Enhancement of the brain delivery of methotrexate with administration of mid-chain ester prodrugs: In vitro and in vivo studies

Article abstract of DOI:10.1016/j.ijpharm.2021.120479

In the present study, with the aim of improving the permeability of methotrexate (MTX) to the brain, the lipophilic MTX prodrugs containing the ester functional moiety were synthesized. The chemical structure of synthesized prodrugs was characterized and confirmed by FT-IR, NMR and mass spectral studies. Based on the results of in vitro cytotoxic studies, all of the synthesized prodrugs led to decrease in the IC50 in 72 h on U87 cancer cell line and the best result was observed for dihexyl methotrexate (MTX-DH) in comparison with free MTX, which led to decrease the IC50 amount up to 6 folds. In addition, in vivo toxicity on Artemia salina (A. salina) showed that the lipophilic MTX prodrugs have been able to partially mask the toxic profile of free MTX, at the same concentrations. These findings were also in compliance with hemolysis assay results, which confirm that the conjugates has not made the drug more toxic. Furthermore, in vivo study in rat model, was employed to determine the simultaneous drug concentration in brain and plasma. According to the obtained results, the brain-to-plasma concentration ratios (Kp values) of MTX-DH and dioctyl methotrexate (MTX-DO) groups were significantly higher compared with free MTX. Moreover, the uptake clearance of MTX by brain parenchyma increased significantly (3.85 and 9.08-time increased for MTX-DH and MTX-DO prodrugs, respectively). These findings indicate that the synthesized lipophilic MTX prodrugs are non-toxic and able to enhance brain penetration of MTX.

Full text of DOI:10.1016/j.ijpharm.2021.120479

International Journal of Pharmaceutics 600 (2021) 120479
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Enhancement of the brain delivery of methotrexate with administration of
mid-chain ester prodrugs: In vitro and in vivo studies
,
b
,
,
d
,
e,
f,
,
c,
g
,
h
,
,j
Nadia Fattahia c, Ali Ramazania
*, Mehrdad Hamidib
*, Maliheh Parsai ,
Kobra Rostamizadehk,l, Hamid Rashidzadehl
a Department of Chemistry, Faculty of Science, University of Zanjan, Zanjan 45371-38791, Iran
b Department of Pharmaceutics, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran
c Trita Nanomedicine Research Center (TNRC), Trita Third Millennium Pharmaceuticals, 45331-55681 Zanjan, Iran
d Department of Biotechnology, Research Institute of Modern Biological Techniques (RIMBT), University of Zanjan, Zanjan 45371-38791, Iran
e Department of Agronomy, Research Institute of Modern Biological Techniques (RIMBT), University of Zanjan, Zanjan 45371-38791, Iran
f Department of Animal Science, Research Institute of Modern Biological Techniques (RIMBT), University of Zanjan, Zanjan 45371-38791, Iran
g Zanjan Pharmaceutical Nanotechnology Research Center (ZPNRC), Zanjan University of Medical Sciences, Zanjan, Iran
h Department of Pharmaceutical Nanotechnology, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran
i Department of Toxicology and Pharmacology, Faculty of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran
j Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
k Department of Medicinal Chemistry, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran
l Department of Pharmaceutical Biomaterials, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the present study, with the aim of improving the permeability of methotrexate (MTX) to the brain, the
lipophilic MTX prodrugs containing the ester functional moiety were synthesized. The chemical structure of
synthesized prodrugs was characterized and confirmed by FT-IR, NMR and mass spectral studies. Based on the
results of in vitro cytotoxic studies, all of the synthesized prodrugs led to decrease in the IC50 in 72 h on U87
cancer cell line and the best result was observed for dihexyl methotrexate (MTX-DH) in comparison with free
MTX, which led to decrease the IC50 amount up to 6 folds. In addition, in vivo toxicity on Artemia salina (A.
salina) showed that the lipophilic MTX prodrugs have been able to partially mask the toxic profile of free MTX, at
the same concentrations. These findings were also in compliance with hemolysis assay results, which confirm
that the conjugates has not made the drug more toxic. Furthermore, in vivo study in rat model, was employed to
determine the simultaneous drug concentration in brain and plasma. According to the obtained results, the brain-
to-plasma concentration ratios (Kp values) of MTX-DH and dioctyl methotrexate (MTX-DO) groups were
significantly higher compared with free MTX. Moreover, the uptake clearance of MTX by brain parenchyma
increased significantly (3.85 and 9.08-time increased for MTX-DH and MTX-DO prodrugs, respectively). These
findings indicate that the synthesized lipophilic MTX prodrugs are non-toxic and able to enhance brain pene-
tration of MTX.
Methotrexate
Blood-brain barrier
Prodrug
Cell culture
Brain delivery
1. Introduction
BBB is comprised of brain capillary endothelial cells that are surrounded
by astrocytes, neurons, and pericytes which create a microenvironment
In spite of extensive advances in brain research, the central nervous
system (CNS) and brain diseases, still remain the world’s leading cause
of disability (Hosokawa et al., 2007). The main challenge in CNS disease
treatment, is the presence of blood-brain barrier (BBB) that is a func-
tional and structural barrier between neural tissue and circulating blood.
that is crucial to BBB function (Abbott et al., 2010; Hawkins and Davis,
2005). Therefore, owing to its particular features, penetration across the
BBB, is the major limiting factor in the delivery of therapeutics to
diseased sites for the treatment of CNS disorders or brain tumors. Thus,
CNS therapeutic agents should be designed and developed with suitable
* Corresponding authors at: Department of Chemistry, Faculty of Science, University of Zanjan, Zanjan 45371-38791, Iran (A. Ramazani). Department of Phar-
maceutics, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran (M. Hamidi).
E-mail addresses: aliramazani@gmail.com (A. Ramazani), hamidim@zums.ac.ir (M. Hamidi).
https://doi.org/10.1016/j.ijpharm.2021.120479
Received 25 December 2020; Received in revised form 5 March 2021; Accepted 7 March 2021
Available online 17 March 2021
0378-5173/© 2021 Elsevier B.V. All rights reserved.

N. Fattahi et al.
International Journal of Pharmaceutics 600 (2021) 120479
brain penetration attributes (Liu et al., 2008).
2.2. Synthesis of lipophilic MTX prodrugs
Recently, various drug delivery vehicles are being developed in order
to achieve enough CNS penetration. Among them, prodrug strategy is
one of the most effective and promising approaches to attain good BBB
permeability and low brain non-specific tissue binding, without losing
the pharmacological efficiency (Cacciatore et al., 2018; Pavan et al.,
2008; Rautio et al., 2008a; Rautio et al., 2008b; Sutera et al., 2017).
Nowadays, the fabrication and design of novel prodrugs are pro-
posed as one of the most promising and significant strategies for both the
time-controlled and site-specific drug delivery. Prodrugs are chemically
modified derivatives of drugs that will convert to active drug molecules
in biological systems. The main goal of utilizing prodrugs in drug de-
livery is to overcome various pharmaceutical, physicochemical, bio-
pharmaceutical, and/or pharmacokinetic limitations of the free drug,
which otherwise would prevent its optimum clinical use (Najjar and
Karaman, 2019; Rautio et al., 2018; Zawilska et al., 2013).
MTX was chemically attached to alkyl halide based on the literature
reported method with slight modification (Moura et al., 2011). In brief,
0.3 mmol of MTX was dissolved in 5 mL of dimethyl sulfoxide (DMSO).
Then, cesium carbonate (0.32 mmol) and alkyl halide (0.73 mmol) were
added to the reaction vessel and the progress of the reaction was checked
by thin-layer chromatography. After completion of the reaction, 4 mL of
water was added in order to quench the reaction. The final product was
extracted with (3 × 5 mL) of chloroform and purified by preparative
layer chromatography plates (Merck silica gel (F254) powder); meth-
anol–chloroform (10:90, v/v). The chemical structure of conjugates was
characterized by the Fourier transform infrared spectroscopy on a Jasco
6300 FTIR spectrometer in the range of 400–4000 cmꢀ 1 and nuclear
magnetic resonance spectroscopy in CDCl3 on a Bruker DRX-250 Avance
spectrometer at 250.13 MHz 1H NMR and 62.90 MHz 13C NMR. Mass
spectra were recorded on a FINNIGAN-MAT 8430 mass spectrometer
operating at an ionization potential of 70 eV. The melting points were
measured on an Electrothermal 9100 apparatus and reported without
correction.
It is noteworthy that ester bonds are among the most fundamental
and numerous chemical linkages in nature that can be found in bulk
polymers, natural products, pharmaceuticals, and many other sub-
stances (Ahankar et al., 2018; Fattahi et al., 2019). This chemical bond
can be utilized in the conjugation of therapeutic agents to the various
promoieties (Fattahi et al., 2020b; Irby et al., 2017).
2.2.1. Dibutyle methotrexate (MTX-DB)
MTX, a folate analogue, functions as a strong inhibitor of dihy-
drofolate reductase, a key enzyme involved in tetrahydrofolate synthe-
sis. Thus, it can interfere in the synthesis of RNA, DNA, and consequently
proteins (Bertino, 1993; Chen et al., 2007; Fattahi et al., 2020a). MTX is
a hydrophilic anticancer drug (MW, 454.5 and log P, ꢀ 1.8) widely uti-
lized in the treatment of autoimmune diseases and various cancers
especially brain cancer. However, some drawbacks limit the use of MTX
such as high toxicity, low specificity, short plasma half-life, low solu-
bility, drug resistance by target cells and rapid diffusion throughout the
body (Abolmaali et al., 2013). In addition, its brain permeability at
conventional doses is weak and should be applied in high-doses (1–8 g/
m2) to achieve the desired amount in the brain (Batchelor et al., 2003).
Considering limitations of MTX and complexities of BBB, various
methods have been developed for improving its brain delivery,
including osmotic BBB disruption (NEUWELT et al., 1981), cetuximab
dendrimer bioconjugates(Wu et al., 2006), transnasal delivery (Shingaki
et al., 2010) and intracarotid administration of short-chain alkylgly-
cerols (Erdlenbruch et al., 2003). In continuous of our research on MTX
brain delivery (Azadi et al., 2013; Azadi et al., 2015), herein, with the
goal of demonstrating the full potential of the prodrug approach in drug
delivery, lipophilic MTX prodrugs were synthesized via an esterification
reaction and characterized by FT-IR, 1HNMR, 13C NMR and mass spec-
troscopy. The synthesized prodrugs were examined for their in vitro
stability in phosphate buffer (pH 7.4), and in the presence of rat serum.
Furthermore, their cytotoxic effects against U-87 glioma cells as well as
in vivo on A. salina were investigated. In addition, in vivo capability of
prodrugs in MTX brain delivery was examined in the rat model.
Yellow solid, m.p: 154–157 ◦C, IR (KBr) 3386, 3179, 2959, 2855,
1736, 1630, 1511, 1441, 1206, 1016, 828 cmꢀ 1; 1H NMR (250.13 MHz,
CDCl3) δ 0,85 (t, J = 6.5 Hz, 6H), 1.24–1.66 (m, 8H), 2.07–2.15 (m, 1H),
2.25–2.34 (m, 1H), 2.43–2.61 (m, 2H), 3.17 (s, 3H), 4.02 (t, J = 6.00 Hz,
2H), 4.16 (t, J = 6.5 Hz, 2H), 4.72 (s, 2H), 4.77–4.80 (m, 1H), 5.49 (s,
2H), 6.73 (2H, d, J = 8.25 Hz), 6.90 (d, J = 6.5 Hz,1H), 7.70 (d, J = 8.0
Hz, 2H), 8.63 (s, 1H) ppm; 13C NMR (62.90 MHz, CDCl3) δ 13.6, 19.0,
27.5, 30.5, 39.2, 52.2, 55.9, 64.6, 65.5, 111.5, 121.8, 128.8, 147.2,
149.7, 151.5, 162.9, 166.8, 172.5, 173.4 ppm; MS m/z: calculated for
C28H38N8O5, 566.30; found 566.50.
2.2.2. Dihexyl methotrexate (MTX-DH)
Yellow solid, m.p: 152–155 ◦C, IR (KBr) 3328, 3200, 2929, 2857,
1736, 1632, 1509, 1448, 1200, 1098, 827 cmꢀ 1; 1H NMR (250.13 MHz,
CDCl3) δ 0.86 (t, J = 6.0 Hz, 6H), 1.24–1.27 (m, 12H); 1.54–1.62 (m,
4H), 2.12–2.14 (m, 1H), 2.26–2.28 (m, 1H), 2.31–2.46 (m, 2H), 3.13 (s,
3H), 4.00 (t, J = 6.5 Hz, 2H), 4.13 (t, J = 6.5 Hz, 2H), 4.68 (s, 2H),
4.77–4.79 (m, 1H), 5.73 (bs, 1H), 6.70 (d, J = 5.5 Hz, 2H), 6.96 (bs, 1H),
7.68 (d, J = 5.5 Hz, 2H), 8.59 (s, 1H) ppm; 13C NMR (62.90 MHz, CDCl3)
δ 14.0, 22.5, 25.5, 27.4, 28.4, 30.6, 31.3, 39.2, 52.2, 55.8, 64.9, 65.7,
111.4, 121.6, 128.8, 147.1, 151.4, 162.9, 166.8, 172.5, 173.3 ppm; MS
m/z: calculated for C32H46N8O5, 622.36; found 622.50.
2.2.3. Dioctyl methotrexate (MTX-DO)
Yellow solid, m.p: 139–141 ◦C, IR (KBr) 3458, 3318, 2953, 2855,
1736, 1644, 1509, 1448, 1201, 1099, 827 cmꢀ 1; 1H NMR (250.13 MHz,
CDCl3) δ 0,85 (t, J = 6.5 Hz, 6H), 1.23 (s, 20H); 1.56–1.60 (m, 4H), 2.12
(m, 1H), 2.26 (m, 1H), 2.44–2.47 (m, 2H), 3.13 (s, 3H), 3.98 (t, J = 6.5
Hz, 2H), 4.11 (t, J = 6.5 Hz, 2H), 4.67 (s, 2H), 4.77 (m, 1H), 5.83 (bs,
1H), 6.70 (2H, d, J = 5.5 Hz), 6.98 (bs, 1H), 7.68 (d, J = 5.5 Hz, 2H),
8.57 (s, 1H) ppm; 13C NMR (62.90 MHz, CDCl3) δ 14.0, 22.6, 25.8, 27.5,
28.5, 29.1, 30.6, 31.7, 39.1, 52.2, 55.9, 65.0, 65.8, 111.4, 121.6, 128.9,
151.4, 162.4, 162.9, 166.9, 172.6, 173.3 ppm; MS m/z: calculated for
2. Materials and methods
2.1. Materials
Methotrexate sodium (MTX) was kindly provided by Loghman
Pharmaceutical Co. (Tehran, Iran). Butyl bromide, hexyl bromide (HB),
octyl bromide, and cesium carbonate were purchased from Merck
Chemical Co. All cell culture media and their supplements, including
RPMI cell culture medium, fetal bovine serum, penicillin/streptomycin,
phosphate buffer saline (PBS), L-glutamine and non-essential amino
acids were obtained from Atocel Co. (Hungry). Cell culture flasks and
cell culture plates were obtained from Nest Co. (China). 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained
from Sigma-Aldrich Co. (Germany). All other solvents, reagents, and
chemicals utilized were of analytical grade or chemically pure grade in
quality.
C36H54N8O5, 678.42; found 678.60.
2.3. Stability studies in phosphate buffer solution
The synthesized prodrugs were investigated for their chemical sta-
bility in phosphate buffer solution at 37 ◦C (0.05 M, pH 7.4). The kinetic
studies were performed by adding 50 µL of a stock solution of the pro-
drugs (1.0 mg/mL in DMSO) to 1.95 mL of the buffer solution. After
vortexing, the samples were maintained in a 37 ◦C water bath. At
appropriate time intervals, 200 µL samples were withdrawn, filtered
(0.22 µm), and then analyzed by HPLC. Experiments were performed in
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N. Fattahi et al.
International Journal of Pharmaceutics 600 (2021) 120479
triplicate, and the pseudo-first-order rate constants were calculated from
the slopes of the linear plot of the logarithms of the residual concen-
trations of compounds against time.
method (Rajabi et al., 2015). A. salina eggs were kindly gifted by
Aquatic Animal Research Center, Urmia University, Urmia, Iran. Briefly,
in a bottle containing artificial sea water (35 g/L NaCl solution), dried
cysts were collected. After incubation at room temperature during
36–48 h under the direct light and air conditioner, the larvae hatched
and the Nauplii are ready for the test. The experiment was conducted on
larvae of brine shrimp (A. salina Leach.). A stock solution of 10 mg of
MTX and synthesized prodrugs in distilled water (1 mL) was made. Next,
various concentrations of MTX and synthesized prodrugs were prepared
via serial dilution of the stock samples of synthesized prodrugs and free
2.4. Stability studies in rat serum solution
The in vitro stability of synthesized prodrugs was also performed in
physiological medium containing 50% v/v of rat serum. 100 µL of the
stock solution of prodrugs in DMSO (1 mg/mL) was added to 1.9 mL of
serum solution. After mixing, the samples were kept in a 37 ◦C water
bath. At different time points over a period of 0–120 min, aliquots of
100 µL of each sample were removed and added to 500 µL of cold
acetonitrile for deproteinization of serum. Then, the samples were
MTX in artificial sea water instantly before use. Then, 20
samples were added at various concentrations to each well of the 96-
well plates which contained 160 L RPMI (with a concentration of 35
μL of test
μ
g/L NaCl). Then, 10 nauplii were transferred into each well of the 96-
well plates using Pasteur pipet followed by incubation at 25 ◦C for 24
h. After then, the numbers of surviving nauplii were counted in each
well using a stereoscopic microscope. For each concentration, the test
was carried out in triplicate. Also, the negative control wells contained
only artificial sea water and 10 nauplii. The percentages of deaths were
estimated by counting the number of surviving nauplii in both control
and test wells.
centrifuged (4000g, 15 min) and the supernatant (20 μL) was analyzed
by HPLC.
2.5. Evaluation of safety
2.5.0.1. Hemolysis assay
The blood samples were received from healthy volunteers and placed
in EDTA-containing tubes, then centrifuged during 5 min at 3000 × g.
After then, plasma was separated, and erythrocytes were washed with
PBS solution (pH 7.4) three times, followed by resuspending them in the
PBS, a ratio of 20: 1 PBS and pure erythrocyte was utilized for preparing
final hematocrit of 5% (HCT 5%). The erythrocyte solution with the
desired HCT value was then placed into a 2 mL microtube in triplicate. A
two-fold serial dilution of MTX, MTX-DB, MTX-DO, and MTX-DH were
The lethality was obtained using Abbott’s formula as follows:
% Lethality = [(Test ꢀ Control)/Control] × 100
2.7.2. Neuropharmacokinetic study
2.7.2.1. Animals. Male Sprague-Dawley rats weighing from 250 to 300
g were procured from Pasteur Institute, Iran Branch (Karaj, Iran). The
rats were placed in individual standard cages (54 × 33 × 20 cm) at 25 ±
2 ◦C under 12-hours light–dark period and relative humidity of 60 ± 5%.
The care of animals was in compliance with the guidelines established
by the ethics committee of Zanjan University of Medical Sciences
(Zanjan, Iran).
tested, starting at concentration of 1000 μg/mL. The deionized water
and PBS were utilized as positive (100% hemolysis) and negative (0%
hemolysis) controls, respectively. Then the microtubes with rotator
shaker were incubated at 37 ± 1 ◦C for 4 h, and were centrifuged at
3000 × g for 5 min. Finally, 100 μL of supernatant was taken and
introduced to a 96-well microtiter plate and the content of free hemo-
globin was analyzed at a wavelength of 540 nm by UV–Vis spectro-
photometry. The percent of hemolysis was determined using the
following formula:
2.7.2.2. Experimental procedure and sample collection. The rats were
anesthetized one day before the experiments, by an intraperitoneal in-
jection of xylazine (10 mg/kg) and ketamine (100 mg/kg) mixture, and
a polyethylene-silicone rubber cannula was implanted in the right ju-
gular vein of rats according to a standard surgical protocol (Harms and
Ojeda, 1974; Waynforth, 1992). Details of the surgery protocol have
been previously described (Azadi et al., 2015).
Hemolysis (%) = [(Asample - Anegative)/ (Apositive - Anegative)] × 100
In the above formula, Asample stands for the samples absorbance, and
Apositive and Anegative represent the absorbance of the positive and the
negative control samples, respectively.
2.6. In vitro cytotoxicity and anticancer activity
On the day of the experiments, 48 rats were divided randomly into
three groups (n = 16). Each group received a dose of MTX (1 mg/mL)
from the following formulations: MTX (dissolved in deionized water)
was injected intravenously to group 1 (control) via the cannula, while to
groups 2 and 3 was injected the equi-dose of the MTX-DH and MTX-DO
prodrugs dissolved in absolute ethanol, respectively. During the entire
drug administration and sampling, animals remained unrestrained in
their cages. At time intervals of 0.25, 0.5, 1, 2 and 4 h followed by the
injection, a 1-mI blood sample was taken via the cannula and then an-
imals were immediately anesthetized and decapitated. The whole brain
samples were harvested, washed externally with saline and stored frozen
until the time of drug assay. Plasma was separated from the rest of the
blood components by centrifuging blood samples for 20 min at 2000g.
Brain and plasma samples were stored frozen at ꢀ 80 ◦C until drug
analysis by high performance liquid chromatography (HPLC). The pro-
tocol of the animal experiment was approved by the Ethics Committee of
Zanjan University of Medical Sciences (Zanjan, Iran).
Effects of the lipophilic MTX prodrugs and free MTX on cell viability
were investigated on U87 glioblastoma and cancer cells using MTT
assay. Firstly, the cells were cultured in RPMI supplemented with 1%
penicillin/streptomycin and 10% PBS and incubated at cell culture
conditions (5% CO2, 37 ◦C). U87 cells were then seeded in 96-well plates
at 104 cells/well. After 24 h, different concentrations of free MTX, MTX-
DB, MTX-DH, and MTX-DO prodrugs (0.625, 1.25, and 2.5 µg/mL) were
added to the cells. Cell culture medium was preferably utilized to ach-
ieve favorable concentrations, if necessary, DMSO (up to 1% V/V) was
added. Cell viability was determined using MTT reagent after 72 h.
Briefly, MTT solution (5 mg/mL in PBS, 20 µL) was added into each well
and the plates were incubated for next 4 h. After that, the MTT solution
was carefully aspirated and DMSO (100 µL) was added to each well for
dissolving formazan crystals and eventually the absorbance was deter-
mined at 570 nm by plate reader. The reference wavelength was set to
690 nm. The cell viability (percent) was measured as the ratio of the
samples to the control group.
2.7.2.3. Drug assay. The concentrations of MTX in both plasma and
brain tissue homogenates were measured by HPLC technique developed
in our previous work [21] adopted and modified for this project. The
brain samples were weighed and homogenized in normal saline by high-
speed homogenizer (Heidolph, Silent Crusher M, Schwabach, Germany).
To 225 µL of brain homogenates or plasma samples, trichloroacetic acid
2.7. In vivo study
2.7.1. Artemia test
The general toxicity of MTX and synthesized lipophilic MTX pro-
drugs on A. salina was performed according to our previously reported
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N. Fattahi et al.
International Journal of Pharmaceutics 600 (2021) 120479
(40 µL, 2 N in ethanol) and para-amino acetophenone (25 µL, 2 µg/mL
aqueous solution; internal standard) were added; the mixture was then
shaken during 30 min and centrifuged (12,000g, 15 min). Finally, the
clear supernatant (50 µL) was injected to HPLC system which was
composed of a Rheodyne injector (Rheodyne, Model 7725, USA)
equipped with a 20 l loop and a pump-controller unit (Knauer, Well-
chrom®, model k-1001, Berlin, Germany). A C18 column (Eurospher
100–5 C18, 150 mm × 4.6 mm, Knauer, Germany) as the stationary
phase and a mixture of 0.01 M phosphate buffer (pH 4.0) and acetoni-
trile (90:10, v/v) as the mobile phase were used with the flow rate of 1.0
mL/min and a detection wavelength of 307 nm (UV-detector; Knauer,
model k-2600, Berlin, Germany). The limits of detection (LOD) and
quantitation (LOQ) of the proposed method for plasma analysis were 18
and 50 ng/ml, and for brain analysis were 10 and 25 ng/ml,
respectively.
groups at the range of 1.25–3.34 ppm. MTX gives two doublet signals at
δ 7.61 (J = 8.00 Hz) and 6.81 (J = 8.75 Hz) ppm for protons on benzene
ring and the singlet signal at 8.55 ppm, which is assigned to the proton
on the pyrimidine ring (Fig. 2b). On the other hand, Fig. 2c indicates the
1H NMR of MTX-DH prodrug. The presence of characteristic signals of
both HB and MTX confirms the synthesis of prodrug. In addition, from
the 13C NMR spectra in Fig. 3, the specific signals of MTX and HB could
all be found in MTX-DH prodrug.
3.2. Lipophilicity study
Lipophilicity is a fundamental physicochemical parameter of bioac-
tive compounds, related to the ability of molecule to be transported
through biological membranes. Lipophilicity parameters like LogP and
CLogP of synthesized prodrugs as well as free MTX were calculated using
ChemBiodraw ultra 14.0 software and the values are presented in
Table 1. It was found that all synthesized compounds having values of
LogP and CLogP are in acceptable range and appeared to be suitably
lipophilic to cross the BBB.
2.8. Statistical analysis
All data were presented as mean ± standard deviation. Data were
analyzed by Kruskal–Wallis and Mann–Whitne U tests. Outcomes were
considered statistically significant at P value ≤ 0.05.
3.3. Stability studies
The chemical stability of synthesized prodrugs was investigated in
phosphate buffer solution (0.05 M, pH 7.4) at 37 ◦C; the physiological
stability was also carried out using a dilute rat serum solution (50% v/v)
at 37 ◦C. The half-lives were computed by the disappearance of the
starting compounds and are presented in Table 1. It was found that
prodrugs were stable enough in pH 7.4 phosphate buffer solution. But
the stability in serum was decreased, most likely owing to the presence
of enzymes in serum which are active in hydrolyzing ester prodrugs. The
obtained results also indicated that increasing the molecular weight of
the alkyl chain, decreased the hydrolysis of the prodrug. However, it
needs further investigation to find out the pattern of enzymatic degra-
dation for the synthesized prodrugs.
3. Results and discussion
3.1. Synthesis and characterization of lipophilic MTX prodrugs
The lipophilic MTX prodrugs were prepared by esterification of the
α
- and γ-carbonyl moieties of the glutamic acid of MTX with alkyl ha-
lides in the presence of cesium carbonate (Fig. 1). The isolated yields
were found to be over 97%. The chemical structure of obtained prodrugs
was characterized and confirmed by 1H NMR and 13C NMR. For
example, the 1H NMR and 13C NMR spectra of MTX-DH are shown in
Figs. 2 and 3, respectively. As clearly seen, the characteristic signals of
MTX and HB could be clearly observed in MTX-DH prodrug. As indicated
in Fig. 2a, the typical 1H NMR spectrum of HB gives a triplet signal for
methyl protons at 0.86 ppm (J = 6.00 Hz) and signals for the methylene
3.4. Hemolysis assay
In order to utilize diverse drugs and drug conjugates in biomedicine,
it is necessary to evaluate its hemocompatibility. Hemolytic toxicity is
based on the disruption of erythrocytes’ membrane which resulted in
release of hemoglobin to surrounded media. To get a clue of the he-
molytic toxicity of the drugs and drug conjugates that are directly
administrated and exposure to the human blood, hemolysis test is of
significant importance. The MTX and lipophilic MTX prodrugs were well
tolerated as suggested by the toxicity studies done. The major limitation
of drugs and drug conjugates for using in biomedicine is its hemolytic
toxicity. Therefore, hemolytic toxicity was carried out in terms of
percent RBC hemolysis to monitor toxicity of the free drug and its
conjugates. As presented in Fig. 4, both free MTX and lipophilic MTX
prodrugs exhibited does-dependent hemolysis. Moreover, the lipophilic
MTX prodrugs exhibited relatively less hemolytic toxicity in comparison
with free MTX at same concentrations. Indeed, these results indicated
that the lipophilic MTX prodrugs have an ability to partially reduce the
toxic profile of free MTX, at the same concentration. Since the hemolysis
value of lipophilic MTX prodrugs were close to that of free MTX at low
concentrations (<250 μg/mL), the hemolytic toxicity profiles can be
considered similar to that of free MTX, at the same time conjugate has
also not made it more toxic.
3.5. Cell viability assay
The obtained data from MTT cell viability assays on U87 cells is
presented in Fig. 5. Results were reported compared to the control
group. The IC50 values of MTX-DB, MTX-DH, MTX-DO, and free MTX
were 0.609, 0.254, 1.51 and 1.7 µg/mL, respectively. As observable, in
comparison with free MTX, all of the synthesized prodrugs led to
Fig. 1. Schematic representation of chemical synthesis of lipophilic
MTX prodrugs.
4

N. Fattahi et al.
International Journal of Pharmaceutics 600 (2021) 120479
Fig. 2. 1H NMR (250.13 MHz) spectra of (a) HB in CDCl3, (b) MTX sodium in DMSO and (c) MTX-DH prodrug in CDCl3.
Fig. 3. 13C NMR (62.90 MHz) spectra of (a) HB in CDCl3, (b) MTX sodium in DMSO and (c) MTX-DH prodrug in CDCl3.
decrease the IC50 in 72 h on U87 cancer cell line and the best result was
Table 1
observed for MTX-DH prodrug which led to decrease the IC50 amount
up to 6 folds.
LogP and ClogP values and stability studies in phosphate buffer solution and in
Diluted Rat Serum.
Compound
LogP
ClogP
t1/2(h) PBS
t1/2(min) Diluted Rat Serum
3.6. Artemia assay
MTX-DB
MTX-DH
MTX-DO
MTX
3.95
5.62
7.71
0.94
3.23
5.34
7.46
ꢀ 0.52
41 ± 0.11
64 ± 0.21
79 ± 0.18
–
46 ± 0.11
59 ± 0.21
67 ± 0.13
–
Since the A. salina is characterized by ease of handling, low cost,
high sensitivity, and the fact that it can be observed with the naked eye
and without requiring additional or advanced equipment, the use of this
microcrustacean as an animal model for the evaluation of acute toxicity
LogP and ClogP calculated by using ChemBioDraw Ultra version 14.0.
5

N. Fattahi et al.
International Journal of Pharmaceutics 600 (2021) 120479
catheter inserted intravenously followed by brain and plasma sampling.
The pharmacokinetic approach used was such that a total of 4 rats were
utilized to collect the data for each time point since the need for the
decapitation of animals does not allow any of the animals to survive for
the further time points. Fig. 7a and b show the plasma and brain con-
centrations of MTX at various time points following intravenous (IV)
injection of MTX-DH, MTX-DO, as well as the equi-dose free MTX. As
presented in Fig. 7a, the MTX plasma concentration at 15 min was
significantly different between the free drug and the synthesized pro-
drugs. Also, during all test times, the MTX plasma concentration in both
status, MTX-DH and MTX-DO, is less than the free drug. This is probably
due to the different tissue distribution of the precursors due to their
higher lipophilicity.
Hemolysis Assay
10
9
8
7
6
5
4
3
2
1
0
1000
500
250
125
62.5
Concentrations (μg/ml)
The concentration of MTX in the free drug mode at 15, 30, and 60
min is higher than in both MTX-DH and MTX-DO modes. Also, during
120 min, the brain concentration of MTX in both MTX-DH and MTX-DO
modes is significantly higher than in the free drug, indicating that
despite the low plasma concentrations of MTX in both MTX-DH and
MTX-DO cases, the brain concentration has been increased. It shows that
MTX-DH and MTX-DO prodrugs increase the ability of MTX to pass
through BBB due to their lipophilic properties. During 240 min,
medullar concentration of MTX was higher in both MTX-DH and MTX-
DO modes than the free drug. At this point, there is no significant dif-
ference in brain concentration in MTX-DH and MTX-DO prodrugs
(Fig. 7b).
MTX
MTX-DH
MTX-DO
MTX-DB
Fig. 4. In vitro hemolysis of synthesized prodrugs and free MTX at different
concentrations.
has been unexpectedly increased. So, in this study, inexpensive and
reliable acute toxicity of lipophilic MTX prodrugs and free MTX were
carried out against A. salina. The percentage of lethality can be utilized
as a bioassay indicator for the acute toxicity of MTX and lipophilic MTX
prodrugs. The acute toxicity was evaluated by measuring the number of
dead nauplii after 24 h exposure to MTX and lipophilic MTX prodrugs
and the mortality rates of A. salina nauplii are shown in Fig. 6. As
indicated, at high concentrations of MTX and lipophilic MTX prodrugs
In addition, by considering the Kp values for three groups, it is clear
that MTX in the free drug state has a lower shelf life in brain than
plasma, which is probably due to the limited entry into the brain owing
values from 1000 to 250
μg/mL, the concentration-dependent toxicity
was observed, whereas no mortality was observed at lower concentra-
tion) < 250 μg/mL) for both MTX and lipophilic MTX prodrugs. In
addition, there was no significant difference of mortality was observed
among the lipophilic MTX prodrugs. Also the percentage of dead nauplii
Artemia assay
reduced from 13.3% at 1000
250
drugs reduced from 8% at 1000
g/mL. Indeed lipophilic MTX prodrugs in all cases exhibited less
μg/mL of MTX to a little more than 3% at
14
μ
g/mL and the percentage of dead nauplii for lipophilic MTX pro-
12
10
8
μg/mL to a little more than 0.9% at 250
μ
toxicity in comparison with free MTX, but the values were close to that
of free MTX. These results suggest that the lipophilic MTX conjugate has
been able to partially mask the toxic profile of free MTX, at the same
concentration. These results were also repeated in the hemolysis assay,
which confirms that the conjugates has not made the drug more toxic.
6
4
2
0
1000
500
250
125
62.5
3.7. Neuropharmacokinetic study
Concentrations (μg/ml)
MTX
MTX-DH
MTX-DO
MTX-DB
In order to obtain the simultaneous drug concentrations in brain and
plasma and the corresponding Kp values, a cross-sectional pharmaco-
kinetic study was performed by drug administration to rats via the
Fig. 6. General toxicity of various formulations to A.salina in sea water.
Fig. 5. Effect of lipophilic MTX prodrugs on viability of U87 cells which were exposed with appropriate concentrations of the various formulations and incubated for
72 h at cell culture conditions.
6

N. Fattahi et al.
International Journal of Pharmaceutics 600 (2021) 120479
Fig. 7. (a) Plasma and (b) brain concentration-time profiles of MTX after 1 mg IV administration of free MTX and equi-dose of the MTX-DH and MTX-DO prodrugs in
male Sprague-Dawley rats.
to its hydrophilicity. In addition, higher Kp is observed in both MTX-DH
and MTX-DO compared to the free drug (Fig. 8).
Briefly, Kp was plotted against AUC/Cp (the ratio of the AUC in the
plasma to the corresponding plasma concentrations) from zero to a
given time. The slope of the plot, which shows the apparent brain uptake
Integration plot analysis was carried out for the calculation of brain
uptake clearance of free MTX and synthesized formulations. The theo-
retical basis for this analysis was illustrated in previous reports (Azadi
et al., 2015; Hamidi, 2006; Karami et al., 2019; Nasiri et al., 2019).
clearance, was computed (Fig.
Information).
9 and Table S1 in Supporting
Brain uptake clearance shows clearly the efficiency of MTX-DH and
MTX-DO in increasing MTX levels in the brain. In this case, MTX-DH and
MTX-DO have reached 3.85 and 9.08 times the clearance value of brain
uptake in comparison with the free drug. These data besides the plasma
AUC of the free drug, which is probably 16.76 and 28.84 times higher
than the MTX-DH and MTX-DO, respectively, which is due to the
different tissue distribution of the prodrugs, can confirm the scenario of
the MTX drug delivery system to the brain. Considering the lipophilic
properties of MTX-DH and MTX-DO, the role of ester-prodrugs in
penetration through BBB could be the main reason for the increasing the
level of MTX.
4. Conclusion
In summary, the lipophilic MTX prodrugs containing the ester
functional moiety were synthesized and characterized by FT-IR and
NMR analyses. In vitro cytotoxic studies showed that all of the synthe-
sized prodrugs led to decrease in the IC50 in 72 h on U87 cancer cell line.
In addition, in vivo toxicity on A. salina indicated that the lipophilic MTX
prodrugs have been able to partially mask the toxic profile of free MTX,
Fig. 8. Kp (Brain-to-plasma concentration–time) profile of MTX after 1 mg IV
administration of free MTX and equi-dose of the MTX-DH and MTX-DO pro-
drugs in male Sprague-Dawley rats.
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N. Fattahi et al.
International Journal of Pharmaceutics 600 (2021) 120479
Fig. 9. Brain uptake plot of MTX after 1 mg IV administration of (a) free MTX and equi-dose of the (b) MTX-DH and (c) MTX-DO prodrugs; in male Sprague-
Dawley rats.
at the same concentrations. These findings were also in compliance with
hemolysis assay results, which confirm that the conjugates has not made
the drug more toxic. Meanwhile, results from in vivo study on rats
showed that the Kp values of MTX-DH and MTX-DO groups were
significantly higher compared with free MTX. Moreover, the uptake
clearance of MTX by brain parenchyma increased significantly (3.85 and
9.08-time increased for MTX-DH and MTX-DO prodrugs, respectively).
Taken together, our results indicate that the synthesized lipophilic MTX
prodrugs are non-toxic and able to enhance brain penetration of MTX.
National Science Foundation: INSF”. Also, the present work has been
done in line with Nadia Fattahi PhD Thesis.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijpharm.2021.120479.
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