In Vivo Ocular Pharmacokinetics of Acyclovir Dipeptide
Ester Prodrugs by Microdialysis in Rabbits
Banmeet S. Anand, Suresh Katragadda, Sriram Gunda, and Ashim K. Mitra*
DiVision of Pharmaceutical Sciences, School of Pharmacy, UniVersity of
MissourisKansas City, Kansas City, Missouri 64110
Received November 4, 2004
Abstract: In vivo corneal absorption of the dipeptide prodrugs of acyclovir (ACV) was evaluated
using microdialysis in rabbits. A corneal well was placed on the cornea of the anesthetized
New Zealand White rabbits with implanted linear probes into the aqueous humor. Two hundred
microliters of a 1% solution of L-valine-ACV (VACV), glycine-valine-ACV (GVACV), valine-valine-
ACV (VVACV), and valine-tyrosine-ACV (VYACV) was placed in the corneal well and was allowed
to diffuse for a period of 2 h, following which the drug solution was aspirated and well removed.
Samples were collected every 20 min throughout the infusion and postinfusion phases and were
analyzed by HPLC to obtain the aqueous humor concentrations. Absorption rate constants of
all the compounds were found to be lower than the elimination rate constants. GVACV exhibited
highest absorption rate (ka) compared with other prodrugs, but all the prodrugs showed similar
terminal elimination rate (ìz). The time of maximum absorption (Tmax) of ACV after administration
of VACV and the dipeptide prodrugs did not vary significantly (p < 0.05). GVACV exhibited the
highest concentration (Cmax) and area under curve (AUC) upon absorption (p < 0.05) compared
to VACV, VVACV, and VYACV. Dipeptide prodrugs of ACV were absorbed through the cornea
at similar rates but to varying extents. The dipeptide prodrug GVACV owing to its enhanced
absorption of ACV seems to be a promising candidate for the treatment of ocular HSV infections.
Keywords: Acyclovir; dipeptide prodrugs; ocular absorption; microdialysis
Introduction
Herpes simplex keratitis is the leading cause of blindness
in the United States as well as the most frequent cause of
corneal opacities in developed countries.1 Nucleoside ana-
logues developed initially for the treatment of herpes simplex
virus (HSV) infections (HSV keratitis) include trifluridine
(TFT), idoxuridine (IDU), and cytosine arabinoside (Ara-
A), all of which were found to be too toxic for systemic use
and were, therefore, restricted to topical use for herpetic
keratitis.2 Acyclovir (ACV), also a nucleoside analogue, has
been shown to be clinically effective against herpes viruses,
but due to poor aqueous solubility and low corneal perme-
ability, the drug is not very effective against ocular herpes
infections.3
Ocular availability of drugs is restricted due to pharma-
cological, pharmacokinetic, or pharmaceutical barriers. Chemi-
cal approach to designing bioreversible prodrugs can be
useful in the optimization of drug absorption properties.4
Prodrugs are designed to overcome the undesirable properties
of drugs but are themselves biologically inactive. Further
* Corresponding author. Mailing address: School of Pharmacy,
University of MissourisKansas City, 5005 Rockhill Road,
Kansas City, MO 64110-2499. Phone: 816-235-1615. Fax:
816-235-5190. E-mail: mitraa@umkc.edu.
(1) Turner, J.; Turner, O. C.; Baird, N.; Orme, I. M.; Wilcox, C. L.;
Baldwin, S. L., Influence of increased age on the development of
herpes stromal keratitis. Exp. Gerontol. 2003, 38 (10), 1205-12.
(2) Infectious Diseases: Antiviral Drugs. The Merck Manual of
Diagnosis and Therapy, 17th ed.; Merck Research Laboratories,
Division of Merck & Co., Inc.; John Wiley & Sons: New York,
1999.
(3) Hughes, P. M.; Mitra, A. K. Effect of acylation on the ocular
disposition of acyclovir. II: Corneal permeability and anti-HSV
1 activity of 2′-esters in rabbit epithelial keratitis. J. Ocul.
Pharmacol. 1993, 9 (4), 299-309.
articles
10.1021/mp0498998 CCC: $33.50 © 2006 American Chemical Society VOL. 3, NO. 4, 431-440 MOLECULAR PHARMACEUTICS 431
Published on Web 05/25/2006
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prodrug strategy can be utilized to specifically target
membrane transporters expressed on epithelial cells. In that
direction transporter-targeted prodrug derivatization strategy
seems to be one of the most exciting of all the current drug
delivery strategies.5
Recently peptide transporters attracted a lot of attention
as drug delivery targets. Due to their broad substrate
specificity, peptide transporters contribute to the intestinal
absorption of several drug compounds.6-12 In the past,
peptide transporters have been utilized successfully to
improve the bioavailability of the nucleoside analogues
acyclovir13 and zidovudine (AZT) by designing 5′-amino acid
ester prodrugs.9,14 Previously in our lab PepT1 was identified
on the corneal epithelium for the first time and successfully
targeted using valacyclovir.15
In an earlier report the ACV dipeptide prodrugs were
shown to be substrates for peptide transporter (PEPT1) on
the rabbit cornea.16 Also the prodrugs exhibited excellent
solution stability relative to valacyclovir (VACV), a drug
of choice for oral and genital herpes.16 The dipeptide
prodrugs also showed significantly lower cytotoxicty on the
Statens Seruminstitut rabbit corneal cell line and rabbit
primary corneal epithelial cells in comparison with TFT and
ACV itself and exhibited excellent in vitro antiviral efficacy
against HSV-1 in comparison with ACV. Finally the pro-
drugs were highly soluble and permeable across the cornea
in comparison with ACV.17 The dipeptide ACV prodrugs
can be formulated into 1-3% eye drops and seem, therefore,
to be promising drug candidates for the treatment of HSV
keratitis with stromal involvement. In this study we have
utilized the dipeptide prodrugs to target the PepT1 transporter
on the corneal epithelium for enhanced absorption of ACV.
Topical administration is the preferred mode to treat
diseases that affect the anterior chamber of the eye. Unfor-
tunately, the disposition of drugs administered in this manner
is not well understood. Several pharmacokinetic models of
varying complexity have been proposed to predict absorption
and disposition of drugs applied topically to the eye.18-20
Pharmacokinetics of topically applied pilocarpine in the
albino rabbit eye has been described using a four-compart-
ment classical model represented by a four exponential
equation yielding eight equation parameters.19 Another
pharmacokinetic model has been applied to pilocarpine
pharmacokinetics that uses a physiologically based model.18,20
However, both modeling approaches are complex with regard
to numerical analyses.
Two basic problems in determining anterior chamber
kinetics are as follows. (i) Determination of ka is difficult
due to the presence of precorneal kinetic events. (ii)
Absorption across the cornea is often a slower process than
elimination from the eye, and an erroneous assignment of
slopes is possible. To simplify the approach and correctly
estimate ocular absorption rate constant, a “topical infusion”
(4) Stella, V. J.; Charman, W. N.; Naringrekar, V. H. Prodrugs. Do
they have advantages in clinical practice? Drugs 1985, 29 (5),
455-73.
(5) Dey, S.; Anand, B. S.; Patel, J.; Mitra, A. K. Transporters/receptors
in the anterior chamber: pathways to explore ocular drug delivery
strategies. Expert Opin. Biol. Ther. 2003, 3 (1), 23-44.
(6) Dantzig, A. H.; Bergin, L. Uptake of the cephalosporin, cephal-
exin, by a dipeptide transport carrier in the human intestinal cell
line, Caco-2. Biochim. Biophys. Acta 1990, 1027 (3), 211-7.
(7) Hashimoto, N.; Fujioka, T.; Toyoda, T.; Muranushi, N.; Hirano,
K. Renin inhibitor: transport mechanism in rat small intestinal
brush-border membrane vesicles. Pharm. Res. 1994, 11 (10),
1448-51.
(8) Kiss, A.; Farah, K.; Kim, J.; Garriock, R. J.; Drysdale, T. A.;
Hammond, J. R. Molecular cloning and functional characterization
of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2)
equilibrative nucleoside transporters from mouse brain. Biochem.
J. 2000, 352 (Part 2), 363-72.
(9) Han, H.; de Vrueh, R. L.; Rhie, J. K.; Covitz, K. M.; Smith, P.
L.; Lee, C. P.; Oh, D. M.; Sadee, W.; Amidon, G. L. 5′-Amino
acid esters of antiviral nucleosides, acyclovir, and AZT are
absorbed by the intestinal PEPT1 peptide transporter. Pharm. Res.
1998, 15 (8), 1154-9.
(10) Inui, K.; Yamamoto, M.; Saito, H. Transepithelial transport of
oral cephalosporins by monolayers of intestinal epithelial cell line
Caco-2: specific transport systems in apical and basolateral
membranes. J. Pharmacol. Exp. Ther. 1992, 261 (1), 195-201.
(11) Temple, C. S.; Stewart, A. K.; Meredith, D.; Lister, N. A.; Morgan,
K. M.; Collier, I. D.; Vaughan-Jones, R. D.; Boyd, C. A.; Bailey,
P. D.; Bronk, J. R. Peptide mimics as substrates for the intestinal
peptide transporter. J. Biol. Chem. 1998, 273 (1), 20-2.
(12) Doring, F.; Will, J.; Amasheh, S.; Clauss, W.; Ahlbrecht, H.;
Daniel, H. Minimal molecular determinants of substrates for
recognition by the intestinal peptide transporter. J. Biol. Chem.
1998, 273 (36), 23211-8.
(13) Steingrimsdottir, H.; Gruber, A.; Palm, C.; Grimfors, G.; Kalin,
M.; Eksborg, S. Bioavailability of aciclovir after oral administra-
tion of aciclovir and its prodrug valaciclovir to patients with
leukopenia after chemotherapy. Antimicrob. Agents Chemother.
2000, 44 (1), 207-9.
(14) Han, H. K.; Oh, D. M.; Amidon, G. L. Cellular uptake mechanism
of amino acid ester prodrugs in Caco-2/hPEPT1 cells overex-
pressing a human peptide transporter. Pharm. Res. 1998, 15 (9),
1382-6.
(15) Anand, B. S.; Mitra, A. K. Mechanism of corneal permeation of
L-valyl ester of acyclovir: targeting the oligopeptide transporter
on the rabbit cornea. Pharm. Res. 2002, 19 (8), 1194-202.
(16) Anand, B.; Nashed, Y.; Mitra, A. Novel dipeptide prodrugs of
acyclovir for ocular herpes infections: Bioreversion, antiviral
activity and transport across rabbit cornea. Curr. Eye Res. 2003,
26 (3-4), 151-63.
(17) Anand, B. S.; Hill, J. M.; Dey, S.; Maruyama, K.; Bhattacharjee,
P. S.; Myles, M. E.; Nashed, Y. E.; Mitra, A. K. In vivo antiviral
efficacy of a dipeptide acyclovir prodrug, val-val-acyclovir, against
HSV-1 epithelial and stromal keratitis in the rabbit eye model.
InVest. Ophthalmol. Visual Sci. 2003, 44 (6), 2529-34.
(18) Lee, V. H.; Robinson, J. R. Mechanistic and quantitative evaluation
of precorneal pilocarpine disposition in albino rabbits. J. Pharm.
Sci. 1979, 68 (6), 673-84.
(19) Makoid, M. C.; Robinson, J. R. Pharmacokinetics of topically
applied pilocarpine in the albino rabbit eye. J. Pharm. Sci. 1979,
68 (4), 435-43.
(20) Miller, S. C.; Himmelstein, K. J.; Patton, T. F. A physiologically
based pharmacokinetic model for the intraocular distribution of
pilocarpine in rabbits. J. Pharmacokinet. Biopharm. 1981, 9 (6),
653-77.
articles Anand et al.
432 MOLECULAR PHARMACEUTICS VOL. 3, NO. 4
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Administrator
高亮
model has been described previously.21 In this model, a
constant concentration of the drug is maintained on the
cornea so that the effect of tear dynamics is minimized and
simpler equations can be applied independent of compartment
modeling. During constant input of the drug through the
cornea, absorption, distribution, and elimination can be
determined independent of the number of peripheral com-
partments that are operative. Constant concentration was
maintained through the use of a plastic cylindrical well
containing the drug solution.
A major constraint in the determination of ocular phar-
macokinetics of drugs is the inaccessibility of ocular fluids
such as aqueous humor and vitreous humor for continuous
serial sampling. Furthermore, adding to the problem in
assessing ocular pharmacokinetics is the fact that a single
rabbit must be used for a single time point. Complete
pharmacokinetic profiles are usually constructed by sacrific-
ing 6-20 rabbits at each time point. Microdialysis has been
proven to be beneficial over conventional sampling tech-
niques in determining ocular pharmacokinetics by both
reducing the number of subjects and providing statistically
robust data. It has been applied in aqueous and vitreous drug
disposition and delivery studies.3,22-25 In this study we have
employed the use of microdialysis for sampling the aqueous
humor. We conceptualized the use of a combination of the
topical well infusion model and aqueous humor microdialysis
sampling for precise prediction of ocular absorption.
In this report, we have examined the in vivo corneal
absorption of the dipeptide prodrugs through a topical
infusion model along with aqueous humor microdialysis in
New Zealand White rabbits. The aqueous humor kinetics of
the dipeptide prodrugs GVACV, VVACV, and VYACV was
compared with that of VACV, which is transported across
cornea owing to its recognition by the oligopeptide trans-
porter on the corneal epithelium.15
Materials and Methods
Materials. VACV was a gift from GlaxoSmithKline Inc.,
Research Triangle Park, NC. All other chemicals were
obtained from Sigma Chemical Company (St. Louis, MO).
The solvents were of analytical grade and obtained from
Fisher Scientific (St. Louis, MO). The dipeptide prodrugs,
namely, Val-Val-ACV (VVACV), Gly-Val-ACV (GVACV),
and Val-Tyr-ACV (VYACV) (Figure 1), were synthesized
in our laboratory.16 Linear microdialysis probes (MD-2000,
0.32 � 10 mm, polyacrylonitrile membrane and 0.22 mm
tubing) employed for aqueous humor sampling were pur-
chased from Bioanalytical Systems (West Lafayette, IN). A
microinjection pump (CMA/100) for perfusing the isotonic
buffer saline was procured from CMA/Microdialysis (Acton,
MA). Ketamine HCl was supplied by Fort Dodge animal
health and xylazine by Bayer animal health. Nembutal
sodium was purchased from Abbott Laboratories (Abbott
Park, Chicago, IL). Topical wells (Figure 2A) were custom
made by Hansen Ophthalmic Development Corporation
(Iowa City, IA) according to special instructions.
Animals. New Zealand White male rabbits weighing
between 5 and 5.5 lb were obtained from Myrtle’s Rabbitry
(Thompson Station, TN). Animal care and treatment in this
investigation was in compliance with the Association for
Research in Vision and Ophthalmology (ARVO) Statement
for the Use of Animals in Ophthalmic and Vision Research.
In Vivo Absorption Experiments. (1) Probe Implanta-
tion. Aqueous humor sampling to assess the ocular absorp-
tion of the dipeptide prodrugs was carried out using
microdialysis. The animals were anesthetized prior to the
surgery by administration of ketamine (50 mg/kg) and
xylazine (5 mg/kg) intramuscularly. Pupils were dilated by
topical instillation of 1% tropicamide prior to the probe
implantation. The linear microdialysis probe was placed in
the anterior chamber using a 25G needle. It was inserted
across the cornea preventing any damage to the iris-ciliary
body, and the outlet of the linear probe was placed into the
needle at the bevel edge. Then the needle was slowly
withdrawn such that the probe remained fixed in the anterior
chamber (Figure 2B). The microdialysis probe was perfused
with isotonic phosphate buffer saline at a flow rate of 2 íL/
min by a microinjection pump. The animals were kept under
anesthesia throughout an experiment with ketamine HCl and
xylazine given intramuscularly every 40 min. After probe
implantation, the animals were allowed to stabilize for 2 h
before the initiation of any study. This duration has been
shown to be sufficient for the restoration of intraocular
pressure and replenishment of the aqueous humor originally
lost during probe implantation.26
(2) Microdialysis. Subsequent to probe implantation and
recovery of the animal, the eyelids of the rabbits were
mechanically retracted with Colibri retractors, and the
precorneal well (Hansen Ophthalmic Development Corpora-
tion, Iowa City, IA), designed to fit over the sclera of the
rabbit eye, was mounted. Care was taken to avoid contact
with the entry and exit ports of the aqueous humor microdi-
alysis probe (Figure 2B). The base of the device fitted over
the eye with the central portion forming a well, thereby
(21) Eller, M. G.; Schoenwald, R. D.; Dixson, J. A.; Segarra, T.;
Barfknecht, C. F. Topical carbonic anhydrase inhibitors IV:
Relationship between excised corneal permeability and pharma-
cokinetic factors. J. Pharm. Sci. 1985, 74 (5), 525-9.
(22) Rittenhouse, K. D.; Pollack, G. M. Microdialysis and drug delivery
to the eye. AdV. Drug DeliVery ReV. 2000, 45 (2-3), 229-41.
(23) Waga, J.; Ohta, A.; Ehinger, B. Intraocular microdialysis with
permanently implanted probes in rabbit. Acta Ophthalmol. 1991,
69 (5), 618-24.
(24) Waga, J.; Nilsson-Ehle, I.; Ljungberg, B.; Skarin, A.; Stahle, L.;
Ehinger, B. Microdialysis for pharmacokinetic studies of ceftazi-
dime in rabbit vitreous. J. Ocul. Pharmacol. Ther. 1999, 15 (5),
455-63.
(25) Stempels, N.; Tassignon, M. J.; Sarre, S. A removable ocular
microdialysis system for measuring vitreous biogenic amines.
Graefe’s Arch. Clin. Exp. Ophthalmol. 1993, 231 (11), 651-5.
(26) Macha, S.; Mitra, A. K. Ocular pharmacokinetics in rabbits using
a novel dual probe microdialysis technique. Exp. Eye Res. 2001,
72 (3), 289-99.
Ocular PK of ACV Dipeptide Prodrugs articles
VOL. 3, NO. 4 MOLECULAR PHARMACEUTICS 433
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allowing the drug/prodrug solution to remain in direct contact
with the cornea. The outer edge of the precorneal well was
coated with silicone grease to prevent its movement. The
rabbit was placed on its left side, and the base of the well
was positioned on the sclera of the right eye such that the
empty well was above the cornea. Subsequent to placement
of the well, the animals were allowed to stabilize for another
45 min to maintain proper intraocular pressure. After this
time period, 200 íL of isotonic phosphate-buffered saline
containing drug/prodrug was added to the well at time zero
and samples were collected at predetermined time points by
microdialysis. The compounds were allowed to diffuse for
a period of 120 min, after which the drug solution was
aspirated from the well, which was subsequently removed.
The corneal surface was washed clean with a few drops of
distilled water. Samples were collected every 20 min
throughout the infusion and postinfusion phases over a period
of 8 h. At the end of an experiment, euthanasia was
performed under deep anesthesia with an intravenous injec-
tion of sodium pentobarbital through the marginal ear vein.
Samples obtained in the study were analyzed by HPLC.
In Vitro Probe Calibration. In vitro probe calibration
was performed by placing the probe in isotonic phosphate
buffer saline (IPBS) solution, pH 7.4, of the appropriate
prodrug/drug of known concentration. The probe was per-
fused at a flow rate of 2 íL/min with IPBS, and the dialysate
was collected every 20 min. Relative recovery of the
respective prodrug was calculated by eq 1. Cd is the dialysate
Figure 1. Structure of acyclovir and prodrugs of acyclovir: (a) ACV; (b) VACV; (c) VVACV; (d) GVACV; (e) VYACV.
recovery ) Cd/Cs (1)
articles Anand et al.
434 MOLECULAR PHARMACEUTICS VOL. 3, NO. 4
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