Computational Prodrug Design Methodology for Liposome Formulability Enhancement of Small-Molecule APIs

Abstract

Encapsulation into liposomes is a formulation strategy that can improve efficacy and reduce side effects of active pharmaceutical ingredients (APIs) that exhibit poor biodistribution or pharmacokinetics when administered alone. However, many APIs are unsuitable for liposomal formulations intended for parenteral administration due to their inherent physicochemical properties─lipid bilayer permeability and water-lipid equilibrium partitioning coefficient. Too high permeability results in premature leakage from liposomes, while too low permeability means the API is not able to pass across biological barriers. There are several options for solving this issue: (i) change of the lipid bilayer composition, (ii) addition of a permeability enhancer, or (iii) modification of the chemical structure of the API to design a prodrug. The latter approach was taken in the present work, and the effect of small changes in the molecular structure of the API on its permeation rate across a lipidic bilayer was systematically explored utilizing computer simulations. An in silico methodology for prodrug design based on the COSMOperm approach has been proposed and applied to four APIs (abiraterone, cytarabine, 5-fluorouracil, and paliperidone). It is shown that the addition of aliphatic hydrocarbon chains via ester or amide bonds can render the molecule more lipophilic and increase its permeability by approximately 1 order of magnitude for each 2 carbon atoms added, while the formation of fructose adducts can provide a more hydrophilic character to the molecule and reduce its lipid partitioning. While partitioning was found to depend only on the size and type of the added group, permeability was found to depend also on the added group location. Overall, it has been shown that both permeability and lipid partitioning coefficient can be systematically shifted into the desired liposome formulability window by appropriate group contributions to the parental drug. This can significantly increase the portfolio of APIs for which liposome or lipid nanoparticle formulations become feasible.

Full text

Computational Prodrug Design Methodology for Liposome
Formulability Enhancement of Small-Molecule APIs
Martin Balouch, Katerina Storchmannová, Frantisek Stepánek,*and Karel Berka*
Cite This: https://doi.org/10.1021/acs.molpharmaceut.2c01078
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ABSTRACT: Encapsulation into liposomes is a formulation strategy that can improve ecacy and reduce side eects of active
pharmaceutical ingredients (APIs) that exhibit poor biodistribution or pharmacokinetics when administered alone. However, many
APIs are unsuitable for liposomal formulations intended for parenteral administration due to their inherent physicochemical
properties�lipid bilayer permeability and water−lipid equilibrium partitioning coecient. Too high permeability results in
premature leakage from liposomes, while too low permeability means the API is not able to pass across biological barriers. There are
several options for solving this issue: (i) change of the lipid bilayer composition, (ii) addition of a permeability enhancer, or (iii)
modification of the chemical structure of the API to design a prodrug. The latter approach was taken in the present work, and the
eect of small changes in the molecular structure of the API on its permeation rate across a lipidic bilayer was systematically explored
utilizing computer simulations. An in silico methodology for prodrug design based on the COSMOperm approach has been
proposed and applied to four APIs (abiraterone, cytarabine, 5-fluorouracil, and paliperidone). It is shown that the addition of
aliphatic hydrocarbon chains via ester or amide bonds can render the molecule more lipophilic and increase its permeability by
approximately 1 order of magnitude for each 2 carbon atoms added, while the formation of fructose adducts can provide a more
hydrophilic character to the molecule and reduce its lipid partitioning. While partitioning was found to depend only on the size and
type of the added group, permeability was found to depend also on the added group location. Overall, it has been shown that both
permeability and lipid partitioning coecient can be systematically shifted into the desired liposome formulability window by
appropriate group contributions to the parental drug. This can significantly increase the portfolio of APIs for which liposome or lipid
nanoparticle formulations become feasible.
KEYWORDS: lipid bilayer, permeability, partitioning coecient, prodrug, COSMOperm
1. INTRODUCTION
Since the first liposomal formulation of an active pharmaceut-
ical ingredient (API) was approved in 1995,
1
lipid nano-
formulations have rapidly developed, leading to the recent
rollout of mRNA vaccines.
2,3
The encapsulation of macro-
molecules such as antibodies or nucleic acids
4
within liposomal
structures is facilitated mainly by combining steric factors and
electrostatic interactions with charged lipids. Regarding small
molecules, formulability into liposomes for parenteral
application strongly depends on the lipid/water partitioning
and the permeation rate across the liposome bilayer, which is
unfavorable for many APIs. Only about 20 liposomal
formulations of small-molecule APIs are currently approved
in the EU and the U.S.A. Liposomal formulations in clinical
trials are often only previously used APIs in dierent
combinations or strengths
5
rather than new chemical entities.
Received: December 15, 2022
Revised: March 6, 2023
Accepted: March 9, 2023
Article
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The development of liposomal formulations of new APIs is
limited because many molecules in their native form are
inherently unsuitable for liposome encapsulation and release.
An API′s partitioning and permeation properties need to be
properly balanced to be suitable for liposome formulation
intended for parenteral administration. The permeability across
the liposome membrane should be suciently high to allow
the API to be released from the liposomes in the target tissue
or cell but suciently low to avoid premature spontaneous
leakage. The lipid/water partitioning coecient should be
suciently high to allow the API to overcome the energy
barrier represented by the lipid bilayer but suciently low to
prevent the API from being trapped in the membrane. The
phenomena of API partitioning and permeability were
investigated computationally and validated experimentally in
our recent publication.
6
Systematic rules for the formulability
of small-molecule APIs in liposomes have been proposed using
the liposome biochemical classification system (LBCS). LBCS
was designed as a two-dimensional (2D) diagram with
approximative areas for the partitioning and permeation
constants for each pair of API and lipid bilayer composition
that enable or prevent successful liposome formulation. The
meaning of these areas is explained in Figure 1.
To change the API/membrane behavior (i.e., the API
position in the LBCS parametric space), there are, in principle,
three options: (i) changing the membrane composition, (ii)
using a permeation enhancer, or (iii) modifying the structure
of the API. The membrane composition can be changed, e.g.,
by adding cholesterol or specific phospholipids to influence the
membrane phase transition temperature and lipid bilayer
structure. The key characteristics governing permeability
through membranes were published recently.
7
The bilayer
thickness, the lipid phase, and the sterol type were found to
play the main role. In the specific case of ceramide bilayers, the
length of the ceramide was found to play a crucial role as well.
8
Quantitatively, a change of lipid bilayer composition is
generally suitable for fine-tuning the permeation/partitioning
values but not for achieving profound, orders-of-magnitude
shifts within the LBCS diagram for a given API.
Permeation enhancers can achieve more dramatic shifts in
permeability, substances added to the API−lipid bilayer system
to modify the permeation rate
9,10
by various mechanisms such
as the disruption of phospholipid packing in the bilayer,
solubilization of the API,
11,12
or facilitating transport.
13
Permeation enhancers are mostly studied in the context of
skin permeation, where the lipidic membrane is easily
accessible. Also, there are studies into permeation enhancers
for the oral route of administration, e.g., for improving the oral
administration of peptides.
14
Examples of permeability
enhancers for oral peptide administration include citric acid,
sodium caprate, dodecyl-β-D-maltopyranoside, and others.
15
The third option for aecting permeation and partitioning
properties is to modify the molecular structure of the API itself,
i.e., to design a prodrug. In the pharmaceutical practice,
prodrugs are used for various reasons, such as to modify an
API′s solubility or dissolution rate. Examples include the
antipsychotic drug paliperidone, whose ester paliperidone
palmitate
16
controls the dissolution rate in an intramuscular
depot formulation. Another example is abiraterone, an
anticancer drug whose ester abiraterone acetate
17
is used for
improving solubility in the gastrointestinal tract. However,
there are no reports of designing prodrugs specifically to enable
liposomal formulations of small-molecule drugs. When
considering the prodrug design, there comes a question of
whether and how the API can be modified to obtain the
desired lipid/water partitioning and liposome bilayer perme-
ation behavior. Generally, the partition coecient and
permeation rate are considered to be strongly correlated
based on the Meyer−Overton rule.
18
They correlate strongly
in the case of very simple molecules
19
(molar mass around and
under 50 Da), but in the case of more complex molecules, this
approximation works poorly.
20
Moreover, the combination of
prodrug synthesis and permeation enhancement is also
possible. The key principle is the modification of API and its
incorporation into the liposome.
21
This approach includes
diglyceride conjugate of doxorubicin
22
or cholesterol conjugate
of topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothe-
cin.
23
Therefore, the present work aims to propose a general
methodology for computational prodrug design by systemati-
cally investigating the relationship between API molecular
structure modification and its position in the LBSC diagram.
To this end, four APIs with dierent initial positions in the
LBSC diagram have been chosen (cytarabine, fluorouracil,
paliperidone, abiraterone) and systematically modified by
adding either lipophilic or hydrophilic groups of dierent
properties (e.g., length of acyl chain) or position. These
molecules represent general situations where an API is initially
unsuitable for liposomal formulation either because of too low
or too high permeability or due to an unsuitable partitioning
coecient, as shown in Figure 1. Apart from their position in
the LBCS diagram, the rationale for choosing these four APIs
is that they all provide opportunities for substituting functional
groups in their molecular structure without impacting the
pharmacophore responsible for their biological action. In the
Figure 1. Parametric space showing the sorting mechanism for LBCS
classification, where log Kis the membrane/water partition coecient
and log Perm is the permeation rate through the liposome membrane at
the designated temperature. Gray bin: APIs too permeable for
liposome formulation; blue bin: neutral APIs too permeable but
possible to move to the green bin if ionizable in liposome cavity by
pH; green bin: APIs suitable for liposome formulation and even for
thermally induced release; red bin: APIs suitable for liposome
formulation but not for thermally induced release; yellow bin:
lipophilic APIs suitable for entrapment in the liposome membrane.
Positions of the four APIs investigated in this work are marked by the
squares.
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case of paliperidone
16
and abiraterone,
17
the ability to form
ester prodrugs without negatively influencing bioactivity has
even been shown experimentally, and these ester prodrugs are
successfully marketed and clinically used. We aim to provide
more general guidelines for rational prodrug design by
systematically exploring the relationship between API molec-
ular structure modification and its position in the perme-
ability−partitioning diagram.
2. MATERIALS AND METHODS
2.1. Preparation of Molecular Structures. Each
considered molecule was drawn in MarvinSketch 21.9
24
and
saved as a SMILES file. From the SMILES, the LigPrep and
MacroModel packages from Schrodinger Release 2017-2
software generated neutral conformers of all compounds in
vacuum using the OPLS_2005 force field.
25
These compounds
were cytarabine, its ester, amide, and alkyl chain analogues, as
described in Figure 2, and four APIs and their fructose adducts,
as shown in Figure 5. For each compound, a maximum of 10
conformers were selected based on MCMM/LMC2 con-
formation searching algorithm with Monte Carlo structure
selection with the MacroModel package from the Schrodinger
2017-2 software suite. The conformers were selected within 5
kcal/mol of the conformer with the lowest energy and RMSD
of at least 0.2 nm between individual conformers. A
subsequent DFT/B-P/cc-TZVP vacuum and COSMO water
optimization with fine grid option were carried out for each
conformer using Turbomole 6.3.
26
The COSMO files for each
conformer were obtained from this procedure and sub-
sequently used for partitioning and permeation calculation.
2.2. COSMOperm Permeability Calculation. For the
purpose of the present work, the membrane with a fixed
composition (DPPC/DPPG/cholesterol = 75:10:15 mol %) at
293 K was used as the permeation barrier. The lipid bilayer
structure was obtained by molecular dynamics (MD)
simulations, as described in detail in our recent work.
6
Briefly,
a membrane bilayer containing 128 preordered lipid molecules
was created by an in-house script. Slipids
27
parameters and the
TIP3P
28
water model were used for the simulation, and the
membrane was simulated at 293 K under periodic boundary
conditions for approx. 250 ns to ensure the thermodynamic
equilibrium of the membrane. The simulation was performed
using Gromacs 4.5.4. software.
29
From the MD simulation, 5
randomly chosen conformations from the last 10 ns were
chosen and saved as.pdb files. Using COSMOtherm X8
software and.cosmo files of all membrane components, the five
separate.mic files with a σ-profile representation of the
membrane sliced into 50 horizontal layers and its electron
density and charge of each layer were calculated using the
COSMO-RS approach.
30
For each combination of the lipid membrane (five snapshots
from the DPPC/DPPG/cholesterol membrane) and each
conformer of each calculated molecule (26 molecular variants
in total), the partition coecient between the membrane and
the water phase was calculated using the COSMOmic
31
approach, and the permeation rate was calculated using the
COSMOperm approach.
32
Briefly, the chemical potential of
the molecule in that part of the membrane was calculated using
the σ-profile of a permeating molecule and the σ-profile of a
specific layer of the membrane. Therefore, the energy profile
through the membrane can be obtained (an example of
calculated profiles for several cytarabine prodrugs is shown in
Figure 3). Then, the membrane/water partitioning coecient
can be calculated from the energy minima of the chemical
potential profile directly using the following equation
=Klog e G RT(min)/
where log Kis the lipid/water partition coecient, ΔG(min)
is the free energy change for permeating molecule between the
water environment and the energy minimum, Ris the universal
gas constant, and Tis the temperature in K. The permeation
rate across the liposome membrane was calculated using the
Diamond and Katz model for the steady-state flux of a solute
through the membrane
33
=
P D z K z
z
1 1
( ) ( )d
L
L
erm
where 2Lis the thickness of the membrane, D(z) is the
diusivity of the permeating molecule in the membrane layer,
and K(z) is the partition coecient of the permeating
Figure 2. Cytarabine molecule (A) and its ester (B), amide (C), and alkyl (D) prodrugs that were included in a systematic permeation study using
COSMOperm calculations. R1 = ethyl (CytO2), butyl (CytO4), hexyl (CytO6), octyl (CytO8), decyl (CytO10), dodecyl (CytO12), tetradecyl
(CytO14), and hexadecyl (CytO16); R2 = ethyl (CytN2), butyl (CytN4), hexyl (CytN6), octyl (CytN8), and decyl (CytN10); and R3 = ethyl
(CytC2), butyl (CytC4), hexyl (CytC6), octyl (CytC8), and decyl (CytC10).
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C

molecule in the membrane layer. Finally, the results for all
investigated molecular structures were incorporated into the
MolMeDB database.
34
3. RESULTS AND DISCUSSION
3.1. Molecule Selection and Case Study Strategy.
Recently,
6
the permeability and partitioning coecients of 56
APIs from the DrugBank database were calculated and sorted
into the LBCS diagram. Out of these, four APIs were identified
as particularly poorly permeating: allosamidin, azacitidine,
decitabine, and cytarabine. For the sake of case studies in the
present work, cytarabine has been selected. Cytarabine is an
antineoplastic agent used to treat acute myeloid leukemia,
acute lymphocytic leukemia, chronic myelogenous leukemia,
meningeal leukemia, and other meningeal neoplasms. Cytar-
abine is on the WHO list of essential medicines due to its
therapeutic significance and is usually administered by i.v.
infusion, intrathecal injection, or subcutaneous injection as a
solution, but liposomal formulations have also been proposed,
e.g., DepoCyte
35
(now discontinued) or Vyxeos liposomal.
36
There are numerous cytarabine prodrugs in development.
These comprise mostly cytarabine amino acids, cytarabine
phosphates, and cytarabine fatty acids,
37
also considered in this
work. Cytarabine amino acid prodrugs improve cytarabine
permeability,
38
and they have already achieved satisfactory
results in clinical trials.
39
Fatty acid conjugation to cytarabine
was done with various fatty acid chain lengths, even with some
non-common ones like 24-carbon long double acid chain.
40
When palmitic acid was attached, the octanol partition
coecient increased by 4 orders of magnitude.
41
As can be seen from the examples above, adding ester-
attached fatty acid is one of the common strategies to enhance
cytarabine permeability (and bioavailability). However, a
rational guideline for the choice of the fatty acid chain length
and its position seems to be lacking. Therefore, various lengths
of fatty acids were systematically investigated in this study. To
ensure the versatility of our approach, the fatty acids were
attached to two dierent cytarabine parts: nitrogen on the
benzene ring and oxygen on the fructose C5 carbon. The
temperature for the case study was chosen to be 293 K to
mimic the temperature of liposomal preparation during the
manufacture. To ensure sucient API retention in liposomes,
the API must not leak spontaneously out of the liposome at the
temperature at which it is manufactured. There can be
applications where also, at the body temperature, the API must
not permeate through liposomes instantaneously but slowly
over time. Though the absolute values of permeation rate will
dier with temperature, the trends and principles for
permeation modification drafted in this publication will stay
relevant and be modifiable for a specific situation.
3.2. Prodrug Design for Permeability Enhancement.
The permeability of the unmodified cytarabine molecule
obtained by COSMOperm calculation was log Perm =−13.2,
which is approx. 4−5 orders of magnitude lower than the ideal
range for liposomal formulation. Hence, cytarabine has been
chosen for the present study as a representative candidate of a
poorly permeating substance to test the computational prodrug
design methodology. To improve the permeability of such a
poorly permeating molecule, prodrugs containing nonpolar
aliphatic hydrocarbon chains attached via a hydroxyl or amine
group have been systematically created in silico, and their
permeability and partitioning coecients have been calculated.
Cytarabine contains one amine group and three hydroxyl
groups (Figure 2A), which can be theoretically used for
Figure 3. Free energy profiles of cytarabine and its ester prodrugs
from acetyl to hexadecyl diering by two carbon lengths between
neighboring analogues.
Table 1. Calculated Permeability and Partitioning Coecients for Dierent Cytarabine Prodrugs through DPPC/DPPG/Chol
(75:10:15) Membrane at 293 K, Using the Mean of 5 Calculations through Randomly Chosen MD Snapshots from the Last 10
ns of Simulation
molecule log K(mol/mol) log Perm (cm/s) molecule log K(mol/mol) log Perm (cm/s)
cytarabine (Cyt) −0.68 ±0.17 −13.19 ±0.04
substitution on −OH group using esterification substitution on −NH2group to make amides
CytO2 −1.08 ±0.16 −11.43 ±0.04 CytN2 −1.15 ±0.26 −11.33 ±0.03
CytO4 −1.31 ±0.05 −10.21 ±0.06 CytN4 −1.35 ±0.04 −10.18 ±0.04
CytO6 −0.25 ±0.25 −8.67 ±0.10 CytN6 −1.37 ±0.04 −8.85 ±0.04
CytO8 0.16 ±0.57 −7.22 ±0.17 CytN8 0.00 ±0.29 −7.42 ±0.05
CytO10 2.08 ±0.60 −5.47 ±0.23 CytN10 1.37 ±0.36 −5.77 ±0.06
CytO12 3.90 ±0.77 −3.55 ±0.45 alkyl chain substitution on pyrimidine ring
CytO14 5.64 ±0.98 −1.93 ±0.53 CytC2 −0,92 ±0.11 −11.65 ±0.05
CytO16 8.05 ±0.99 0.08 ±0.28 CytC4 −0.27 ±0.13 −10.42 ±0.08
CytC6 0.47 ±0.17 −8.78 ±0.14
CytC8 1.91 ±0.22 −7.75 ±0.21
CytC10 3.58 ±0.40 −5.48 ±0.42
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D

prodrug preparation. The amine group and the pentose C5′
hydroxyl group (Figures 2B,C) have been chosen due to their
opposite locations. To test the robustness of the computational
prodrug design approach, the alkylation of the pyrimidine ring
at position 5 next to the amine group (Figure 2D) has also
been considered. Esters with carboxylic acids containing 2, 4, 6,
8, 10, 12, 14, and 16 carbon atoms and amides with 2, 4, 6, 8,
and 10 carbon long carboxylic acid were created computa-
tionally as cytarabine prodrugs. Regarding the pyrimidine ring,
2, 4, 6, 8, and 10 carbons were added.
The COSMOperm method is based on calculating the
energy profile through the membrane. The energy profile of
cytarabine and its ester prodrugs calculated through one of the
randomly chosen snapshots from the last 10 ns of the MD
simulation of the membrane is shown in Figure 3. With
increasing acyl chain length attached to cytarabine, the energy
barrier within the membrane monotonically decreases,
resulting in higher permeability. Concurrently, the energy
minimum located around 20 Å from the membrane center
becomes more pronounced with increasing acyl chain length. A
lower minimum leads to a higher membrane/water partition-
ing coecient. The permeability and partitioning coecients
have been calculated as described in Section 2.2, and the
resulting values are summarized in Table 1.
The three prodrug families derived from cytarabine show
distinct trajectories in the LBCS parametric space of
partitioning and permeability coecient values (Figure 4). It
is clearly visible from this graphical representation that even a
substance that was initially far from the liposome formulability
window can be brought into the feasible range by appropriate
modification of its molecular structure. In the specific case of
cytarabine, its butyrate prodrugs seem to be the most
promising candidates for encapsulation into liposomes and
their thermal release. On the other hand, if the formulation
needs to be a liposome or lipid particle where the API is
supposed to remain dissolved in the membrane, prodrugs with
longer hydrocarbon chains (dodecylate, tetradecylate, hexade-
cylate, or higher) would be the appropriate choice in this case.
It is interesting to observe that while the permeability of the
N- and O- and C-substituted prodrugs seems to depend only
on the length of the added hydrocarbon chain, the water/
membrane partitioning coecient is also aected by the
position of the substitution. From the calculation results, the
acetylation of the hydroxyl and the amine group has led to an
almost identical increase in the permeation coecient (1.71
and 1.81 in log Perm, respectively), and the addition of each
further two carbons then resulted in an almost uniform
increase in log Perm (1.44 ±0.21 for acyls and 1.52 ±0.48 for
alkyls). The monotonous and nearly linear increase of log Perm
with the number of carbons added to the original molecular
structure continued until the end of the investigated range. A
more complex pattern could be seen in the partitioning
coecient. For the first 2−6 carbons added to the structure,
there was only a negligible eect on the values of log K,
followed by the onset of a sustained increase (approx. 1.9 in
log Kfor every two carbons).
This observation provides guidelines for the modification of
API behavior by the formation of a prodrug: it is possible to
increase permeability by several orders of magnitude, either
with or without a simultaneous increase in the partitioning
coecient, simply by choosing an appropriate length of the
carbon chain and its substitution position.
3.3. Prodrug Design for Permeability Reduction. The
case discussed above represented a situation where the API
permeability was initially too low, and the objective of prodrug
design was to improve the permeation. However, if the API
permeation rate is too high, such API cannot be successfully
encapsulated in liposomes due to premature leakage during
manufacturing and storage before administration. The
permeation rate through the membrane should be slowed
down to prevent excessive spontaneous leakage from lip-
osomes. This can be, in principle, achieved by adding polar
groups to the API structure. The choice of possible polar
groups is broad, the main limitation being that they should be
biocompatible and either not interfere with the pharmaco-
phore or be metabolizable. For the sake of the present work,
fructose has been chosen as an example of such a structure.
Four APIs with a dierent initial location in the LBCS space
(i.e., dierent initial permeability/partitioning combinations)
have been selected to demonstrate the computational prodrug
design for permeability reduction: anticancer drugs abirater-
one, cytarabine, and 5-fluorouracil and an antipsychotic drug
paliperidone. Their fructose adducts were formed computa-
tionally, and the eect on permeability has been investigated,
as described in Section 2.2. The structures are shown in Figure
5.
The free energy profiles of native drugs and their fructose
adducts, calculated by COSMOperm,
32
are shown in Figure 6.
All fructose adducts generally have an energy barrier higher by
approximately 5 kcal/mol than the corresponding free drugs.
Although the absolute increase of the energy barrier in the
middle of the membrane (position 0 Å in Figure 6) due to
fructose addition was identical for all four investigated APIs,
the consequence on permeability was not the same. For the
two pyrimidine derivates (cytarabine and 5-fluorouracil) that
already had a high energy barrier before fructose addition (19
and 11 kcal/mol, respectively) and no or small energy
minimum within the membrane, the fructose addition
increased the overall energy barrier. Thus, the permeability
of both molecules has decreased significantly (by approx. 4
orders of magnitude), as can be seen in Table 2.
Figure 4. Cytarabine and its prodrugs in LBCS parametric space. The
squares, circles, and triangles represent dierent locations of
cytarabine substitution as indicated; the numbers represent the
length of the hydrocarbon chain (acyls for O- and N-positions, alkyls
for C-position).
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E

On the other hand, when considering the two already rapidly
permeating molecules (abiraterone and paliperidone), a
dierent pattern was found. Their energy in the membrane
center increased by approximately 5 kcal/mol, as in the case of
cytarabine and 5-fluorouracil. However, the fructose addition
also aected the position and magnitude of an energy
minimum within the membrane (at approx. 15−20 Å from
the membrane center, as shown in Figure 6). In the case of
paliperidone, the minimum was higher by 3 kcal/mol.
Therefore, there was still an additional energy barrier which
decreased permeability by about 2 orders of magnitude.
However, abiraterone showed an increase of the energy
minimum by the same amount as in the middle of the
membrane, and consequently, the predicted permeation rate of
abiraterone and its fructose adduct did not dier significantly.
When plotting the positions of the original drugs and their
fructose prodrugs in the LBCS parametric space (Figure 7),
the combined eect of fructose addition on permeability and
partitioning coecient can be visualized. The expected
permeability reduction due to the addition of a polar substance
(fructose) resulted in a shift along the log Perm axis, which was
rather uneven depending on the starting structure. As
Figure 5. Structures of fructose adducts with cytarabine (A), abiraterone (B), 5-fluorouracil (C), and paliperidone (D).
Figure 6. Calculated free energy profiles of the selected molecules and
their fructose prodrugs within a lipid bilayer (Abi = abiraterone, FU =
5-fluorouracil, Pali = paliperidone, Cyt = cytarabine, X-fru = fructose
prodrug of compound X).
Table 2. Calculated Partition and Permeation Coecients
for Dierent APIs and Their Fructose Prodrugs through a
DPPC/DPPG/Chol (75:10:15) Membrane at 293 K, Using
5 Calculations through Randomly Chosen MD Snapshots
from the Last 10 ns of Simulation
molecule log K(mol/mol) log Perm (cm/s)
cytarabine (Cyt) −0.68 ±0.17 −13.19 ±0.04
Cyt-fru −1.35 ±0.05 −17.31 ±0.04
fluorouracil (FU) 0.69 ±0.11 −7.28 ±0.04
FU-fru −1.13 ±0.06 −11.21 ±0.04
abiraterone (Abi) 5.95 ±0.98 −4.59 ±0.79
Abi-fru 2.70 ±0.72 −4.00 ±0.61
paliperidone (Pali) 2.95 ±0.43 −4.24 ±0.58
Pali-fru 0.63 ±0.38 −6.59 ±0.97
Figure 7. Selected drugs and their corresponding fructose prodrugs in
LBCS parametric space as defined in Figure 1. Black squares with ″P/
A/U/C″represent the position of the studied drug in LBCS. Each
molecule representing a square leads an arrow to the square, with ″f″
inside representing the corresponding fructose adduct.
Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Article
https://doi.org/10.1021/acs.molpharmaceut.2c01078
Mol. Pharmaceutics XXXX, XXX, XXX−XXX
F

discussed above, cytarabine and 5-fluorouracil show the most
significant permeability reduction after fructose addition (4
orders of magnitude), the permeability reduction of paliper-
idone was intermediate (about 2 orders of magnitude), and the
permeability of abiraterone was practically unaected by
fructose addition. Thus, fructose addition would appear to
be sucient for moving 5-fluorouracil into a feasible region in
the LBCS space, whereas, for abiraterone and paliperidone, this
might not be the case. Regarding the partitioning coecient, a
systematic shift to lower log Kvalues can be seen for all four
substances, but the extent to which this happened was again
uneven. In general, the more hydrophobic the original
substance, the more pronounced the eect of fructose addition
due to the increased polarity of the adduct. Thus, for
abiraterone and paliperidone, the addition of fructose resulted
in a reduction of log Kby 3.2 and 2.3, respectively, whereas for
5-fluorouracil and cytarabine, the log Kreduction was a more
moderate 1.8 and 1.3, respectively (Figure 7 and Table 2).
In summary, these results illustrate that the attachment of
fructose to the drug molecule can be an eective strategy to
change its liposomal formulation suitability. Depending on the
position of the initial molecule in the LBCS space, fructose
addition can change either permeability or partitioning
coecient alone or both parameters simultaneously, improving
the liposome formulability of the prodrug.
4. CONCLUSIONS
The ability of small-molecule APIs to be successfully
formulated into liposomal carriers intended for parenteral
administration depends on the combination of their water/
lipid equilibrium partitioning coecient and permeability
across the lipid bilayer. As only a few APIs in their native
form fall into the ideal formulability window, the possibilities
for permeability and partitioning coecient modification by a
systematic change of the API molecular structure have been
investigated in this work. We have investigated the feasibility of
moving the position of an API in the LBCS formulation
diagram in either direction, i.e., permeability enhancement or
permeability suppression using a computational approach
based on COSMOperm. We have created a systematic line
of prodrugs derived from a poorly permeating API
(cytarabine) to study the eects on permeability enhancement
by calculating partitioning and permeation coecients. These
prodrugs were esters and amides of cytarabine with various
carbon chain lengths. Various carbon chains were also added to
compare these results with simple carbon chain addition. A
linear dependence of permeability coecients on the length of
a hydrocarbon chain added to the molecule was found,
independently of the substitution location. However, the
dependence of the membrane partitioning coecient on the
substitution location and the hydrocarbon chain length was not
monotonic, especially for shorter carboxyls. Based on the
computational results, it was possible to identify potential
cytarabine prodrugs that should exhibit enhanced permeability
and yet avoid overarching membrane partitioning that would
block the drug′s availability to the aqueous phase. This means
that using our approach, on-demand permeability can be
reached by the addition of a proper length of carboxylic acid to
form a prodrug.
Prodrug design strategies for permeability reduction have
been investigated as well. From the various possibilities of
polar molecules that can be added to the API to form a
prodrug, fructose was chosen as an example of a very polar and
biocompatible molecule. The eect of adding fructose as a
representative polar structure to four APIs with the dierent
initial positions in the LBCS diagram (cytarabine, abiraterone,
5-fluorouracil, and paliperidone) was explored. It was found
that although fructose addition resulted in a nearly identical
increase of the free energy barrier represented by the lipid
bilayer, the eect on permeation rate was strongly API-specific.
Three scenarios were identified depending on the initial API:
(i) reduction of permeability only, (ii) reduction of
partitioning coecient only, and (iii) simultaneous reduction
of both permeability and partitioning coecient. Therefore,
the addition of fructose to API into prodrug formation does
not universally lead to permeation reduction. Rather, it is
dependent on the API structure and the initial position of the
substance in the LBCS diagram.
In summary, a computational methodology for virtual
prodrug design has been developed. It has been demonstrated
that by systematically modifying the molecular structure of the
original API, it is possible to change permeability and
partitioning coecient either simultaneously or individually
and thus move the position of the API in the LBCS diagram in
the desired direction. Hence, an API with initially unfavorable
properties (either too high or too low permeability) can be
converted into a prodrug more suitable for a liposomal
formulation. Of course, the computational selection of
potential prodrug candidates is only the first step, which
must be followed by the actual synthesis of the proposed
structures, their physicochemical characterization, and phar-
macological evaluation.
■AUTHOR INFORMATION
Corresponding Authors
Frantis
ek S

te
pánek −Department of Chemical Engineering,
University of Chemistry and Technology, Prague, 166 28
Prague 6, Czech Republic; orcid.org/0000-0001-9288-
4568; Email: frantisek.stepanek@vscht.cz
Karel Berka −Department of Physical Chemistry, Faculty of
Science, PalackyUniversity Olomouc, 771 46 Olomouc,
Czech Republic; orcid.org/0000-0001-9472-2589;
Email: karel.berka@upol.cz
Authors
Martin Balouch −Department of Chemical Engineering,
University of Chemistry and Technology, Prague, 166 28
Prague 6, Czech Republic
Kater
ina Storchmannová −Department of Physical
Chemistry, Faculty of Science, PalackyUniversity Olomouc,
771 46 Olomouc, Czech Republic
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.molpharmaceut.2c01078
Author Contributions
M.B. and K.S.: methodology, investigation, data analysis, and
manuscript writing. F.S.: conceptualization, methodology,
supervision, and manuscript writing. K.B.: methodology,
supervision, data analysis, and manuscript writing.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
F.S. would like to acknowledge support from the Czech
Science Foundation (Project No. 19-26127X). M.B. would like
Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Article
https://doi.org/10.1021/acs.molpharmaceut.2c01078
Mol. Pharmaceutics XXXX, XXX, XXX−XXX
G

to acknowledge support from the Pharmaceutical Applied
Research Center (PARC). K.S. and K.B. acknowledge support
from Palacky University Olomouc Project
IGA_PrF_2023_018 and FunGIM project DSGC-2021-0060
within OP VVV CZ.02.2.69/0.0/0.0/19_073/0016713. K.S.
and K.B. acknowledge support from the ELIXIR-CZ infra-
structure (Project LM2023055). Open Access funding
provided by the CzechELib project.
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