Antiviral Research 121 (2015) 138144

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Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral

Effect of consecutive alternating administration (CAA) of a triple

anti-enteroviral combination on Coxsackievirus B1 neuroinfection
in mice
Adelina Stoyanova, Ivanka Nikolova, Angel S. Galabov
Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Acad. G. Bonchev Str., 1113 Soa, Bulgaria

a r t i c l e

i n f o

Article history:
Received 26 March 2015
Revised 15 July 2015
Accepted 16 July 2015
Available online 18 July 2015
Keywords:
CVB1
Mice
Pleconaril
Oxoglaucine
Guanidine-HCl
Drug sensitivity

a b s t r a c t
Currently, clinically effective antivirals for use in the treatment of enteroviral (EV) infections do not exist.
The main reason is the development of drug resistance, the principle obstacle in the development of EV
infection chemotherapy, based til now on monotherapy. The most important achievement of our previous studies was the development of a novel scheme for in vivo application of a triple combination of EV
inhibitors with different modes of action against Coxsackievirus B (CVB) infections in mice. It consists of
consecutive alternating administration (CAA) of the substances in the combination. Here, we tested the
effect of the triple combination pleconaril, guanidine-HCl, and oxoglaucine (PGO) via CAA in newborn
mice infected with a neurotropic strain of CVB1 (20 LD50 per mouse). This combination manifested a considerable protective effect with pleconaril doses of 25200 mg/kg: it decreased mortality rate (protection
index, PI, between 31.3% and 67.7%) and increased mean survival time (MST) by 46 days. Pleconaril
monotherapy demonstrated activity similar to that of PGO via CAA, as measured by PI values, but MST
values were slightly lower. However, it also greatly suppressed growth of infected suckling mice, especially at 200 mg/kg. This toxic effect was avoided with CAA of PGO at pleconaril doses of 25100 mg/kg.
Pleconaril monotherapy administered every 3 days was ineffective. The PGO with CAA treatment course
decreased infectious virus content, whereas pleconaril monotherapy did not. Analysis of drug-sensitivity
in brain samples from CVB1 infected mice, based on IC50 (50% inhibitory concentration) values from cell
culture experiments, showed that the CAA course counteracted the development of drug resistance to
pleconaril and oxoglaucine in the triple PGO combination and increased drug sensitivity. In contrast, pleconaril and oxoglaucine monotherapies resulted in drug resistance. This data clearly proves the effectiveness of the proposed novel approachthe CAA treatment coursefor combined application of EV
replication inhibitors.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Human enteroviruses, distributed worldwide, are causative
agents of a broad spectrum of diseases with enormously high morbidity, including a series of severe illnesses that involve pathologies
of the CNS, heart, b-cells of pancreas, skeletal muscles, and so on.
With the common cold, they contribute to the development of
chronic respiratory diseases, including chronic obstructive pulmonary disease. It should be stressed that there is signicantly high
morbidity and mortality in children and high-risk populations (people with immunodeciencies, neonates) (Pallansch and Roos, 2006;
Tan, 2005; Mallia et al., 2007). The unusually large number of
Corresponding author.
E-mail address: galabov@microbio.bas.bg (A.S. Galabov).
http://dx.doi.org/10.1016/j.antiviral.2015.07.004
0166-3542/ 2015 Elsevier B.V. All rights reserved.

enterovirus serotypes that are causative agents of clinical illness

hamper the establishment and introduction of anti-enterovirus
vaccines. All these facts point to chemotherapy as the main tool
for controlling enterovirus infections. The high prevalence of inapparent cases (more than 85%) (Morens and Pallansch, 1995;
Pallansch and Roos, 2006; Strauss and Strauss, 2008) is a strong
argument for beginning administration of specic anti-enteroviral
chemotherapeutic agents during the disease latency period. Such
urgent prophylaxis is especially indicated for all children during
enteroviral infection outbreaks in childcare settings (nursery
schools, kindergartens, primary schools, pediatric hospital units,
and so on) (Morens and Pallansch, 1995). Doing so could result in
reduced risk of acquired diabetes and other severe forms of
enterovirus-induced infections (Hyty et al., 1995; Hyty and
Taylor, 2002; Galabov and Angelova, 2006).

A. Stoyanova et al. / Antiviral Research 121 (2015) 138144

Currently, clinically effective antivirals for treating enteroviral

infections do not exist. Despite the large number of compounds
that are effective in vitro (Barnard, 2006; De Palma et al., 2008),
anti-enteroviral chemotherapeutics for clinical use have not yet
been approved, and the results of clinical trials of the most active
antivirals could be considered as modest. The unsatisfactory
in vivo effectiveness of enteroviral replication inhibitors has been
ascribed to the rapid development of drug resistance (Loddo,
1980; Melnick et al., 1961) by initially drug-sensitive viruses.
This phenomenon is related to the quasispecies composition of
the enteroviral population (Eigen and Biebricher, 1988; Domingo
et al., 2008), which is a consequence of the accumulation of resistant quasispecies populations that result from countless point
mutations due to errors introduced by the viral RNA-dependent
RNA polymerase during viral replication (Chumakov et al., 2007).
As a result, drug-resistant mutants have developed for almost
every specic enterovirus replication inhibitor.
The failure of hitherto prevailing antiviral trials for enterovirus
infections is considered to be due to the monotherapy approach. If
so, use of antiviral drug combinations might be an efcient approach
to overcoming the disadvantages of monotherapy (i.e., development
of resistance to each or all of the partners in the combination). By
using a combined therapy, a greater effect could be achieved at
lower concentrations than those required with monotherapies; this
could prevent the so-called pressure of the dose effect, which favors
the rapid development of virus-drug resistance.
A main result of our previous studies in experimental
chemotherapy of enterovirus infections was the introduction and
further development of a new treatment course: consecutive (not
simultaneous) alternating administration (CAA) of a triple combination of enterovirus replication inhibitors with different modes
of actiondisoxaril, guanidine hydrochloride (guanidine-HCl),
and oxoglaucine (Vassileva-Pencheva and Galabov, 2010; Galabov
et al., 2012). The results obtained from the application of CAA
against Coxsackievirus B (CVB) infections in mice demonstrated
that this novel treatment scheme prevents the development of
drug resistance and provides signicant antiviral activity
(Vassileva-Pencheva and Galabov, 2010). The CAA course we developed is new to antiviral chemotherapy.
In the current study, we further investigated the CAA approach
for combined administration of anti-enteroviral compounds with
proven effect by replacing disoxaril with pleconaril, both of which
are WIN compounds. Pleconaril has been proven experimentally to
be the most effective WIN compound (Pevear et al., 1999; Hayden
et al., 2003), and it has been included in clinical trials. WIN compounds are considered the most active anti-enteroviral inhibitors.
As VP1 hydrophobic pocket binders, they attack in the early stages
of enteroviral replication. The second compound in the pleconari
l/guanidine-HCl/oxoglaucine (PGO) combination, guanidine-HC1,
is a classic enteroviral inhibitor that interferes with the function
of the 2C protein, thereby preventing initiation of negative RNA
strand synthesis (Loddo et al., 1962; Barton and Flanegan, 1997).
The third partner, oxoglaucine, is a compound of plant origin that
specically inhibits replication of a broad spectrum of enteroviruses (Nikolaeva-Glomb et al., 2008). Its mechanism of action is
enviroxime-like: it targets the 3A coding region (Arita et al., 2015).
We studied the in vivo effect of the PGO combination, applied
following the CAA treatment course, on neurotropic
Coxsackievirus B1 (CVB1) infection in newborn mice.
2. Materials and methods
2.1. Cells
Monolayer cultures of human epithelial type 2 (HEp-2) cells
(National Bank for Industrial Microorganisms and Cell Cultures,

139

Soa), 3.54.0x105 cells per ml, in 6-well plates (CELLSTAR,

Greiner Bio-One GmbH, Frickenhausen, Germany), were used for
both plaque purication of the viral isolates from mouse brains
and determination of IC50. The cells were grown in Dulbeccos
MEM (minimal essential medium; Gibco BRL, Paisley, Scotland,
UK) containing 10% fetal bovine serum (Gibco BRL) and supplemented with 10 mM HEPES buffer (Merck, Darmstadt, Germany)
and antibiotics (penicillin: 100 IU/ml; streptomycin: 100 lg/ml)
in a CO2 incubator (HERA cell 150, Heraeus, Germany) at 37 C,
5% CO2, and high humidity. The maintenance solution was 0.5%
heated fetal bovine serum in Dulbeccos MEM.
2.2. Virus
Coxsackievirus B1 (Connecticut-5 strain) for in vivo experiments
was obtained through intracerebral passages (0.02 ml/mouse) in
newborn albino mice (ICR line) and prepared as a 10% brain suspension in phosphate-buffered saline (PBS). The virus underwent
multiple intracerebral passages (without intermediary passages
in cell cultures).
2.3. Mice
ICR random-bred newborn albino mice (obtained from the
Experimental and Breeding Base for Laboratory Animals of the
Bulgarian Academy of Sciences, Slivnitza, Bulgaria) were used.
This random-bred stock was originally developed at the Institute
for Cancer Research (Fox Chase Cancer Center) by Dr. T.S.
Hauschka, beginning in 1948.
2.4. Compounds tested
Pleconaril, 3-{3,5-dimethyl-4-[3-(3-methyl-1,2-isoxazol-5-yl)
propoxy]phenyl}-5-(triuoromethyl)-1,2,4-oxadiazole (VP 63843,
WIN 63843, Picovir), was synthesized by Dr. Vadim Makarov from
the State Research Center for Antibiotics, Moscow, Russia. It was
formulated as a suspension in 0.5% xanthan gum and 1% Tween
80 (Pevear et al., 1999).
Oxoglaucine {1,2,9,10-tetramethoxy-7h-dibenzo[de,g]quinolin7-one}, an aporphinoid alkaloid from Glaucium avum Cranz
(yellow horn poppy), was obtained by Dr. Stefan Philipov from
the Institute of Organic Chemistry with Centre of Phytochemistry,
Bulgarian Academy of Sciences. The compound was dissolved in
1:9 v/v DMSO/saline.
Guanidine-HC1 was provided by Eastman Organic Chemicals
(New York). The compound was dissolved in saline solution.
2.5. Coxsackievirus B1 infection in newborn mice
2.5.1. Testing of triple combination of anti-enteroviral compounds
Groups of newborn mice (4050 mice per group) were inoculated subcutaneously with CVB1, 20 LD50. Beginning 1 h after virus
inoculation (Day 1), mice received a combined treatment course of
three compounds (pleconaril, guanidine-HCl, and oxoglaucine
(PGO)) over 12 consecutive days, with one compound administered each day. Thus, each individual compound was administered
every third day, either orally (pleconaril) or subcutaneously
(guanidine-HC1 and oxoglaucine) (see Table 1 for the order of
administration). The course continued through Day 12
postinoculation.
Pleconaril was tested at the following daily doses: 12, 25, 50,
100, and 200 mg/kg. Daily doses of the other compounds were
selected as optimal (guanidine-HCl: 48 mg/kg; oxoglaucine:
25 mg/kg), based on literature data and previous experiments
in our laboratory (Herrmann et al., 1982; Galabov and Spasov,
unpublished data).

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A. Stoyanova et al. / Antiviral Research 121 (2015) 138144

Table 1
Daily administration scheme for compounds applied in combination or alone.
Compounds in combination or as monotherapies

Days
Compounds used in combination
1

Consecutive combination P/G/O

Simultaneous triple combination
Monotherapies of the partner substances
Placebo

Ple
Gua
Oxo
Ple
Gua
Oxo
Ple
Gua
Combination of Ple, Gua and Oxo is simultaneously applied every day
Each drug partner is applied every day for 12 days
Saline solution every day

10

11

12

Oxo

Ple

Gua

Oxo

Ple (P), pleconaril per os; Oxo (O), oxoglaucine s.c.; Gua (G), guanidine-HCl s.c. Compounds were applied once per day, beginning 1 h postinfection.

In addition to the placebo group, the following control groups

were tested: pleconaril, oxoglaucine, and guanidine-HCl
monotherapies administered every day, every other day, and every
third day. An overview of the treatment courses is presented in
Table 1. Cumulative mortality (percentage), mean survival time
(MST; days) and body weight (g) were measured.
2.5.2. Preparation of virus brain isolates from treated mice
Virus samples (34 brain samples from each group) were collected daily, beginning on Day 4 postinoculation and continuing
through 1 day after treatment ended (Day 13). For each group,
samples were combined, and brain isolates were prepared as 10%
brain suspensions in phosphate-buffered saline (PBS).
2.5.3. Virus assay and plaque purication of virus isolates
Viral content (PFU/ml) of brain isolates was determined by the
plaque method (Dulbecco, 1952). Monolayer HEp-2 cell cultures in
6-well CELLSTAR plates were inoculated with 10-fold dilutions of
each virus stock and left for 1 h at 37 C for virus adsorption. The
agar overlay (1.75 ml per dish) was 1% puried agar (Oxoid Ltd.,
Basingstoke, Haunts, UK) in Dulbeccos MEM, supplemented with
10% heated calf serum, 1.65 g/ml sodium bicarbonate, and antibiotics (penicillin: 100 IU/ml; streptomycin: 100 g/ml). After 48-h
incubation at 37 C, a neutral-red containing agar overlay (1.5%
agar with 0.02% neutral red (Fluka, Buchs, Switzerland) in physiological saline) was added, and plates were kept at room temperature. PFU was recorded twice, after 4 and 24 h. A single plaque
from each viral stock was isolated and resuspended in Dulbeccos
MEM containing 0.5% heated calf serum.
2.5.4. Virus isolate sensitivity to pleconaril and oxoglaucine
Herrmanns (1961) and Siminoffs (1961) plaque-inhibition test
was used to determine the drug sensitivity of plaque-puried virus
progeny. Monolayer HEp-2 cell cultures in 6-well CELLSTAR microplates were inoculated with 5060 PFU/ml of virus per well, left for
1 h at 37 C for virus adsorption, and covered with an agar overlay
(described in 2.5.3). The test compounds were included in the
overlay at the following concentrations: pleconaril = 0.001,
0.0032, 0.01, 0.032, 0.1, and 0.32 lM; oxoglaucine = 0.1, 0.32, 1,
3.2, and 10 lM. Following 48-h incubation at 37 C, a neutral-red
containing agar overlay (described in 2.5.3) was added, and microplates were kept at room temperature for at least 4 h. The percentage of PFU inhibition was evaluated in comparison to the control
(no test compound in the agar overlay). IC50 of compound (i.e.,
the concentration required to inhibit the plaque titer by 50%)
was determined for each virus sample. These experiments were
performed three times.
2.5.5. Statistical analysis
Mortality was followed until Day 12, and survival time was
measured as the period from the day of virus inoculation until
one day before death. The protection index (PI) was determined
by the equation PI = [(PC 1)/PC]  100, where PC is the

protection coefcient (that is, % mortality in placebo group/% mortality in the drug-treated group). Two-tailed Fishers exact test was
used to compare survival rates between the experimental groups.
P-values less than 0.05 were considered statistically signicant.
One-way ANOVA with Bonferronis post-test was used to determine statistical differences between virus titer values from brain
samples, mean survival time (MST), and IC50 of pleconaril alone,
the PGO combination, and the placebo.
3. Results
3.1. Effects of consecutively applied combination PGO on experimental
CVB1 infection in newborn mice
We tested the effect of the triple combination PGO with CAA
treatment course in newborn mice infected with a neurotropic
strain of CVB1 at massive virus inoculum (s.c. 20 LD50 per mouse).
The course started on the day of virus inoculation (1 h postinoculation) and continued through the end of the observation period
(Day 12). In parallel, we studied (i) the effect of the combination
PGO applied simultaneously every day postinfection, and (ii) the
individual effect of the three partners in the combination administered every day postinoculation. Pleconaril, as a representative of
one of the most active antipicornavirus inhibitorsWIN compounds (from Winthrop Co.)was studied at several doses.
Guanidine-HCl and oxoglaucine were tested at a single daily dose
of 48 mg/kg and 25 mg/kg, respectively.
As Table 2 shows, the CAA course with PGO manifested a considerable protective effect when pleconaril was administered in
doses of 25200 mg/kg. There was a dose-response decrease in
mortality rate (PI between 31.3% and 67.7%) and an increase in
MST by 46 days. Activity of the CAA course with PGO is shown
in Fig. 1.
The simultaneous daily administration of PGO had no effect.
Interestingly, the pleconaril monotherapy demonstrated activity
analogous to that of PGO with CAA, as measured by PI values, at
doses of 25100 mg/kg (Fig. 2). However, it manifested a
well-expressed suppression of growth in the infected suckling
mice, especially at 200 mg/kg (Table 3). This toxic effect was
avoided in the CAA course at pleconaril doses of 25100 mg/kg;
evidently, this was due to the compound being administered only
once every 3 days.
The single administration of pleconaril (25 mg/kg) given every
3 days was ineffective (Table 2). Oxoglaucine (25 mg/kg) and
guanidine-HCl (48 mg/kg) monotherapies were also ineffective.
3.2. Effect of the PGO combination on infectious virus content in brains
of treated mice
Fig. 3 shows the infectious virus content of mouse brain isolates
infected with CVB1 and treated with combination PGO administered consecutively. For the experiments described in Sections
3.2, 3.3, and 3.4, pleconaril was administered at a daily dose of

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A. Stoyanova et al. / Antiviral Research 121 (2015) 138144

Table 2
Effects of combination PGO, applied consecutively or simultaneously, compared to individual compound effects against experimental Coxsackievirus B1 neuroinfection in
newborn mice.
Compounds

Survivors/totala

Mortality, %

MST SD, daysb

PI%

P12.5GO consecutively
P25GO consecutively
P50GO consecutively
P100GO consecutively
P200GO consecutively
P12.5GO simultaneously
P25GO simultaneously
P200GO simultaneously
Pleconaril 12.5 mg/kg
Pleconaril 25 mg/kg
Pleconaril 50 mg/kg
Pleconaril 100 mg/kg
Pleconaril 200 mg/kg
Oxoglaucine 25 mg/kg
Guanidine-HCl 48 mg/kg
Pleconaril 25 mg/kg 2 days apart
Placebo PBS
Placebo per os
Placebo consecutively
Placebo simultaneously

0/36
10/32***
8/30***
14/34***
34/50***
0/27
0/33
0/24
0/26
14/45^^^
8/29^^^
14/32^^^
10/29^^^
0/47
1/20
0/23
0/48
0/29
0/35
0/35

100.0
68.8
73.3
58.8
32.0
100.0
100.0
100.0
100.0
68.9
72.4
56.3
65.5
100.0
95.0
100.0
100.0
100.0
100.0
100.0

7.9 0.3
9.1 0.4***,hhh
8.3 0.2***
8.1 0.1
10.4 0.2
4.8 0.2
4.2 0.4
3.8 0.5
7.4 0.2
7.9 0.6^^
7.5 1.2
7.9 0.7
9.9 0.8
5.8 0.3
4.5 0.6
4.4 0.2
4.6 0.9
4.6 0.3
4.0 0.2
3.9 0.6

0
31.3
26.7
41.2
68.0
0
0
0
0
31.1
27.6
43.8
34.5
0
5.0
0
0
0
0
0

Data are from three independent experiments (averaged). The treatment regimen details are given in Table 1. MST, mean survival time; PBS, phosphate-buffered saline; PC,
protection coefcient; PI, protection index; SD, standard deviation; P#GO, triple combination of pleconaril, guanidine-HCl, and oxoglaucine. MST calculations counted living
mice as being dead on the last observation day (Day 12).
a
Two-tailed Fishers exact test.
b
One-way ANOVA (Bonferronis multiple comparison post-test).
***
P < 0.001 vs. placebo consecutively.
^^^
P < 0.001 vs. placebo per os.
hhh
P < 0.001 vs. pleconaril 25 mg/kg 2 days apart.
^^
P < 0.01 vs. placebo per os.

25 mg/kgthe lowest dose showing a marked antiviral effect and a

lack of toxicity in in vivo tests. Pleconaril monotherapy did not signicantly inhibit the infectious virus load. As shown, the combination of PGO in the CAA treatment course markedly decreased the
infectious virus content, especially at Days 1013 postinoculation.
The placebo group experienced 100% mortality following Day 6
postinoculation.

3.3. Virus brain isolate sensitivity to pleconaril following the PGO

combination via CAA treatment course
The plaque-inhibition test was used in HEp-2 cells to determine
the pleconaril sensitivity of virus progeny in brain samples from
mice infected with CVB1 and treated with CAA of combination
PGO or with pleconaril monotherapy. The results show that sensitivity to pleconaril gradually decreased in the virus isolated from

the pleconaril monotherapy group; this decrease was especially

pronounced on Days 811 postinoculation (Table 4).
In contrast, when combination PGO was administered consecutively, not only was there an increased inhibitory effect, but a
marked increase in sensitivity to pleconaril occurred on Days 5
13 postinoculation, with a maximum on Day 8. This was especially
clear in comparisons between IC50 values of the PGO via CAA
course and pleconaril monotherapy groups.
Simultaneous daily application of the PGO combination showed
a quick emerging of pleconaril resistance, at Day 3 postinoculation.
3.4. Virus brain isolate sensitivity to oxoglaucine following the PGO
combination via CAA treatment course
A marked increase in viral sensitivity to oxoglaucine, another
partner in the triple combination, was seen in the
virus-containing brain samples from mice treated with the PGO

Fig. 1. Effect of combination PGO, applied consecutively, against experimental neurotropic infection with Coxsackievirus B1 in newborn mice.

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A. Stoyanova et al. / Antiviral Research 121 (2015) 138144

Fig. 2. Individual effects of pleconaril against experimental neurotropic infection with Coxsackievirus B1 in newborn mice.

Table 3
Individual body weight of newborn mice at the end of therapeutic courses (mean
values of 23 experiments).
Pleconaril oral
daily dose
(mg/kg)

25
50
100
200

4. Discussion
This work presents the further development and conrmation of
consecutive alternating administration of a triple combination of
enterovirus replication inhibitors with different modes of action
(Vassileva-Pencheva and Galabov, 2010; Galabov et al., 2012).
Results from a previous study, which used the combination disox
aril/guanidine-HCl/oxoglaucine, applied following a CAA treatment
course, in neurotropic-CVB1-infected mice, demonstrated that this
novel treatment scheme prevents the development of drug
resistance
and
provides
signicant
antiviral
activity
(Vassileva-Pencheva and Galabov, 2010).
Here, we tested the activity of a triple combination in which
disoxaril was replaced by another WIN compound, pleconaril
(WIN63843). The main reason for this change was pleconarils
leading position among WIN enterovirus replication inhibitors
(Pevear et al., 1999; Groarke and Pevear, 1999; Schiff and
Sherwood, 2000). Pleconaril displayed a marked efcacy in 2 of 3
neonates with enteroviral hepatitis (Aradottir et al., 2001) and a
marked efcacy against chronic meningoencephalitis (78%

Animal body weight on Day 11 post infectiona (g)

Infected and treated animals
Pleconaril
monotherapyb

PGO combination
via CAA treatment
course

5.0 0.9
4.9 1.1
4.2 0.3
2.7 0.3***,^^^

5.9 0.5
5.8 0.3
6.1 0.8
4.9 0.5

Non-infected
and untreated
animals

6.2 1.1

The placebo groups are not presented due to animal mortality within the rst half
of the studied period.
b
One-way ANOVA (Bonferronis multiple comparison post-test).
***
P < 0.001 vs. non-infected and untreated animals.
^^^
P < 0.001 vs. P200GO combination with CAA treatment course.

via CAA course, especially beginning on Day 6 postinoculation

(Table 5). Simultaneous application of the PGO combination
showed decreased drug sensitivity.

10

Placebo
Pleconaril 25 mg/kg
P25GO consecutively

**
***

Virus Titer (PFU/ml)

***
10

10

10

10

10

Days
Fig. 3. Infectious virus titer of brain samples.

11

12

13

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A. Stoyanova et al. / Antiviral Research 121 (2015) 138144

Table 4
Sensitivity to pleconaril in plaque-inhibition tests of virus brain isolates from newborn mice infected with Coxsackievirus B1.
Group

Pleconaril IC50 values (lM) of viral brain samples

Taken on day (after viral inoculation)

Placebo
Pleconaril 25 mg/kg
PGO consecutively
PGO simultaneously

10

11

12

13

0.0689
0.0904
0.0651
0.1316

0.0475
0.164***
0.0075***,^^^
0.1297***

0.0532
0.1249
0.0076
a

a
0.1519
0.0077
a

a
0.2217
0.0042
a

a
0.2156
0.0075
a

a
0.2587
0.0083
a

a
0.1748
0.0098
a

a
0.1093
0.0066
a

a
0.1139
hhh
0.0072
a

a After Day 6 there was no animal alive, 100% mortality rate.

The statistical analysis was performed with one-way ANOVA (Bonferronis multiple comparison post-test).
***
P < 0.001 vs. placebo Day 5.
^^^
P < 0.001 vs. pleconaril 25 mg/kg Day 5.
hhh
P < 0.001 vs. pleconaril 25 mg/kg Day 13.

Table 5
Sensitivity to oxoglaucine in plaque-inhibition tests of virus brain isolates from newborn mice infected with Coxsackievirus B1.
Group

Oxoglaucine IC50 values (lM) of viral brain samples

Taken on day (after viral inoculation)

Placebo
Oxoglaucine 25 mg/kg
PGO consecutively
PGO simultaneously

10

11

12

13

1.0192
1.1984
1.596
1.7757

1.379
1.163
1.5982
1.9184

1.0633
a
0.3604
a

a
a
0.322
a

a
a
0.4945
a

a
a
0.8553
a

a
a
0.3407
a

a
a
0.5936
a

a
a
0.9983
a

a
a
0.7749**
a

a After Day 6 there was no animal alive, 100% mortality rate.

The statistical analysis was performed with one-way ANOVA (Bonferronis multiple comparison post-test).
**
P < 0.01 vs. PGO consecutively Day 5.

improvement with minimal adverse effects) (Rotbart and Webster,

2001), though it was not effective in a double-blind trial of children
with enteroviral meningitis, where twice as many adverse effects
were registered (Abzug et al., 2003). Nevertheless, in a phase III
double-blind placebo-controlled trial, pleconaril showed some efcacy against Coxsackievirus A21-induced respiratory infections
(common cold) when treatment began within 24 h of symptom
onset (Hayden et al., 2003). However, in 2002 the FDA stopped further trials due to drug-increased levels of the CYP3A4 liver microsomal enzyme (Senior, 2002). In 20062007, Schering-Plough
conducted a placebo-controlled study of the effects of pleconaril
nasal spray on common cold symptoms and asthma exacerbations
following rhinovirus exposure (Hayden et al., 1995; Clinical
Trials.gov.U.S. NIH, 2007), the results of which have not yet been
announced. Meanwhile, pleconaril showed virucidal properties in
hand treatments for prevention of rhinovirus infection (Turner
and Hendley, 2005). Evidently, the efcacy of pleconaril has not
been sufciently strong to move it forward in clinical development.
It is well known that clinical trials of the previously synthesized
and characterized WIN compound disoxaril (WIN51711) were
stopped during phase I. Further clinical development was required
because disoxaril had shown poor bioavailability and crystalluria
in healthy volunteers (De Palma et al., 2008).
Our previous and current studies found a signicant difference
between the single effects of pleconaril and disoxaril against
experimental CVB1 neuroinfection. Pleconaril exhibited a pronounced protective effect with oral daily doses of 25200 mg/kg,
while disoxaril was inactive.
However, the pleconaril course with a daily dose of 200 mg/kg
manifested a well-expressed suppression of suckling mouse
growth. In addition, some growth suppression was recorded with
daily doses of 25100 mg/kg. Evidently, the in vivo antiviral effect
of this compound is characterized by comparatively low selectivity.
The CAA course of triple combination PGO manifested a marked
protective effect (decreased mortality rate and increased MST) in

mice with experimental CVB1 neuroinfection (20 LD50). The efcacy of the PGO CAA course was approximately of the same order
as that of the pleconaril monotherapy, based on comparisons of
protection index percentages and mean survival times.
This study also demonstrated a signicant advantage of the PGO
combination applied following the CAA treatment course: the negative effect (animal growth suppression) was overcome within the
whole range of daily therapeutic doses. Evidently, administering
pleconaril every 3 days avoids the compounds potential toxic
effect.
We have emphasized that there was a signicant difference
between the single effects of pleconaril and disoxaril against
experimental CVB1 neuroinfection. However, when applied in triple combination with guanidine-HCl and oxoglaucine, following
the CAA scheme, both combinations showed a clear protective
effect, with the disoxaril combination (DGO) showing a slightly
better efcacy (Vassileva-Pencheva and Galabov, 2010).
This effect was also clearly distinguished from the lack of activity seen with simultaneous daily PGO, as well as from the effects of
the oxoglaucine and guanidine-HCl monotherapies, the other partners in the PGO combination.
A signicant part of our study was the analysis of drug sensitivity in the brain samples from mice with induced neurotropic CVB1
infection. Based on longitudinal daily IC50 values from cell culture
experiments, the CAA course markedly counteracted the development of drug resistance to pleconaril as a component of the triple
PGO combination. In contrast, pleconaril monotherapy resulted in
drug resistance, beginning on Day 5 postinoculation and peaking
on Day 10. The drug sensitivity of CVB1 brain isolates from mice
receiving the CAA course with the pleconaril combination was
analogous to that of brain isolates from CVB1-infected mice that
received the disoxaril combination (Vassileva-Pencheva and
Galabov, 2010). The same characteristic, an increase in drug sensitivity, was observed. The mechanism of this phenomenon is actually unclear and requires further investigation.

144

A. Stoyanova et al. / Antiviral Research 121 (2015) 138144

The great therapeutic advantage of the CAA course as a barrier

to drug-resistance development was seen when comparing its
results to those of the PGO triple combination administered simultaneously every day. In that case, pleconaril resistance was
observed as early as Day 4 postinfection.
Thus, the CAA course for both WIN compound-containing triple
combinationspleconaril and disoxarilshowed similar advantages as a therapeutic approach for CVB1 infection in mice: antiviral activity and suppression of the drug resistance process.
In summary, these data clearly prove the effectiveness of the
proposed novel approach for combined application of enteroviral
replication inhibitors: the consecutive alternative administration
treatment course. By following this approach, other
triple-combination models could be studied and developed, resulting in possible bases for new therapeutic strategies.
Acknowledgements
The authors are grateful to the National Science Fund of
Bulgaria (Project B 01-13/2012: Novel Approach for
Highly-Effective Chemotherapy of Enterovirus Infections) for their
nancial support. We also thank Mme. Lucia Mukova, MS, for the
preparation of cell cultures, and Mme. Petya Stoyanova, DVM,
Eleni Aksioti, and Ivanka Zahova, for their technical assistance with
the 1.51.9 animal experiments.
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