Note: Descriptions are shown in the official language in which they were submitted.
UTFC:lg
ANTI-HIV ACTIVITY OF
LIPOSOMAL NYSTATIN AND AMPHOTERICIN B
The United States may own certain rights in this
invention as the development of part of the present
invention was supported by grant number NIH-NOl-AI-72639
from the National Institutes of Health, Department of
Health and Human Services.
This application is a continuation in part of U.S.
application serial number 314,710, filed on February 23,
1989. That application is incorporated here by reference.
Acquired immunodeficiency syndrome (AIDS), resulting
from infection by the human immunodeficiency virus (HIV),
affects thousands of people around the world, and is
uniformly fatal. Extensive research efforts are underway
in an attempt to develop methods of curing or preventing
this disease, but as of the present, no completely
satisfactory solution has been found.
One group of drugs with known antibiotic properties
is the polyene macrolides, which are secondary metabolites
produced by various species of Streptomyces. Without
being bound by any particular theory of activity, the
previously-known antibiotic properties of the polyenes
appears to be based on their interaction with sterols in
the cell membranes of infectious agents, such as fungal
cell membranes. As a result of this interaction, the
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membranes are rendered selectively permeable to the
outflow of vital constituents, such as potassium.
Nystatin and amphotericin B are two specific examples
of polyenes. Although they have previously demonstrated
some antibiotic activity, they have not been previously
demonstrated to have a practical use with respect to
treatment or prophylaxis of AIDS. The present invention
is based on the surprising discovery that certain
formulations comprising nystatin or amphotericn B do in
fact have practical and useful activity against HIV-1
virus.
The present invention, in one aspect, concerns a
method for inhibiting the replication of HIV-1 virus,
comprising the step of administering to a subject which is
infected with HIV-l a pharmaceutically effective amount of
a liposomal composition which comprises a polyene selected
from nystatin and amphotericin B. The composition will
comprise liposomal vesicles, formed of at least one
phospholipid, in which a polyene selected from nystatin
and amphotericin B is entrapped or ~ncapsulated. The
composition can optionally also comprise a sterol, such as
cholesterol.
In another aspect, the pr~sent invention relates to a
pharmaceutical composition comprising a cytotoxicity-
exclusionary amount of a liposomal polyene selected from
nystatin and amphotericin B. In this respect, the
invention is based on the therapeutic window created by
the selective activity of liposomal nystatin and
amphotericin B, whereby a dose of such compositions that
is toxic to HIV-infected cells is not substantially toxic
to uninfected cells. "Cytotoxicity-exclusionary" means a
composition or dosage which has an LD50 for HIV-infected
target cells above the IDso for noninfected target cells.
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In another aspect, the present invention concerns a
method of inhibiting the replication of HIV-l virus,
comprising the step of administering to a subject which is
infected with HIV-l a vector-interdicting amount of a
composition which comprises liposomal vesicles in which a
polyene selected from nystatin and amphotericin B is
incorporated. A "vector-interdicting amount" refers to
the amount which will result in the inhibition, blocking,
or elimination of a vector pathway for HIV proliferation
and/or transmission in the treatment of an animal.
"Vector pathway" refers to organ and organelle (i.e.,
nonfluid) replication-based pathways of HIV proliferation
and transmission.
In preferred embodiments of the invention, the lipids
will make up between about 25% and about 90% by weight of
the total amount oP lipid and nystatin. If a sterol is
included, it will preferably make up between about 10% and
about 75% by weight, most preferably between about 30~ and
about 60%, of the total amount of nystatin, lipid, and
sterol. In an especially preferred embodiment, the
composition consists essentially of nystatin, dimyri_coyl
phosphatidyl choline, and dimyristoyl phosphatidyl
glycerol, with the weight ratio of DMPC:DMPG being about
7:3. The weight ratio of nystatin to lipids is preferably
about 1:10.
The present invention permits the selectively toxic
treatment of an animal that is infected with HIV-1 virus,
whereby infected cells are selectively killed, while
avoiding substantial toxicity to other cells. The present
invention takes advantage of the surprising ability of
liposomal nystatin and amphotericin B to inhibit the
replication and/or transmission of HIV-1. This activity
is especially surprising in view of the lack of such
activity on the part of liposomal formulations of other
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polyenes, such as mepartricin and hamycin. The
compositions and methods of the present invention should
be useful for the treatment and/or prophylaxis of AIDS in
animals, including humans.
In the method of the present invention, the agent to
be administered comprises a polyene selected from the
group consisting of nystatin and amphotericin B, at least
one phospholipid, and optionally a sterol, such as
cholesterol. The phospholipid takes the form of lipid
vesicles or liposomes, in which ~he polyene is entrapped
or encapsulated. (For the sake of simplicity, in this
patent all lipid -~esicles in which polyene is entrapped or
encapsulated will be referred to as "liposomes".) The
liposomes can be unilamellar, multilamellar, or can have
an undefined lamellar construction.
The phospholipids are preferably selected from the
group consisting of phosphatidyl choline, phosphatidyl
serine, phosphatidyl glycerol, sphingomyelin, and
phosphatidic acid. The most preferred phospholipids are
dimyristoy~ phosphatidyl choline and dimyristoyl
phosphatidyl glycerol. However, other lipids can be used.
When the liposomes comprise dimyristoyl phosphatidyl
choline and dimyristoyl phosphatidyl glycerol, those two
lipids are preferably present in a weight ratio of between
1:10 and 10:1, with the most preferred ratio being 7:3
DMPC: DMPG. The lipids preferably comprise from about 25%
to about 90 % by weight o~ the overall composition of
lipid, nystatin, and optionally, sterol.
It is useful to include sterol in the composition in
order to reduce the toxicity that is typically associated
with free nystatin. The sterol, for example cholesterol,
can be included in an amount equal to from 10% to 75% by
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weiyht of the overall composition (on a dry basis). A
more preferred range is 30% to 60%.
The mode of administration is preferably parenteral,
i.e., by intravenous, intraarterial, intramu~cular,
intralymphatic, intraperitoneal, subcutaneous,
intrapleural, or intrathecal injection or infusion. The
administration could also be through inhalation or topical
or oral means. The composition can be employed in
admixture with conventional excipients, i.e~,
pharmaceutically acceptable carrier substances which do
not deleteriously react with the active compositions. The
composition is preferably formulated as a pharmaceutically
acceptable solution, such as a sterile isotonic aqueous
solution. A pharmaceutically effective amount will be
administered, preferably in a dosage between about 0.1 mg
of nystatinlkg of body weight and about 80 mg/kg. ~he
presently preferred dosage is about 4 mg/kg, administered
three times a week for about six months.
Further details regarding the making and use of the
present invention can be determined from the following
examples.
Examples
Druas. Nystatin (Nys) was obtained from American Cyanamid
Company. Liposomal nystatin (L-Nys) was prepared as
previously described. Briefly, the drug in methanol and
the phospholipids, dimyristoyl phosphatidyl choline (DMPC)
and dimyristoyl phosphatidyl glycerol (DMPG) dissolved in
chloroform (DMPC:DMPG, 7:3 by weight) were mixed in a
ratio of 1:10 (nys:phospholipid) and the organic solvents
were evaporated. The dried lipid film was dispersed with
o.9% NaCl solution.
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Another procedure for preparing the composition is as
follows. 7 g of DMPC (Lipoid KG, Ludwigshafen, West
Germany) and 3 g of DMPG (Lipoid KG~ are mixed with 250 cc
of t-butyl alcohol tFisher Scienti~ic, Fairlawn, New
Jersey). 250 cc of water are added and mixed until
dissolved. This solution is then passed through a 0.45 ~M
pore size filter cartridge. Separately, 1.1 g Nys is
dissolved in 7 cc of methyl sulfoxide (Fisher Scientific~,
and mixed until dissolved. The nys solution is then
passed through a 0.45 ~M pore size filter cartridge, and
the lipid solution and nys solution are then mixed. The
concentration is adjusted to 1.1 mg/ml by adding an
appropriate volume of diluent, and the solution is then
passed through a sterile, 0.22 ~M membrane. The solvents
can be removed by lyophilization, and the resulting
preliposomal powder can be reconstituted whe~ desired by
adding water or saline solution.
Virus. HIV-l (HTLV-III/B) was prepared from HIV infected
H9 cells in culture. Cells were harvested and the virus
isolated from the culture medium by banding on sucrose
gradients. The virus band sediment had a density of 1.16
to 1.19 g/ml, and was collected by pelleting. The pellet
was resuspended in TNE (lOmM Tris. HCl, pH 7.5, 0.lM NaCl,
0.001 EDTA) at 2xlOIl virus particles/ml.
H9 Cells. H9 cells are a transformed line of human
lymphocytes permissive for HIV-l. The cultures were
maintained using RPMI supplemented with 20% fetal bovine
serum, 2% glutamine and 1% gentamicin. Cells were
harvested in log phase of growth for testing and seeded at
5x105 cells/test culture.
Culture Conditions. HIV-l at 5X108 particles/ml was added
to the test cultures (5x105 cells/ml) simultaneously with
compounds to be tested. The cultures were incubated for 4
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days at 37 C in an atmosphere containing 5% C02. Cells
were then harvested and assayed for virus growth as
described below.
~mmunofluorescer.ce Assays. The cells were collected by
centrifugation, washed and resuspended in O.OlM phosphate
buffered saline (PBS), to approximately lx106 cells/ml.
An aliquot was used for determining cell viability by the
exclusion of trypan blue dye. The remaining cells were
added to wells on a toxoplasmosis slide, allowed to dry
for one hour and fixed in acetone methanol (1:1, v/v) for
15 minutes, as described. After fixation, the slides were
pretreated with 10% normal goat serum at room temperature
for 30 minutes. The slides were rinsed four times with
PBS, 15 minutes each wash. Mouse monoclonal antibody to
HIV-1 p24 antigen was added to each slide and incubated
for 30 minutes at 37C, in a humidified chamber. The
cells were then rinsed with PBS, and washed 4 times as
above. The cells were then stained with FITC labeled goat
anti-mouse IgG by incubating for 30 minutes as above.
The slides were rinsed with P8S, washed 4 times and
given a final wash overnight with fresh PBS. The slides
were counterstained with 0.02% Evans blue for 1 minute
followed by rinsing with distilled water. After air-
drying the slides were mounted with 50% glycerol, and
fluorescence measurements were done with a Zeiss
fluorescence microscope. The results are expressed as the
percent of fluorescent positive cells derived from the
average of several microscopic fields.
Reverse TranscriPtase Activity. The supernatant fractions
of the test cultures were assayed for reverse
transcriptase activity as described previously (Sarin,
1985, 1986). This was measured by the incorporation of
2~ 7 h
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3H-deoxythymidine triphosphate into trichloracetic acid-
insoluble DNA using (dT)~5:(A)n, as a template primer.
Antibody Induction Assay. C57BL/6 mice were primed with
5 lx107 sheep red blood cells (SRBC's) ip 10 days prior to
the experiment. The mice were sacrificed, spleens removed
and a homogeneous cell population prepared as using a
tissue homogenizer. Cultures were set up with lx107
primed spleen cells, lx105 SRBC's and 0.1 ml of the test
compound. The cultures were incubated for 6 days in
Click's medium (Click, 1972) under the same conditions as
the H9 cultures. After incubation, the supernatant fluids
were collected by centrifugation and assayed for the
presence oP antibody to sheep red blood cells by the
complement dependent immune hemolysis assay.
Complement Dependent Immune Hemolysis. Various dilutions
tl:10, l:100, 1:1000) of the culture supernatants were
added to lx108 SRBC's for 30 minutes at room te~perature.
~0 The cells were collected by centrifugation, washed with
vernal buffer, and mixed with an excess of complement
(Gibco) determined by hemolysin titration. The
suspensions were incubated for 1 hour in a 37 C water
bath. After incubation the supernatants were collected by
centrifugation and read spectrophotometrically at 541 nm
fo~ the presence of hemoglobin from the lysed SRBC's.
This direct test measures the amount of IgM in the
cultures. To measure IgG, total antibody was measured by
an indirect technique using rabbit anti-mouse IgG. The
antibody was added to the suspensions before the addition
of complement to facilitate complement binding. The value
obtained from the indirect technique is subtracted from
the total antibody and the result is the amount of IgG in
the culture. The unit of antibody is defined as the
amount of antibody which can sensitize 50% of the SRBC's
lysed in the presence of complement.
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g
Table 1. Anti-HlV-1(111C) activity of liposomal-nystatin
Drug
Drug Combination H9 cells Percent inhibition of HIV expression
~g/ml tlOu/ml) p17 p24 RT
H9 cells (no virus) 2.52
H9 cells infected 1.72
1 5 Expæri~ent 1
Empty liposomes 0.01 0 0
0.1
1.0 0 0 0
2 0 10.0 0
100.0 0
Liposr~nal- 0.01 2.36 0 0 26
Nystatin 0.1 2.72 35 33 27
2 5 1.0 1.76 50 40 30
10.0 1.6 90 93 70
100.0 ToxicToxic to cells
Experincnt 2
3 0
Empty liposomes 0.01 0
0.1
1.0 0 0 0
10.0
3 5 100.0 0 0 0
Liposomal- 0.01 0 0
Nystatin 0.1 39 31 44
1.0 54 37 35
4 0 10.0 92 99 78
100.0 Toxic to cells
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Table 2. Anti-HlV-l,~ activity of liposomal-nystaein
Drug
Drug Combination Percent inhibition of HIV e~lpression
Syncytia
I~g/mlinhibitionp17 PZ4 RT
Experi lent 1
Empty liposomes 7.5 0 0 0 0
0 0 0 0
0 0 0 0
ZO O O O O
Liposomal- 7.5 0 Z9 57 52
Nystatin 10 60 55 83 69
88 81 90 n
89 81 93 88
2 5 E~periment 2
Empty liposomes 7.5 0 0 0 0
lo o o o n
0 0 0 0
ZO o o o o
Liposomal- 7.518 39 60 SZ
~Iystatin 10 58 61 77 62
82 74 83 80
82 90 83
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Anti-HIV ~IIIB) activity of L-Nys in H9 cells
In the first set of experiments, in which the effects
of L-Nys on HIV (IIIB) infected H9 cells was tested~ the
viral antigens pl7 and p24 were inhibited in a dose
dependent fashion by L~Nys (see Table 1). RT activity was
also inhibited to a general degree. Empty liposomes had
no effect on any of the parameters studied. L-Nys was not
toxic to uninfected H9 cells at concentrations up to 10
~gtml; however, at concentrations between 10.0-100.00, L-
Nys was toxic to HIV infected cells. HIV-expression was
inhibited by L-Nys at concentrations which are not toxic
to uninfected H9 cells.
Anti-llIV-lcr activity of L-Nys in Molt-3 cells
When the effects of L-Nys on HIV-1cc infected Molt-3
cells was tested, similar to H9 cells, a dose response
effect on pl7, p24 and RT was observed (see Table 2).
Furthermore, syncytia formation was also inhibited. Empty
liposomes did not have an effect on the parameters
studied. L-Nys was not toxic to non-infected Molt-3 cells
at the concentrations studied.
The results obtained indicate that L-Nys displayed a
selective toxicity for HIV infected cells. L-Nys had
direct anti-HIV activity as reflected in the inhibition of
syncitia formation, and on HIV infected cells. L-Nys was
not toxic even at the higher concentrations used on non-
infected cells. The HIV inhibition observed was dose
dependent for all parameters tested.
The increased cholesterol observed in HIV-infected
cells may make the infected cells more vulnerable to the
membrane effects of Nys. We have previously shown that L-
Nys was far less toxic to human red blood cells than free
Nys, thus liposomal incorporation prevented the direct
toxic effects of Nys to mammalian cells. See U.S. patent
2 ~ ,9 9 ~
-12-
4,812,312, which is incorporated here by reference. Such
was also the case for non-infected l,vmphocytes.
* * *
The preceding description is intended to illustrate
specific embodiments of the present invention. It i5 not
intended to be an exhaustive list of all possible
embodiments. Person skilled in this field will recognize
that modifications could be made to the compositions and
methods described above that would remain within the scope
of the present invention.
