Comparison of specific antiviral agents

in herpes simplex keratitis

Herbert E. Kaufman, Emily D. Maloney, and A. B. Nesburn

Several compounds were studied for possible therapeutic activity in experimental herpes simplex
keratitis. Of the 5-halogenated 2'-deoxyuridines the iodo and bromo were active and their
activity was antagonized by thymidine. The chloro was less effective and the fluoro was not
active. Iodo- and fiuorouracil and 5-aminouracil mustard were also inactive. These results are
in agreement with the hypothesis that a requisite for antiviral activity is the ability to block
the final step of nucleotide incorporation into DNA. Blockade of more preliminary steps is not
sufficient. The incorporation of drug into fraudulent DNA may also be important for antiviral
activity. The final assembly of nucleic acids into DNA is the one step that must be virus
specific and it may be here that therapeutically active antivirals must act.

U. ntil recently no specific agent had been

reported for the therapy of any "true" virus
disease. Approaches to the problem of the
therapy of virus disease have included the
use of antibody, interferon, antimetabolites,
and other substances. Until recently, how-
ever, although many of these substances
could prevent infection when given at the
time of inoculation, or as long as 24 hours
after inoculation, none of these substances
could cure or significantly improve disease
once it was clinically evident. The dis-
covery that 5-iodo-2'-deoxyuridine (IDU)
and 5-bromo-2'-deoxyuridine (BDU) were
effective in the treatment of keratitis caused
by vaccinia virus and herpes simplex virus
marked the first specific treatment of any
From the Division of Ophthalmology, University
of Florida, Gainesville, Fla.
Supported in part by United States Public Health
Sendee Grant B 3538 from the National In-
stitute of Neurological Diseases and Blindness
of the National Institutes of Health.
686
"true" virus disease. Unlike previous
therapy which depended upon the re-
moval of all comeal epithelium sometimes
combined with the precipitation of proteins
by various denaturing agents, these drugs
act within the corneal cells to prevent the
synthesis of infectious virus.T> 2? 3
The use of antimetabolites to interfere
with synthesis of virus is not new. As early
as 1948 Thompson4 found that certain
antimetabolites could prevent infection of
tissue culture by vaccinia or inhibit the
production of infectious vaccinia virus by
diseased cultures. In this case, as in others,
the effect on the course of infection could
be observed only when the antimetabolite
was given before signs of disease were ap-
parent. Braley5'll sought to employ this
approach in the treatment of ocular herpes
simplex and reported the trial of folic acid
antagonists. These agents do not alter the
course of experimental herpes simplex kera-
titis. Tamm,7 in 1960, reported that 5-fluoro-
2/-deoxyuridine (FDU) inhibited the pro-
duction of herpes simplex virus by infected

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 Volume 1 Specific antiviral agents in herpes simplex keratitis 687
Number 5
HeLa cell cultures, but reiterated the fact
that this was similar to observations made
on other antimetabolites, and that this
agent had no effect on the course of dis-
ease, even in tissue culture, once disease
was manifest.
Shortly after Tamm's studies, we em-
ployed FDU in the study of experimental
herpes simplex keratitis, and found it to
be without any effect on the course of
herpetic keratitis in the rabbit.
Herrmann8 added 5-iodo-2'-deoxyuridine
(IDU) and 5-bromo-2'-deoxyuridine
(BDU) to tissue overlays at the time of
infection of the culture, and found that
these drugs inhibited the formation of
plaques. This finding, at least in our mind,
did not clearly differentiate these agents
from the many other antimetabolites which
had been shown to prevent infection, but
closer study of these drugs indicated that
they might well be unique.
When a virus infects a cell, it is likely
that it uses the nucleotides produced by
the cell in its synthesis of virus nucleic
acids. That is, it is unlikely that new path-
ways for the synthesis of the nucleotides
which are already being produced by the
cell are contributed by the virus. One step
in this synthesis of virus nucleic acids which
must be virus specific, however, is the
final assembly of the components of the
nucleic acid into the specific virus DNA
molecule. Since this polymerase system
must be unique for the virus, it seemed
likely that the best chance for specific
antiviral activity would lie with compounds
acting at this site. In addition, interference
with the final steps in the synthesis of
DNA results in a metabolic block that is
different from the blockade of more pre-
liminary steps in that it is not easily over-
come noncompetitively by the diffusion of
substances from other cells, or the forma-
tion of alternate metabolic pathways. With
an effect at the final phosphorylation or
polymerization of nucleotides into nucleic
acids, noncompetitive bypass of the anti-
metabolite effect would be nearly impos-
sible. Since IDU and BDU act at these
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sites, they seemed to warrant intensive
study.
The effect of these compounds as thera-
peutic antiviral agents has already been
documented but the validity of the hypoth-
esis which first prompted their trial has
been without confirmatory evidence. If it
were possible to ascertain that antiviral
agents, in order to be effective, must act
at the final stages of nucleic acid synthesis,
many types of antimetabolites could im-
mediately be eliminated from study, and
a new intensive investigation of compounds
acting at this site could be made. SuflScient
methods of study are as yet unavailable to
prove or disprove such a hypothesis con-
clusively, but the purpose of this paper is
to document the trial of antimetabolites re-
lated to IDU and BDU in a search for
evidence which could tend to confirm or
refute this initial hypothesis.
Methods and materials
The strains of virus used are described else-
where and the method of infection and treatment
is as previously described, treatment being ad-
ministered as drops every 2 hours around the
clock, with the exception of the 5-bis (2-chloro-
ethyl)-aminouracil, which was administered as
indicated below.1' 2> 3 Treatment was begun 3
days after infection and continued for 48 hours.
Cultures were taken by complete corneal
curettage in rabbit kidney epithelial tissue cultures
as previously described.1' 2> 3 All experiments were
done "blind," each experiment being completed
by a single observer.
Results
The results are listed in Table I and
summarized in Table II. From this it is
apparent that not only is IDU effective
against several strains of herpes simplex
virus, both as far as clinical disease is con-
cerned and as far as eradicating culturable
virus, but 5-bromo-2'-deoxyuridine is also
effective against herpes simplex virus. This
BDU compound was used in higher con-
centration than the IDU, appeared slightly
less effective, and in some rabbits seemed
to produce conjunctivitis and iritis. Iritis
was more common when the BDU was
used than when the rabbits went without
 688 Kaufman, Maloney, and Nesburn Investigative Ophthalmology
October 1962

Table I. Treatment of herpes keratitis

No ulcers Ulcers Virus remaining No virus
A. Sullivan strain
IDU* 10 6 6 10
BDU 6 5 2 9
FDU 1 4 5 0
CDU 0 10
FU 0 8 8 0
IU 2 6 7 1
Mustard, 0.01 %f 1 9 4 0
Mustard, 0.1% f 1 9 4 1
Control 2 32 12 1
B. McKrae strain
IDU 5 0 4 1
BDU 4 1 3 2
FDU 4 5 0
i—i

CDU 5 6 5 0
Control 0 15
C. Keith strain
IDU 8 2 3 1
BDU 6 6 2 3
FDU 1 4 4 1
IDU + BDU 4 4
IDU + FDU 3 4
Control 0 4 4 0
D. Virtue strain
IDU 20 4 9 10
BDU 18 7 10 7
FDU 2 3 5 0
IDU + Thymidine 3 3 5 1
Control 0 10 5 0
°1DU, 5-iodo-2'-deoxyuridine 0.1%.
BDU, 5-bromo-2'-deoxyuridine 1%.
CDU, 5-chloro-2'-deoxyuridine 1%.
FDU, 5-fluoro-2'-deoxyuridine 1%.
FU, 5-fliiorourncil saturated.
IU, 5-iodourncil saturated.
{One drop administered every 2 hours for 10 hours.
tOne drop administered every 2 hours for 6 hours.

Table II. Summary (all strains combined) virus was serially passaged, the observed
infection became progressively more mild.
No Virus No
ulcers Ulcers remaining virus This was especially true with the Virtue
IDU 42 12 22 22
strain of virus. It is also of great interest
BDU 35 19 27 21 that, as the epithelial disease in the rabbits
FDU 5 15 19 1 became less severe, it appeared to become
Control 2 51 26 1 more difficult to treat rather than less diffi-
cult to treat. That is, the IDU appeared to
be less effective as the virulence of the
treatment, and no iritis was seen when the infection decreased. It is possible that this
IDU alone was used. The chlorine sub- correlation between virulence and the effi-
stituted compound was effective against cacy of antimetabolite drugs is similar to
only one strain and appeared less effective that which has been found with Toxo-
than the IDU. plasma gondii, where metabolic antagonists
It was of great interest that as work appear much more effective against organ-
with these viruses progressed and the isms which are virulent and multiply

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 Volume 1 Specific antiviral agents in herpes simplex keratitis 689
Number 5
rapidly.9 Curiously, in the case of herpes
simplex, although the virus became atten-
uated as to virulence in clinical infection,
it appeared much better adapted to tissue
culture and its cytopathogenic effect ap-
peared more promptly.
Just as very mild infection was difficult
to treat, so infection by very virulent en-
cephalitogenic strains of virus was less suc-
cessfully treated. Encephalitogenic strains
infect not only corneal epithelium, but also
the corneal stroma, and, in these nonim-
mune rabbits, severely infect extraocular
structures such as conjunctiva, lids, and
probably lacrimal gland tissue. The infected
adnexa permit reinfection of treated cor-
neas even if virus has been initially eradi-
cated from the epithelium.3 Not only was
virulence of the virus a variable but these
findings indicated that it was absolutely
essential in any experiment which was
designed to compare various drugs to com-
pare all the desired drugs at one time and
to have controls at the same time. Con-
siderable variation was noticed from one
batch of rabbits to another in regard to
their susceptibility to infection and the in-
tensity of infection.
Because of the possibility that IDU and
BDU might be active by their incorpora-
tion into fradulent DNA, thereby resulting
in noninfectious virus, 5-bis(2-chloroethyl)-
aminouracil was tested. This is an agent
which attaches to nucleoprotein and is
incorporated into DNA and RNA. The at-
tachment to nucleic acid is somewhat er-
ratic, and biologic effects have not always
been well correlated with the uptake, but
it seemed possible that such an agent might
have antiviral properties.10 When this agent
was administered as indicated above in
concentrations which are similar to those of
mustards previously studied, it was found
to be totally ineffective in improving the
disease or eradicating virus. This concen-
tration of mustard was sufficient to produce
appreciable keratoconjunctivitis, and it was
felt that additional amounts would lead
to frank corneal necrosis. Despite the lack
of effect of the aminouracil mustard, how-
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Table III. Effect of N-methylisatin thio-
semicarbazone on vaccinia virus
No ulcers Ulcers
Drug 13
Control 12
ever, the mechanism of attachment of this
agent is sufficiently unclear that a negative
result is not proof that incorporation into
DNA is not the essential mechanism by
which IDU and BDU are effective.
Since FDU inhibits the synthesis of
thymidine, it might be anticipated that,
although in itself it is not effective as a
virucide, its addition to IDU might poten-
tiate the effect of the latter drug by de-
creasing the source of available thymidine.
Our results do not confirm the potentia-
tion of IDU by FDU. Further studies of
this relationship may be worthwhile, how-
ever. Similarly, our studies do not indicate
that the combination of IDU and BDU is
more effective than the IDU alone, altliough
the combination appeared to cause more
local irritation and iritis.
It was to be expected that thymidine
would reverse the therapeutic action of
IDU, and results summarized in Table I
indicate that, in fact, with the addition of
thymidine the proportion of eyes with re-
sidual ulcers was higher (P < .01) and
virus was not eradicated as efficiently as
when IDU alone was used (P < .01).
Against vaccinia IDU is effective, but
N-methylisatin thiosemicarbazone (an
agent preventing vaccinia infection in vitro)
administered as above in saturated saline
solution had no effect on vaccinia keratitis
(Table III).
Discussion
For at least thirteen years the effect of
antimetabolites on virus multiplication has
been studied,4 and agents have been found
that prevent infection or alter virus titers
in vitro after infection. Until the descrip-
tion of the drugs reported above, however,
no agent had been found which would
 690 Kaufman, Maloiiey, and Nesburn
cure any animal, or even prevent the pro-
gression of cytopathogenic changes in tissue
culture once signs or symptoms of virus
disease were present.
Our present knowledge permits only a
superficial understanding of the mechanism
of action of effective antiviral agents, but
even this information is of interest. IDU
and BDU act in a manner very different
from that of the other compounds tested.11
Of the analogs, the iodine and bromine
atoms most resemble the methyl group
of thymidine in effective radius (Fig. 1).
As such, they compete with thymidine for
enzymes required in phosphorylation and
polymerization to DNA and inhibit the
utilization of thymidine even if residual or
exogenous thymidine is present. In being
similar to thymidine they can be incor-
porated into a fraudulent and presumably
R - I.35A
CH
o-c.
DR-5-P
FLUORODEOXYURONE 5*-PHOSPHATE
THYMIDTUC SYNTHETASE
R- 2.I5A
N N
O«C
IODODEOXYURIDINE 5'" PHOSPHATE
THYMIDYLIC PHOSPHORYLASE
Fig. 1.
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Investigative Ophthalmology
October 1962
noninfectious DNA. As expected, their
action is antagonized by thymidine (Table
I). The chloro substituted uridine is further
removed in size from the methyl group and
perhaps this is why it is therapeutically
less effective.
The efficacy of 5-iodo-2'-deoxycytidine
has recently been reported by Perkins and
associates.12 It is deaminated to the 5-iodo-
2'-deoxyuridine and is active as the IDU.
The 5-iodo-2'-deoxycytidine may be more
soluble and more stable in solution than
IDU but is difficult to synthesize and its
relative potency must be studied.12
Replacing the methyl group of thy-
midine by a fluorine atom results in a totally
and predictably different site of action
(Fig. 1). The radius of fluorine is similar
to that of hydrogen so that the FDU mole-
cule resembles deoxyuridine rather than
R-L2A
R = VAN DER WAAL RADIUS
oc CHgOHFH = HYDROXYMETHYL-
FOUNIC ACID
DEOXYURIDINE 5'-PHOSPHATE
CH2OHFH
R-I.8IA
R-1.9 5 A
N
O»C CH O«C CH
N N Nx
DR-5-P DR-5-P
L
THYMIDINE 5 PHOSPHATE BROMODEOXYURIDINE 5'" PHOSPHATE
-t-
DNA POLYMERASE
DNA
 Volume 1 Specific antiviral agents in herpes simplex keratitis 691
Number 5
the methylated base thymidine. It is, as the
phosphate, similar to uridine phosphate
and in competing with the uridylic acid
for thyrridylic synthetase inhibits its methy-
lation to thymidylic acid. Thus, the fluoro
substituted compound acts to prevent the
synthesis of thymidine but if thymidine is
available from other sources or if any re-
sidual thymidine is present, the FDU is
not effective.
Compounds such as fluorouracil (FU),
although similarly reducing the titers of
virus produced by some infected tissue
cultures, act even earlier in their inter-
ference with the synthetic chain. It seems
reasonable, therefore, that such interfer-
ence would be more easily overcome by
supplies of any of the subsequent products
of synthesis diffusing from the blood stream
or produced by alternate synthetic path-
ways.
In the use of compounds which can in-
terfere with the synthesis of normal cells
it is vital to know whether toxicity to' the
virus is selective or whether host toxicity
is implied. Recently Delamore and Prusoff13
have shown that IDU has different effects
in different types of cells in vitro. In some
cells it primarily affects the transport of
thymidine into cells and its early phos-
phorylation. In others it affects the forma-
tion of di- and triphosphates and the ac-
tion of DNA polymerase which incorporates
the thymidylic acid into DNA. The im-
portance of this finding is that it suggests
that in different DNA synthesizing systems
the site of action and toxicity of IDU may
be different and that toxicity may well be
selective, inhibiting virus production far
more than normal cellular DNA produc-
tion. In fact, preliminary results in our
laboratory indicate that IDU in virucidal
concentrations does not inhibit multiplica-
tion or thymidine uptake of normal cells.
The most likely site of activity for a
specific antiviral agent seems, therefore,
to be at the final enzymatic step, the poly-
merization of the nucleotides. Agents that
act earlier in the synthetic chain such as
FDU and FU can reduce virus production
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in a nonspecific way by reducing the avail-
able nucleotide pool. Since viral DNA
polymerases must have a very high affinity
for the nucleotides, these less specific
agents, apparently, cannot halt virus pro-
duction by diseased tissues without killing
the normal cells.
Many questions remain to be solved: (1)
It is unknown whether it is the inhibition
of thymidine uptake or the incoi'poration
of IDU into abnormal DNA that is most
important for therapeutic antiviral efficacy.
Unfortunately the lack of effect of 5-
aminouracil mustard sheds little light on
this question. (2) Similarly, in the search
for more potent agents and for broad-
spectrum antiviral agents that cure infec-
tion a reliable in vitro system for testing
drugs is required. (3) Little is known
about the penetration of these compounds
or their breakdown.
No matter how humble the start, several
compounds active against at least two
viruses have already been described.1'2> 12
The defenses of the virus have been
broached and rapid progress toward the
treatment of other virus disease may soon
result.
REFERENCES
1. Kaufman, H. E.: Clinical cure of herpes
simplex keratitis by 5-iodo-2'-deoxyuridine
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251, 1962.
2. Kaufman, H. E.: Treatment of herpes simplex
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bromo-2'-deoxyuridine, Perspectives in Vi-
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3. Kaufman, H. E., Nesburn, A. B., and
Maloney, E. D.: Treatment of herpes simplex
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4. Thompson, R. L., Wilkin, M. L., Hitchings,
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49: 283, 1952.
7. Tamm, I.: Symposium on the experimental
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October 1962
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