher than CPT-ALA, the
di�erence becoming especially pronounced in cortical cells
(Table 1). This is demonstrated in Figures S4 and S5
Table 1. IC50 Values for CPT and CPT-ALA after 72 h
a
Incubation with the Di�erent Cell Lines Tested
3
.3. In Vivo Study. 3.3.1. Animal Supervision. To ensure
cell line
C6
U251
U87
cortical cells
astrocytes
CPT (�g/mL)
CPT-ALA (�g/mL)
t
animal welfare, weight and pain symptoms were constantly
monitored during the experiment. Table 2 shows the evolution
of animal weight and Morton and Gri�ths scale score in both
treated and nontreated groups. Animal weight was kept mostly
constant throughout the whole experiment (weight loss was
always lower than 20% with regard to the initial value). Face
signs and Morton and Gri�th’s test results did indicate no
intense su�ering in any animal. Finally, both groups did not
present symptoms such as alopecia, hematuria, or diarrhea.
3.3.2. Drug Cytotoxicity. Figure 5 shows representative
histological sections of analyzed organs from tumor-bearing
rats injected with 0.8 mg of CPTeq/kg for 2 weeks and the
corresponding control with no treatment. No structural or
cellular abnormalities were observed in the kidney, heart, and
spleen of treated animals. Moreover, no alteration or
hemorrhages were found in all examined structures of those
tissues. Heart histology (Figure 5a−d) did not present changes
0.016 ± 0.002
1.389 ± 1.037
2.290 ± 1.740
2.733 ± 1.305
4.734 ± 1.032
0.099 ± 0.015
0.914 ± 0.013
2.770 ± 1.785
7.486 ± 1.400
7.521 ± 1.371
ns
ns
ns
p < 0.01
p < 0.05
a
Statistical di�erence according to Student’s t test. ns = not-
signi�cant.
(
Supporting Information), showing the stronger cytotoxic
e�ect of CPT-ALA with increased concentration when
comparing U87 cell line (Figure S4) to cortical cells and
astrocytes (Figure S5).
3
.2.3. Apoptosis Rate. Apoptosis analysis by FC was carried
out over C6 cells treated for 24 h with CPT-ALA and stained
with annexin V-FITC and PI (PE �lter). The cellular
population was sorted out into four groups: Q1, early
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Figure 3. Flow cytometry results after incubation of C6 cells for 24 h with increasing concentrations of CPT-ALA, CPT+5-ALA, and CPT. (A−D)
Flow cytometry results after incubation of C6 cells for 24 h with CPT-ALA. X-axis: PI �uorescence (PE �lter). Y-axis: FITC �uorescence.
Representative scatter plots (Q1, early apoptotic cells; Q2, late apoptosis cells; Q3, healthy cells; Q4, necrotic cells) of cells with no treatment (A),
0.002 �g of CPTeq/mL (B), 0.4 �g of CPTeq/mL (C), and 1.6 �g of CPTeq/mL (D). (E) Percentage of viable, apoptotic, and necrotic cells
treated with CPT-ALA, CPT+5-ALA, and CPT (n = 5). Untreated cells were used as negative controls. Data are expressed as the mean ± SD.
Viable cells showed signi�cant di�erences between CPT-ALA (***p < 0.001), CPT+5-ALA (*p < 0.05), and CPT (***p < 0.001) groups and the
untreated group. Apoptotic cells showed signi�cant di�erences between CPT-ALA (***p < 0.001), CPT+5-ALA (*p < 0.05), and CPT (*p <
0.05) groups and the untreated group.
in myocardium, endocardium, and pericardium between
treated and untreated animals. Similarly, kidney histology
In addition, histological analysis of the CPT-ALA group did
not show any hemorrhage or damage in the liver (Figure 5m−
p), but the hepatic tissue showed areas where cells presented
pigment deposits inside cells, which were not localized at
untreated animals. Furthermore, we have to report that lung
alveoli histology showed some hemorrhagic damage in CPT-
ALA treated animals (Figure 5q−t), although no chronic issue,
as in�ammation or �brosis, was present. Indeed, although these
hepatic and lung alteration cases and respiratory and hepatic
(Figure 5e−h) did not show any alteration of renal corpuscles
and tubules when compared to the control group. Also, spleen
histologic images (Figure 5i−l) indicated that the tissue is
intact after CPT-ALA administration. No signi�cant immuno-
logic system activity was observed at the spleen, and neither
pulp (both red and white) abnormalities nor lymph node was
detected.
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Figure 4. Cell cycle phase study of C6 cells after incubation for 6 and 24 h with increasing concentrations of CPT-ALA, CPT+5-ALA, and CPT.
A−D) Cell cycle phase reports after 24 h incubation with CPT-ALA: control (A), 0.002 �g of CPTeq/mL (B), 0.4 �g of CPTeq/mL (C), and 1.6
g of CPTeq/mL (D). (E, F) Percentage of cells in each phase of the cell cycle (G0/G1, S, and M) vs CPT-ALA, CPT+5-ALA, and CPT after 6 h
(
�
incubation (E) and 24 h incubation (F) (n = 5). Legend: P4, phase G0/G1; P5, phase S; P6, phase M. Data are expressed as the mean ± SD.
Statistical signi�cance (***p < 0.001) compared to the untreated group for CPT-ALA group; statistical signi�cance (***p < 0.001) compared to
the untreated group for CPT+5-ALA group; statistical signi�cance (***p < 0.001) compared to the untreated group for CPT group.
Table 2. Main Parameters Monitored in Animals during
CPT-ALA Toxicity Study
orthotopic (intracranial) animal model of glioblastoma
generated with cell line C6. For this purpose, tumor volume
was monitored during 2 weeks in Wistar rats administered with
four doses of CPT-ALA (0.8 mg of CPTeq/kg) (CPT-ALA
group) or DMSO/PBS (control group). Results (Figure 6)
indicated a signi�cant decrease of 30% tumor volume in CPT-
ALA injected animals with regard to the control group (p <
0.05) (Figure 6G). Modeling of tumor reduction under CPT-
ALA administration illustrated tumor recession under CPT-
ALA therapy (Figure 6H).
a
parameter
control
CPT-ALA (0.8 mg/kg)
weight loss (%)
pain
physical alterations
behavior disorders
0
5
−
−
+
+
−
+
a
According to Morton and Gri�ths test score: +++ � 3; ++ � 2; + �
; − ≡ 0.
1
Brain histology con�rmed no damage over nontumor tissue
at the brain or meningeal in�ammation in the CPT-ALA
group, although we observed the cystic glioblastoma formation
in one of the treated animals (Figure 7A−C). In addition,
immuno�uorescence images (Figure 7D−I) showed that
alterations were not observed, there was no variation in food
and water intake, and animals did not present malaise, weight
loss, or dehydration symptoms.
3
.3.3. Antitumor Activity. To assess the antitumor e�ects of
prodrug CPT-ALA, we studied its activity against an
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Figure 5. Hematoxylin and eosin staining of histological slices from CPT-ALA and control groups. The therapy was extended for 2 weeks (0.8 mg
of CPTeq/kg, 4 doses): (a−d) heart; (e−h) kidney; (i−l) spleen; (m−p) liver; (q−t) lung.
GFAP expression around tumor was higher in the CPT-ALA
group than in the control group. This indicated an increase in
astrocytes recruitment in tumor surrounding area, with normal
surrounding tissue being well-de�ned in all cases.
described that CPT-ALA promotes apoptosis in glioblastoma
C6 cells after 24 h treatment.
4. DISCUSSION
Quanti�cation of immunohistochemistry was used to detect
Ki67 expression in glioblastoma cells at brain slices, and the
results are presented in Figure 7J. Proliferation assay indicated
that cells expressing Ki67 decreased 20% in the CPT-ALA
group. This demonstrated that the number of cells in division
was signi�cantly reduced following CPT-ALA administration
with regard to the control group (p < 0.05), delaying cell
proliferation inside glioblastoma. These data were consistent
with the results presented in the cell cycle analysis section
Despite decades of research, GBM remains incurable. Its
aggressiveness depends mostly on the protection given by the
BBB and its high levels of proliferation. On one hand, BBB
6
blocks the arrival of most drugs to the CNS, hampering
pharmacological therapy and demand for drugs with the
potential to go across it. On the other hand, the high rate of
division of GBM cells does not only increase its size faster than
45
any other brain tumo, but also promote a high rate of
mutations, which makes GBM quickly resistant to medicines
46
3
.2.4, where it was observed that CPT-ALA produced an arrest
of glioblastoma C6 cell cycle in the G0/G1 phase.
.3.4. TUNEL Assay. Quanti�cation of TUNEL apoptosis
and which contributes to its malignant phenotype.
In this work, a new CPT prodrug, CPT-ALA, is proposed for
GBM therapy through intravenous administration. As a
powerful inhibitor of DNA topoisomerase I, CPT can stop
3
assay in brain slices from tumor area is presented in Figure 8.
This test showed a signi�cant increase in the percentage in
apoptotic cells inside glioblastoma after 2 weeks of treatment
with CPT-ALA (control group 15.1%, CPT-ALA group 35.1%,
p < 0.05). Conversely, no increase of cellular apoptosis was
observed in healthy tissue. These data con�rm what is
presented in the apoptosis rate section 3.2.3, where it was
47
GBM cell growth. For this reason, CPT structural derivative
irinotecan is a known therapy against GBM, which is usually
administrated together with bevacizumab in the recurrent
GBM, as well as in patients with unmethylated MGMT gene
48
promoter. In this context, the conjugation of CPT with 5-
ALA should boost the activity of the therapeutic molecule
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Figure 6. Antitumor activity of CPT-ALA against glioblastoma in Wistar rats. (A−C) Brain slices tissue reconstruction of the control group (n = 3).
(
D−F) Brain slices tissue reconstruction of CPT-ALA group (0.8 mg of CPTeq/kg, 4 doses in 2 weeks) (n = 5). Nuclei were stained with
HOECHST (blue) and astrocytes with GFAP (red). Scale bar: 1 mm. (G) Normalized tumor volume comparison: control group (n = 3); CPT-
ALA group (n = 5). Data are expressed as the mean ± SEM: *p < 0.05. (H) 3D representation of normalized tumor volume. The blue outer sphere
corresponds to the control group, whereas the green inner sphere refers to the CPT-ALA group.
against GBM for two reasons: (i) 5-ALA improves the
hydrophilicity of CPT molecule, providing the drug with the
ability to go across the BBB, as CPT has shown little e�cacy to
di�use through the BBB even when it is formulated with a
However, both CPT and its 5-ALA derivative were more
toxic in all glioblastoma cell types, but especially in C6 cells,
than in cortical cells and astrocytes cultures, and we attribute
these results �rst to the GBM cells accelerated division rate
compared to primary cultures. At this point, the doubling time
1
7
nanocarrier; (ii) 5-ALA promotes CPT internalization in
GBM cells. Indeed, 5-ALA is a key molecule in the
50
for those cancer cells ranges approximately from 16 h (C6)
26
51,52
protoporphyrin IX route, which is overexpressed in GBM.
to 24 h (U87, U251),
whereas astrocytes and cortical cells
53
Then, CPT release is expected to take place in the cytosol by
present doubling times above 4 days. Furthermore, di�er-
ences in cytotoxicity between glioblastoma and healthy cells
were signi�cantly broadened for CPT-ALA prodrug, due to the
promoted cell internalization in cancer cells caused by the 5-
ALA moiety. As commented, 5-ALA is a porphyrin
metabolized by cells where the heme synthesis pathway is
active (e.g., GBM cells but not non-neoplastic CNS cells) to
the action of speci�c carboxylases that cleave the ester bond
36
between 5-ALA and CPT. Consequently, glioblastoma cells
present an increasing uptake of 5-ALA, which has been used in
photodynamic therapies and as tumor marker during surgical
2
4
removal.
We studied the anticancer properties of the new prodrug
both in vitro and in vivo. First, viability assays by MTT proved
an increase in tumor cell mortality depending on the
incubation time for both CPT and CPT-ALA. This is due to
the cell needing to be in the cell cycle S phase, thus expressing
24,25
the �uorescent metabolite protoporphyrin IX (PpIX).
In
this sense, metabolic targeting of 5-ALA takes place over GBM
54,55
cells,
which is enough to promote the superior activity of
CPT-ALA over malignant cells with regard to cortical cells and
astrocytes cultures. Indeed, 5-ALA targets the prodrug against
cells with an increased intake of this molecule (e.g.,
glioblastoma cell lines) and then reduces the intake by cells
15
topoisomerase I, to be vulnerable to CPT activity. It is
noteworthy that no signi�cant di�erences in the cytotoxic
e�ect were observed for CPT and CPT-ALA over the di�erent
GBM cell lines. Indeed, this is not surprising, as in vitro models
usually provide very favorable conditions for drug intake by
26
with low 5-ALA demand (e.g., normal brain cells). Hence,
the studied prodrug had a strong and similar e�ect over
di�erent GBM cells but limited cytotoxicity over healthy tissue.
4
9
cells with accelerated metabolism (e.g., cancer cells).
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Figure 7. Brain histology and immunohistochemistry after administration of CPT-ALA against glioblastoma in Wistar rats. (A−C) Cystic
glioblastoma brain sample reconstruction in a Wistar rat specimen treated with CPT-ALA (0.8 mg of CPTeq/kg, 4 doses in 2 weeks). Scale bar 50
�
m. (D−I) GFAP expression in glioblastoma and brain tissue in control group (n = 3) (D−F) and CPT-ALA group (n = 5) (G−I). Scale bar: 250
�
m. (J) Percentage of proliferative cells inside tumor in control group (n = 3) and CPT-ALA group (n = 5). Data are expressed as the mean ±
SEM: *p < 0.05. Nuclei were stained with HOECHST (blue) and astrocytes with GFAP (red) except (C) where nuclei were stained with
hematoxylin and cytoplasm with eosin.
5
6
This has promising clinical applications, as the lower toxicity
should be translated into minimized secondary e�ects, also
allowing the administration of higher CPT-ALA doses.
the physical mixture CPT+5-ALA shows that CPT-ALA
promotes clearly better the apoptosis process in the C6 cell
line at moderate exposure time (e.g., 24 h). We hypothesize
that this is another evidence of the metabolic targeting that the
5-aminolevulinic moiety imposes on the prodrug molecule in
glioblastoma cells, which promotes internalization.
In vivo testing over Wistar rats has shown that the
administration of therapeutic doses of CPT-ALA does not
a�ect the well-being of the studied animals. This is di�erent
from what has been observed for other CPT prodrugs, such as
topotecan and irinotecan, which normally produce very
harmful side e�ects, like lack of appetite, diarrhea, and
The results obtained from the in vitro viability were further
supported by additional studies over the cell cycle. Flow
cytometry results at 24 h incubation indicated that CPT-ALA
arrested cell cycle of C6 rat glioblastoma cell line in G0/G1
phase by topo-I inhibition. This causes destabilization of the
DNA chain, and �nally the cascade of cell apoptosis is
activated. Thus, we could con�rm that the decrease in cell
viability shown in the MTT assays was produced by apoptosis
and not by necrosis. However, the comparison with CPT and
5
7
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Figure 8. Apoptotic cells stained with TUNEL label (green) in brain slices of control and CPT-ALA treated animals. Nuclei were stained with
HOECHST (blue).
1
3,58
alopecia,
and is consistent with the rationale of 5-ALA as a
this type of tumor grows more slowly and has a better life
expectancy because it has nondi�use limits.
64
molecule that promotes metabolic targeting, increasing CPT
selectivity to GBM cells and reducing drug accumulation in
other proliferative healthy tissues. Indeed, literature involving
in vivo testing and clinical trials of topotecan and irinotecan for
GBM therapy shows that the standard dose range (�5−10
As an additional remark, it must be taken into account that
the BBB increases its permeability in the tumor area due to
uncontrolled growth of GBM cells and decreases the
expression of ocludin and claudin-1 proteins, which play a
1
0,59
mg/kg)
is about 1 order higher than CPT-ALA (0.8 mg of
65
key role in BBB structure and organization. Consequently, it
is di�cult to con�rm experimentally whether CPT-ALA can
cross BBB or whether its entry is due to this increase in
permeability. At this point, drug di�usion through BBB is
usually monitored by HPLC determination in the CNS �uid,
but in this case, due to the di�erences in BBB permeability in
the tumor area with regard to healthy tissue, the results
obtained might not be representative. Eventually, our in vitro
and in vivo models supported that CPT-ALA can go across the
BBB (even considering the possible role of a partially disrupted
BBB due to tumor proliferation) and block the reproduction of
GBM cells, leading to tumor size recession. At this point,
nowadays, GBM treatment remains a challenge due to the
di�culty of designing drugs with combined abilities of going
through the BBB and killing cancer cells. Although these
results are preliminary, we expect to improve tumor collapse by
extending the treatment for a longer time and combining CPT
chemotherapeutic activity with the photodynamic performance
of 5-ALA.
CPTeq/kg), which claims the promising potential of this novel
prodrug.
Histology results showed no histological damage indicator in
kidneys, liver, and spleen after 2 weeks of treatment. However,
liver tissue showed pigment deposits inside hepatocytes in the
group treated with CPT-ALA, which may consist of heme
group accumulation. This would increase the expression of
enzymes for the degradation of protoporphyrins and heme
60
group without any harmful e�ect to the organism. Moreover,
we found some alveolar morphological alterations after CPT-
ALA administration, but no breath issue was observed during
the treatment. Future studies will be focused on the study of
possible long-term respiratory problems, which have also been
61
reported for irinotecan and topotecan. In this sense, it is
noticeable that the treatment with CPT-ALA prodrug has
similar side e�ects as the free 5-ALA molecule, which has
60
already been approved for clinical uses.
Furthermore, the analysis of CPT-ALA anticancer activity in
the intracranial GBM rat showed signi�cant di�erences
between control and treated (CPT-ALA) groups, con�rming
the anticancer activity previously described in in vitro assays.
Tumor size and cell proliferation values were signi�cantly
reduced in the treated animals (30%). Furthermore, an
increase of 20% apoptotic cells in tumor tissue was seen, and
there was no e�ect in healthy cortical tissue, which supports
the ability of CPT-ALA to induce apoptosis by topo-I
5. CONCLUSION
The success of pharmacological approaches in GBM therapy
depends on two crucial steps: (i) the di�usion of the drug
through the BBB and (ii) the selective targeting to cancer cells.
In this work we have shown by in vitro and in vivo studies that
the novel prodrug CPT-ALA is able to accomplish these
points, based on the solubility improvement, the increased
permeability of BBB due to tumor proliferation, and the
metabolic-based targeting capability of the 5-ALA moiety,
which signi�cantly reduces tumor growth with very few side
e�ects. This new prodrug opens the door to combine selective
chemo- and photodynamic therapies against GBM in one go
due to the speci�c performance of the 5-ALA molecule.
57
inhibition during cell division.
GFAP expression increased around the carcinoma, which
indicated the existence of a glial scar encapsulating the
6
2
tumor. Indeed, one of the main reasons for recurrence in
GBM is its high in�ltrative properties in the healthy cortical
6
3
tissue, but the appearance of a glial scar following CPT-ALA
treatment can reduce the in�ltrative capacity of GBM and
therefore enhance the success of surgical removing, improving
life expectancy by reducing tumor recurrence. In addition, it is
also noticeable that in one of the treated animals the tumor
evolved into a cystic glioblastoma, as it has been reported that
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Classification of Tumors of the Central Nervous System: A Summary.
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.molpharma-
ceut.0c00968.
�
Acta Neuropathol. 2016, 131, 803−820.
*
(2) Omuro, A.; DeAngelis, L. M. Glioblastoma and Other Malignant
Gliomas: A Clinical Review. JAMA 2013, 310, 1842−1850.
(3) Preusser, M.; De Ribaupierre, S.; Wöhrer, A.; Erridge, S. C.;
Hegi, M.; Weller, M.; Stupp, R. Current Concepts and Management
Full synthesis and characterization details for CPT-ALA,
release kinetics in human serum, protoporphyrin IX
of Glioblastoma. Ann. Neurol. 2011, 70, 9−21.
(
4) Kesari, S. Understanding Glioblastoma Tumor Biology: The
Potential to Improve Current Diagnosis and Treatments. Semin.
Oncol. 2011, 38, S2−S10.
�uorescence images, and phase-contrast microscope
images of tumor and normal cells (PDF)
(5) Furnari, F. B.; Fenton, T.; Bachoo, R. M.; Mukasa, A.; Stommel,
J. M.; Stegh, A.; Hahn, W. C.; Ligon, K. L.; Louis, D. N.; Brennan, C.;
et al. Malignant Astrocytic Glioma: Genetics, Biology, and Paths to
Treatment. Genes Dev. 2007, 21, 2683−2710.
AUTHOR INFORMATION
Corresponding Authors
Pablo Botella − Instituto de Tecnología Química, Universitat
Polite c�n ica de Vale n� cia-Consejo Superior de Investigaciones
Cientí�cas, 46022 Valencia, Spain; orcid.org/0000-0003-
�
(
6) Abbott, N. J.; Rönnbäck, L.; Hansson, E. Astrocyte-Endothelial
Interactions at the Blood-Brain Barrier. Nat. Rev. Neurosci. 2006, 7,
1−53.
7) Uribe, D.; Torres, A.; Rocha, J. D.; Niechi, I.; Oyarzu n� , C.;
4
(
�
2
141-3069; Email: pbotella@itq.upv.es
Sobrevia, L.; San Martín, R.; Quezada, C. Multidrug Resistance in
Glioblastoma Stem-like Cells: Role of the Hypoxic Microenvironment
and Adenosine Signaling. Mol. Aspects Med. 2017, 55, 140−151.
Eduardo Fernández − Institute of Bioengineering, Universidad
Miguel Hernández, Elche, Spain and Centre for Network
Biomedical Research (CIBER-BBN), 03202 Elche, Spain;
Email: e.fernandez@umh.es
(8) Stupp, R.; Mason, W. P.; van den Bent, M. J.; Weller, M.; Fisher,
B.; Taphoorn, M. J. B.; Belanger, K.; Brandes, A. A.; Marosi, C.;
Bogdahn, U.; Curschmann, J.; Janzer, R. C.; Ludwin, S. K.; Gorlia, T.;
Allgeier, A.; Lacombe, D.; Cairncross, J. G.; Eisenhauer, E.;
Mirimanoff, R. O. Radiotherapy plus Concomitant and Adjuvant
Temozolomide for Glioblastoma. N. Engl. J. Med. 2005, 352, 987−
Authors
Elisa Checa-Chavarria − Institute of Bioengineering,
Universidad Miguel Hernández, Elche, Spain and Centre for
Network Biomedical Research (CIBER-BBN), 03202 Elche,
Spain
9
(
96.
9) Feng, E.; Sui, C.; Wang, T.; Sun, G. Temozolomide with or
Eva Rivero-Buceta − Instituto de Tecnología Química,
Universitat Polite c� nica de Vale n� cia-Consejo Superior de
Investigaciones Cientí�cas, 46022 Valencia, Spain
Miguel Angel Sanchez Martos − Institute of Bioengineering,
Universidad Miguel Hernández, Elche, Spain and Centre for
Network Biomedical Research (CIBER-BBN), 03202 Elche,
Spain
Gema Martinez Navarrete − Institute of Bioengineering,
Universidad Miguel Hernández, Elche, Spain and Centre for
Network Biomedical Research (CIBER-BBN), 03202 Elche,
Spain
without Radiotherapy in Patients with Newly Diagnosed Glioblasto-
ma Multiforme: A Meta-Analysis. Eur. Neurol. 2017, 77, 201−210.
(10) Friedman, H. S.; Prados, M. D.; Wen, P. Y.; Mikkelsen, T.;
Schiff, D.; Abrey, L. E.; Yung, W. K. A.; Paleologos, N.; Nicholas, M.
K.; Jensen, R.; et al. Bevacizumab Alone and in Combination with
Irinotecan in Recurrent Glioblastoma. J. Clin. Oncol. 2009, 27, 4733−
4740.
(11) Van Tellingen, O.; Yetkin-Arik, B.; De Gooijer, M. C.;
Wesseling, P.; Wurdinger, T.; De Vries, H. E. Overcoming the Blood-
Brain Tumor Barrier for Effective Glioblastoma Treatment. Drug
Resist. Updates 2015, 19, 1−12.
(12) Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail,
Cristina Soto-Sánchez − Institute of Bioengineering,
Universidad Miguel Hernández, Elche, Spain and Centre for
Network Biomedical Research (CIBER-BBN), 03202 Elche,
Spain
A. T.; Sim, G. A. Plant Antitumor Agents. I. The Isolation and
Structure of Camptothecin, a Novel Alkaloidal Leukemia and Tumor
Inhibitor from Camptotheca Acuminata. J. Am. Chem. Soc. 1966, 88,
3888−3890.
(13) Botella, P.; Rivero-Buceta, E. Safe Approaches for Camptothe-
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.molpharmaceut.0c00968
cin Delivery: Structural Analogues and Nanomedicines. J. Controlled
Release 2017, 247, 28−54.
(14) Liu, L. F.; Duann, P.; Lin, C. T.; D’Arpa, P.; Wu, J. Mechanism
Notes
of Action of Camptothecin. Ann. N. Y. Acad. Sci. 1996, 803, 44−49.
15) Pizzolato, J. F.; Saltz, L. B. The Camptothecins. Lancet 2003,
61, 2235−2242.
The authors declare no competing �nancial interest.
(
3
ACKNOWLEDGMENTS
�
(16) Bala, V.; Rao, S.; Boyd, B. J.; Prestidge, C. A.; Wall, M. E.;
Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A. T.; Sim, G. A.;
et al. Plant Antitumor Agents. I. The Isolation and Structure of
Camptothecin, a Novel Alkaloidal Leukemia and Tumor Inhibitor
from Camptotheca Acuminata. J. Controlled Release 2013, 172, 3888−
Financial support from Spanish Ministry of Economy and
Competitiveness (Projects PID2019-111436RB-C21 and SEV-
2
016-0683) and the Generalitat Valenciana (Project PROM-
ETEO/2017/060) is gratefully acknowledged. We thank Prof.
Luis Fernández (Group of Structural Mechanics and Materials
Modellings-GEMM, University of Zaragoza, Spain) for
donation of human GBM cell lines. We are grateful to Dr.
Lawrence Humphreys (CIBER-BBN) for critical reading of the
manuscript.
3890.
(17) Aparicio-Blanco, J.; Sanz-Arriazu, L.; Lorenzoni, R.; Blanco-
Prieto, M. J. Glioblastoma Chemotherapeutic Agents Used in the
Clinical Setting and in Clinical Trials: Nanomedicine Approaches to
Improve Their Efficacy. Int. J. Pharm. 2020, 581, 119283.
(18) Wang, C. Y.; Pan, X. D.; Liu, H. Y.; Fu, Z.-d.; Wei, X. Y.; Yang,
L. X. Synthesis and Antitumor Activity of 20-O-Linked Nitrogen-
Based Camptothecin Ester Derivatives. Bioorg. Med. Chem. 2004, 12
(13), 3657−3662.
(19) Hu, X.; Hu, J.; Tian, J.; Ge, Z.; Zhang, G.; Luo, K.; Liu, S.
Polyprodrug Amphiphiles: Hierarchical Assemblies for Shape-
REFERENCES
�
(
1) Louis, D. N.; Perry, A.; Reifenberger, G.; von Deimling, A.;
Figarella-Branger, D.; Cavenee, W. K.; Ohgaki, H.; Wiestler, O. D.;
Kleihues, P.; Ellison, D. W. The 2016 World Health Organization
1
570
https://dx.doi.org/10.1021/acs.molpharmaceut.0c00968
Mol. Pharmaceutics 2021, 18, 1558−1572

Molecular Pharmaceutics
pubs.acs.org/molecularpharmaceutics
Article
Regulated Cellular Internalization, Trafficking, and Drug Delivery. J.
Am. Chem. Soc. 2013, 135 (46), 17617−17629.
and Its Biological Evaluation. J. Controlled Release 2011, 156, 246−
257.
(
20) Lin, H. M.; Lin, H. Y.; Chan, M. H. Preparation,
(37) Langford, D. J.; Bailey, A. L.; Chanda, M. L.; Clarke, S. E.;
Drummond, T. E.; Echols, S.; Glick, S.; Ingrao, J.; Klassen-Ross, T.;
Lacroix-Fralish, M. L.; et al. Coding of Facial Expressions of Pain in
the Laboratory Mouse. Nat. Methods 2010, 7, 447−449.
(38) Morton, D. B.; Griffiths, P. H. Guidelines on the Recognition of
Pain, Distress and Discomfort in Experimental Animals and an
Hypothesis for Assessment. Vet. Rec. 1985, 116, 431−436.
(39) Schmidt, K. F.; Ziu, M.; Schmidt, N. O.; Vaghasia, P.; Cargioli,
T. G.; Doshi, S.; Albert, M. S.; Black, P. M.; Carroll, R. S.; Sun, Y.
Volume Reconstruction Techniques Improve the Correlation between
Histological and in Vivo Tumor Volume Measurements in Mouse
Models of Human Gliomas. J. Neuro-Oncol. 2004, 68, 207−215.
Characterization, and in Vitro Evaluation of Folate-Modified
Mesoporous Bioactive Glass for Targeted Anticancer Drug Carriers.
J. Mater. Chem. B 2013, 1 (44), 6147−6156.
(21) Ha, W.; Yu, J.; Song, X. Y.; Chen, J.; Shi, Y. P. Tunable
Temperature-Responsive Supramolecular Hydrogels Formed by
Prodrugs as a Codelivery System. ACS Appl. Mater. Interfaces 2014,
6
(
(13), 10623−10630.
22) Ayuso, J. M.; Virumbrales-Mun �o z, M.; Lacueva, A.; Lanuza, P.
M.; Checa-Chavarria, E.; Botella, P.; Fernández, E.; Doblare, M.;
Allison, S. J.; Phillips, R. M.; et al. Development and Characterization
of a Microfluidic Model of the Tumour Microenvironment. Sci. Rep.
(40) Zhao, H.; Lee, C.; Sai, P.; Choe, Y. H.; Boro, M.; Pendri, A.;
2
(
016, 6, 36086.
23) Novotny, A.; Stummer, W. 5-Aminolevulinic Acid and the
Blood-Brain Barrier - A Review. Med. Laser Appl. 2003, 18, 36−40.
24) Inoue, H.; Kajimoto, Y.; Shibata, M. A.; Miyoshi, N.; Ogawa,
Guan, S.; Greenwald, R. B. 20-O-Acylcamptothecin Derivatives:
Evidence for Lactone Stabilization. J. Org. Chem. 2000, 65 (15),
4
(
601−4606.
(
41) Liu, X.; Zhang, J.; Song, L.; Lynn, B. C.; Burke, T. G.
N.; Miyatake, S. I.; Otsuki, Y.; Kuroiwa, T. Massive Apoptotic Cell
Death of Human Glioma Cells via a Mitochondrial Pathway
Following 5-Aminolevulinic Acid-Mediated Photodynamic Therapy.
J. Neuro-Oncol. 2007, 83, 223−231.
Degradation of Camptothecin-20(S)-Glycinate Ester Prodrug under
Physiological Conditions. J. Pharm. Biomed. Anal. 2004, 35 (5),
1
(
113−1125.
42) Botella, P.; Abasolo, I.; Fernández, Y.; Muniesa, C.; Miranda,
(25) Smith, S. J.; Rowlinson, J.; Estevez-Cebrero, M.; Onion, D.;
S.; Quesada, M.; Ruiz, J.; Schwartz, S.; Corma, A. Surface-Modified
Silica Nanoparticles for Tumor-Targeted Delivery of Camptothecin
and Its Biological Evaluation. J. Controlled Release 2011, 156, 246−
Ritchie, A.; Clarke, P.; Wood, K.; Diksin, M.; Lourdusamy, A.;
Grundy, R. G.; et al. Metabolism-Based Isolation of Invasive
Glioblastoma Cells with Specific Gene Signatures and Tumorigenic
Potential. Neuro-Oncol. Adv. 2020, 2 (1), vdaa087.
2
(
57.
43) Chu, E. S. M.; Wu, R. W. K.; Yow, C. M. N.; Wong, T. K. S.;
(26) Johansson, A.; Palte, G.; Schnell, O.; Tonn, J. C.; Herms, J.;
Chen, J. Y. The Cytotoxic and Genotoxic Potential of 5-Amino-
levulinic Acid on Lymphocytes: A Comet Assay Study. Cancer
Chemother. Pharmacol. 2006, 58 (3), 408−414.
Stepp, H. 5-Aminolevulinic Acid-Induced Protoporphyrin IX Levels in
Tissue of Human Malignant Brain Tumors. Photochem. Photobiol.
2
(
010, 86, 1373−1378.
(44) Fahmy, U. A.; Fahmy, O. In Vitro Evaluation of Cytotoxic
27) Teng, L.; Nakada, M.; Zhao, S. G.; Endo, Y.; Furuyama, N.;
Properties of 5-Aminolevulinic Acid (5-ALA) on Bladder Cancer
Cells. Photodiagn. Photodyn. Ther. 2020, 30 (March), 101714.
Nambu, E.; Pyko, I. V.; Hayashi, Y.; Hamada, J. I. Silencing of
Ferrochelatase Enhances 5-Aminolevulinic Acid-Based Fluorescence
and Photodynamic Therapy Efficacy. Br. J. Cancer 2011, 104, 798−
(
45) Wen, P. Y.; Kesari, S. Malignant Gliomas in Adults. N. Engl. J.
Med. 2008, 359, 492−507.
46) Ng, K.; Kim, R.; Kesari, S.; Carter, B.; Chen, C. C. Genomic
8
(
07.
(
28) Leroy, H. A.; Vermandel, M.; Lejeune, J. P.; Mordon, S.; Reyns,
Profiling of Glioblastoma: Convergence of Fundamental Biologic
N. Fluorescence Guided Resection and Glioblastoma in 2015: A
Tenets and Novel Insights. J. Neuro-Oncol. 2012, 107, 1−12.
Review. Lasers Surg. Med. 2015, 47, 441−451.
(47) Venditto, V. J.; Simanek, E. E. Cancer Therapies Utilizing the
(29) Blume, J. E.; Oseroff, A. R. Aminolevulinic Acid Photodynamic
Camptothecins: A Review of the in Vivo Literature. Mol.
Therapy for Skin Cancers. Dermatol. Clin. 2007, 25, 5−14.
Pharmaceutics 2010, 7, 307−349.
(30) Fisher, C. J.; Niu, C.; Foltz, W.; Chen, Y.; Sidorova-Darmos, E.;
(48) Herrlinger, U.; Schäfer, N.; Steinbach, J. P.; Weyerbrock, A.;
Eubanks, J. H.; Lilge, L. ALA-PpIX Mediated Photodynamic Therapy
of Malignant Gliomas Augmented by Hypothermia. PLoS One 2017,
Hau, P.; Goldbrunner, R.; Friedrich, F.; Rohde, V.; Ringel, F.;
Schlegel, U.; et al. Bevacizumab Plus Irinotecan versus Temozolomide
in Newly Diagnosed O6-Methylguanine-DNA Methyltransferase
Nonmethylated Glioblastoma: The Randomized GLARIUS Trial. J.
Clin. Oncol. 2016, 34, 1611−1619.
1
(
2, e0181654.
31) Villate-Beitia, I.; Puras, G.; Soto-Sánchez, C.; Agirre, M.; Ojeda,
E.; Zarate, J.; Fernández, E.; Pedraz, J. L. Non-Viral Vectors Based on
Magnetoplexes, Lipoplexes and Polyplexes for VEGF Gene Delivery
into Central Nervous System Cells. Int. J. Pharm. 2017, 521, 130−
(
49) Wang, Y.; Xia, Y.; Lu, Z. Metabolic Features of Cancer Cells.
Cancer Commun. 2018, 38 (1), 65.
50) Naus, C. C. G.; Elisevich, K.; Zhu, D.; Belliveau, D. J.; Del
1
(
40.
(
32) Battah, S. H.; Chee, C.; Nakanishi, H.; Gerscher, S.; Macrobert,
Maestre, R. F. In Vivo Growth of C6 Glioma Cells Transfected with
A. J.; Edwards, C. Synthesis and Biological Studies of 5-Amino-
levulinic Acid-Containing Dendrimers for Photodynamic Therapy.
Bioconjugate Chem. 2001, 12, 980−988.
Connexin43 CDNA. Cancer Res. 1992, 52, 4208−4213.
(51) Torsvik, A.; Stieber, D.; Enger, P. O.; Golebiewska, A.; Molven,
A.; Svendsen, A.; Westermark, B.; Niclou, S. P.; Olsen, T. K.;
Chekenya Enger, M.; et al. U-251 Revisited: Genetic Drift and
Phenotypic Consequences of Long-Term Cultures of Glioblastoma
Cells. Cancer Med. 2014, 3, 812−824.
(52) Lane, R.; Simon, T.; Vintu, M.; Solkin, B.; Koch, B.; Stewart,
N.; Benstead-Hume, G.; Pearl, F. M. G.; Critchley, G.; Stebbing, J.;
Giamas, G. Cell-Derived Extracellular Vesicles Can Be Used as a
Biomarker Reservoir for Glioblastoma Tumor Subtyping. Commun.
Biol. 2019, 2, 315.
(53) Geisert, E. E.; Yang, L. J.; Irwin, M. H. Astrocyte Growth,
Reactivity, and the Target of the Antiproliferative Antibody, TAPA. J.
Neurosci. 1996, 16, 5478−5487.
(54) Mathupala, S. P. Metabolic Targeting of Malignant Tumors:
Small-Molecule Inhibitors of Bioenergetic Flux. Recent Pat. Anti-
Cancer Drug Discovery 2011, 6 (1), 6−14.
(33) Greenwald, R. B.; Pendri, A.; Conover, C. D.; Lee, C.; Choe, Y.
H.; Gilbert, C.; Martinez, A.; Xia, J.; Wu, D.; Hsue, M. Camptothecin-
0-PEG Ester Transport Forms: The Effect of Spacer Groups on
Antitumor Activity. Bioorg. Med. Chem. 1998, 6, 551−562.
34) Jacobs, V. L.; Valdes, P. A.; Hickey, W. F.; de Leo, J. A. Current
2
(
Review of in Vivo GBM Rodent Models: Emphasis on the CNS-1
Tumour Model. ASN Neuro 2011, 3, 171−181.
(35) Brehar, F. M.; Ciurea, A. V.; Chivu, M.; Zarnescu, O.;
Radulescu, R.; Dragu, D. The Development of Xenograft
Glioblastoma Implants in Nude Mice Brain. J. Med. Life 2008, 1,
2
(
75−286.
36) Botella, P.; Abasolo, I.; Fernández, Y.; Muniesa, C.; Miranda,
S.; Quesada, M.; Ruiz, J.; Schwartz, S.; Corma, A. Surface-Modified
Silica Nanoparticles for Tumor-Targeted Delivery of Camptothecin
1
571
https://dx.doi.org/10.1021/acs.molpharmaceut.0c00968
Mol. Pharmaceutics 2021, 18, 1558−1572

Molecular Pharmaceutics
pubs.acs.org/molecularpharmaceutics
Article
(55) Badawy, A. A. B. Hypothesis: Metabolic Targeting of 5-
Aminolevulinate Synthase by Tryptophan and Inhibitors of Heme
Utilisation by Tryptophan 2,3-Dioxygenase as Potential Therapies of
Acute Hepatic Porphyrias. Med. Hypotheses 2019, 131, 109314.
(56) Jakobsen, J. N.; Hasselbalch, B.; Stockhausen, M.-T.; Lassen,
U.; Poulsen, H. S. Irinotecan and Bevacizumab in Recurrent
Glioblastoma Multiforme. Expert Opin. Pharmacother. 2011, 12,
8
(
25−833.
57) Krishnan, P.; Rajan, M.; Kumari, S.; Sakinah, S.; Priya, S. P.;
Amira, F.; Danjuma, L.; Pooi Ling, M.; Fakurazi, S.; Arulselvan, P.;
et al. Efficiency of Newly Formulated Camptothecin with Beta-
Cyclodextrin-EDTA-Fe3O4 Nanoparticle-Conjugated Nanocarriers
as an Anti-Colon Cancer (HT29) Drug. Sci. Rep. 2017, 7, 10962.
(58) Creemers, G. J.; Bolis, G.; Gore, M.; Scarfone, G.; Lacave, A. J.;
Guastalla, J. P.; Despax, R.; Favalli, G.; Kreinberg, R.; Van Belle, S.;
et al. Topotecan, an Active Drug in the Second-Line Treatment of
Epithelial Ovarian Cancer: Results of a Large European Phase II
Study. J. Clin. Oncol. 1996, 14, 3056−3061.
(59) Pratesi, G.; De Cesare, M.; Carenini, N.; Perego, P.; Righetti, S.
C.; Cucco, C.; Merlini, L.; Pisano, C.; Penco, S.; Carminati, P.; Vesci,
L.; Zunino, F. Pattern of Antitumor Activity of a Novel
Camptothecin, ST1481, in a Large Panel of Human Tumor
Xenografts. Clin. Cancer Res. 2002, 8 (12), 3904−3909.
(60) Offersen, C. M.; Skjoeth-Rasmussen, J. Evaluation of the Risk
of Liver Damage from the Use of 5-Aminolevulinic Acid for Intra-
Operative Identification and Resection in Patients with Malignant
Gliomas. Acta Neurochir. 2017, 159, 145−150.
(61) Weekes, J.; Lam, A. K.-Y.; Sebesan, S.; Ho, Y.-H. Irinotecan
Therapy and Molecular Targets in Colorectal Cancer: A Systemic
Review. World J. Gastroenterol. 2009, 15, 3597−3602.
(62) Fawcett, J. W.; Asher, R. A. The Glial Scar and Central Nervous
System Repair. Brain Res. Bull. 1999, 49, 377−391.
63) Ironside, A. J.; Ironside, J. W. Pathology of Tumours of the
Central Nervous System. Surgery 2012, 30, 107−115.
64) Utsuki, S.; Oka, H.; Suzuki, S.; Shimizu, S.; Tanizaki, Y.;
(
(
Kondo, K.; Tanaka, S.; Kawano, N.; Fujii, K. Pathological and Clinical
Features of Cystic and Noncystic Glioblastomas. Brain Tumor Pathol.
2
(
006, 23, 29−34.
65) Komarova, Y. A.; Kruse, K.; Mehta, D.; Malik, A. B. Protein
Interactions at Endothelial Junctions and Signaling Mechanisms
Regulating Endothelial Permeability. Circ. Res. 2017, 120 (1), 179−
206.
1
572
https://dx.doi.org/10.1021/acs.molpharmaceut.0c00968
Mol. Pharmaceutics 2021, 18, 1558−1572