Enhancing the intestinal membrane permeability of zanamivir: A carrier mediated prodrug approach

Article abstract of DOI:10.1021/mp200291x

The purpose of this study was to improve the membrane permeability and oral absorption of the poorly permeable anti-influenza agent, zanamivir. The poor oral bioavailability is attributed to the high polarity (cLogP ～ -5) resulting from the polar and zwitterionic nature of zanamivir. In order to improve the permeability of zanamivir, prodrugs with amino acids were developed to target the intestinal membrane transporter, hPepT1. Several acyloxy ester prodrugs of zanamivir conjugated with amino acids were synthesized and characterized. The prodrugs were evaluated for their chemical stability in buffers at various pHs and for their transport and tissue activation by enzymes. The acyloxy ester prodrugs of zanamivir were shown to competitively inhibit [³H]Gly-Sar uptake in Caco-2 cells (IC₅₀: 1.19 ± 0.33 mM for l-valyl prodrug of zanamivir). The l-valyl prodrug of zanamivir exhibited ～3-fold higher uptake in transfected HeLa/hPepT1 cells compared to wild type HeLa cells, suggesting, at least in part, carrier mediated transport by the hPepT1 transporter. Further, enhanced transcellular permeability of prodrugs across Caco-2 monolayer compared to the parent drug (P_app = 2.24 × 10^-6 ± 1.33 × 10^-7 cm/s for l-valyl prodrug of zanamivir), with only parent zanamivir appearing in the receiver compartment, indicates that the prodrugs exhibited both enhanced transport and activation in intestinal mucosal cells. Most significantly, several of these prodrugs exhibited high intestinal jejunal membrane permeability, similar to metoprolol, in the in situ rat intestinal perfusion system, a system highly correlated with human jejunal permeability. In summary, this mechanistic targeted prodrug strategy, to enhance oral absorption via intestinal membrane carriers such as hPepT1, followed by activation to parent drug (active pharmaceutical ingredient or API) in the mucosal cell, significantly improves the intestinal epithelial cell permeability of zanamivir and has the potential to provide the high oral bioavailability necessary for oral zanamivir therapy.

Full text of DOI:10.1021/mp200291x

ARTICLE
pubs.acs.org/molecularpharmaceutics
Enhancing the Intestinal Membrane Permeability of Zanamivir: A
Carrier Mediated Prodrug Approach
†
†
†
§
†
‡
Sheeba Varghese Gupta, Deepak Gupta, Jing Sun, Arik Dahan, Yasuhiro Tsume, John Hilfinger,
†
,†
Kyung-Dall Lee, and Gordon L. Amidon*
†
Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109,
United States
§
Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev,
Beer-Sheva 84105, Israel
‡
TSRL, Inc., 540 Avis Drive, Ann Arbor, Michigan 48108, United States
ABSTRACT: The purpose of this study was to improve the
membrane permeability and oral absorption of the poorly perme-
able anti-influenza agent, zanamivir. The poor oral bioavailability is
attributed to the high polarity (cLogP ∼ ꢀ5) resulting from the
polar and zwitterionic nature of zanamivir. In order to improve the
permeability of zanamivir, prodrugs with amino acids were devel-
oped to target the intestinal membrane transporter, hPepT1. Several
acyloxy ester prodrugs of zanamivir conjugated with amino acids
were synthesized and characterized. The prodrugs were evaluated
for their chemical stability in buffers at various pHs and for their
transport and tissue activation by enzymes. The acyloxy ester
prodrugs of zanamivir were shown to competitively inhibit
3
[
H]Gly-Sar uptake in Caco-2 cells (IC : 1.19 ( 0.33 mM for L-
50
valyl prodrug of zanamivir). The L-valyl prodrug of zanamivir exhibited ∼3-fold higher uptake in transfected HeLa/hPepT1 cells
compared to wild type HeLa cells, suggesting, at least in part, carrier mediated transport by the hPepT1 transporter. Further,
ꢀ
6
enhanced transcellular permeability of prodrugs across Caco-2 monolayer compared to the parent drug (P = 2.24 ꢁ 10
app
(
ꢀ
7
1
.33 ꢁ 10 cm/s for L-valyl prodrug of zanamivir), with only parent zanamivir appearing in the receiver compartment, indicates
that the prodrugs exhibited both enhanced transport and activation in intestinal mucosal cells. Most significantly, several of these
prodrugs exhibited high intestinal jejunal membrane permeability, similar to metoprolol, in the in situ rat intestinal perfusion system,
a system highly correlated with human jejunal permeability. In summary, this mechanistic targeted prodrug strategy, to enhance oral
absorption via intestinal membrane carriers such as hPepT1, followed by activation to parent drug (active pharmaceutical ingredient
or API) in the mucosal cell, significantly improves the intestinal epithelial cell permeability of zanamivir and has the potential to
provide the high oral bioavailability necessary for oral zanamivir therapy.
KEYWORDS: prodrugs, carrier-mediated transport, hPepT1, zanamivir
’
INTRODUCTION
Zanamivir and oseltamivir are the two NA inhibitors approved
by the FDA for clinical use. Zanamivir has been shown to be a
potent inhibitor of both influenza A and influenza B as well as
Influenza is an acute viral infection of the human respiratory
tract. Four drugs are recommended for the treatment and
prophylaxis of the influenza infection: the ion channel inhibitors
3
active against emerging resistant strains. There are reported
4
incidences of resistance against oseltamivir, however, there are
(amantadine and rimantadine) and the neuraminidase (NA)
no reports of resistance to zanamivir to date. This lack of
resistance development for zamamivir is thought to be due to
the fact that zanamivir is structurally very similar to sialic acid, the
natural substrate of NA. Thus, any drug-resistant mutations in
NA would likely compromise the binding of sialic acid to NA and
inhibitors (zanamivir and oseltamivir). Amantadine and riman-
tadine act by interfering with the viral coating process and are
effective against only influenza A. Additionally, there has been a
rapid increase in the resistance of influenza A virus against these
ion channel inhibitors, notably, including the bird flu virus H5N1
5
is likely compromise the viral propagation.
1
responsible for the influenza outbreak in Asia. The neuramini-
dase inhibitors act via a different mechanism, by inhibiting the
neuraminidase enzyme essential for viral propagation. Among
the recommended drugs to treat influenza infection, neuramini-
dase inhibitors are considered to be the drugs of choice princi2-
pally due to their low toxicity and low incidences of resistance.
Received: June 7, 2011
Accepted: September 9, 2011
Revised:
September 2, 2011
Published: September 09, 2011
r 2011 American Chemical Society
2358
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|Mol. Pharmaceutics 2011, 8, 2358–2367

Molecular Pharmaceutics
ARTICLE
tert-butyloxycarbonyl (Boc) protected amino acids Boc-L-Val-
OH, Boc-L-Ile-OH, and Boc-D-Val-OH were obtained from
Calbiochem-Novabiochem (San Diego, CA). HPLC (high-per-
formance liquid chromatography) grade acetonitrile and
LCꢀMS (liquid chromatographyꢀmass spectrometry) grade
methanol were obtained from Fisher Scientific. All other reagents
were purchased from Sigma Aldrich (Milwaukee, WI) unless
otherwise mentioned. All solvents used for chemical syntheses
were anhydrous and were obtained in Sure/Seal bottles unless
otherwise mentioned. Deuterated solvents for nuclear magnetic
resonance (NMR) spectroscopy were purchased from Cambridge
Isotopes, Inc. (Andover, MA). Valacyclovir was a gift from
GlaxoSmithKline, Inc. (Research Triangle Park, NC). Cell
culture reagents were purchased from Invitrogen (Carlsbad,
CA), and the cell culture supplies were obtained from Corning
Figure 1. Zanamivir.
The absolute oral availability of zanamivir is very low, approxi-
6
mately 2%, and zanamivir is administrated clinically by inhala-
tion. The major drawback with this route of administration is lack
of patient compliance, and therapy is not recommended in
patients with chronic respiratory conditions such as asthma. It
is also difficult to administer this drug by inhalation to the
pediatric population, which is at high risk for influenza infection.
Thus, the poor oral bioavailability limits the clinical utility of
zanamivir to treat influenza infections.
The principal reason for the poor oral availability of zanamivir
is its polar nature, contributed particularly by the guanidine,
carboxylic, and hydroxyl functional groups in the molecule
(
Corning, NY) and Falcon (Lincoln Park, NJ). All the NMRs
were taken using a Varian 500 MHz NMR spectrometer. The
HPLC analyses were done using an Agilent 1200 series HPLC
system (Agilent Technologies, Inc. Santa Clara CA). The
LCꢀMS studies were carried out using Shimadzu LCꢀMS
system. The molecular weights of the compounds were con-
firmed by positive-mode electrospray ionization mass spectra
(Figure 1). A common approach to overcome this type of oral
delivery problem is to chemically modify zanamivir to make a
more lipophilic molecule and, thus, potentially increase its mucosal
membrane permeability and oral bioavailability. An analogue ap-
proach to modification of zanamivir’s polarity, generating a new
chemical entity (NCE), has the obvious limitation of requiring full
safety and efficacy testing and development of a NCE. Since
zanamivir itself has a well established toxicity profile, has excellent
antiviral properties, and has shown little or no resistance develop-
ment, we developed a prodrug approach targeting the intestinal
(
ESI-MS) on a Waters electronspray ionization spectrometer.
Boc-Zanamivir and Prodrug Synthesis . Boc-zanamivir was
synthesized from sialic acid according to the procedures men-
tioned in the literature. Briefly, sialic acid 1 was converted to the
+
13
methyl ester 2, in the presence of Dowex H . The hydroxyl
groups of 2 were protected with acetyl groups to give compound
3
, which was then converted to the oxazoline derivative 4 in the
14
presence of trimethylsilyl trifluoromethanesulfonate. Azide 5
was synthesized from 4 in presence of azidotrimethylsilane.
1
4
7
epithelial cell hPepT1 transporter to orally deliver zanamivir. The
The azide is reduced to the corresponding amine 6 by using
Lindlar’s catalyst; the amine (6) is then converted to the
prodrug approach has the advantage of preserving the safety and8
efficacy of the parent zanamivir. However, both prodrug transport
15
9
guanidine derivative 7. The final step involved the deprotection
of the methyl ester and acetyl groups in the presence of
methanolic sodium hydroxide to give Boc-protected zanamivir
and activation to parent have to occur for a prodrug strategy to be
successful. Amino acid prodrugs of zanamivir were chosen based on
our prior studies with carrier mediated transport of prodrug.
7b,10
15
8
. The synthetic procedure is shown in Scheme 1. Acyloxy ester
Here we report the synthesis and transport of prodrugs of
zanamivir linked to amino acids. The acyloxy group is often used
as linker in prodrug strategies in order to improve the biophar-
maceutical and/or pharmacokinetic properties of the drug.
Examples of successful acyloxy prodrugs include pivampicillin
and cefuroxime axetil. Another example is tranexamic acid, a
clinically used antifibrinolytic agent that is not well absorbed
orally due to the amphoteric nature of the drug. Acyloxy esters of
tranexamic acid have shown increased absorption compared to
prodrugs of zanamivir, 4aꢀg, were synthesized from intermedi-
1
6
ate 8 and the α-chloro methyl ester of the respective R group.
The alkyl α-chloro methyl ester was synthesized from the
respective carbonyl chloride in the presence of zinc chloride
16
and acetaldehyde. For the compounds with amino acid sub-
stitution instead of an alkyl group, the α-chloro methyl ester was
synthesized from the cesium salt of respective Boc-protected
1
7
amino acid in presence of 1-chloro-1-bromoethane. The
α-chloro methyl ester of amino acid was reacted with Boc-
protected zanamivir in the presence of triethylamine in dimethyl-
formamide (DMF) at 50 °C for 4 days. The reaction mixture was
concentrated in vacuo, and the crude mixture was purified using
flash column chromatography with silica gel (60 Å, 230ꢀ400
mesh) by eluting with chloroform:methanol (20:1) to yield the
pure compound as oil. The Boc-protected prodrugs were depro-
tected by reacting with 50% trifluoroacetic acid (TFA) in
dichloromethane and stirring for 2 h at room temperature, the
reaction mixture was concentrated in vacuo and the residues
were constituted in water and were lyophilized. The TFA salts of
amino acid prodrugs of zanamivir were obtained as white
hygroscopic amorphous powder. The synthetic steps for prodrugs
1
1
the parent drug. In this manuscript we report the synthesis and
permeability evaluations of acyloxy ester prodrugs of zanamivir.
We have evaluated the cellular transport and activation of the
3
prodrugs and their ability to compete with [ H]Gly-Sar uptake in
Caco-2 cells and in direct uptake in HeLa/hPEPT1 cells. We also
report the chemical and enzymatic (cell homogenates) stability
of the prodrugs. Furthermore, several of the prodrugs that
3
exhibited good [ H]Gly-Sar uptake inhibition and uptake in
HeLa/hPepT1 cells were studied for transmembrane perme-
ability in Caco-2 monolayers and the in situ rat perfusion system
that is highly correlated with human jejunal permeability.
12
’
MATERIALS AND METHODS
Boc-zanamivir was synthesized from N-acetylneuraminic acid
sialic acid) purchased from TCI America Ltd. Zanamivir was
purchased from Waterstone Technology (Carmel, IN). The
4
aꢀg are summarized in Scheme 2.
1
Boc-Zanamivir (8). H NMR (CD OD): δ (ppm) 5.6 (d, J =
3
(
2.0 Hz, 1H), 5.01 (dd, J = 9.6, 2.1 Hz, 1H), 4.25 (dd, J = 10.8,
1.1 Hz, 1H), 4.18 (dd, J = 10.6, 9.6 Hz, 1H), 3.89 (ddd, J = 9.4, 6.2,
2
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Molecular Pharmaceutics
ARTICLE
a
Scheme 1. Synthesis of Boc-Zanamivir
a
(
a) Dowex H, methanol; (b) acetic anhydride, 4-dimethylaminopyridine (DMAP), pyridine; (c) trimethylsilyl trifluoromethanesulfonate, ethyl
0
acetate; (d) azidotrimethylsilane, butanol; (e) Lindlar's catalyst, ethanol; (f) N,N -bis-tert-butoxycarbonyl-1H-pyrazole-1-carboxamidine, tetrahydro-
furan; (g) sodium hydroxide, methanol.
1
Scheme 2. Synthesis of Acyloxy Ester Prodrugs of
Zanamivir
Zan-L-Ile (4b). H NMR (CD OD): δ (ppm) 7.0 (m, 1H),
3
a
5.9 (d, J = 2.0 Hz, 1H), 5.5 (m, 1H) 4.9 (m, 1H) 4.22 (m, 1H)
.07 (m, 1H), 3.85 (m, 2H), 3.70 (m, 2H) 3.2 (q, 1H), 2.03
4
(
(
s, 6H), 1.3 (m, 2H), 1.02ꢀ1.03 (m, 6H). ESI-MS: 490.21
+
M + H) .
1
Zan-D-Val (4c),. H NMR (CD OD): δ (ppm) 6.88 (q, 1H),
3
5
4
2
.85 (d, J = 2.0 Hz, 1H), 5.2 (m, 1H), 4.9 (m, 1H), 4.3 (m, 2H),
.1 (m, 2H), 3.9 (m, 1H), 3.7 (m, 1H), 2.22 (m, 1H), 2.1 (s, 3H),
.0 (s, 3H), 0.96 (d, J = 6.0 Hz, 3H), 0.92 (d, J = 6.0 Hz, 3H). ESI-
+
MS: 476 (M + H) .
Cell Culture. Caco-2 cells (passages 22ꢀ34) and HeLa cells
(
(
passages 20ꢀ33) from American Type Culture Collection
Rockville, MD) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% FBS, 1% nonessential amino
acids, 1 mM sodium pyruvate and 1% L-glutamate. Cells were
grown in an atmosphere of 5% CO and 90% relative humidity at
2
3
7 °C.
3
[
H] Gly-Sar Uptake Inhibition. Caco-2 cells were grown to
be confluent in 12 well plates. The cells were grown for 10 days
after seeding. On the day of experiment, cells were washed with
uptake buffer (pH 6.0, 145 mM NaCl, 3 mM KCl, 1 mM
NaH PO , 1 mM CaCl , 0.5 mM MgCl , 5 mM D-glucose,
2
4
2
2
3
and 5 mM MES) and incubated with 10 μmol/L [ H]Gly-Sar
9.94 μmol/L Gly-Sar and 0.06 μmol/L [ H]Gly-Sar) and
3
(
different concentrations (0.05ꢀ5 mM) of zanamivir or its
prodrugs in 0.3 mL of the uptake buffer for 30 min at 37 °C.
After 30 min, the drug solution was aspirated and the cells were
washed with ice-cold uptake buffer. Methanol:water (50:50)
a
R COCH(CH )Cl, triethylamine, dichloromethane; (b) R OH, 1-ethyl-
-(3-dimethylaminopropyl)carbodiimide (EDC), 4-dimethylaminopyridine
DMAP), dimethylformamide; (c) trifluoroacetic acid, dichloromethane.
1
3
2
3
(
(
500 μL) was added to each well, and the cells were scraped
2
5
1
.7 Hz, 1H), 3.84 (dd, J = 11.3, 2.8 Hz, 1H), 3.67 (dd, J = 11.3,
.8 Hz, 1H), 3.57 (d, J = 9.3 Hz, 1H), 1.9 (s, 3H), 1.55 (s, 9H),
and dissolved in the scintillation cocktail (ScintiVerse* LC
Cocktail, Fisher Chemicals). The radioactivity was measured
by scintillation counter (Beckman LS-9000, Beckman Instru-
ments, Fullerton, CA). IC50 values were determined using non-
linear data fitting (Graph Pad Prism v4.0).
+
.50 (s, 9H). ESI-MS: 533 (M + H) .
1
Zan-L-Val (4a),. H NMR (CD OD): δ (ppm) 6.9 (q, 1H), 5.8
3
(
(
2
d, J = 2.0 Hz, 1H), 5.2 (m, 1H), 4.9 (m, 1H), 4.2 (m, 2H), 4.02
m, 2H), 3.9 (m, 1H), 3.7 (m, 1H), 2.14 (m, 1H), 2.08 (s, 3H),
.01 (s, 3H), 0.98 (d, J = 6.8 Hz, 3H), 0.90 (d, J = 6.5 Hz, 3H).
Uptake Studies. Carrier mediated prodrug transport was
7b
evaluated in HeLa/hPepT1 as described earlier. HeLa cells
were transfected by adenovirus containing hPepT1 as described
+
ESI-MS: 476 (M + H) .
2
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ARTICLE
1
8
previously. Two days postinfection the cell culture medium was
removed and washed with uptake buffer (pH 6.0) and was
incubated with 0.5 mL of test compounds (1 mM) in uptake
buffer at 37 °C for 45 min. After 45 min the drug solutions were
removed and the cells were washed with ice-cold uptake buffer.
Methanol:water (50:50) (500 μL) was added to each well, and
the incubated at room temperature for 1 h. The cells were
collected after 1 h and were vortexed and centrifuged. The
supernatant was filtered (0.22 μm) and analyzed by LCꢀMS.
Control experiments were performed in nontransfected HeLa
cells. The protein amount of each sample was determined with
the Bio-Rad DC Protein Assay using bovine serum albumin as
the standard.
apical layer medium was aspirated and replaced with 1.5 mL of
drug (0.25 mM) solution in MES buffer (pH 6.0), and the
basolateral medium was replaced with 2.6 mL of fresh HEPES
buffer. At every 15 min interval 200 μL aliquot was taken from
the basolateral receiving medium and was replaced with equal
amount of HEPES buffer. The degradation of prodrugs due to
monolayer or buffer in the apical layer was determined by taking
10 μL aliquots of the apical solution and quenching with 90 μL of
0.1% (w/v) TFA in acetonitrile. The epithelial integrity of the
representative monolayers was checked by monitoring TEER
values at the end of the experiment. Both aliquots from apical and
basolateral sides were analyzed by LCꢀMS.
Rat Perfusion. All animal experiments were conducted using
protocols approved by the University Committee of Use and
Care of Animals (UCUCA), University of Michigan, and the
animals were housed and handled according to the University of
Michigan Unit for Laboratory Animal Medicine guidelines. Male
albino Wistar rats (Charles River, IN) weighing 250ꢀ280 g were
used for all perfusion studies. Prior to each experiment, the rats
were fasted overnight (12ꢀ18 h) with free access to water.
Animals were randomly assigned to the different experimental
groups.
Hydrolysis in Caco-2 Homogenates. Caco-2 cells 22 days
after seeding were washed with phosphate buffer saline (pH 7.4).
The cells were scraped from the plate using a cell scraper
(
Corning Small Cell Scraper). The cells were collected in
phosphate buffer (pH 7.4, 100 mM) and spun down by
centrifugation. The cells were resuspended in phosphate buffer
and were lysed by sonication. The cell lysate was centrifuged at
7
150g for 15 min, and the supernatant was collected. The protein
amount of supernatant was determined with the Bio-Rad DC
Protein Assay using bovine serum albumin as the standard. The
hydrolysis reactions were carried out in eppendorf tubes with
total reaction volume of 1 mL containing 1 mM drug solution
and cell lysate corresponding to 500 μg/mL protein concentra-
tion in 100 mM pH7.4 phosphate buffer. The assay mixture was
incubated at 37 °C, and 100 μL of the reaction mixture was
removed at various time points (0, 5, 10, 15, 20 and 30 min) and
was quenched with 100 μL of ice-cold TFA (10% w/v). The
samples were filtered (0.45 μM), and the filtrate was assayed for
prodrug and parent drug by HPLC. The extent of hydrolysis for
the prodrug at each time point was calculated. The measure-
ments were done in triplicate.
The procedure for the in situ single-pass intestinal perfusion
12,20
followed previously published reports.
Briefly, rats were
anesthetized with an intramuscular injection of 1 mL/kg of
ketamineꢀxylazine solution (9%:1%, respectively) and placed
on a heated surface maintained at 37 °C (Harvard Apparatus Inc.,
Holliston, MA). The abdomen was opened by a midline incision
of 3ꢀ4 cm. A proximal jejunal segment, midsmall intestine, or a
distal ileal segment, of approximately 10 cm was carefully
exposed and cannulated on two ends with flexible PVC tubing
(2.29 mm i.d., inlet tube 40 cm, outlet tube 20 cm, Fisher
Scientific Inc., Pittsburgh, PA). Care was taken to avoid dis-
turbance of the circulatory system, and the exposed segment was
kept moist with 37 °C normal saline solution. The perfusate was
incubated in a 37 °C water bath to maintain temperature, and a
perfusion solution containing 10 mM MES buffer, pH 5.5,
135 mM NaCl, 5 mM KCl, and 0.1 mg/mL phenol red with
an osmolarity of 290 mosm/L was pumped through the intestinal
segment (Watson Marlow Pumps 323S, Watson-Marlow Bredel
Inc., Wilmington, MA). The isolated segment was rinsed with
blank perfusion buffer, pH 5.5 at a flow rate of 0.5 mL/min in
order to clean out any residual debris.
At the start of the study, perfusion solution containing the
tested drug was perfused through the intestinal segment at a flow
rate of 0.18 mL/min. Phenol red was added to the perfusion
buffer as a nonabsorbable marker for measuring water flux.
Metoprolol was coperfused with the other test compounds as
well, as a compound with known permeability that serves as a
marker for the integrity of the experiment, and as a reference
standard for permeability in close proximity to the low/high
permeability class boundary. The concentrations of the test
compounds used in the perfusion studies were determined by
dividing the highest prescribed oral dose by 250 mL, the accepted
gastric volume, in order to represent the maximal drug concen-
tration present in the intestinal segment, and were within their
intrinsic solubility reported at pH 5.5. The perfusion buffer was
first perfused for 1 h, in order to ensure steady state conditions
(as also assessed by the inlet over outlet concentration ratio of
phenol red, which approaches 1 at steady state). After steady
state was reached, samples were taken in 10 min intervals for 1 h
(10, 20, 30, 40, 50, and 60 min). All samples including perfusion
Chemical Stability. The hydrolysis profiles of the prodrugs
were determined in 100 mM hydrochlorate buffer (pH 1.2),
1
(
00 mM acetate buffer (pH 4), and 100 mM phosphate buffer
pH 6.5, and 7.4) corresponding to physiologically relevant pHs.
The choice of buffer types and strength was based on information
from the data published earlier. Hydrolysis of the prodrugs
was studied in respective buffers at 37 °C for a period of 4 h.
At every time point, 50 μL aliquots of the samples were taken and
diluted with 0.1% ice-cold trifluoroacetic acid and was analyzed
by HPLC.
1
9
Caco-2 Monolayer Permeability. The Caco-2 permeability
for the prodrugs and parent drug was determined as previously
7
b
described. Briefly, Caco-2 cells were seeded on 6-well format
collagen-coated transwell inserts (Corning, NY, 0.4 μm pore
size: area 4.7 cm ) at a density of 100,000 cells/mL and cultured
in DMEM containing 10% FBS, 1% nonessential amino acids,
mM sodium pyruvate and 1% L-glutamine (Invitrogen, Carls-
bad CA). Cells were grown in 5% CO and 90% relative humidity
at 37 °C. The culture medium was replaced as needed. Trans-
endothelial electrical resistance (TEER) measurements were
performed on all monolayers. Monolayers with TEER values
250 Ω/cm were used for the study. 1.5 mL of MES buffer pH
.0 (5 mM D-glucose, 5 mM MES, 1 mM CaCl , 1 mM MgCl ,
50 mM NaCl, 3 mM KCl and 1 mM NaH PO ) was applied to
the apical side, and 2.6 mL of HEPES buffer pH 7.4 (1 mM
CaCl , 1 mM MgCl , 150 mM NaCl, 3 mM KCl and 1 mM
NaH PO ) was applied to the basolateral side. The plates were
2
1
2
2
>
6
1
2 2
2
4
2
2
2
4
incubated at 37 °C for 15 min for equilibration. After 15 min the
2
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ARTICLE
samples at different time points, original drug solution, and inlet
solution taken at the exit of the syringe were immediately
assayed by HPLC. Following the termination of the experiment,
the length of the perfused intestinal segment was accurately
measured.
HPLC Analysis. The samples from enzymatic and chemical
stability studies of zanamivir and its prodrugs were analyzed by
Agilent 1200 Series HPLC system (Agilent Technologies, Inc.
Santa Clara CA) equipped with two Agilent 1200 pumps, an
Agilent autosampler and a Agilent UV detector. The system was
operated by Agilent 1100 software. Waters Xterra C18 reversed-
phase column (5 μm, 4.6 ꢁ 250 mm) was used for sample
analysis. The analytes were eluted using a gradient method. The
mobile phases (water and acetonitrile) contained 0.1% TFA as
modifier.
Cout/C ratio was corrected for water transport (to obtain the
in
0
0
corrected ratio C /C ) according to the following equation:
out
in
0
C
C
Cout
inðphenol redÞ
out
¼
ꢁ C
0
C
Cin
in
out phenol red
ð
Þ
where Cin(phenol red) is equal to the concentration of phenol red in
the inlet sample, and Cout(phenol red) is equal to the concentration
of phenol red in the outlet sample.
The effective permeability (P ) through the rat gut wall in the
eff
single-pass intestinal perfusion studies was determined assuming
2
1
the “plug flow” model expressed in the following equation:
0
ꢀ
Q lnðC0 =C Þ
out
in
Peff ðcm=sÞ ¼
2πRL
LCꢀMS Analysis. The prodrug and parent drug samples from
uptake and transepithelial permeability studies were analyzed
using a LCꢀMS 2010A system (Shimadzu Scientific Instru-
ments, Columbia MD). The LCꢀMS unit consisted of two
pumps (LC-20AD), autosampler (SIL 20A HT) and QoQ
Optical System. Positive-mode electrospray ionization (ESI)
was used to ionize the molecules. The system was operated by
LCMSsolution Ver. 3 software. The CDL temperature used was
0
where Q is the perfusion buffer flow rate (0.18 mL/min), C out/
0
C
is the ratio of the outlet concentration and the inlet or
in
starting concentration of the tested compound that has been
adjusted for water transport, R is the radius of the intestinal
segment (set to 0.2 cm), and L is the length of the intestinal
segment.
’
RESULTS
2
50 °C, and the detector voltage was maintained at 1.5 kV.
Samples were analyzed by Restek Allure Aqueous C column
18
Synthesis of Zanamivir Prodrugs. Boc-protected zanamivir
(
5 μm, 2.1 ꢁ 50 mm) equipped with a guard column. The
compounds were eluted using a gradient system. Mobile phases
water and methanol) contained 0.1% formic acid as modifier,
was synthesized from sialic acid following well established
synthetic procedures from the literature (Scheme 1) and then
was used as the starting material for prodrug syntheses. For
acyloxy ester prodrugs the ethyl bridge was chosen over the
methyl bridge as our studies with the model compounds have
shown that the ethyl bridge imparts more chemical stability to the
prodrugs as compared to the methyl bridge (results not shown).
The amino acid starting material was synthesized from the
corresponding aliphatic amino acid and 1-chloro-1-bromoethane
to give amino acid α-chloromethyl ester. The amino acid α-
chloromethyl ester was reacted with Boc-protected zanamivir.
Boc-protected prodrug of zanamivir was purified using flash
column chromatography, and the Boc-group was deprotected
using TFA. The final product was lyophilized to yield the desired
compounds as amorphous powder. The overall yield of the
prodrug was 20ꢀ25%. The purity of the compounds was
determined by HPLC, and all the prodrugs showed >90% purity.
Although TLC showed the presence of a single spot, the HPLC
chromatogram showed two peaks for the prodrugs due to the
presence of two isomers resulting from the formation of a chiral
center at the acyloxy ethyl linker. The racemic mixture of the
prodrug was used for the studies. The starting material and
intermediates were sterically pure as evidenced by observation of
a single peak in HPLC and LCꢀMS systems. Zanamivir also
showed a single peak in HPLC. The structural identities of the
prodrugs were confirmed by positive-mode ESI mass spectro-
(
and the flow rate was 0.2 mL/min.
Data Analysis. Hydrolysis Studies. Apparent first-order ki-
netics and rate constants were determined by using initial rates
of hydrolysis. The apparent first-order degradation rate constants
of zanamivir prodrugs at 37 °C were determined by plotting the
logarithm of prodrug remaining as a function of time. The
relation between the rate constant, k, and slopes of the plots
are explained by the equation
k ¼ 2:303 ꢁ slope ðlog C vstimeÞ
ð1Þ
The degradation half-lives were then calculated by the equation
t1=2 ¼ 0:693=k
ð2Þ
Caco-2 Monolayer Permeability. The apparent permeability
Papp) was calculated using the following equation:
(
Vr dCr
papp
¼
AC0 dt
where V is the receiver volume, A is the surface area of the
r
exposed monolayer, C is the concentration of the prodrug in the
0
donor solution and dC /dt is the rate of change of concentration
r
1
in the receiver solution. The concentration of zanamivir and its
prodrugs is analyzed by LCꢀMS.
metry (ESI-MS) and H NMR. ESI-MS spectra showed a single
molecular ion peak for all the prodrugs, and the H NMR spectra
wereincompleteagreementwiththe structures of thecompounds.
[ H] Gly-Sar Uptake Inhibition Studies. An inhibition con-
stant of the acyloxy ester prodrugs of zanamivir for hPepT1 was
determined by studying their ability to inhibit the uptake of
[ H]Gly-Sar in Caco-2 cell lines. All prodrugs showed competi-
1
The stability of the prodrugs in the apical layer was analyzed by
taking samples in the beginning, during and at the end of the
experiments and the concentration was determined by LCꢀMS.
Rat in Situ Perfusion. The net water flux in the single-pass
intestinal perfusion studies, resulting from both water absorption
and efflux in the intestinal perfused segment, was determined by
measurement of phenol red, a nonabsorbed, nonmetabolized
marker. Phenol red (0.1 mg/mL) was included in the perfusion
buffer and coperfused with the tested compound. The measured
3
3
3
tion with [ H]Gly-Sar, while zanamivir exhibited no competition
in the tested concentration range (0.05 mM to 5 mM). All the
experiments were compared with the positive control, valacyclo-
vir, a known, well absorbed hPepT1 substrate. The valyl acyloxy
2
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3
Table 1. [ H]Gly-Sar Uptake Inhibition in Caco-2 Cells
a
b
Mean and SD from 3 experiments. No inhibition over concentration range of 0.05ꢀ5 mM.
ester prodrug of zanamivir (Zan-L-Val) showed an IC50 compar-
able to that of valacyclovir. The isoleucyl prodrug of zanamivir
which did not demonstrate any inhibition. The remaining
prodrugs noted in Table 1 showed inhibitory constants in the
range of 1ꢀ3 mM. The substitution with promoiety groups at
different positions of zanamivir did not markedly improve the
(Zan-L-Ile) showed a slightly higher IC50 value compared to
valacyclovir but still significantly better than that of zanamivir,
2
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Figure 3. Apparent apical-to-basolateral permeability coefficient (Papp
)
across Caco-2 monolayers. All values are expressed as mean ( SD, n = 3,
at 37 °C. Apical side concentration of prodrugs and zanamivir was
250 μM; only zanamivir was detected on the basolateral side in the case
of prodrugs.
Figure 2. (a) Comparison of zanamivir prodrugs uptake in hPepT1
transfected and nontransfected HeLa cells. The uptake is measured as
the concentration of zanamivir/mg of protein over 45 min period in the
cell lysates. The results are expressed as mean ( SD (n = 3). (b)
Comparison of valacylcovir uptake in hPepT1 transfected and non-
transfected HeLa cells. The uptake is measured as the concentration of
acyclovir/mg of protein over 45 min period in the cell lysates. The results
are expressed as mean ( SD (n = 3).
Table 2. Estimated Hydrolysis Half-lives (t1/2) of Zanamivir
Prodrugs Determined in 100 mM Hydrochlorate Buffer
(pH 1.2), 100 mM Acetate Buffer (pH 4), and 100 mM
Phosphate Buffer (pH 6.5 and 7.4) as Well as in Caco-2
Homogenate Corresponding to 500 μg/mL Protein Con-
a
centration in 100 mM of pH 7.4 Phosphate Buffer
t
1/2 (min)
prodrug pH 1.2 pH 4
pH 6 pH 7.4 Caco-2 homogenate
Zan-L-Val
Zan-L-Ile
>240 >240 150 ( 17.7 35 ( 4.2
>240 >240 165 ( 17.0 48 ( 11.3
15 ( 3.3
25 ( 4.2
35 ( 7.3
Figure 4. (a) Comparison of effective permeabilities of drug and
prodrugs in in situ rat perfusion studies. The results are expressed as
mean ( SD (n = 4). (b) Percentage hydrolysis of prodrugs in perfusate
at pH 6.5 and 5.5 respectively for 2 h at 37 °C.
Zan-D-Val >240 >240 155 ( 15.4 36 ( 8.1
All the values are expressed as mean ( SD, n = 3.
a
3
[
H]Gly-Sar uptake inhibition of the prodrugs. We chose Zan-L-
Val, Zan-D-Val and Zan-L-Ile for further studies. Zan-L-Val had
the lowest IC50 of all the prodrugs tested while Zan-L-Ile
exhibited a roughly 3-fold higher IC50 than Zan-L-Val, so it was
deemed to be interesting how they would fare in permeability
and uptake studies. Zan-D-Val was also chosen for further studies
to see the effect of stereochemistry of the promoiety on uptake
and permeability of the prodrugs. Compounds 4dꢀg mentioned
in Table 1 were not chosen for further evaluations as they were
double prodrugs and the presence of an additional promoiety did
not enhance their affinity for hPepT1 transporter. However,
compounds 4dꢀg are important as they offered important
structure activity relationship insights on zanamivir prodrugs.
The significant affinity for the hPepT1 transporter by the
prodrugs is in marked contrast to the parent drug itself, which
lacked any apparent affinity for the transporter.
Uptake in HeLa/hPepT1 Cells. Increased hPepT1 mediated
uptake in HeLa/hPepT1 cells was observed in the case of Zan-L-
Val (3-fold) compared to that of nontransfected HeLa cells
(Figure 2a). The uptake of Zan-D-Val and Zan-L-Ile in HeLa/
hPepT1 was not significantly enhanced as compared to the
nontransfected cells. However, in most cases the uptake of the
prodrugs in the cells was higher than the uptake of parent drug
under similar experimental conditions. Valacyclovir used as the
positive control showed enhanced uptake in HeLa/hPEPT1 cells
(∼5-fold) compared to that in nontransfected HeLa cells
(Figure 2b).
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2
3
Stability Studies. Buffer Hydrolysis. The estimated half-lives
solubility, dissolution and oral absorption limitations. Trans-
porters in the intestinal epithelial cell membrane function to
absorb polar nutrients. The endogenous transporters efficiently
transport nutrients containing considerable polarity and aqueous
solubility. Oligopeptide transporters, e.g. hPepT1, are present in
the gastrointestinal tract, and their apparently relatively broad
substrate specificity makes them attractive targets for oral drug
(
t1/2) obtained from the linear regression of prodrug concentra-
tion vs time for zanamivir prodrugs in various buffers at pH 1.2, 4,
.5, and 7.4 at 37 °C are shown in Table 2. As expected the pH
had a significant effect on hydrolysis of the prodrugs. The
prodrugs were significantly more stable at acidic pHs. The
isoleucyl prodrug was modestly more stable compared to the
valyl prodrug of zanamivir.
Prodrug Hydrolysis in Caco-2 Cell Homogenate. The
hydrolysis of prodrugs in Caco-2 homogenate was determined
at 37 °C. The half-lives (t1/2) were estimated from linear
regression of prodrug concentrations vs time (Table 2). Zan-L-
Val and Zan-L-Ile prodrugs were hydrolyzed at a faster rate when
incubated with Caco-2 homogenate compared to buffer alone.
There was no significant difference in hydrolysis for Zan-D-Val in
Caco-2 homogenate and buffer. All prodrugs had half-lives of less
than 40 min in homogenates. Isoleucyl prodrug appeared some-
what more stable than the L-valyl prodrug.
6
1
0b,24
delivery.
humans has been shown to be the result of enhanced intestinal
The improved oral bioavailability of valacyclovir in
7
b,8,25
transport by the hPepT1 peptide transport systems.
In this report, the linked amino acid prodrugs of zanamivir
exhibited affinity for the hPepT1 transporter in [ H]Gly-Sar
uptake inhibition experiments, indicating binding but not (yet)
transport, while zanamivir itself did not exhibit any affinity or
inhibition at all the tested concentrations. The inhibitory con-
3
3
stant for [ H]Gly-Sar uptake inhibition for the ethoxy linked L-
valyl ester of zanamivir was comparable to that of valacyclovir,
while the isoleucyl prodrug exhibited a somewhat higher IC50
(∼3 mM). This observation is consistent with previous results
Caco-2 Permeability. The comparison of apparent transcel-
lular permeabilities of zanamivir and the prodrugs in the apical to
basolateral (AP to BL) direction (absorption direction) is shown
in Figure 3. The permeability of L-valyl prodrug of zanamivir was
approximately 9-fold higher than that of zanamivir whereas the
isoleucyl prodrug showed more than a 2-fold increase in the
apparent permeability. The hydrolysis of the prodrugs on the
apical compartment side of the diffusion cell was less than 15%
during the course of the experiment. Irrespective of the promoi-
ety all the prodrugs were completely hydrolyzed following
transport across the cell monolayers with only zanamivir de-
tected on the basolateral membrane side of the diffusion cell.
Rat in Situ Perfusion. The effective permeabilities of zanami-
vir and its prodrugs were determined by the in situ single-pass
intestinal perfusion system in rats. This experimental method
is known to correlate very well with human intestinal
26
on prodrugs targeted to this transporter.
Direct uptake studies in hPepT1 transfected HeLa cells
compared to the nontransfected HeLa cells indicated that the
L-valyl prodrug of zanamivir exhibited an approximately 3-fold
increase in permeability (membrane transport) in transfected
cells compared to the nontransfected cells; while the uptake of
isoleucyl prodrug was not (statistically) significant different
between transfected and nontransfected HeLa cells. However,
in most cases the uptake was higher for prodrugs in both cell
types when compared to the uptake of parent drug.
The chemical stability analysis showed that the prodrugs are
more stable at acidic pHs and, as expected, the stability decreases
with increase in pH. While the prodrugs have a half-life of less
than 1 h at pH 7.4, the half-life at pH = 6 is nearly 3 h, sufficient
half-life to allow good oral absorption in the upper small
intestine. The isoleucyl prodrug was shown to be somewhat
more stable than the L-valyl prodrug in buffer. Activation
(hydrolysis) studies of the prodrugs by Caco-2 cell homogenates
indicates that the prodrugs, with the exception of Zan-D-Val, are
more rapidly activated to parent drug in tissue. The Caco-2
homogenate results show the half-lives for the prodrugs to be less
than 40 min, somewhat faster than the hydrolysis results in buffer
at pH = 7.4. At any given time the hydrolysis of the prodrugs
(Zan-L-Val and Zan-L-Ile) in Caco-2 homogenate was signifi-
cantly higher than the corresponding buffer hydrolysis at pH 7.4,
suggesting that these prodrugs are effectively activated by the
enzymes present in Caco-2 cells. Using the previously identified
recombinant valacyclovirase enzyme, the hydrolysis kinetics of
the prodrugs by valacyclovirase indicates that the prodrugs are
not as good substrates for this enzyme as valacyclovir. However,
the rapid hydrolysis of prodrugs in the Caco-2 cell homogenate
suggests that there are additional cellular enzymes capable of
activating the zanamivir prodrugs in vivo.
1
2
permeability. The effective permeability is measured as the
disappearance of prodrug and parent drugs from the intestinal
perfusate. The experiment was performed at pH 5.5 to minimize
chemical hydrolysis of the prodrug (Figure 4b). Hydrolysis of the
prodrugs by the intestinal epithelium was also taken into con-
sideration when calculating the disappearance of the prodrugs
from the intestinal perfusate in order to avoid overestimation of
permeability of the prodrugs. Zanamivir showed absolutely no
permeability in this in situ system whereas the valyl ester of
zanamivir exhibited a significant permeability comparable to that
of well absorbed metoprolol. The isoleucyl prodrug also exhib-
ited an increase in permeability compared to zanamivir. The
comparison of effective permeabilities of zanamivir and its
prodrugs is shown in Figure 4a.
’
DISCUSSION
Zanamivir has low absolute oral bioavailability, which limits its
clinical use despite being very effective against both influenza A
and B, with low toxicity and low incidences of resistance.
However, the cumbersome mode and route of delivery of
zanamivir by Diskhaler limits compliance especially in patients
with underlying pulmonary conditions and in children.
The comparison of apparent permeabilities of zanamivir and
its prodrugs in Caco-2 monolayers indicates that the L-valyl
prodrug had a significantly higher apparent permeability than the
parent zanamivir. Most significantly, only the parent drug
zanamivir appeared in the receiver compartment, indicating
complete hydrolysis during transport through the mucosal cell.
The L-valyl prodrug was approximately 3-fold more permeable
than the isoleucyl prodrug, which was consistent with the
The prodrug approach has been used to address various drug
delivery problems, including poor bioavailability, low solubility
22
and metabolic instability. The rationale for prodrug design to
increase oral absorption is usually based on passive transport of a
more lipophilic compound. This approach may in turn lead to
3
[ H]Gly-Sar uptake inhibition results, where there was an
approximately 3-fold difference between the affinities of L-valyl
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ARTICLE
and isoleucyl prodrugs. The D-Val prodrug had a lower perme-
ability compared to its L-Val counterpart, indicating a significant
stereochemical dependence of the carrier mediated transport
mechanism.
(5) Zurcher, T.; Yates, P. J.; Daly, J.; Sahasrabudhe, A.; Walters, M.;
Dash, L.; Tisdale, M.; McKimm-Breschkin, J. L. Mutations conferring
zanamivir resistance in human influenza virus N2 neuraminidases
compromise virus fitness and are not stably maintained in vitro.
J. Antimicrob. Chemother. 2006, 58 (4), 723–32.
The in situ rat perfusion system has the best correlation with
human oral jejunal mucosal transport for carrier mediated
(6) Cass, L. M.; Efthymiopoulos, C.; Bye, A. Pharmacokinetics of
zanamivir after intravenous, oral, inhaled or intranasal administration to
1
2
compounds. The transport results in this system indicated that
zanamivir, as expected, has a very low permeability while the
L-valyl prodrug was much more permeable. The isoleucyl and D-
valyl prodrugs also showed an increase in permeability though
lower than that of L-valyl prodrug. Thus the carrier mediated
transport and the cellular activation of the amino acid linked
prodrugs of zanamivir have considerable promise in improving
the oral bioavailability of zanamivir.
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shown that the prodrug strategy of targeting a carrier mediated
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described in this work has been very successful in markedly
improving the transport, uptake and membrane permeability of
zanamivir. The increase in transport and uptake of prodrugs
appears to be mediated by the PepT1 transporter, the design
target. However, the role of other transporters in facilitating
membrane transport cannot be ruled out. The in situ intestinal
perfusion results demonstrating an intestinal membrane perme-
ability of the linked valine ester of zanamivir comparable to that
of metoprolol, combined with the significant mucosal cell
hydrolysis (activation), suggest that both good oral absorption
and cellular (first-pass, intestinal) activation may result in good
systemic availability in humans.
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’
AUTHOR INFORMATION
(11) Svahn, C. M.; Merenyi, F.; Karlson, L.; Widlund, L.; Gr €a lls, M.
Corresponding Author
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1986, 29 (4), 448–53.
*University of Michigan, College of Pharmacy, Department of
Pharmaceutical Sciences, 428 Church Street, Ann Arbor, MI.
Tel: +1 734 764 2440. Fax: +1 7347636423. E-mail: gla.cmdt@
umich.edu.
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ACKNOWLEDGMENT
This work was supported by NIH RO1 GM 037188.
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dx.doi.org/10.1021/mp200291x |Mol. Pharmaceutics 2011, 8, 2358–2367