Bioscience Reports, Vol. 7, No. 3, 1987
Comparison of the Interaction of the AntiViral Chemotherapeutic Agents Amantadine
and Tromantadine with Model Phospholipid
Membranes
James J. Cheetham and Richard M. Epand 1
Received May 10, 1987
amantadine;tromantadine; virus chemotherapy;phospholipid; phase transition; bilayerhexagonal change.
KEY WORDS:
ABBREVIATIONS: DEPE, dielaidoyl phosphatidylethanolamine; POPE, 1-palmitoyl-2-oleoyl
phosphatidylethanolamine; DMPC, dimyristoyl phosphatidylcholine; DSC, differential scanning
calorimetry; PIPES, piperazine-N,N'-bis(2-ethanesulphonicacid); NMR, nuclear magnetic resonance;
tromantadine, N-l-adamantyl-N-[2-(dimethylamino)ethoxy]acetamide-hydrochloride;amantadine, (ladamantamine)-hydrochloride;HSV, Herpes SimplexVirus.
Amantadine and tromantadine are agents used against influenza and herpes infections,
respectively. Tromantadine raises the bilayer to hexagonal phase transition
temperature of synthetic phosphatidylethanolamines and is less disruptive to
phospholipid packing. Tromantadine acts similar to cyclosporin A, previously
demonstrated to inhibit viral-induced cell-cell fusion. We suggest the balance between
the hydrophobic and hydrophilic group sizes would allow tromantadine to prevent
membrane fusion more than amantadine and thus inhibit infection by viruses such as
Herpes, which l~use with the plasma membrane. Study of agents which stabilize the
bilayer phase of membranes may lead to efficacious inhibitors of viral infections
requiring cell fusion events.
Department of Biochemistry, McMaster University, Health Sciences Centre, 1200 Main Street West,
Hamilton, Ontario, Canada, L8N 3Z5.
J To whom correspondenceshould be sent.
225
0144-8463/87/0300-0225505.00/0 9 1987 Plenum Publishing Corporation
226
Cheetham and Epand
INTRODUCTION
Tromantadine is an amantadine derivative reported to have inhibitory activity on both
HSV I and HSV II replication (1-3). It is believed to act both early in the viral infection,
preceeding macromolecular synthesis, and also later in the assembly of the virion or its
release (2). Amantadine is a prophylactic and therapeutic agent effective against
Influenza A infections, but has no activity against HSV I or HSV II infections (4, 5).
The ability of viruses to induce membrane fusion has been extensively studied
(6, 7, 8) and the phenomenon of lipid polymorphism is also well documented (9-12).
Many agents which induce the formation of non-bilayer structures also promote
membrane fusion (10, 11, 13). Viral induced cell-cell fusion can be inhibited by
cyclosporin A, an agent that stabilizes the bilayer phase of membranes (14, 15). Some
other peptides with anti-viral activity, including carbobenzoxy-D-Phe-L-Phe-Gly
(16), also stabilize model membranes against hexagonal phase formation (17). We
wished to compare the effects of two structurally related anti-viral agents,
tromantadine and amantadine (Fig. 1), on model membranes with their activity in
inhibiting viral replication.
0
NHa
XI-ICCH20CH~CH~N(CHs)~
Amantadine
Tromantadine
Fig. 1. Structuresof amantadineand tromantadine.
MATERIALS AND METHODS
L-c~-Dielaidoyl phosphatidylethanolamine (DEPE) and 1-Palmitoyl-2-oleoyl
phosphatidylethanolamine (POPE) were obtained from Avanti Polar Lipids Inc.
Amantadine (1-Adamantanamine HC1), Gold Label was obtained from Aldrich,
> 99 % pure. Tromantadine was a gift from Merz Laboratories of West Germany.
H P L C grade chloroform and methanol were used to dissolve the samples.
Sample Preparation
The phospholipid was dissolved in a chloroform:methanol solution (2:1, v/v) and
divided into several aliquots. The additives were also dissolved in a 2:1 chloroform:
methanol solution and added to the phospholipid solution to generate a series of
Amantadine and'Tromantadine with Membranes
227
samples with increasing concentrations of additive. After mixing, the solvent was
evaporated with a stream of dry nitrogen gas causing the solutes to form films inside
the glass test tubes. The samples were then placed in a vacuum evaporator with a liquid
nitrogen trap for one hour to remove the remainder of the solvent. The dried
phospholipid film was suspended by vigorous vortexing at about 45~ in 20 m M
PIPES, 150 m M NaC1, 0.02 mg/mL N a N 3, 1 m M EDTA buffer at pH = 7.40.
Differential Scanning Calorimetry (DSC)
Samples were degassed under vacuum. Buffer was loaded into the reference cell
and the lipid suspension into the sample cell of an MC-2 high sensitivity scanning
calorimeter (Microcal Co., Amherst, MA). The calorimeter was calibrated electrically.
The samples were scanned at a rate of 0.7 K/rain and the transition temperatures and
enthalpies were calculated by fitting the observed transitions to a single Van't Hoff
component using the DA2 software package from Mierocal.
31P.NMR
Lipid and drug were deposited inside a i0 mm N M R tube and the chloroform/
methanol solvent evaporated with dry nitrogen gas. Any residual solvent was removed
when the sample was put under high vacuum for 8 hours. The lipid film was suspended
at a concentration of 0.1 mg/mL in PIPES buffer at pH 7.4 for N M R studies. A Bruker
WM-250 N M R spectrometer running at 101.2 MHz was employed using broad band
proton decoupling. A spectral bandwidth of 30 kHz was used along with an aquisition
time of 0.28 sec, a relaxation delay of 0.3 sec and a pulse width of 25 #sec (90 deg).
Typically 800 accumulated free induction decays were used to obtain an adequate
signal to noise ratio.
RESULTS
Representative DSC scans of P O P E with varying amounts of tromantadine
demonstrate that as the mol fraction of drug is increased, the bilayer to hexagonal
phase transition temperature also increases and the temperature range over which the
transition occurs is broadened (Fig. 2). The enthalpy of the transition remains at
500 + / - 150 cal/mol phospholipid for all of the concentrations of tromantadine and
amantadine examined. Neither the enthalpy nor the temperature of the gel to liquid
crystalline phase transition of DEPE, P O P E or D M P C (data not shown) are affected
to any large extent by the concentrations of drug used in these experiments. For
example at 0.30mol fraction amantadine the transition temperature is changed
- 0.57~ and at 0.33 mol fraction of tromantadine the change is - 0.46~ for DEPE.
The mol fractions of tromantadine and amantadine used are plotted against the
change in the bilayer to hexagonal phase transition temperature (Fig. 3).
Tromantadine increases the bilayer to hexagonal phase transition temperature in both
D E P E and POPE, while amantadine lowers it slightly in both the phospholipids used.
The slopes of the lines shown in Fig. 3, are summarized in Table 1.
228
Cheetham and Epand
01
,,-t
B.358
tn
!
9.271
.
L~
.
.
.
A
I
/
7P
75
.
TL~'ERflTU~E
,
I
8B
85
(DE'GREE5 C,1
Fig. 2. Representative DSC scan of POPE with various tool fractions oftromantadine
added. Numbers for each curve indicate the tool fraction of tromantadine in that sample.
Lipid concentration is 7mM in 20raM PIPES, 150ram NaC1, O.02mg/ml NAN3,
1 mM EDTA, at pH 7.4. Heating scan rate 0.7 K/rain.
s~
7,
+,.,,
IF
U
s
9
9
'0
~.~
<
+
-F
3.
1o
-2
.I
I
.l
'1 o,
.2
I
o~
1
.+
I
.s
I
.e
M o l l ~ " z ' a e t . i o "r, o..f A d d i t i v e
Fig. 3. Dependence of the bilayer to hexagonal phase transition temperature of
DEPE on added tromantadine (+) or amantadine {aN)and of POPE on added
tromantadine (A), or amantadine (9
Amantadine and Tromantadine with Membranes
Table 1.
229
Comparison of slopes of ATm versus Mol Fraction plots (see Fig. 3)
Drug
Phospholipid
Tromantadine
Tromantadine
Amantadine
Amantadine
Slope (~
DEPE
POPE
DEPE
POPE
16.56
26.85
- 2.11
- 0.59
fraction)
+ / - 1.01
+ / - 1.44
+ / - 0.48
+ / - 1.43
The 31p-NMR results (Fig. 4), correlate with the DSC observations.
Tromantadine raises the temperature at which the chemical shift anisotropy
characteristic of the bilayer phase is converted to one indicative of the hexagonal
phase. For example at 1 0 ~ tromantadine there is still a bilayer component at 58~
while it has completely disappeared from the 58~ spectrum at 10 ~ amantadine. As
noted previously (18), the temperature at which the NMR spectrum changes is a few
degrees lower than the transition temperature observed with DSC. Both tromantadine
and amantadine markedly narrow the bilayer spectrum at higher drug concentrations
and induce the formation of an isotropic spectrum at higher temperatures. These
effects are exhibited more strongly by amantadine than by tromantadine. For example
at 10~o drug at 50~ the amantadine spectrum is much narrower than the one for
tromantadine and at 54~ the isotropic signal near 0ppm is prominent for the
amantadine but is not a significant component of the tromantadine spectrum. The
isotropic signal could arise from small vesicles, micelles or cubic phase.
PURE DEPE
5% TROMANTADINE
10% TROMANTAblNE
20% TROMANTAD~NE
t~
i\
20% AMANTADINE
IL_
\
10% AMANTADZNE
]
j a
j.~l
"~'
:',
L
i 'IV"
58'
,/I
Jl
pp~
o
~
pp~
o
pp~
pp~
pp~
pp~
o
Fig. 4. 31p-NMR scans of DEPE with varying amounts of tromantadine and amantadine at
different temperatures. Concentrations above each column refer to weight ~o of additive. Chemical
shift is measured with respect to an external phosphoric acid reference.
230
Cheetham and Epand
DISCUSSION
T r o m a n t a d i n e is shown to be a potent stabilizer of the bilayer phase of model
phospholipid membranes. This is consistent with the idea that stabilizers of the bilayer
phase m a y prevent m e m b r a n e fusion necessary for the infection or spreading of some
enveloped viruses. Amantadine however, does not show activity against H S V virus
infections where fusion of the viral capsid to the plasma m e m b r a n e plays a critical role
(2). Amantadine has been reported to act at the uncoating stage of the virion (4, 5), as
has the amantadine analog rimantadine (19), which is m o r e effective against Influenza
A replication in vitro.
We propose that the relative sizes of the h y d r o p h o b i c and hydrophilic regions of
the t r o m a n t a d i n e molecule are more balanced than amantadine making t r o m a n t a d i n e
better able to interact with the phospholipids and impart stability to the bilayer phase.
The a 1P - N M R indicates that amantadine is m u c h more perturbing to the organization
and motional properties of phospholipids in the bilayer phase than is tromantadine.
Further studies on the mechanism of the action of t r o m a n t a d i n e and localization
of the c o m p o u n d within cells should be undertaken to determine the exact mechanism
of inhibition of viral replication. Studies on c o m p o u n d s which are potent stabilizers of
the bilayer phase of phospholipid membranes m a y lead to the development of
efficacious inhibitors of viral-infections requiring cell fusion events.
ACKNOWLEDGEMENTS
We are grateful to Dr K. H u m m e l of Merz and Co., Frankfurt am Main, for
generously providing us with a sample of tromantadine. This w o r k was supported by
grant MT-7654 from the Medical Research Council of Canada.
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