Long-term stability of the anti-influenza A
Christoph Scholtissek, Robert G. Webster *
Department of Virology and Molecular Biology
. Jude Children
’s Research Hospital
, 332 N
Received 14 June 1997; accepted 5 September 1997
Amantadine and rimantadine hydrochloride were tested for stability after storage at different temperatures and
under different conditions for extended periods of time. Both compounds were quite stable after storage for at least25 years at ambient temperature; they both retained full antiviral activity after long-term storage or after boiling andholding at 65 – 85°C for several days. Thus, amantadine and rimantadine could be synthesized in large quantities andstored for at least one generation without loss of activity in preparation for the next influenza A pandemic in humans.
1998 Elsevier Science B.V. All rights reserved.
: Anti-influenza compounds; Amantadine; Rimantadine
Amantadine is a derivative of adamantane that
necessary for fusion of the endosomal and viral
efficiently inhibits replication of influenza A
membranes (Daniels et al., 1985; Steinhauer et al.,
viruses (Davies et al., 1964). This drug inhibits
1991). Because influenza viruses (like other RNA
replication by interfering with the ion channel
viruses) have a relatively error-prone polymerase
activity of the matrix (M)-2 protein (Sugrue and
and no repair system, drug-resistant variants can
Hay, 1991; Pinto et al., 1992; Schroeder et al.,
be obtained easily by passing the virus in the
1994) and/or with the pH-dependent conforma-
presence of amantadine (Cochran et al., 1965;
tional change of the hemagglutinin (HA), which is
Appleyard, 1977). The strains obtained by passingthe virus in the presence of relatively low concen-trations of amantadine (
* Corresponding author. Tel.: + 1 901 4953400; fax: + 1
901 5232622; e-mail: [email protected]
amino acid replacements at one of four specific
0166-3542/98/$19.00 1998 Elsevier Science B.V. All rights reserved.
38 (1998) 213–215
Table 1Inhibition of plaque formation by amantadine and rimantadine hydrochloride
Amantadine or rimantadine hydrochloride in overlay (vg/ml)
Number of plaque-forming units (log ) after treatment with
the following amantadine or rimantadine hydrochloride prepa-rations
Preparations: 1, newly purchased amantadine hydrochloride (Sigma); 2, amantadine stored at room temperature ( :20°C) atSJCRH for at least 25 years; 3, amantadine stored at 4°C in Giessen for at least 20 years; 4, amantadine hydrochloride (Giessen)made up to 1 mg/ml in PBS, heated at 100°C for 60 min and held at 65°C for 5 days; 5, newly purchased rimantadine hydrochloride(Forest Pharmaceuticals); 6, rimantadine stored at room temperature ( :20°C) at SJCRH for at least 25 years; and 7, rimantadineheated at 100°C for 120 min and held at 80°C for 4 days.
A/Singapore/1/57 (H2N2) was diluted to produce approximately 8.0 (log ) plaques in MDCK cells and treated with amantadine
and rimantadine hydrochloride in the overlay at the above concentrations.
NT, not tested.
aVery small plaques (:95% reduction) in all drug preparations tested.
bPlaques reduced in size by :50% or more in all drug preparations tested.
cA few minute plaques in each drug preparation tested.
dPlaques reduced in size by :75% or more in all drug preparations tested.
sites in the membrane-spanning region of the M2
ture ( :20°C) at St. Jude Children’s Research
Hospital (SJCRH), Memphis, TN, and another
In preparation for the next human influenza A
batch of amantadine hydrochloride (Sigma, St.
pandemic, strategic plans have been developed in
Louis, MO) was kept at 4°C for at least 20 years
many countries on steps that can be taken to
at the Institute of Virology in Giessen, Germany.
prepare for this event. One possibility considered
These drugs were compared with freshly pur-
was the preparation and storage of the two li-
chased amantadine (Sigma, St. Louis, MO) and
censed anti-influenza A compounds — amantadine
rimantadine hydrochloride (Forest Pharmaceuti-
and rimantadine. At a symposium entitled Pan-
cals, USA). Stock solutions of 1 mg/ml in phos-
phate buffered saline (PBS) were prepared and 1
Threat, held in Bethesda, MD in December 1995,
ml of the Giessen amantadine stock solution was
questions were raised about the stability of aman-
boiled for 1 h and kept for 5 days at 65°C, and 1
tadine and rimantadine. Currently these two
ml of the Memphis rimantadine stock solution
drugs have a short FDA approval time of only 2
was boiled for 2 h and kept for 4 days at 80°C.
To investigate the stability of these two com-
were used throughout the study and were infected
pounds, studies were done on drug preparations
with A/Singapore/1/57 (H2N2) at appropriate di-
available in two different laboratories. One batch
lutions starting with approximately 108 plaque-
of amantadine hydrochloride and one of riman-
forming units/ml and overlaid with agar in Eagle’s
tadine hydrochloride (Dupont, Wilmington, DE)
minimal essential medium containing 4% bovine
were kept for at least 25 years at room tempera-
serum albumin, 0.5 vg/ml trypsin, and the drugs
38 (1998) 213–215
at various concentrations as given in Table 1. For
tute of Allergy and Infectious Disease, Cancer
each dilution one monolayer was used. Each ex-
Center Support (CORE) Grant CA-21765 from
periment was conducted at least twice. The maxi-
the National Cancer Institute, and the American
mal difference in two independent experiments
Lebanese Syrian Associated Charities. We thank
Janice Cole and Dayna Baker for typing the
Each of the preparations of amantadine hy-
drochloride inhibited plaque formation by A/Sin-gapore/1/57 (H2N2) influenza virus and therewere no detectable differences between the prepa-
rations (Table 1). It is noteworthy that each of thepreparations caused plaque size reduction at the
Appleyard, G., 1977. Amantadine-resistance as genetic marker
same concentrations and that heating to 100°C
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and storage at 65°C for 5 days also failed to
Cochran, K.W., Maassab, H.R., Tsunoda, A., Berlin, B.S.,
1965. New studies of the antiviral activity of amantadine
decrease the biological activity of amantadine hy-
hydrochloride. Ann. NY Acad. Sci. 130, 432 – 439.
drochloride. Comparable results were obtained
Daniels, R.S., Downie, J.C., Hay, A.J., Knossow, M., Skehel,
with rimantadine. At the lowest dose tested (0.05
J.J., Wang, M.L., Wiley, D.C., 1985. Fusion mutants of
vg/ml), only a few micro plaques were observed
the influenza virus hemagglutinin glycoprotein. Cell 40,
with all three samples. Higher doses completely
Davies, W.L., Grunert, R.R., Haff, R.F., McGahen, J.W.,
inhibited plaque formation by A/Singapore/1/57
Neumayer, E.M., Paulshok, M., Watts, J.C., Wood, T.R.,
Herrmann, E.C., Hoffmann, C.E., 1964. Antiviral activity
These results indicate that both amantadine
of 1-adamantanamine (amantadine). Science 144, 862 – 863.
and rimantadine are extraordinarily stable, and
Hay, A.J., Wolstenholm, A.J., Skehel, J.J., Smith, M.H., 1985.
would be ideal for long-term storage for use in the
The molecular basis of the specific anti-influenza action ofamantadine. EMBO J. 4, 3021 – 3024.
initial stages of a new influenza A pandemic.
Pinto, L.H., Holsinger, L.J., Lamb, R.A., 1992. Influenza
According to our estimates, it would be difficult
virus M2 protein has ion channel activity. Cell 69, 517 –
even for our grandchildren to determine the ex
vivo decay time of these compounds. After a new
Schroeder, C., Ford, C.M., Wharton, S.A., Hay, A.J., 1994.
influenza A pandemic has appeared, and while a
Functional reconstitution in lipid vesicles of influenza virus
vaccine is under development, amantadine and
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rimantadine are the only FDA-approved drugs
Steinhauer, D.A., Wharton, S.W., Skehel, J.J., Wiley, D.C.,
Hay, A.J., 1991. Amantadine selection of a mutant influ-enza virus containing an acid-stable hemagglutinin glyco-protein: evidence for virus-specific regulation of the pH of
glycoprotein transport vesicles. Proc. Natl. Acad. Sci. USA88, 11525 – 11529.
Sugrue, R.J., Hay, A.J., 1991. Structural characteristics of the
This work was supported by US Public Health
M2 protein of influenza A viruses: evidence that it forms a
Service Grants AI-08831 from the National Insti-
tetrameric channel. Virology 180, 617 – 624.
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