ANTIMICROBAL AGENTS AND CHEMOTHERAPY, Sept. 1973, p. 294-298 Vol. 4, No.

3
Copyright 0 1973 American Society for Microbiology Printed in U.S.A.

Antiviral Effects of Aphidicolin, a New Antibiotic

Produced by Cephalosporium aphidicola
R. A. BUCKNALL, H. MOORES, R. SIMMS, AND B. HESP
Imperial Chemical Industries Ltd., Pharmaceuticals Division, Alderley Park, Macclesfield, Chesire, England
Received for publication 14 June 1973

Aphidicolin is an antibiotic of novel structure produced by the mold Cephalos-

porium aphidicola. It is a potent inhibitor of cellular deoxyribonucleic acid
synthesis, and it also strongly inhibits the growth of herpes simplex virus both in
tissue culture and in the rabbit eye. Aphidicolin is active against iododeoxyuri-
dine-resistant herpes virus, and does not itself readily induce the formation of
drug-resistant strains of herpesvirus.

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In 1972, Brundret et al. (1) described the
structure of an antibiotic produced by Ce-
phalosporium aphidicola Petch. The antibiotic
was named aphidicolin and has the structure
shown in Fig. 1. During the biological evalua-
tion of aphidicolin, it was shown to have a
marked inhibitory effect on the growth of deoxy-
ribonucleic acid (DNA) viruses. This paper
describes these effects in vitro and in vivo.
MATERIALS AND METHODS
Aphidicolin. C. aphidicola Petch was grown on a
medium containing Czapek-Dox salts plus 5% cere-
lose and 0.1% yeast extract. Aphidicolin was extracted
and purified by methods of Hesp et al. (manuscript in
preparation). The solubility of aphidicolin in water is
extremely low and in the order of a few micrograms
per milliliter.
Tissue cultures. Human embryonic lung cells and
primary calf kidney cells were grown in stoppered
tubes (3 by 0.5 in, approximately 7.5 by 1.25 cm)
medium containing 8% inactivated calf serum. When
confluent, monolayers were changed to Eagle medium
containing 2% calf serum or 0.2% bovine plasma
albumin.
Viruses. The following strains of viruses were used:
Herpes simplex types 1 and 2 (clinical isolates ob-
tained from the Public Health Laboratory, Manches-
ter, England), influenza A, (DSP), influenza A,
(CAM), influenza A2 (Hong Kong), rhinovirus type 2,
coronavirus type 229E, parainfluenza type 1, and
vaccinia. Virus infectivity was titrated in tissue cul-
ture tubes of appropriate cells.
The methods for measuring the effects of
aphidicolin and other drugs on the DNA and ribonu-
cleic acid RNA synthesis of tissue culture cells by
uptake of 3H-thymidine and 'H-uridine have already
been described (2).
Herpetic keratitis in rabbit eyes. Separately
caged Dutch female rabbits (1-2 kg) were used
throughout. One eye in each of six rabbits formed an
experimental group.
294
The cornea was anesthetized with one drop of 4%
Xylocaine and the epithelium was lightly scratched in
a cross-hatch pattern by using a 27-gauge needle, care
being taken not to disturb the stromal layer. A 0.1-ml
virus suspension was then instilled into the lower
cul-de-sac and the lids were gently rubbed together
for several seconds. The corneal epithelium was
examined daily after staining with fluorescein and
washing with phosphate-buffered saline. Corneal le-
sions were graded 0 to 4, grade 0 representing no
abnormality and grade 4 representing complete cor-
neal involvement. The allocation of scores was done
by one person throughout the whole of this study and
was organized so that he had no knowledge of the
therapy each animal was receiving.
In order to make a statistical comparison between
the response of groups of animals on different treat-
ments, the following procedure was used. The lesion
scores for each group of animals were summed from
the first day of treatment up to day 9 postinfection.
Scores beyond 9 days were not included because after
this time the lesions in the control groups usually
began to heal (at least with herpes simplex type 1),
and the difference between the control and the treated
group became less.
The mean sum of the lesion scores was calculated
for each group, and the difference between the control
group mean and each drug-treatment mean was
tested for statistical significance by means of Stu-
dent's t test. The significance of each comparison at
the 1% level of confidence is given in the figure
legends.
RESULTS
Spectrum of antiviral activity. Aphidicolin
was not visibly toxic to confluent human em-
bryonic lung cells at concentrations up to 200
ug/ml for periods of up to 7 days. In human lung
cultures infected with approximately 100 mean
tissue culture doses (TCD50) of various viruses,
aphidicolin caused a 50% inhibition of growth of
herpes types 1 and 2 at 0.2 Ag/ml and a 50%
VOL 4, 1973 ANTIVIRAL EFFECTS OF APHIDICOLIN 295
-

inhibition of vaccinia growth at 4 ug/na]. It was
not active against adenovirus type ;i. In the
same test system, it showed no activitiy against
rhinovirus type 2 or coronavirus type 229E. In
confluent primary calf kidney cultures,
aphidicolin was not active against infltienza AO,
A,, or A2, or parainfluenza type 1.
Mode of action. The specific inhi ,bition of
DNA viruses by aphidicolin suggested that the
antibiotic is an inhibitor of DNA syntiiesis. We
therefore compared the effects of aphidlicolin on
the nucleic acid synthesis of culture'd human
H17
pH
lung cells with two well-known inhibitors of
DNA synthesis: 5-iodo-2-deoxyuridine (IUdR)
and cytosine arabinoside (CA). The results (Fig.
2) show that aphidicolin, like IUdR and CA, is a
powerful inhibitor of cellular DNA synthesis.
None of the compounds had any effect on
cellular RNA synthesis at 12.5 jg/ml, the high-
est concentration tested. Parallel cultures were
infected with approximately 100 TCD,. of
herpes simplex type 1, treated with the same
range of concentrations of IUdR, CA, or
aphidicolin, and harvested 24 h later to measure
virus yields.
It is clear that aphidicolin depresses virus
yield and cellular DNA synthesis in parallel,
and it is therefore probable that it exerts its
antiviral effects by inhibiting herpes virus DNA
synthesis. IUdR, on the other hand, is antiviral
at concentrations much lower than those which
inhibit cellular DNA synthesis, suggesting a
considerable degree of specificity of IUdR for

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viral, rathe'r than cellular, DNA synthesis. The
figures for CA show that cellular DNA synthesis
is affected rather more than is virus DNA
synthesis.
The activity of aphidicolin against ocular
herpes. Aphidicolin was active against experi-
mental herpes type 1 infections of the rabbit
eye. When applied as aqueous drops hourly for 7
FIG. 1. Structure of aphidicolin. h each day for 5 days, aphidicolin at 1 mg/ml
Aphidicolin lododeoxyuridine Cytosine Arabinoside

100-i 100- R.N.Ao -a, 'X.

- .. . 14r---CtD-.-Zs
/ ?
III i
fII
W;

c
75-
RAA

I.
75- .iI
II
._
'U
%A

._

I
U
50-
II I.
50-
4rIi Ii
at
e Virus&4-d~~~~~~~
Il
. . . . .
25- 25- D.N.A4 |

D.N.A_-
Virus6-
I I I
125 31 0:8 0'2 0.b50.6125 12.5 3:1 0:8 0:2 650.01125 1d.5 3:1 0:8 0;2 0.05 0.0125
big/ml iggml ug/ml
FIG. 2. Effect of aphidicolin, IUdR, and CA on RNA (0) and DNA (0) synthesis of uninfected human
embryonic lung cells, and on the growth of herpes simplex type 1 (A) in infected cells. All results are expressed
as a percentage of appropriate controls for ease of comparison.
296 BUCKNALL ET AL.
was somewhat less effective against an ocular
challenge of 400 eye mean infective dose (ID50)
than was IUdR at 1 mg/ml (Fig. 3). By increas-
ing the concentration of aphidicolin to 10 mg/
ml, or by reducing the challenge virus to 40 eye
ID,,, the activity of aphidicolin could be made
comparable to that of IUdR at 1 mg/ml.
In human cases, the corneal herpetic lesion is
often well advanced before therapy is begun; to
simulate these conditions, groups of rabbits
were infected with 400 eye ID,0 of type 1 herpes
and left untreated for 48 or 72 h before begin-
ning 5 days of eye drops of either 1 mg of IUdR
per ml or 10 mg of aphidicolin per ml. When
treatment was begun 48 h after infection, both
drugs reduced the maximal lesion score to
approximately half that achieved in the controls
(Fig. 4a). Treatment begun 72 h after infection
was less effective, and neither drug produced a
statistically significant reduction (at the 1%
level of confidence) in the development of
lesions.
In an experiment where rabbits were infected
in the eye with herpes simplex type 2 and left for
Challenge Virus - 400 Eye I.D.50
4 was well controlled by 5 days of treatment with
3-
Control
c2
o 2- d b'
\ri
1- ld R X,C b.Aphidicolin
10\ mg/Iml
S>/ b IL.U.D.R.1mg/ml
1 3 5 7 9 11 Days
Treatment
FIG. 3. Effect of aphidicolin (0) and IUdR (0) on
the growth of herpes simplex virus on the rabbit
comea. Treatment was begun immediately after in-
fection and was continued for 5 days. Each score is the
mean of six eyes in a single experiment. Statistics:
Control versus IUdR, days 0 to 9, calculated t = 15.6;
difference is significant (1%) Control versus
aphidicolin, days 0 to 9, calculated t = 8.40; differ-
ence is significant (1%). IUdR versus aphidicolin,
days 0 to 9, calculated t = 7.20; difference not
significant (1%).
ANTIMICROB. AG. CHEMOTHER.
' bControl
' b
I* Aphidicolin 10mg/mi
I.I..D.R. I mg/Iml
-Treatment-
Days
-Treatment-
FIG. 4. Effect of aphidicolin (0) and IUdR (0) on
the growth of herpes simplex virus on the rabbit
cornea. Treatment commenced 48 or 72 h after
infection and continued for 6 days. Each score is the
mean of four or six eyes in a single experiment.
Statistics: (a) Control versus IUdR, days 2 to 9,
calculated t = 8.50; difference is significant (1%).
Control versus aphidicolin, days 2 to 9, calculated t =
6.60; difference is significant (1%). IUdR versus
aphidicolin, days 2 to 9, calculated t = 1.917; differ-
ence not significant (1%). (b) Control versus IUdR,
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days 3 to 9, calculated t = 1.66; difference not
significant (1%). Control versus aphidicolin, days 3 to
9, calculated t = 1.33; difference not significant (1%).
IUdR versus aphidicolin, days 3 to 9, calculated t =
0.481; difference not significant (1%).
72 h before treatment was begun, the disease
either drug (Fig. 5).
Activity of aphidicolin against IUdR-re-
sistant herpes simplex type 1. The growth of
our experimental strain of herpes simplex type 1
virus in vitro was reduced by 50% in the
presence of 0.2 to 0.4 Mg of IUdR per ml.
However, when virus which had been grown in
the presence of 0.4 jig of the drug per ml was
harvested and tested again for susceptibility to
IUdR, it then required 12 ,g/ml to produce 50%
inhibition. The virus was thus 30 times less
sensitive to inhibition after one passage in the
presence of the drug, but further passages in the
drug did not increase the resistance. The devel-
opment of IUdR-resistant strains of herpes virus
is well known (5, 6) and may be one reason why
IUdR therapy of human ocular herpesvirus is
occasionally found to be ineffective (7). Unlike
IUdR, aphidicolin does not readily give rise to
drug-resistant strains of virus. After six pas-
sages in the presence of 0.8 Mg of aphidicolin
per ml, there was no decrease in the drug-sus-
ceptibility of the virus. Also, IUdR-resistant
herpes virus is fully susceptible to aphidicolin.
As may be expected from these results, when
an IUdR-resistant strain of herpesvirus type 1
was inoculated into the rabbit eye, the severity
of the keratitis could be reduced with
aphidicolin, but not with IUdR (Fig. 5).
The effects of combined IUdR and
VOL. 4, 1973 ANTIVIRAL EFFECTS OF APHIDICOLIN 297
Aphidicolin and herpetic encephalitis. The
relatively low animal toxicity of aphidicolin
makes it possible to test the compound systemi-
cally in guinea pigs inoculated intracerebrally
with herpes simplex virus. Pairs of guinea pigs
.0
were dosed subcutaneously or intraperitoneally
J1 with a suspension of aphidicolin at 200 mg/kg
from the day of virus inoculation (100 guinea pig
mean lethal dose intracerebrally) for 12 days.
The drug dose was reduced to 100 mg/kg on day
\.O11 L.U.R.lImg/ ml 6 because the animals looked unwell and were
Aph idicol in 10 mg / mI not gaining weight at the same rate as were the
infected, but undosed, controls. However, there
3 5 7 9 11 13 15 Days
1
was no evidence of any protective effect of the
-Treatment- compound in either the time of onset of symp-
FIG. 5. Effect of aphidicolin (0) and IUdR (0) on toms or the mortality rate.
the growth of herpes simplex type 2 on the rabbit Derivatives of aphidicolin. Because of the
cornea. The infecting inoculum was approximately low solubility of aphidicolin in water, the drug
100 TCD,O. Each score is the mean of four eyes in a used at 1 and 10 mg/ml in the rabbit eye
single experiment. Statistics: Control versus IUdR, experiments was in the form of a fine suspen-
days 3 to 9, calculated t = 3.50; difference is signifi- sion. Two water-soluble derivatives were made,

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cant (1%). Control versus aphidicolin, days 3 to 9, the C17 C18 diphosphate and the C,7 monohe-
calculated t 4.00; difference is significant (1%).
=
misuccinate sodium salt, but only the latter
IUdR versus aphidicolin, days 3 to 9, calculated t = showed any activity in vitro, and this was only
0.479; difference not significant (1%). about one-quarter of the activity of aphidicolin
4-
itself (Table 1). The monohemisuccinate ester
sodium salt is highly soluble in water, but when
it was tested in the rabbit eye, a concentration
of 30 mg/ml was needed to achieve a therapeutic
3- ~~~~Control effect comparable to a suspension of 1 mg/ml of
aphidicolin. It seems, therefore, that conferring
water-soluble properties on the molecule does
not increase its efficacy.
A large number of derivatives of aphidicolin
have been synthesized (Hesp et al., manuscript
in preparation) and tested in vitro, but those
which showed activity against herpes-virus were
A hAphidicolin 10mg/ml much less active than was aphidicolin itself.
I.U.D.R. 1mg / ml Only the C17 monoacetate was comparable in
1 3, 5 7 9 11 13 15 Days activity, and this was possibly because it may
-Treatment- be hydrolyzed to aphidicolin in the test system.
FIG. 6. Effect of aphidicolin (0) and IUdR (0) on TABLE 1. Concentration of aphidicolin and
the growth of an IUdR-resistant herpes simplex virus derivatives causing 50% cytotoxicity in uninfected
on the rabbit cornea. Treatment was begun immedi- human embryonic lung cells and 50% inhibition of
ately after virus infection. Each score is the mean of herpes simplex type 1 growth
four eyes in a single experiment. Statistics: Control
versus IUdR, days 0 to 9, calculated t = 1.34; differ- Amt (iug/ml) causing
ence not significant (1%). Control versus aphidicolin, Drug 50% Cyto- 50% Virus
days 0 to 9; calculated t 4.90; difference is sig-
=

nificant (1%). IUdR versus aphidicolin, days 0 to 9, toxic concn inhibition

calculated t 3.56; difference is significant (1%).
=

Aphidicolin > 200 0.25

Aphidicolin C17, C18 bis > 45 Not active
aphidicolin on the growth of herpesvirus in vitro primary phosphate
seem to be simply additive, and the effects of (tetraammonium salt)
the two drugs together on the progress of her- Aphidicolin C17 mono- >45 1
hemisuccinate
petic keratitis in the rabbit cornea is only (sodium salt)
slightly greater than those either drug alone.
298 BUCKNALL ET AL.
DISCUSSION
Although IUdR is probably the most widely
used drug in the treatment of human herpetic
infections, it is far from an ideal medicine. It is
an inhibitor not only of virus growth but also of
celIular DNA synthesis (albeit at a higher
concentration), and it is also incorporated into
mammalian DNA (4). Herpes simplex rapidly
becomes resistant to it in vitro and may also do
so in vivo (7), and it is not without toxicity when
administered systemically to humans (3). There
is therefore a need for better anti-herpes drugs,
and as part of our research program for develop-
ing such agents, we have evaluated aphidicolin,
a novel antibiotic produced by C. aphidicola.
The antibiotic is highly active against herpes
virus growth in vitro, and this activity could not
be improved by considerable chemical manipu-
lation of the parent molecule. Indeed, most
chemical modifications gave a large reduction
in the antiviral activity.
In therapeutic experiments in the rabbit eye,
the curative effects of 1 mg of aphidicolin and
IUdR per ml were equivalent against 40 eye ID,0
of herpesvirus. Although virtually equimolar
amounts of aphidicolin and IUdR were used in
these experiments (the molecular weights being
354 and 338, respectively), because aphidicolin
is so poorly soluble in water, the effective
concentration in true solution would be very
low. Thus, on a molar basis, aphidicolin is
probably considerably more potent than IUdR,
and its effectiveness is limited only by its
extremely low solubility in water. Unfortu-
nately, chemical modification of aphidicolin to
increase its water solubility led to a con-
siderable loss of intrinsic anti-herpes activity.
The basis of the anti-herpes activity of
aphidicolin is almost certainly an inhibition of
virus DNA synthesis, although this has not
ANTIMICROB. AG. CHEMOTHER.
been demonstrated directly. The concentration
of antibiotic required to reduce herpesvirus
growth by 5o0% in vitro is very close to that re-
quired to inhibit cellular DNA synthesis by
50%. Thus, the action of the antibiotic is not
specific for virus DNA synthesis.
The high intrinsic potency of aphidicolin, its
ability to inhibit the growth of IUdR-resistant
herpesvirus, and the apparent lack of the devel-
opment of drug resistance are all valuable
properties of a potential anti-herpes drug. How-
ever, because its action is not specific for
virus-directed DNA synthesis, aphidicolin may
not offer any advantages for clinical use over
other DNA antagonists.
ACKNOWLEDGMENTS
We thank J. Harrad and A. Toal for their valuable
technical contribution to this work.
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LITERATURE CITED
1. Brundret, K. M., W. Dalziel, B. Hesp, J. A. J. Jarvis, and
S. Neidle. 1972. X-ray crystallographic determination of
the structure of the antibiotic Aphidicolin: a tetracycline
diterpenoid containing a new ring system. J. Chem. Soc.
D. Chemical Commun. 1027-1028.
2. Bucknall, R. A. 1967. The effects of substituted ben-
zimidazoles on the growth of viruses and the nucleic acid
metabolism of host cells. J. Gen. Virol. 1:89-99.
3. Calabresi, P. 1965. Clinical studies with systemic adminis-
tration of antimetabolites of pyrimidine nucleosides in
viral infections. Ann. N. Y. Acad. Sci. 131:192-208.
4. Prusoff, W. H. 1960. Incorporation of IUdR into DNA of
mouse Erlich ascites cells. Biochim. Biophys. Acta
39:327.
5. Smith, K. 0. 1963. Some biological aspects of herpes virus
cell interactions in the presence of 5-iodo-2'-deoxyuri-
dine. J. Immunol. 91:582-590.
6. Underwood, G. E. 1962. Activity of 1-P-D-
arabinofuranosylcytosine hydrochloride against herpes
simplex keratitis. Proc. Soc. Exp. Biol. Med.
111:660-664.
7. Underwood, G. E., G. A. Elliot, and D. A. Buthala. 1965.
Herpes keratitis in rabbits: pathogenesis and effect of
antiviral nucleosides. Ann. N.Y. Acad. Sci. 131:151-167.