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Antimicrobial Agents and Chemotherapy, July 2000, p. 1964-1969, Vol. 44, No. 7
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Antiviral Activities of Oral
1-O-Hexadecylpropanediol-3-Phosphoacyclovir and Acyclovir in
Woodchucks with Chronic Woodchuck Hepatitis Virus
Infection
Karl Y.
Hostetler,1,*
James R.
Beadle,1
William E.
Hornbuckle,2
Christine A.
Bellezza,2
Ilia A.
Tochkov,2
Paul J.
Cote,3
John L.
Gerin,3
Brent E.
Korba,3 and
Bud C.
Tennant2
Department of Medicine, University of
California, San Diego, La Jolla, California 92093-0676, and the
Veterans Affairs Medical Center, San Diego, California
921611; Department of Clinical Science,
College of Veterinary Medicine, Cornell University, Ithaca, New York
148532; and the Division of Molecular
Virology and Immunology, Georgetown University School of Medicine,
Rockville, Maryland 208523
Received 18 October 1999/Returned for modification 11 January
2000/Accepted 7 April 2000
 |
ABSTRACT |
Acyclovir triphosphate is a potent inhibitor of hepatitis B virus
DNA polymerase, but acyclovir treatment provides no benefit in patients
with hepatitis B virus infection. This is due in part to the fact that
hepatitis B virus, unlike herpes simplex virus, does not code for a
viral thymidine kinase which catalyzes the initial phosphorylation of
acyclovir. We synthesized
1-O-octadecyl-sn-glycero-3-phospho (3-P)-acyclovir and found that it was highly active in reducing hepatitis B virus replication in 2.2.15 cells, while acyclovir was
inactive. The greater antiviral activity of
1-O-octadecyl-sn-glycero-3-P-acyclovir appeared
to be due to liver cell metabolism of the compound to acyclovir
monophosphate (K. Y. Hostetler et al., Biochem. Pharmacol. 53:1815-1822, 1997). However, a closely related compound without a
hydroxyl group at the sn-2 position of glycerol,
1-O-hexadecylpropanediol-3-P-acyclovir, was more active and
selective in 2.2.15 cells in vitro. In this study, we treated
woodchucks chronically infected with woodchuck hepatitis virus with
increasing oral doses of
1-O-hexadecylpropanediol-3-P-acyclovir and assessed the
response to therapy versus acyclovir or a placebo. At a dosage of 10 mg/kg of body weight twice a day, the test compound significantly
inhibited viral replication in vivo, as indicated by a 95% reduction
in serum woodchuck hepatitis virus DNA levels and by a 54% reduction
in levels of woodchuck hepatitis virus replicative intermediates in the
liver. Higher doses were somewhat less effective. In contrast, 20 mg of
acyclovir/kg twice daily, a 5.3-fold-higher molar dosage, had no
demonstrable activity against woodchuck hepatitis virus. Oral
1-O-hexadecylpropanediol-3-P-acyclovir appeared to be safe
and effective in chronic woodchuck hepatitis virus infection.
| |
INTRODUCTION |
Acyclovir [ACV;
9-(2-hydroxyethoxymethyl)guanine] is remarkably effective against
herpes simplex virus (HSV) infection (7, 27). ACV is
phosphorylated by an HSV-coded thymidine kinase (9) and is
subsequently converted to ACV triphosphate by cellular enzymes
(22). ACV triphosphate inhibits the DNA polymerase of HSV
(6) and is incorporated into viral DNA, causing chain
termination because ACV lacks the equivalent of a 3'-hydroxyl group.
ACV triphosphate also inhibits the DNA polymerase of hepatitis B virus
(HBV) and the DNA polymerase of woodchuck hepatitis virus (WHV) by 50%
at 0.9 and 0.7 µM, respectively (13). Nevertheless, ACV
treatment of patients with HBV infection had no additional effect on
serum HBeAg levels in patients also treated with alpha interferon
(3). ACV given intravenously for 28 days had only a weak
effect on viral replication and did not significantly increase the rate
of seroconversion to anti-HBe in chronically infected patients
(1). Zidovudine (AZT) triphosphate inhibits HBV DNA polymerase by 50% at 0.3 µM (4), but AZT treatment had no
effect on serum HBV DNA levels in AIDS patients with concomitant HBV infection (12, 17). Poor phosphorylation of ACV has been
noted previously in HepG2 cells (15) and in 2.2.15 cells
(29). Thus, the lack of clinical efficacy of AZT and ACV in
chronic HBV infection is presumably due to inefficient hepatic
phosphorylation of AZT and ACV.
To develop an orally bioavailable form of ACV and other
nucleosides which can bypass thymidine kinase phosphorylation, we synthesized several 1-O-alkylglycerol- or
1-O-alkylpropanediol phosphate analogs of ACV and AZT and
found them to have substantial antiviral activity in HBV-producing
2.2.15 cells, while AZT and ACV were inactive (15).
Metabolic studies with HepG2 cells using radiolabeled compounds
indicated that the antiviral activity in 2.2.15 cells was most likely
due to a bypass of thymidine kinase. Furthermore, the
1-O-octadecylglycerol-3-phosphate analogs of ACV and AZT
were 100% orally bioavailable in mice (15). The low
toxicity of ACV in herpes simplex infection is due primarily to the
fact that ACV is not phosphorylated to a significant degree in cells
which are not infected with HSV. However, there is 30- to 60-fold
selectivity of ACV triphosphate for inhibition of HSV DNA polymerase
versus human DNA polymerase alpha (16, 25, 26, 30),
suggesting that 1-O-hexadecylpropanediol-3-phospho-ACV (HDP-P-ACV) might be reasonably well tolerated in spite of its ability
to bypass the initial phosphorylation step.
In this paper, we report the results of oral administration of
HDP-P-ACV to woodchucks chronically infected with WHV. The objectives
of the studies were to compare the antiviral activity of orally
administered HDP-P-ACV with that of ACV and to assess antiviral
responses to varying doses of HDP-P-ACV.
| |
MATERIALS AND METHODS |
Chemistry.
HDP-P-ACV was synthesized using the
phosphotriester approach as described previously (2, 15).
The final product was analytically pure by thin-layer chromatography
and high-performance liquid chromatography and gave the expected proton
nuclear magnetic resonance spectrum. Elemental analysis of the final
product (Oneida Research Service, Whitesboro, N.Y.) gave the following
results. Calculated for
C22H49H5O7PNa · 1.15H2O: C, 51.44; H, 8.20; N, 11.1. Found: C, 51.19; H,
8.15; N, 11.07.
Woodchuck hepatitis studies.
The chronic WHV carrier
woodchucks used in these studies were born in laboratory animal
facilities at Cornell University and were inoculated at the age of 3 days with WHV (19, 20, 31). Woodchucks selected for use
developed acute woodchuck hepatitis surface antigen (WHs) antigenemia
and became chronic WHV carriers. The chronic carrier status of all the
woodchucks was confirmed prior to initiation of drug treatment.
(i) Experiment 1.
The 16 experimental woodchucks used in the
first study were assigned on the basis of age, sex, and serum
gamma-glutamyltranspeptidase (GGT) activity to four treatment groups:
(i) HDP-P-ACV at 20 mg/kg of body weight twice a day (b.i.d.), (ii)
HDP-P-ACV at 10 mg/kg b.i.d., (iii) ACV at 20 mg/kg b.i.d., and (iv) a
placebo control. The animals were treated daily for 4 weeks and
observed for an additional 12 weeks.
(ii) Experiment 2.
The eight chronic WHV carrier woodchucks
used in the second study were similar to those used in study 1 and were
assigned on the basis of age, sex, and serum GGT activity to three
treatment groups: (i) HDP-P-ACV at 30 mg/kg once a day (q.d.), (ii)
HDP-P-ACV at 5 mg/kg b.i.d., and (iii) a placebo control. All other
experimental conditions were identical to those in experiment 1.
Each week, sufficient ACV and HDP-P-ACV were weighed to treat the
woodchucks of the various groups for 1 week. The drugs were suspended
in a semisynthetic liquid diet to ensure their complete consumption.
Two separate concentrations of the drug in the diet were made (4 and 8 mg/ml) so that the dosing volume was consistent at 2.5 ml/kg. Volumes
of drug sufficient to treat each individual woodchuck were withdrawn
from the stock suspensions and administered orally twice daily by dose syringe.
Animals were treated for 4 weeks and were monitored while off treatment
for an additional 12 weeks. Blood samples were obtained
under general
anesthesia (ketamine at 50 mg/kg and xylazine at
5 mg/kg) on the 1st
day of treatment and weekly until week 6 (
19,
31).
Thereafter samples were obtained every 2 weeks until week
16. Body
weight was recorded each time the woodchucks were anesthetized
and
bled. Body weights of drug-treated principals were compared
to those of
placebo recipient controls to assess possible drug
toxicity. Drug
dosages for individual woodchucks were adjusted
each time the
woodchucks were
weighed.
Complete blood counts were performed on day 0 and on day 28, the final
day of treatment, to evaluate hematological parameters.
Biochemical
profiles were also performed at day 0 and day 28.
Serum GGT, sorbitol
dehydrogenase (SDH), aspartate aminotransferase
(AST), alanine
aminotransferase and alkaline phosphatase (AP)
activities, the
serum bilirubin concentration, and serum albumin
levels were determined
in order to assess possible hepatocellular
injury and hepatic function.
The serum urea nitrogen (BUN) and
creatinine levels were determined to
assess renal function. Serum
Na, K, Cl, and bicarbonate levels were
determined to assess electrolyte
and acid-base status. Iron status was
evaluated by determining
total serum iron levels, iron binding
capacity, and percent iron
saturation (
19,
31).
Serum WHV DNA levels were measured during treatment and during the
posttreatment follow-up period weekly for 6 weeks, and
then every other
week until week 16. Other serologic markers of
WHV infection, including
WHsAg, anti-woodchuck hepatitis virus
core antigen (anti-WHc), and
anti-WHs, were determined on day
0, after treatment (at 4 weeks), and
after 9 weeks off
treatment.
Liver biopsy specimens were obtained prior to treatment, at the end of
treatment, and at 4 and 12 weeks following drug withdrawal.
Biopsies
were performed under general anesthesia (ketamine at
50 mg/kg and
xylazine at 5 mg/kg) using 16-gauge Bard Biopty-Cut
disposable biopsy
needles directed by ultrasound imaging (
19,
31). These
specimens were stored at
70°C until nucleic acid
analyses were
performed. A second liver sample was fixed in phosphate-buffered
formalin, embedded in paraffin, sectioned and stained with hematoxylin
and eosin for conventional light microscopy, and sectioned and
stained
for WHsAg and WHc detection using immunohistochemical
methods.
The effects of oral ACV and HDP-P-ACV on WHV replication were
determined in two ways. First, the serum WHV DNA levels of treated
groups were determined, and the concentrations before, during,
and
following treatment were compared to those of placebo-treated
control
woodchucks as previously described (
19,
31). Second,
levels
of WHV nucleic acids (WHV DNA, WHV RNA, and WHV DNA monomers)
in
hepatic biopsy tissue were determined by Southern and Northern
blot
analysis before treatment, at the end of treatment, and at
4 and 12 weeks posttreatment (
19,
31), and the respective
values for
drug-treated and control woodchucks were
compared.
| |
RESULTS |
In experiment 1, serum WHV DNA levels did not decline
significantly after 4 weeks in woodchucks treated with ACV at 20 mg/kg twice daily (176 µmol/kg/day) or in placebo-treated animals (Fig. 1). However, HDP-P-ACV at a dose of 10 mg/kg twice daily (33.3 µmol/kg/day) had substantial antiviral
activity, reducing serum WHV DNA levels by 95% (Fig. 1 and Table
1) and hepatic WHV DNA replicative
intermediates (RI) by 50% (Table 2). At
20 mg/kg twice daily, HDP-P-ACV showed somewhat lower antiviral
activity, with percentage decreases of 66% at 4 weeks and 84% at the
nadir, but the difference between the doses was not statistically
significant. Results with both doses of HDP-P-ACV were statistically
significant (P < 0.05) versus results with the placebo
(Table 1). In the group treated with 10 mg of HDP-P-ACV/kg b.i.d., drug
withdrawal after 4 weeks of treatment was associated with a prompt
return of serum WHV DNA levels to baseline, but at 20 mg/kg b.i.d., the serum WHV DNA levels continued to decline for a week after the drug was
withdrawn before returning to baseline (Fig. 1).
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FIG. 1.
Serum WHV DNA levels of woodchucks with chronic WHV
infection treated with HDP-P-ACV, ACV, or placebo for 4 weeks, measured
during treatment and during posttreatment follow-up (experiment 1).
|
|
In experiment 2, we examined HDP-P-ACV doses of 5 mg/kg b.i.d. and 30 mg/kg q.d. versus a placebo control (Fig.
2; Table 1). At week 4, serum WHV DNA
levels were generally slightly higher in the placebo group but had
declined in both the 5-mg/kg b.i.d. and the 30-mg/kg q.d. group, by 72 and 58%, respectively. In experiment 2, serum WHV DNA levels in most
HDP-P-ACV-treated animals reached a nadir 1 or 2 weeks after drug
withdrawal (Fig. 2). The maximum percent declines in serum WHV DNA
levels for animals treated with 5 mg/kg b.i.d. and 30 mg/kg q.d. were
86 and 72%, respectively (Table 1). The percent differences in serum
WHV DNA levels after 4 weeks or at the nadir were highly significant
versus those for the placebo-treated animals (P < 0.001) (Table 1).
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|
FIG. 2.
Serum WHV DNA levels of woodchucks with chronic WHV
infection treated with HDP-P-ACV, ACV, or placebo for 4 weeks, measured
during treatment and during posttreatment follow-up (experiment 2).
|
|
Significant reductions in mean hepatic WHV DNA RI levels were seen
after 4 weeks of treatment at the 10-mg/kg b.i.d. dose level (Table 2).
With one exception, no effects of HDP-P-ACV treatment were observed on
WHV DNA RI at other dosages (Table 2) or on WHV RNA at any dosage (data
not shown). The one exception occurred in hepatic RNA concentrations 12 weeks posttreatment, when the 30-mg/kg q.d. HDP-P-HCV group (56 ± 4 pg of WHV RNA per µg of whole-cell RNA) had a concentration
statistically different (P = 0.02) from that of
controls (70 ± 5). This is possibly a statistical anomaly,
because the woodchuck in the treated group that had consistently had
the highest hepatic RNA levels (woodchuck M3711) died prior to the
final biopsy and therefore did not contribute data at the +12-week time point.
No physical evidence of toxicity was observed in ACV-treated woodchucks
or in HDP-P-ACV-treated woodchucks at any of the dose levels used, and
body weights of drug-treated groups were similar to those of controls.
No drug- or dose-related changes in hepatic enzymes, in bilirubin, in
total serum protein, in serum albumin, or in amylase were apparent in
treated groups compared to controls. Similarly, no treatment-related
changes were observed in comparison with controls in BUN, creatinine,
blood glucose, cholesterol, serum electrolytes, calcium, phosphorus,
iron, iron binding capacity, or percent iron saturation. Hematological
parameters in all treatment groups were similar to those in controls,
including hematocrit, leukocyte count, neutrophil count, and platelet
count (data not shown).
No differences between treatment groups were observed histologically in
biopsy specimens of the liver obtained prior to treatment. Portal and
parenchymal hepatitis in all groups initially was mild to moderate in
nature (median scores, 1 to 2 of 4). At the end of 4 weeks of
treatment, there were no remarkable differences in the histologic
appearance of biopsy specimens from drug-treated and control animals,
and no remarkable differences between groups were observed in the
biopsy specimens obtained at 4 and 12 weeks posttreatment. Similarly,
no differences were observed between treated groups and control
woodchucks in the immunohistochemical expression of either WHcAg or
WHsAg in hepatic biopsy specimens obtained at the end of the 4-week
treatment period or at 4 or 12 weeks posttreatment.
| |
DISCUSSION |
In previous experiments with lipid prodrugs in WHV infection, we
studied intraperitoneal administration of a liposomal phospholipid analog of 2',3'-dideoxyguanosine (ddG), 1,2-dipalmitoylphosphatidyl-ddG (DPP-ddG), to achieve liver targeting. Daily intraperitoneal injections of liposomal DPP-ddG at 2.6 mg/kg/day resulted in a decrease in serum
WHV DNA levels of 96 to 98%, while an equimolar dose of ddG had little
effect (19). While this study clearly demonstrated the
utility of liver targeting using a liposomal antiviral agent, parenteral administration of the formulation was required. DPP-ddG and
phosphatidyl analogs of other nucleosides are poorly absorbed after
oral administration in mice (K. Y. Hostetler, unpublished data,
1999). We next focused on finding a way to develop orally active lipid prodrugs.
To develop an orally bioavailable form of ACV and other poorly
absorbed nucleosides which bypass thymidine kinase phosphorylation, we
synthesized ACV analogs of lysophosphatidylcholine. Early studies on the absorption of dietary fats using radioisotopically labeled lipids showed that a high percentage of lysophosphatidylcholine, a
partially degraded phospholipid, is absorbed intact from the small
intestine (23, 28). We prepared a prodrug of
lysophosphatidylcholine in which the choline moiety was replaced by ACV
(2, 15) or ganciclovir (GCV) (Hostetler et al., submitted),
where the sn-1 fatty acid ester was changed to an alkyl
ether and the hydroxyl at the sn-2 position of the glycerol
backbone was lacking (Fig. 3). The
1-O-alkylglycerol phosphate or alkylpropanediol phosphate analogs of ACV and AZT had substantial antiviral activity in
HBV-producing 2.2.15 cells, while AZT and ACV were inactive
(15). Metabolic studies with HepG2 cells using radiolabeled
compounds suggested that the antiviral activity in 2.2.15 cells was
most likely due to direct formation of ACV monophosphate, bypassing the
necessity for phosphorylation by a nucleoside kinase. Furthermore, the
1-O-octadecyl-glycerol-3-phosphate analogs of ACV and AZT
were 100% orally bioavailable in mice (15), while a GCV
analog was 80% orally bioavailable (Hostetler et al., submitted).
Although the low toxicity of ACV in herpes simplex infection is due in
part to the fact that ACV is not phosphorylated to a significant degree
in cells which are not infected with HSV, there is substantial
selectivity of ACV triphosphate for inhibition of HSV DNA polymerase,
30- to 60-fold, versus human DNA polymerase alpha (16, 25, 26,
30). This suggests that HDP-P-ACV might be well tolerated despite
its ability to bypass the initial phosphorylation by thymidine kinase.
WHV and its natural host, the Eastern woodchuck (Marmota
monax), represent a useful model of HBV disease, including
hepatocellular carcinoma. Previous investigators have used this model
system to evaluate potential antiviral therapies (5, 8, 10, 11, 19, 20, 31). The antiviral activity and toxicity profiles of
several antiviral agents in chronic WHV infection correlated with their
anti-HBV activities in human clinical trials. HDP-P-ACV was well
tolerated in WHV-infected woodchucks at oral doses of 5, 10, and 20 mg/kg b.i.d. and 30 mg/kg q.d. for 4 weeks and produced no physical,
biochemical, or hematological evidence of toxicity at any dose. At an
oral dose of 20 mg/kg b.i.d. (40 mg/kg/day), ACV itself had no
demonstrable antiviral effect against WHV, but HDP-P-ACV at 10 mg/kg
b.i.d. significantly inhibited WHV replication, as shown by a 95%
reduction in serum WHV DNA levels (P < 0.05) and by a
50% reduction in WHV DNA RI levels (P < 0.05). Lower antiviral activity was observed at higher and lower HDP-P-ACV doses.
The reason for the lower antiviral activity of the higher doses, 20 mg/kg b.i.d. and 30 mg/kg q.d., is unclear.
Other workers synthesized and evaluated
bis(S-acyl-2-thioethyl) phosphotriester analogs of ACV to
achieve a bypass of ACV phosphorylation in the liver.
Bis(S-acyl-2-thioethyl) analogs of ACV monophosphate were
very active in 2.2.15 cells in vitro, with 90% effective
concentrations (EC90) of 5.1 to 7.1 µM (24). This degree of activity in 2.2.15 cells compares favorably with that of
HDP-P-ACV, which had an EC90 of 3.9 µM, as we reported previously (15). However, although the
bis(S-acyl-2-thioethyl)phosphate ACV analogs were active
when administered intraperitoneally in ducks with duck HBV infection,
the pronucleotides had no statistically significant effect on serum HBV
levels versus those of controls when administered orally
(14).
In summary, oral HDP-P-ACV was safe and effective in reducing levels of
WHV DNA and WHV DNA RI in serum and in the liver in the woodchuck model
of HBV infection, while a 5.3-fold-higher molar equivalent dose of ACV
had no significant effect. This demonstrates that the antiviral
spectrum of ACV can be extended to HBV using this prodrug strategy.
Optimal activity was seen at HDP-P-ACV doses of 10 mg/kg twice daily,
and higher doses did not provide any further benefit. Additional
studies will be required to establish the relationship of HDP-P-ACV
dose to antiviral effect in the woodchuck model of HBV infection. This
prodrug approach may also be useful for other poorly absorbed
nucleosides such as penciclovir (PCV) and GCV. Studies with HDP-P-GCV
show 80% oral bioavailability (Hostetler et al., submitted), and
preliminary studies with HDP-P-GCV and HDP-P-PCV show good antiviral
activity in 2.2.15 cells which constitutively produce HBV
(18).
| |
ACKNOWLEDGMENTS |
This work was supported in part by NIH grants AI-41928, AI-29614,
and EY11832, by the San Diego Veterans Affairs Medical Center Research
Center for AIDS and HIV Infections (K.Y.H.) and by NIH grants
N01-AI-35164 (B.C.T.) and N01-AI-45179 (J.L.G.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medicine (0676), 9500 Gilman Dr., University of California, San Diego, La Jolla, CA 92093-0676. Phone: (858) 552-8585, ext. 2616. Fax: (858)
534-6133. E-mail: khostetler{at}ucsd.edu.
| |
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Antimicrobial Agents and Chemotherapy, July 2000, p. 1964-1969, Vol. 44, No. 7
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
