Transporter-targeted lipid prodrugs of cyclic cidofovir: A potential approach for the treatment of cytomegalovirus retinitis

Article abstract of DOI:10.1002/jps.23140

Cidofovir (CDF) and its cyclic analogue (cCDF) have shown potential in vitro and in vivo antiviral activity against cytomegalovirus (CMV) retinitis. However, hydrophilic nature of CDF may affect cell permeation across lipophilic epithelium and thus limit its effectiveness in the treatment of CMV retinitis. In the present study, we have tested a novel hypothesis, which involves chemical derivatization of cCDF into lipophilic transporter-targeted prodrug [via conjugation with different carbon chain length of lipid raft and targeting moiety (biotin) for sodium-dependent multivitamin transporter (SMVT)]. We have synthesized and characterized three derivatives of cCDF including biotin B-C2-cCDF, B-C6-cCDF, and B-C12-cCDF. Physicochemical properties such as solubility, partition coefficient (n-octanol/water and ocular tissue), bioreversion kinetics, and interaction with SMVT transporter have been determined. Among these novel conjugates, B-C12-cCDF has shown higher interaction to SMVT transporter with lowest half maximal inhibitory concentration value, higher cellular accumulation, and high tissue partitioning. Improvement in physicochemical properties, lipophilicity, and interaction with transporter was observed in the trend of increasing the lipid chain length, that is, B-C12-cCDF > B-C6-cCDF > B-C2-cCDF. These results indicate that transporter-targeted lipid analogue of cCDF exhibits improved cellular accumulation along with higher transporter affinity and hence could be a viable strategy for the treatment of CMV retinitis.

Full text of DOI:10.1002/jps.23140

RESEARCH ARTICLE
Transporter-Targeted Lipid Prodrugs of Cyclic Cidofovir:
A Potential Approach for the Treatment of Cytomegalovirus
Retinitis
MITAN R. GOKULGANDHI, MEGHA BAROT, MAHUYA BAGUI, DHANANJAY PAL, ASHIM K. MITRA
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri 64108
Received 31 January 2012; revised 25 February 2012; accepted 16 March 2012
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23140
ABSTRACT: Cidofovir (CDF) and its cyclic analogue (cCDF) have shown potential in vitro and
in vivo antiviral activity against cytomegalovirus (CMV) retinitis. However, hydrophilic nature
of CDF may affect cell permeation across lipophilic epithelium and thus limit its effectiveness
in the treatment of CMV retinitis. In the present study, we have tested a novel hypothesis,
which involves chemical derivatization of cCDF into lipophilic transporter-targeted prodrug
[
via conjugation with different carbon chain length of lipid raft and targeting moiety (biotin) for
sodium-dependent multivitamin transporter (SMVT)]. We have synthesized and characterized
three derivatives of cCDF including biotin B-C2–cCDF, B-C6–cCDF, and B-C12–cCDF. Physic-
ochemical properties such as solubility, partition coefficient (n-octanol/water and ocular tissue),
bioreversion kinetics, and interaction with SMVT transporter have been determined. Among
these novel conjugates, B-C12–cCDF has shown higher interaction to SMVT transporter with
lowest half maximal inhibitory concentration value, higher cellular accumulation, and high tis-
sue partitioning. Improvement in physicochemical properties, lipophilicity, and interaction with
transporter was observed in the trend of increasing the lipid chain length, that is, B-C12–cCDF
>
B-C6–cCDF > B-C2–cCDF. These results indicate that transporter-targeted lipid analogue of
cCDF exhibits improved cellular accumulation along with higher transporter affinity and hence
could be a viable strategy for the treatment of CMV retinitis. © 2012 Wiley Periodicals, Inc. and
the American Pharmacists Association J Pharm Sci
Keywords: transporters; bioavailability; drug transport; HIV/AIDS; lipids; MDCK cells;
membrane transport; ophthalmic drug delivery; prodrugs; targeted drug delivery
INTRODUCTION
with AIDS, congenital immunodeficiency, or organ
transplant recipients. Although the incidence and
4
Cidofovir (CDF), an acyclic phosphonate nucleotide
analogue of cytosine, exhibits potential antiviral ac-
tivity against herpes simplex viruses (HSVs), vari-
cella zoster viruses, adenoviruses, papillomaviruses,
polyomaviruses, and poxviruses. CDF and its cyclic
analogue (cCDF) are highly potent and clinically in-
prevalence of CMV retinitis in AIDS patients have de-
clined primarily due to the highly active antiretrovi-
ral therapy (HAART), it remains a major cause of the
5
vision loss in 20%–40% AIDS patients. Moreover, a
1
longitudinal study of the ocular complications of AIDS
revealed that ocular findings in patients with newly
diagnosed CMV retinitis in the post-HAART era re-
dicated in the treatment of human cytomegalovirus
2
(
hCMV) infections. In addition to retinal diseases
6
sembled those prior to the introduction of HAART.
including diabetic retinopathy, glaucoma, and age-
In addition, when CDF was directly compared for its
efficacy with other antiviral drugs [acyclovir (ACV) or
ganciclovir (GCV)], it proved clearly superior to ACV
in the treatment of mice infected intracerebrally with
3
related macular degeneration, CMV retinitis is an-
other potential cause of vision impairment. hCMV is
a major cause of an opportunistic infection in patients
4
7
,8
HSV and to GCV in the treatment of both immune-
competent and immune-deficient mice infected with
Correspondence to: Ashim K. Mitra (Telephone: +816-235-1615;
Fax: +816-235-5190; E-mail: mitraa@umkc.edu)
Megha Barot and Mahuya Bagui contributed equally to this
work.
9–11
murine CMV.
Although CDF has shown potential activity against
CMV retinitis, major treatment failure is due to
the lower oral bioavailability and emergence of
Journal of Pharmaceutical Sciences
©
2012 Wiley Periodicals, Inc. and the American Pharmacists Association
JOURNAL OF PHARMACEUTICAL SCIENCES
1

2
GOKULGANDHI ET AL.
resistance.12 Emergence of drug resistance by var-
ious multidrug resistance (MDR)-associated efflux
proteins have been reported as a potential barrier to
Therefore, in the present study to delineate our hy-
pothesis of attaining synergetic effect by simultane-
ously conjugating both transporter-targeted moiety
(biotin) and lipid raft, we have synthesized and char-
acterized several biotin-conjugated lipid prodrugs of
cCDF to target SMVT transporter on the retina for
the treatment of hCMV retinitis. We hypothesized
that the lipid raft of synthesized targeted lipid pro-
drug will facilitate enhanced interaction with the cell
membrane and thereby will assist docking of targeted
moiety (i.e., biotin) into the binding domain of trans-
porter (SMVT). The net effect will be rapid transloca-
tion of targeted lipid prodrug across the plasma mem-
brane, improving cellular absorption. These modifica-
tions will not only help to improve the permeability
(via SMVT transporter and lipid raft) but will also at-
tain desired antiviral efficacy of parent CDF. There-
fore, transporter-targeted lipid prodrug design may
be a promising approach to treat hCMV retinitis.
1
3
the success of ocular therapies. Therefore, there is
an urgency to develop potential analogues of CDF,
which shows high permeability, low toxicity, and si-
multaneously overcomes an incidence of drug resis-
tance. In order to resolve such problems, researchers
have synthesized lipid conjugates of cCDF (octade-
cyloxyethyl–cCDF) for intraocular injection to im-
prove its vitreal half-life, bioavailability, and an-
1
4
tiviral efficacy. However, the major limitation of
lipid conjugates is the higher retention in lipid bi-
layer of plasma membrane and inability to translo-
cate into the cytoplasm due to which it is not ef-
fective in treating deeper tissue infection such as
CMV retinitis. Moreover, the low aqueous solubility
of lipid prodrugs can further restrict the application
1
5
of this approach. Therefore, it is important to bal-
ance the prodrug lipophilicity necessary for transcel-
lular absorption with adequate aqueous solubility. Re-
searchers have opted to synthesize peptide prodrug
of cCDF to target peptide transporter in order to im-
prove its antiviral efficacy and bioavailability follow-
ing oral administration.16 However, the major limita-
tions for transporter-targeted prodrugs are substrate
specificity and transport capacity.17 Therefore, lipid
conjugates and transporter-targeted prodrugs exhibit
partial response since individually these strategies
hold both advantages as well as limitations. Hence,
a balanced approach is needed to produce better per-
meability via transporter-targeted moiety along with
suitable lipophilicity via lipid raft. In this study, we
hypothesized that combination of both transporter-
targeted moiety and lipid raft may provide synergistic
effect.
MATERIALS
Cidofovir was a generous gift from the Gilead
Science (Foster city, California) for this project.
Biotin–N-hydroxysuccinimide (biotin–NHS) and
Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP) were obtained from
Chem-Impex International (Wood Dale, Illinois).
1-Dodecanol, 1-hexanol, 6-amino 1-hexanol, and
12-amino 1-dodecanol were obtained from TCI
America (Portland, Oregon). [3H]-Biotin (35.6
Ci/mmol, radiochemical purity >98.6%) was
obtained from Perkin Elmer (Waltham, Mas-
sachusetts).
N,N-Dimethylformamide
(DMF),
N,N-diisopropylethylamine (DIPEA), methanol,
and dichloromethane were purchased from Sig-
ma–Aldrich (St. Louis, Missouri). All other reagents
of analytical grade were purchased from local sup-
plier and used as such without any purification.
Distilled–deionized water (DDW) was used to pre-
pare buffer and mobile phase. Madin–Darby canine
kidney (MDCK)–multidrug resistance protein 1
(MDR1) cells were donated by P. Borst (Netherlands
Cancer Institute, Amsterdam, the Netherlands).
Dulbecco’s modified Eagle’s medium–Nutrient Mix-
ture F-12 (1:1) (DMEM/F12), DMEM, fetal bovine
serum (FBS), and Non-essential aminoa cids (NEAA)
were purchased from Gibco (Invitrogen Corporation,
Grand Island, New York). Antibiotics and sodium
bicarbonate were purchased from Sigma–Aldrich.
In our laboratory, various transporters have been
investigated on the retina, which can be utilized for
the delivery of therapeutic agents. Transporters such
1
8
19
as peptide/histidine, organic cationic transporter,
amino acid transporter,20,21 folic acid, and sodium-
dependent multivitamin transporter (SMVT) have
been reported on the human retinal pigment epithe-
lium cells (ARPE-19).23 SMVT is an important trans-
port system carrying vitamins such as biotin and co-
factors essential for the ocular tissues. It was observed
in our laboratory that SMVT is a saturable trans-
porter with Km of 138.25 :M and Vmax of 38.85 pmol/
22
2
3
(
min mg) protein on ARPE-19 cells. Biotin prodrugs
were able to utilize this low capacity and high affin-
ity SMVT transporter to enhance the permeability of
therapeutic drug conjugates. We have previously re-
ported that biotin-conjugated GCV (biotin–GCV) was
recognized by SMVT transporter on ARPE-19 cells
and rabbit retina. Moreover, biotin–GCV has shown
therapeutically desirable pharmacokinetics profile as
compared with parent GCV.23
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Transwell
inserts were procured from Fisher
Scientific (Pittsburgh, Pennsylvania), and tenofovir
and tenofovir disoproxil fumarate were purchased
from Santa Cruz Biotechnology (Santa Cruz, Califor-
ꢀ
R
nia). Nonradioactive cytotoxicity kit (CellTiter 96
AQueous Non-Radioactive Cell Proliferation Assay)
was purchased from Promega (Madison, Wisconsin)
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TRANSPORTER-TARGETED LIPID PRODRUGS OF CYCLIC CIDOFOVIR
3
and scintillation cocktail and Bio-Rad Protein Assay
kit was purchased from MP bioscience (Solon, Ohio)
and Bio-Rad (Hercules, California), respectively.
the reaction mixture was stirred overnight under
N2 at room temperature. Solvent was removed un-
der vacuum, and the white residue was purified
by the silica gel column chromatography with 20%
dichloromethane in methanol as the eluent to yield
products (biotin-conjugated lipid moieties, biotin-C2/
biotin-C6/biotin-C12) as white solid.
2
Culture flasks (75 cm growth area) and 12-well
2
plates (3.8 cm growth area per well) were procured
from MidSci (St. Louis, Missouri). Nuclear magnetic
resonance (NMR) spectra were collected by Bruker
4
00 MHz NMR with sample dissolved in deuterated
dimethyl sulfoxide (DMSO-d6).
Biotin–Lipid Conjugates with cCDF
In a separate flask, CDF (1 equiv), 20 mL of anhydrous
DMF, and 2.1 mL of dry DIPEA were added. The re-
action vessel was warmed by a heat gun to facilitate
the dissolution of the CDF–DIPEA salt. The solvent
was then removed under vacuum. To that residue,
ANIMALS
Adult male New Zealand albino rabbits weighing be-
tween 2 and 2.5 kg were obtained from Myrtle’s Rab-
bitry (Thompson Station, Tennessee). Ketamine HCl
and Rompun (Xylazine ) were purchased from Fort
Dodge Animal Health (Fort Dodge, Iowa) and Bayer
Animal Health (Shawnee Mission, Kansas), respec-
tively. Protocol for performing all the surgical proce-
dure was approved by Institutional Animal Care and
Use Committee of the University of Missouri-Kansas
City (Kansas City, Missouri).
2
0 mL of anhydrous DMF, DIPEA (5 equiv), the rele-
ꢀ
R
vant biotin-conjugated lipid (1.5 equiv) in anhydrous
DMF, and PyBOP (2.1 equiv) were added, and the re-
action mixture was stirred under N2 at 45 C for 48–
◦
3
1
60 h. The reaction was monitored by P NMR, and
additional portions of PyBOP were added as neces-
sary. After completion of reaction, solvent was re-
moved under vacuum, and the brownish-red residue
was purified by repetitive silica gel column chro-
matography using gradient solvent combination of
dichloromethane and methanol as the eluent, to yield
products as brownish sticky solid (Figure 1).
METHODS
Synthesis of Biotin-Conjugated Lipid Prodrugs of cCDF
Biotin–Lipid Conjugates
Biotin-C2–cyclic Cidofovir (B-C2–cCDF) was ob-
3
1
tained as brownish sticky solid in 45% yield. P NMR
(400 MHz, DMSO-d6) δ: 13.92 (s), 12.84 (s), electro-
spray ionization–mass spectrometry (ESI–MS): m/z
To biotin–NHS (1 equiv), anhydrous DMF and lipid
moieties i.e. 2-amino 1-ethanol/6-amino 1-hexanol/
+
12-amino 1-dodecanol (1.2 equiv) were added and
calculated 530.5, found 531.4 (M + H) .
Figure 1. Schematic representation of the synthesis of biotin-conjugated lipid prodrugs of
cyclic cidofovir.
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES

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GOKULGANDHI ET AL.
Biotin-C6–cyclic Cidofovir (B-C6–cCDF) was ob-
tained as brownish sticky solid in 56% yield. P NMR
Cell Culture
3
1
MDCK-MDR1 cells (passages 5–15) were cultured
in DMEM supplemented with 10% FBS (heat
inactivated), 1% nonessential amino acids, peni-
cillin (100 U/mL), streptomycin (100 :g/mL), 20 mM
HEPES, and 29 mM sodium bicarbonate adjusting
(
400 MHz, DMSO-d6) δ: 13.12 (s), 11.55 (s), ESI–MS:
+
m/z calculated 586.6, found 587.2 (M + H) .
Biotin-C12–cyclic Cidofovir (B-C12–cCDF) was ob-
tained as brownish sticky solid in 61% yield. 31P NMR
(
400 MHz, DMSO-d6) δ: 13.84 (s), 12.6 (s), ESI–MS:
◦
pH 7.4. Cells were grown and incubated at 37 C in
+
m/z calculated 670.7, found 671.6 (M + H) .
a culture incubator with 5% CO2. The medium was
changed every alternate day. Cells were passaged ev-
ery 4–6 days till it reached 80%–90% confluency. Cells
were seeded at a density of 25,000 per well in 12-
well tissue culture-treated plates and collagen-coated
Synthesis of Lipid Prodrugs of cCDF
To CDF (1 equiv), 20 mL of anhydrous DMF and
ꢀ
R
ꢀR
2
.1 mL of dry DIPEA were added. The reaction flask
Transwell permeable inserts (Costar ) at a density
of 10,000 per well in 96-well plates. These cells were
then allowed to grow for 4–6 days and used for further
studies.
was warmed by a heat gun to facilitate the dissolu-
tion of the CDF–DIPEA salt. The solvent was then
removed under vacuum. To that residue, 20 mL of an-
hydrous DMF, DIPEA (5 equiv), the relevant lipid
moieties (1.5 equiv) in dry DMF, and PyBOP (2.1
equiv) were added, and the reaction mixture was
High-Performance Liquid Chromatography Analysis
High-performance liquid chromatography (HPLC)
methods for simultaneous analysis of CDF, its bi-
otin conjugate (B-C2–cCDF, B-C6–cCDF, and B-C12–
cCDF) and lipid prodrugs (C6-cCDF and C12-
cCDF) were developed (Figure 3). Stability, solubility,
n-octanol/water partition coefficient, and tissue par-
tition coefficient samples were analyzed by an
HPLC system comprising Waters 515 pump (Waters,
Milford, Massachusetts), Alcott 718AL refrigerated
autosampler, ultraviolet detector (RAININ, Dyna-
max, Absorbance Detector Model UV-C), HP 3395 in-
tegrator, Phenomenex C18 (4.6 × 250) column with
C18 guard column. Mobile phases comprised 20 mM
phosphate buffer (pH 6.8) and methanol (90:10, v/v)
for CDF and biotin-conjugated lipid prodrugs of cCDF,
and 20 mM phosphate buffer (pH 6.8) and acetoni-
trile (50:50, v/v) for lipid conjugates of cCDF. All the
compounds were detected at λmax 274 nm and flow
rate of 0.5 mL/min. The analytical method was vali-
◦
stirred under N2 at 45 C for 24 h. The reaction was
3
1
monitored by P NMR, and additional portions of
PyBOP were added as necessary. After completion of
the reaction, solvent was removed under vacuum, and
the brownish-red residue was purified by repetitive
silica gel column chromatography using gradient sol-
vent combination of dichloromethane and methanol
to generate white solid product (Figure 2). The purity
of the prodrugs were confirmed via High-performance
liquid chromatography (HPLC).
C6-cyclic Cidofovir (C6-cCDF) was obtained as
white solid in 65% yield. 31P NMR (400 MHz, DMSO-
d6) δ: 12.92 (s), 11.66 (s), ESI–MS: m/z calculated
3
+
45.3, found 346.2 (M + H) .
C12-cyclic Cidofovir (C12-cCDF) was obtained as
white solid in 62% yield. 31P NMR (400 MHz, DMSO-
d6) δ: 13.47 (s), 12.20 (s), ESI–MS: m/z calculated
4
+
29.5, found 430.3 (M + H) .
dated with respect to precision (% RSD < 1), linearity
2
(
r > 0.998), reproducibility (% Relative standard de-
viation < 2), and selectivity (no interfering peaks were
observed).
Liquid Chromatography–Tandem Mass Spectrometry
Analysis
MDS Sciex API 3200 Triple Quadrupole linear
QTrap mass spectrometry (Applied Biosystems/MDS
Sciex, Foster City, California) system interfaced by
turbo ion spray with positive ion source in Multi-
ple Reaction Monitoring (MRM) mode was used for
detection. Mass-dependent parameters were tuned
and optimized for CDF, B-C2–cCDF, B-C6–cCDF, B-
C12–cCDF, C6–cCDF, C12–cCDF, tenofovir and teno-
fovir diisoproxil fumarate. Tenofovir was used as
an internal standard (IS) for analysis of biotin-
conjugated lipid prodrugs. Initially, full scan mass
spectra were acquired in positive ion mode for all
Figure 2. Schematic representation of the synthesis of
lipid prodrugs of cyclic cidofovir.
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TRANSPORTER-TARGETED LIPID PRODRUGS OF CYCLIC CIDOFOVIR
5
Figure 3. Representative chromatogram of cyclic cidofovir prodrugs (a) B-C2-cCDF,
(b) B-C6–cCDF, (c) B-C12–cCDF, (d) C6-cCDF, and (e) C12-cCDF.
the compounds. During a direct infusion experiment,
the mass spectra for B-C2–cCDF, B-C6–cCDF, B-
C12–cCDF, and tenofovir peaks at mass to charge ra-
tio (m/z) of 280.09, 531.18, 587.24, 671.37, and 288.13,
respectively, as a protonated molecular ions [M +
and 674.10 → 326.10 for tenofovir disoproxil fumarate
2
(IS). The method was developed over the linear (r >
0.99) concentration range 30–1000 ng/mL. LLOQ of
all the compounds was 15.0 ng/mL.
Sample Preparation and Extraction
+
H] . The most stable abundant fragment ions ob-
served in each product tandem mass spectrometry
Frozen samples were thawed at room temperature
and thoroughly mixed using vortex for 1 min. For
biotin-conjugated lipid prodrugs of cCDF: Using cal-
ibrated pipettes, 100 :L of samples was aliquoted
into a 1.5 mL polypropylene microcentrifuge tubes. To
these samples, 20 :L of 10.0 :g/mL freshly prepared
IS working solution (tenofovir) (except for blank sam-
ple) was added. The mixture was vortexed for 30 s.
The samples were diluted with DDW upto 1 mL and
then loaded on to the Bond EluteSCX cartridge, which
is preequilibrated with 2 mL of methanol and 2 mL
of DDW. Following loading of CDF and its biotin-
conjugated lipid prodrugs, the samples were eluted
from Bond EluteSCX cartridge with 2 mL of 3% Am-
monium hydroxide-methanol. The eluent phase was
(MS/MS) spectrum were at m/z 262.30 for CDF, 262.16
for B-C2–cCDF, 262.30 for B-C6–cCDF, 262.13 for B-
C12–cCDF, and 270.03 for tenefovir. Quantitative de-
termination was performed in MRM scan positive ion
mode using the following mass transitions: 280.09 →
2
62.30 for CDF, 531.18 → 262.16 for B-C2–cCDF,
87.24 → 262.30 for B-C6–cCDF, 671.37 → 262.13
5
for B-C12–cCDF, and 288.13 → 270.00 for tenofovir
2
(IS). The method was developed over the linear (r
>
0.99) concentration range 60–1000 ng/mL. Lower
limit of quantification (LLOQ) of all the compounds
was 30.0 ng/mL.
Tenofovir disoproxil fumarate was used as an IS
for the analysis of lipid prodrugs of cCDF. Simi-
larly, a full scan mass spectra were acquired in posi-
tive ion mode for C6-cCDF, C12-cCDF, and tenofovir
disoproxil fumarate peaks at m/z of 346.20, 430.33,
and 674.10, respectively, as a protonated molecular
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◦
evaporated in Speed Vac at 40 C for 2 h, following
reconstitution in 100 :L of mobile phase.
For lipid conjugates of cCDF: To 100 :L of samples
aliquoted into a 1.5 mL polypropylene microcen-
trifuge, 20 :L of 10.0 :g/mL freshly prepared teno-
fovir disoproxil fumarate (IS) working solution (ex-
cept for blank sample) was added. The mixture was
vortexed for 30 s. A 900 :L ice-cold ethyl acetate was
added and vortexed for 1 min, centrifuged, and 800 :L
supernatant was aliquoted in microfuge tube. The
+
ion [M + H] . The most stable abundant fragment
ions observed in each product MS/MS spectrum were
at m/z 262.20 for C6-cCDF, C12-cCDF, and 326.10
for tenefovir disoproxil fumarate. Quantitative deter-
mination was performed in MRM scan positive ion
mode using the following mass transitions: 346.20 →
ꢀ
R
◦
262.20 for C6-cCDF, 430.33 → 262.20 for C12-cCDF,
eluent phase was evaporated in Speed Vac at 40 C
DOI 10.1002/jps
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6
GOKULGANDHI ET AL.
for 4 h, following reconstitution in 100 :L of mobile
phase. A 10 :L of the resulting solution was injected
onto liquid chromatography (LC)–MS/MS for analysis
hydroxide) were prepared within the range of ± 0.05
26
units. The buffer strength was kept constant at
100 mM. To initialize the study, 4.8 mL of buffer was
taken in a glass vial and 200 :L of freshly prepared
prodrug solution (10 mg/mL) in methanol was added.
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using XTerrs MS C18 5 :m column.
Aqueous Solubility and n-Octonal/Buffer Partition
Coefficient
◦
The glass vials were kept in the water bath at 37 C
and 60 rpm. Aliquots (100 :L) were taken at predeter-
◦
mined time interval and immediately freeze at −80 C
Aqueous solubility study was performed by following
2
4
until further analysis by HPLC. Experiments were
performed in triplicate.
previously published protocol from our laboratory.
Briefly, excess amount of CDF and its biotin-
conjugated prodrugs (B-C2–cCDF, B-C6–cCDF, and
B-C12–cCDF) were added to 1 mL of DDW in glass
Ocular Tissues Hydrolysis
Previously published protocol was followed to sepa-
vials. These vials were kept in shaker bath for 24 h at
2
7,28
◦
rate the ocular tissues from the animals.
Briefly,
3
7 C. After 24 h, the content of the vial was carefully
New Zealand white male albino rabbits were euth-
anized with sodium pentobarbital injection through
the marginal ear vein. Both the eyeballs were enucle-
ated immediately and rinsed with DPBS pH 7.4. Both
ocular segments were carefully separated by mak-
ing an incision at the scleral–limbus junction. The
posterior segment ocular tissues such as conjunctiva,
sclera, choroid–retina, and vitreous humor were sepa-
transferred in an Eppendorf tube and centrifuged for
0 min at 10,000X g. The supernatant was collected
and filtered through 0.45 :m Nalgene syringe filter
1
membrane. The supernatant was immediately stored
◦
in −20 C refrigerator until analysis was performed
using HPLC. All the experiments were conducted in
triplicate.
A partition coefficient study was performed with
n-octanol and Dulbecco’s Phosphate-Buffered Saline
◦
rated. These tissues were stored at −80 C prior to use.
Target tissue was retina for bioreversion and the ther-
apeutic action of prodrugs. So, the retina was homog-
enized in ice bath and the homogenate was collected.
Protein concentration of supernatant was measured
with a Bio-Rad protein estimation kit (Bio-Rad). In all
the stability studies, protein concentration of 1.0 mg/
mL was employed. To initiate the study, 2.8 mL of su-
pernatant was taken in a glass vial and 200 :L of
prodrugs solution (1 mg/mL of stock solution of pro-
drugs) was added. At predetermined time interval,
100 :L sample was taken and immediately diluted
with 100 :L of ice-cold methanol containing 0.1% tri-
fluroacetic acid to terminate enzyme activity. Sam-
ples were immediately centrifuged and supernatant
(
DPBS) (pH 7.4) as per previously published pro-
2
5
tocol from our laboratory. Briefly, n-octanol and
DPBS were presaturated for 48 h with each other.
After presaturation, 1 mL aliquots of presaturated
DPBS was placed in glass vial and stock solution
of CDF and its biotin-conjugated lipid prodrugs (100
:
M) were added. The mixture was vortexed and
an initial sample was collected (Caqu). After equili-
bration, 1 mL aliquot of presaturated n-octanol was
added. The glass vial was kept at constant tempera-
◦
ture shaker bath (37 C) for 24 h. Samples were col-
lected from aqueous and organic phase and analyzed
for the determination of partition coefficient and log
P values.
◦
was stored at −80 C until HPLC analysis. Finally,
Stability Studies
Buffer Stability
apparent first-order rate constants (k) from Eq. 1 and
half-life (t1/2) from Eq. 2 were calculated and corrected
for any chemical hydrolysis observed with the control.
This experiment was performed in triplicate.
Prodrugs need to display a good stability and must
be absorbed intact across cell membrane. Simultane-
ously, it should be converted to parent molecule fol-
lowing its active/passive transport in target tissue to
exhibit therapeutic activity. Chemical stability stud-
ies will help to screen an ideal prodrug candidate for
in vivo studies. The chemical stability of prodrugs was
studied in various buffers at different pH (1.4–7.4).
For pH 1.4 hydrochlorate buffer (8.5 mL of 0.2 M
hydrochloric acid and 5.0 mL of 0.2 M potassium chlo-
ride), pH 4.0 acetate buffer (0.3 g sodium acetate tri-
hydrate and 3.9 mL of 2 N acetic acid), pH 6.0 phos-
phate buffer (50 mL of 0.2 M monobasic potassium
phosphate and 5.6 mL of 0.2 M sodium hydroxide),
and pH 7.4 phosphate buffer (50 mL of 0.2 M monoba-
sic potassium phosphate and 39.1 mL of 0.2 M sodium
k = 2.303 × slope(log C vs. time)
(1)
(2)
ꢀ
t1/2 = 0.693 k
Cytotoxicity Studies
Cytotoxicity of CDF and its prodrugs was evaluated
in MDCK –MDR1 (passage no 5–15) using an aque-
ous nonradioactive cytotoxicity kit. This kit is based
on the principle of MTS assay and has been suc-
cessfully used to study cytotoxicity of various pro-
2
9
drugs in our laboratory. MDCK–MDR1 cells were
cultured following procedure mentioned in the Meth-
ods section. Prior to the experiment, a fixed number
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TRANSPORTER-TARGETED LIPID PRODRUGS OF CYCLIC CIDOFOVIR
7
of MDCK–MDR1 cells (10,000 cells per well) were
plated in 96-well plate for 24 h. After 24 h, culture
medium was replaced with 100 :L of freshly pre-
pared solutions of CDF and its biotin-conjugated lipid
prodrugs (B-C2–cCDF, B-C6–cCDF, B-C12–cCDF) in
culture medium at different concentrations and cells
were incubated for 48 h (in a humidified, 5% CO2 at-
mosphere). CDF and its conjugates were tested in
the concentration range of 50, 100, and 250 :M. Af-
ter 48 h, the solution was removed and the above-
mentioned dye was added and incubated for 2 h. At
the end of 2 h, cell viability was calculated using color
determination at 485 nm with a 96-well microtiter
plate reader (SpectraFluor Plus, Tecan, Switzerland).
Five percent Triton-X 100 and culture medium were
used as positive and negative control, respectively.
of [3H]-biotin (in DPBS pH 5.5) in presence and ab-
sence of biotin-conjugated lipid prodrugs of cCDF (all
at 50 :M concentration) toward AP side of cells (donor
chamber). The receiver chamber (BL side) contains
DPBS pH 7.4. The cell monolayer integrity (around
2
250 Ωcm ) was determined by transepithelial electri-
cal resistance measurement. Transport experiment
was conducted for a total time period of 3 h. Sam-
pling (100 :L) from the receiver chamber was carried
out at predetermined time intervals of 15, 30, 45, 60,
90, 120, 150, and 180 min and fresh DPBS pH 7.4
was replaced to maintain sink conditions in receiver
chamber. The samples were then analyzed in a Beck-
man scintillation counter (Model LS-6500, Beckman
Instruments, Inc.). All experiments are performed in
triplicate. The permeability (cm/s) (P) of [3H]-biotin
was determined according to Eq. 3.
Interaction with SMVT Transporter
Inhibition of Uptake of [3H]-Biotin
ꢀ
ꢁ
ꢂ
P = (dM/dt) C × A × 60
(3)
d
This experiment was performed to investigate if
biotin-conjugated lipid prodrugs can be recognized
by the SMVT transporter. For this study, a model
cell line MDCK–MDR1 was selected to delineate in
vitro interaction with SMVT.30 MDCK–MDR1 cells
were cultured on 12-well plates according to an ear-
lier stated protocol from the Methods section. Biotin,
a well-known substrate of SMVT transporter, was se-
lected for this study. After the confluency, cells were
where dM/dt (mol/min) represents the rate of trans-
2
port across the cell monolayer, A (cm ) is the cross-
sectional area available for transport, and Cd (:M) is
the donor concentration.
Affinity Measurements Using [3H]-Biotin on
MDCK–MDR1 Cells
For dose–response studies in which [3H]-biotin
uptake was inhibited in the presence of biotin-
conjugated lipid prodrugs, the inhibitory effect was
calculated by Eq. 4.
washed with DPBS pH 7.4 two times for 10 min at
◦
3
7 C. The uptake study was initiated by adding 1 mL
of [3H]-Biotin (0.25 :Ci/mL in DPBS pH 5.5) in the
presence and absence of biotin-conjugated lipid pro-
drug of cCDF solution (all at 50 :M concentration) as
a competitive inhibitor. Uptake study was carried out
ꢃ
ꢄ
Max − Min
Y= Min
(4)
+ 10(
log IC −x ×H
50
)
1
◦
for 10 min at 37 C. At the end of an incubation pe-
Here, x denotes the logarithm of the concentration
of the biotin-conjugated lipid prodrugs, whereas Y is
the intracellular uptake of [3H]-biotin. IC50 repre-
sents the inhibitor concentration where the uptake
of [3H]-biotin was inhibited by 50%, and H is the Hill
constant. Y starts at a minimum (min) value (at low
inhibitor concentration) and then plateaus at a max-
imum (max) value (at high inhibitor concentration),
resulting in a sigmoidal profile.
riod, drug solutions were removed and the cells were
washed with ice-cold stop solution (200 mM KCl and
2
mM HEPES) three times. Furthermore, cells were
lysed with 1 mL of lysis solution (0.1%, w/v Triton
X-100 in 0.3 N sodium hydroxide) and kept at room
temperature overnight. Next day, 500 :L of aliquots
was transferred to scintillation vials containing 3 mL
scintillation cocktail. Finally, these samples were an-
alyzed with a Beckman scintillation counter (Model
LS-6500, Beckman Instruments, Inc.). Uptake was
normalized to the protein content of each well. Protein
content of cell lysate was quantified using Bio-Rad
assay.
Nonradioactive Cellular Accumulation
Cellular accumulation study was performed on MD-
CK–MDR1. Cells were grown in monolayer on 12-well
plates for 4 –6 days (with confluency of 90%–95%), the
cellular accumulation of CDF, biotin-conjugated lipid
prodrugs, and lipid prodrugs of cCDF were initiated.
Briefly, cells were washed with DPBS pH 7.4 and 1 mL
of CDF, biotin-conjugated lipid prodrugs, and lipid
conjugates of cCDF (all at 50 :M in DPBS pH 5.5)
was added in the presence and absence of competi-
Inhibition of Transport of [3H]-Biotin
Transwell diffusion chamber system was utilized for
this study. Prior to a transport experiment, cell mono-
ꢀ
R
layers grown on the Transwell inserts were rinsed
◦
with DPBS pH 7.4 and incubated at 37 C for 10 min
◦
for two times for both apical (AP) and basolateral
tive inhibitor biotin at 37 C. At the end of an exper-
(BL) sides. Transport was initiated by adding 500 :L
iment, the drug/prodrugs solution was removed and
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8
GOKULGANDHI ET AL.
cell monolayer was washed with ice-cold stop solution.
Cells were lysed by rapid freeze–thaw cycle (three
times). The samples were analyzed by LC–MS/MS af-
ter Bond Elute SCX solid-phase extraction with 3%
Ammonium hydroxide–methanol and cold ethyl ac-
etate extraction. All the experiments were performed
in quadruplicate and cellular accumulation was nor-
malized using protein count.
Table 1. Physicochemical Properties (Aqueous Solubility and
log P) for Biotin-Conjugated Lipid Prodrugs and Lipid Prodrugs
of Cyclic-Cidofovir
Log P
Solubility (mg/mL)
a
Predictedb
Calculatedc
Prodrugs
in DDW
CDF
72.44 ± 3.33
56.08 ± 1.63
17.31 ± 1.02
13.21 ± 0.98
1.27 ± 0.48
0.95 ± 0.37
−2.89
0.23
1.04
2.61
3.64
4.23
−1.86 ± 0.39
−0.64 ± 0.24
−0.47 ± 0.18
−0.16 ± 0.04
2.84 ± 0.56
3.89 ± 1.23
B-C2–cCDF
B-C6–cCDF
B-C12–cCDF
C6-cCDF
Tissue Partition Coefficient
Previously published protocol was followed to sepa-
rate the posterior segment ocular tissues from New
Zealand rabbits.27,28 The retina–choroid and sclera
were removed carefully and placed in preweighed
vials on ice. A 990 :L of isotonic phosphate buffer
saline pH 7.4 was added to the vials containing
C12-cCDF
a
Distilled–deionized water.
b
c
Log P from Chem draw.
Log P = Log [(ConcentrationOctanol)/ConcentrationWater )].
conjugated lipid prodrugs. The reduction in solubil-
ity was about 1.5-, 4.5-, and 5.5-fold for B-C2–cCDF,
B-C6–cCDF, and B-C12–cCDF, respectively. However,
reduction in solubility was about 56- and 74-fold for
C6-cCDF and C12-cCDF, respectively (Table 1). Oc-
tanol–buffer partition studies reveal log P value of
CDF to be −1.86. These results clearly indicate an
improvement in the lipophilicity of prodrugs due to
the presence of lipid raft conjugation.
◦
retina–choroid and incubated at 37 C for 15 min for
tissue equilibration. Ten microliters of drug and pro-
drugs solution was spiked, gently vortexed, and in-
cubated for 6 h. At the end of incubation, the vials
were centrifuged at 5000 rpm for 2 min and the su-
pernatant and tissue pellet were collected in differ-
◦
ent microfuge tubes and stored at −80 C for further
analysis. One hundred microliters of supernatant was
mixed with ice-cold methanol for protein precipita-
tion (CPBS pH 7.4). Tissue pellet was homogenized with
DPBS pH 7.4 and immediately mixed with the ice-cold
methanol for protein precipitation and terminating
enzymatic degradation before analysis (Ctis). Tissue
partition calculation was calculated with Eq. 5. All
the analysis was performed by HPLC or LC–MS/MS.
Buffer and Tissue Homogenate Stability
Buffer (at various pH) and ocular tissue homogenate
stability studies aid in the screening of the ideal pro-
drug candidates for further studies.
Buffer Stability
Chemical stability was determined for all the biotin-
conjugated lipid prodrugs in different pH buffers (pH
ꢁ
ꢀ
ꢂ
Tissue partition = Ctis CPBSpH7.4
(5)
1
.4 to 7.4). We did not observe any appreciable degra-
Statistical Analysis
dation of prodrugs at pH 1.4 (data not shown). Upon
raising pH toward alkaline range, an accelerated
degradation has been observed. Figure 4 and Table 2
summarize the apparent first-order degradation rate
constants at various pH. B-C2–cCDF was found to be
more stable than B-C6–cCDF and B-C12–cCDF.
All experiments were conducted at least in triplicate
and cellular accumulation studies were conducted
in quadruplicate. Results are expressed as mean ±
SD. Student’s t-test was applied to detect statistical
significance between the parameters of the biotin-
conjugated lipid prodrugs, lipid prodrugs, and CDF.
p < 0.05 and p < 0.01 were considered to be statisti-
cally significant.
Ocular Tissue Homogenate Stability
A prodrug molecule is pharmacologically inactive and
hence would not exhibit any therapeutic effect unless
it converts into the parent drug. Therefore, we also
performed the enzymatic stability studies of prodrugs
in presence of retina–choroid and vitreous humor ho-
mogenate (posterior segment tissue). Figure 5 and
Table 3 summarize the apparent first-order degrada-
tion rate constant, indicating the loss of the prodrugs
in the retina–choroid and vitreous.
RESULTS
Aqueous Solubility and n-Octanol–Water Partition
Coefficient
Aqueous solubility of biotin-conjugated lipid prodrugs
was determined in DDW at 37 C. The aqueous solu-
◦
bility and partition coefficient values are shown in
Table 1. As expected, the solubility of biotin-
conjugated lipid prodrugs of cCDF was found to be
lower with increasing carbon chain length of lipid raft.
Drastic reduction in the solubility of lipid prodrugs
of cCDF has been observed compared with biotin-
Cytotoxicity Studies
Cidofovir and all the prodrugs of CDF were studied
for cytotoxicity studies in MDCK–MDR1 cell line. Pro-
drugs were found to be nontoxic up to 250 :M concen-
tration for 48 h. Triton X-100 and culture medium
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TRANSPORTER-TARGETED LIPID PRODRUGS OF CYCLIC CIDOFOVIR
9
Figure 4. pH-dependent degradation of (a) biotin-C2-cCidofovir, (b) biotin-C6-cCidofovir, and
(
c) biotin-C12-cCidofovir. ꢀ, pH 4.0; ᭿, pH 6.0; ♦, pH 7.4. Each data point represents mean ±
SD (n = 3).
were used as a positive and negative control, respec-
tively. Figure 6 summarizes the percentage of viable
cells remaining after exposure of all the compounds
studied.
Interaction with SMVT Transporter
The presence of SMVT transporter on the MDCK–
MDR1 cell line has been characterized previously
3
0
from our laboratory. This cell line is widely used
◦
Table 2. First-Order Rate Constant (k) and Half-Life (t1/2) of Biotin-Conjugated Lipid Prodrugs at Various pH at 37 C
pH 4.0
pH 6.0
pH 7.4
k (h−1)
t1/2 (h)
k (h
−1
)
t1/2 (h)
−1
k (h )
t1/2 (h)
B-C2-cCidofovir
B-C6-cCidofovir
B-C12-cCidofovir
1.4 ± 0.20
6.1 ± 1.1
1.7 ± 0.03
504.80 ± 66.74
115.93 ± 22.71
406.08 ± 8.24
2.2 ± 0.23
1.8 ± 0.051
1.8 ± 0.042
318.28 ± 32.06
390.77 ± 17.30
387.88 ± 9.27
3.6 ± 0.32
6.1 ± 0.20
7.6 ± 0.036
192.97 ± 16.30
486.35 ± 17.86
89.60 ± 12.40
Hydrolysis rate constant values are k × 10−3.
Each data point represents mean ± SD (n = 3).
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GOKULGANDHI ET AL.
Figure 5. Enzymatic degradation biotin-conjugated lipid prodrugs in (a) retina–choroid tissue
homogenate and (b) vitreous humor homogenate. ᭿, B-C2-cCidofovir; ♦, B-C6-cCidofovir; and
, B-C12-cCidofovir. Each data point represents mean ± SD (n = 3).
ꢀ
Table 3. First-Order Rate Constant (k) and Half-Life (t1/2) of Biotin-Conjugated Lipid Prodrugs in
◦
Ocular Tissue Homogenate at 37 C
Retina–Choroid
k (h−1)
Vitreous Humor
−1
t1/2 (h)
k (h
)
t1/2 (h)
B-C2-cCidofovir
B-C6-cCidofovir
B-C12-cCidofovir
2.82 ± 0.0024
2.71 ± 0.0026
3.94 ± 0.0025
24.57 ± 2.02
26.09 ± 2.35
17.85 ± 2.18
1.07 ± 0.0012
1.22 ± 0.00076
1.02 ± 0.00027
65.05 ± 6.87
58.85 ± 3.90
68.23 ± 8.10
Hydrolysis rate constant values are k × 10−2.
Each data point represents mean ± SD (n = 4).
Figure 6. Cytotoxicity study (MTS) for the biotin-conjugated lipid prodrugs on MDCK–MDR1
∗
for 48 h. Each data point represents mean ± SD (n = 3). p < 0.05.
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TRANSPORTER-TARGETED LIPID PRODRUGS OF CYCLIC CIDOFOVIR
11
Figure 7. Cellular accumulation of [3H]-biotin (0.25 :Ci/mL) in presence of biotin-conjugated
lipid prodrugs (50 :M) on MDCK–MDR1 cells. Each data point represents mean ± SD (n = 3).
∗
p < 0.05.
because it possesses tight junctions and it is also
easy to culture.3 Results of this study are shown in
Figure 7. All the biotin-conjugated prodrugs (50 :M)
have shown significant reduction in uptake of [3H]-
biotin (0.25 Ci/mL in DPBS pH 5.5). Uptake of
biotin (0.25 Ci/mL) across MDCK–MDR1 monolayer
1
using transwell. The result clearly indicates signifi-
cant reduction in the transport of [3H]-biotin in pres-
ence of all the biotin-conjugated prodrugs (at 50:M
concentration). However, the reduction in cumulative
transport of [3H]-biotin was higher in presence of B-
C12–cCDF, followed by B-C6–cCDF and B-C2–cCDF.
Cumulative transport of [3H]-biotin across MDCK–
MDR1 at the end of 180 min was reduced by 44%,
58%, and 70% in presence of B-C2–cCDF, B-C6–cCDF,
and B-C12–cCDF, respectively (Fig. 9). We also per-
formed the cellular accumulation of all the prodrugs
on MDCK–MDR1 cells. Cellular accumulation of B-
C2–cCDF, B-C6–cCDF, B-C12–cCDF, C6-cCDF, and
C12-cCDF were 3.5-, 10.5-, 32-, 17.5-, and 42-fold
higher than the parent CDF (Fig. 10).
[3H]-biotin was reduced by 50%, 58%, and 73% for
B-C2–cCDF, B-C6–cCDF, and B-C12–cCDF, respec-
tively on MDCK–MDR1 cells. These results indicate
the effect of lipid chain length of biotin-conjugated
lipid prodrugs on the inhibition of [3H]-biotin uptake.
Moreover, the affinity measurement of these prodrugs
for SMVT transporter shows that all the prodrugs
possess high affinity for SMVT transporters (Fig. 8).
IC50 values are listed in Table 4. These values are
calculated with GraphPad Prism software 5.04. In
addition, we also performed the transport of [3H]-
Figure 8. Dose-dependent inhibition of [3H]-biotin (0.25 :Ci/mL) uptake by (a) biotin-C2-
cCidofovir, (b) biotin-C6-cCidofovir, and (c) biotin-C12-cCidofovir in MDCK–MDR1. Each data
point represents mean ± SD (n = 3).
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GOKULGANDHI ET AL.
Figure 9. Transport of [3H]-biotin (0.25 :Ci/mL) across MDCK–MDR1 cells in absence and
presence of biotin-conjugated lipid prodrugs (50 :M). ♦, Control; ᭿, B-C2-cCidofovir; ꢀ, B-
C6-cCidofovir; X, B-C12-cCidofovir. Each data point represents mean ± SD (n = 3). Insert:
∗
∗∗
Cumulative amount transport at the end of 180 min. p < 0.05 and p < 0.01.
Table 4. IC50 Values from the Graphpad Prism 5.0 for the
Biotin-Conjugated Lipid Prodrugs of Cyclic Cidofovir Toward
SMVT Transporter
Table 5. Tissue (Retina–Choroid) Partition Coefficient for the
Biotin-Conjugated Lipid Prodrug of Cyclic-Cidofovir
Partition Coefficient
Drug
IC50 Value (:M)
Drug
(Retina–Choroid)
Fold Increase
B-C2–cCDF
B-C6–cCDF
B-C12–cCDF
31.42 ± 3.05
11.25 ± 1.48
2.90 ± 0.07
CDF
0.29 ± 0.06
0.89 ± 0.27
1.42 ± 0.45
2.84 ± 0.96
1
B-C2–cCDF
B-C6–cCDF
B-C12–cCDF
3.07
4.90
9.80
Tissue Partition Coefficient
choroid and the results for all the biotin-conjugated
prodrugs are listed in Table 5. Results indicate about
3, 5, and 10-fold improvement in the partition of
these prodrugs into retina–choroid, relative to parent
CDF. This improvement is mainly due to an increase
lipophilicity of these conjugates.
Following intravitreal injection into vitreous body, the
prodrugs needs to partition into retina–choroid in
order to exert their antiviral activity. Therefore, we
have performed the tissue partitioning experiments
with biotin-conjugated lipid prodrugs for retina–
Figure 10. Cellular accumulation of biotin-conjugated lipid prodrugs [alone and in presence
of biotin (25: and 50 :M)] and lipid prodrugs on MDCK–MDR1 cells for 60 min. Each data point
∗
∗∗
represents mean ± SD (n = 4). p < 0.05 and p < 0.01.
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TRANSPORTER-TARGETED LIPID PRODRUGS OF CYCLIC CIDOFOVIR
13
DISCUSSION
lipid prodrugs will enhance their ocular bioavailabil-
ity by overcoming this limitation. Moreover, an en-
hanced lipophilicity of these prodrugs may enhance
the melanin binding in the retina, which will form
the depot in the retina–choroid for longer residence
and, therefore, may prolong the antiviral activity.
Furthermore, we have determined the chemical
stability of all the prodrugs in different pH buffers (pH
Cidofovir is one of the most potent drug molecule indi-
cated in the treatment of HIV-induced opportunistic
infections such as CMV retinitis. Posterior segment
delivery of CDF is challenging because of its high hy-
drophilicity and poor permeability across the retina
3
2
(
target tissue for the CMV retinitis). Retinal pig-
ment epithelium (RPE) cells display high membrane
lipophilicity and tight junctions. So far, different ap-
proaches have been employed to enhance permeabil-
1
.4 to 7.4). We did not observe any appreciable degra-
dation of all the prodrugs at pH 1.4. However, upon
rising pH toward alkaline range, the degradation ap-
pears to be faster. Table 2 summarizes the apparent
first-order degradation rate constant indicating the
loss of the prodrugs. The B-C12–cCDF showed faster
degradation relative to B-C6–cCDF and B-C2–cCDF.
We hypothesized that this could be due to steric hin-
drance caused by the presence of CDF and biotin at
the terminals. This may lead to the low accessibility
of phosphoester group to nucleophilic attack by hy-
droxyl ion. Therefore, it is likely that due to the steric
hindrance, nucleophilic attack on B-C2–cCDF is slow
as compared with B-C6–cCDF and B-C12–cCDF.
We have also performed enzymatic stability studies
of all the prodrugs in the presence of retina–choroid
and vitreous tissue homogenates. A rapid conversion
of these prodrugs into parent drug in the retina—
choroid relative to vitreous homogenates has been
observed (Fig. 5 and Table 3). These results further
indicate that all the prodrugs remain stable for longer
duration upon intravitreal administration. Following
active transport across deeper tissue in the retina,
prodrugs will convert into its parent moiety to exert
its antiviral activity.
Another important objective of the present study
was to target the SMVT transporter. Recognition of
prodrugs by SMVT transporters may enhance their
translocation within inner retinal tissues and even-
tually a higher concentration of drug can be achieved
at the target site. Therefore, we performed in vitro
uptake study using a model MDCK–MDR1 cell line.
The results indicate that all the prodrugs show strong
interaction with the SMVT transporter. An inhibition
trend (of [3H]-biotin) was observed in the following or-
der: B-C12–cCDF > B-C6–cCDF > B-C2–cCDF. These
results clearly indicate that elongation of lipid chain
causes stronger interaction of biotin-conjugated pro-
drugs with cell membrane. Moreover, high percent-
age of transport inhibition of [3H]-biotin in the pres-
ence of B-C12–cCDF in comparison to B-C6–cCDF
and B-C2–cCDF suggested the significant role of lipid
raft. In addition, results of IC50 value clearly indi-
cate the higher affinity of B-C12–cCDF toward SMVT
transporter as compared with other prodrugs. Non-
radioactive cellular accumulation study shows that
there is 3.5-, 10.5-, 32-, 17.5-, and 42-fold increase in
the uptake of B-C2–cCDF, B-C6–cCDF, B-C12–cCDF,
1
4
ity of CDF such as lipid prodrugs or transporter-
1
targeted prodrugs of cCDF . However, individually,
these approaches have exhibited limited advantages.
In the present study, we have synthesized various
transporter-targeted lipid prodrugs of cCDF. We hy-
pothesized that the presence of lipid raft will improve
the interaction of these prodrugs with the cell mem-
brane and transporter-targeting moiety will further
improve the translocation of these prodrugs across the
plasma membrane. This may exhibit synergistic effect
due to the simultaneous conjugation of lipid raft and
transporter-targeted moiety, which may ultimately
improve the uptake of these prodrugs across the cell
layers. Our laboratory has previously confirmed the
presence of SMVT transporter on the AP side of
2
3
RPE. Therefore, we sought to utilize SMVT trans-
porter for our approach. We have successfully synthe-
sized and characterized the biotin-conjugated lipid
prodrugs and lipid prodrugs of cCDF (B-C2–cCDF,
B-C6–cCDF, B-C12–cCDF, C6-cCDF, and C12-cCDF)
3
1
using P NMR, LC–MS/MS, and HPLC. The general
synthesis scheme for the prodrugs has been shown in
Figures 1 and 2. All the prodrugs have exhibited 95%
or more purity along with brownish sticky (biotin-
conjugated prodrugs) or white solid crystalline (lipid
prodrugs) appearance.
It is well known that the rate-limiting barrier
for the uptake and transport of highly hydrophilic
drugs such as CDF is the lipophilic bilayer of the
cell membrane. Hence, we performed the solubil-
ity and n-octanol/buffer partition coefficient studies
of these prodrugs. The results have indicated that
the solubility of biotin-conjugated lipid prodrugs was
significantly lowered, relative to the parent CDF.
However, higher water solubility of biotin-conjugated
lipid prodrugs have been attributed due to the ad-
dition of water-soluble vitamin such as biotin as the
transporter-targeting moiety to the lipid conjugates
of cCDF as compare to lipid prodrug of cyclic cido-
fovir. Moreover, partition coefficient study has shown
an increment in log P value for biotin-conjugated
lipid prodrugs. The lipophilic RPE prevents the en-
try of relatively hydrophilic antiviral drugs follow-
ing systemic or intravitreal administration. The im-
proved lipophilicity of synthesized biotin-conjugated
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GOKULGANDHI ET AL.
C6-cCDF, and C12-cCDF, respectively, as compared
with the parent CDF (Fig. 10). The observed cellular
uptake of B-C6–cCDF and B-C12–cCDF was not more
than their lipid prodrugs (i.e., C6-cCDF and C12-
cCDF). Biotin-conjugated lipid prodrugs have exhib-
ited higher water solubility, which may have lead
to the reduced cell bilayer partitioning relative to
the lipid prodrugs. Cellular uptake of B-C12–cCDF
was found to be comparable to C12-cCDF, wherein
the lipid raft (C12) facilitates the enhanced inter-
action of B-C12–cCDF with membrane protein and,
thereby, assisting docking of biotin into the bind-
ing domain of SMVT transporter. The net effect will
be rapid translocation across RPE cell membrane,
which will not be the case with lipid prodrugs. Thus,
our novel B-C12–cCDF prodrug may significantly en-
hance the delivery of CDF across lipophilic RPE. In
addition, the uptake of biotin-conjugated lipid pro-
drugs across MDCK–MDR1 was significantly inhib-
ited by biotin (due to competitive inhibition). These
results clearly indicate that improvement in uptake
of biotin-conjugated prodrugs was via SMVT inter-
action (Fig. 10). The overall results from the inter-
action of all biotin-conjugated lipid prodrugs with
SMVT transporters suggest that these compounds are
strongly recognized by SMVT transporter. Moreover,
results further clarify that upon increasing the lipid
chain length of the biotin-conjugated lipid prodrugs,
an interaction with the SMVT transporter can be
enhanced.
The major limiting factor for in vivo absorption
of hydrophilic molecules is their low tissue parti-
tioning. The parent drug CDF is highly hydrophilic
in nature and following intravitreal administration,
a large fraction may remain in the vitreous body
and may not partition enough into lipophilic retina–
choroid. However, the tissue–water partition study of
biotin-conjugated lipid prodrugs clearly indicates an
improvement in the uptake into retina–choroid tis-
sue. As all the prodrugs have high lipophilicity; these
compounds may partition into retina–choroid as com-
pared with the parent CDF. Moreover, because of the
strong interaction with SMVT transporter, these pro-
drugs may accumulate into the deeper tissue of retina/
choroid, thereby improving the therapy.
ACKNOWLEDGMENTS
This study was supported by National Institutes of
Health grants R01 EY 09171-14 and R01 EY 10659-
1
2. We would like to thank Gilead Science for the
generous gift of Cidofovir. We would also like to thank
Dr. Ravinder Earla for his assistance in LC–MS/MS
analysis.
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