Antiviral composition

The present invention relates to an antiviral composition and to methods of treating patients with viral infections. The antiviral composition of the present invention comprises prostratin, a phorbol ester derivative, and a pharmaceutically acceptable carrier. The present composition while having antiviral activity does not have substantial tumor promoting activity and does not have other substantial adverse toxicological properties that would preclude its use in antiviral therapy.

BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION 
The present invention relates to an antiviral composition, in particular an 
anti-HIV composition and to methods of treating viral infections. 2. 
Background Information 
A nucleoside class of antiviral agents, a prototype of which is AZT, is 
widely used in the clinical treatment of acquired immune deficiency 
syndrome (AIDS). AZT was initially selected for clinical use based upon an 
in vitro antiviral assay. 
While extremely useful in antiviral therapy, AZT is limited by toxicity and 
a therapeutic index insufficient to make it adequate for therapy. 
Accordingly, new classes of antiviral agents to be used alone or in 
combination with AZT and other agents are urgently needed for effective 
antiviral therapy. 
In the search for new antiviral agents, an extensive screening program to 
identify potential anti-AIDS and anticancer compounds from natural sources 
has been initiated [Boyd M. R.: In AIDS Etiology, Diagnosis, Treatment and 
Prevention, (DeVita V. T. Jr, Hellman S, Rosenberg S. A., eds.) 
Philadelphia: Lippincott, 1988, pp. 305-317]. Ongoing natural product 
collection projects are focusing on unusual or underexplored plant, marine 
and microbial resources. 
As a part of this search, Homalanthus acuminatus, a small endemic tree of 
the primary forests of Samoa, and an important component of Samoan 
ethnopharmacology has been studied. Interviews with "taulasea", or Samoan 
healers, indicated that various parts of the plant are used to treat 
physical ailments. For example, the leaves are used in water infusions to 
treat back pain and abdominal swelling, the roots to suppress diarrhea and 
the stem wood to treat yellow fever [Cox P. A.: Samoan Ethnopharmacology. 
In Economic and Medicinal Plant Research, Vol. 4: Plants and Traditional 
Medicine. (Wagner H., Farnsworth N., eds.) London: Academic Press, in 
press]. Furthermore, related species are used in New Guinea (H. nervosus) 
to treat boils and sores [Holdsworth D. M.: Int. J. Crude Drug Res. 
27:95-100, (1989)] and in Indonesia (H. nutans) for gonorrhea [Perry L. 
M.: Medicinal Plants of East and Southeast Asia. Cambridge: MIT Press, 
1980, p. 149]. Also, the leaves of H. nutans have been used to treat 
circumcision wounds in Samoa [Uhe G.: Econ. Bot. 28:1-30, (1979)]. 
In the present invention, extracts of H. acuminatus are used to exert 
inhibitory effects against the human immunodeficiency virus (HIV-1), the 
causative agent of the AIDS. 
SUMMARY OF THE INVENTION 
Accordingly, it is an object of the present invention to provide a potent 
antiviral agent with minimal adverse toxicological properties. 
It is another object of the present invention to provide an antiviral 
chemotherapeutic agent which has antiviral activity but does not have 
substantial tumor promoting activity. 
In one embodiment, the present invention relates to an antiviral 
composition comprising an antivirally effective amount of prostratin and a 
pharmaceutically acceptable carrier. 
In another embodiment, the present invention relates to a method of 
treating a viral infection comprising administering to a patient with the 
viral infection prostratin in an amount sufficient to effect said 
treatment. 
Various other objects and advantages of the present invention will become 
apparent to one skilled in the art from the drawings and the following 
description of the invention.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to an antiviral composition. The composition 
of the present invention comprises, as the active ingredient, the 
12-deoxyphorbol ester derivative, prostratin and a pharmaceutically 
acceptable carrier. The prostratin of the present composition may be 
purified from a natural source or may be synthetically made. Suitable 
carriers for use in the present invention include, but are not limited to, 
injectable or orally or rectally administerable oils, lipid emulsions or 
aqueous suspensions, or in the case of orally or rectally administerable 
tablets or capsules, a pharmacologically inert excipient. 
Utilizing an in vitro antiviral assay known to accurately predict antiviral 
activity in humans, prostratin was shown to have antiviral activity. In 
addition to its antiviral activity, prostratin substantially lacks tumor 
promoting activity, unlike most other phorbol esters. This lack of 
tumor-promoting activity makes prostratin extremely suitable for use as an 
antiviral agent and more desirable than other agents with anti-HIV 
activity which have tumor-promoting activity. 
The compositions of the present invention inhibit the HIV retrovirus. As 
one skilled in the art will appreciate, prostratin and compositions 
thereof will likely inhibit other retroviruses and may inhibit 
non-retrovirus viruses. 
The present invention further relates to a method of treating viral 
infections comprising administering to a patient an "effective amount", of 
the composition of the present invention. The "effective amount" is 
defined as that amount required to be administered to an individual 
patient to achieve an "effective blood level" of prostratin of 
.gtoreq.3.mu. molar (e.g. the approximate minimum level required for 
maximum antiviral activity--see FIG. 2). Since the fixed "effective blood 
level" is used as the prefered endpoint for dosing, the actual dose and 
schedule for drug administration for each patient may vary, depending upon 
interindividual differences in pharmacokinetics, drug disposition and 
metabolism. The composition can be administered, for example, orally, 
rectally, subcutaneously or intravenously. One skilled in the art can 
easily determine the appropriate method of administration for the exact 
formulation of the composition being used. The composition can be present 
in as a sterile solution suitable, for example, for intravenous 
administration. The composition can also be present in dosage unit form, 
such as, for example, as a tablet or capsule. 
EXAMPLES 
The following non-limiting Examples are provided to aid in the 
understanding of the present invention. It is understood that 
modifications can be made in the procedure set forth, without departing 
from the true spirit of the invention. 
Ethnobotanical Techniques 
Interviews concerning the use of Homalanthus acuminatus (Muell.-Arg.) Pax 
were conducted in the Samoan language with healers in Falealupo and Pesega 
villages, Western Samoa. Bulk samples of stem wood and other parts of H. 
acuminatus were collected and shipped immediately to the NCI Natural 
Products Repository in Frederick, Md. U.S.A. Voucher specimens were 
collected at the same time, verified by healers, and subsequently 
deposited in the herbaria of Brigham Young University (BRG) and Harvard 
University (GH). 
Isolation and Structure Determination 
Large-scale separations, using high-performance liquid chromatography 
(HPLC), were performed initially with a Waters C-18 Prepak.RTM. 500 
cartridge, and final purification was effected on a Rainin Dynamax.RTM. 
C-18 column (1.times.25 cm). .sup.1 H-NMR spectra were recorded on a 
Varian VXR 500 spectrometer and .sup.13 C-NMR spectra were recorded on a 
Varian XL 200 spectrometer. Chemical shifts are given in ppm relative to 
an internal standard of tetramethylsilane (TMS, .delta.=0). Infrared 
spectra were measured on a Perkin-Elmer 267 spectrometer and ultraviolet 
spectra were obtained with a Beckman 34 spectrophotometer. Optical 
rotations were measured on a Perkin-Elmer 241 polarimeter. Mass spectra 
were recorded on a VG Micromass ZAB 2F mass spectrometer. 
Approximately 1.05 kg of fresh stem wood from Homalanthus acuminatus were 
extracted sequentially with ethanol and 1:1 dichloromethane-methanol to 
yield 22.8 g of extract. This crude extract was subjected to a modified 
Kupchan partition protocol [Grode et al.: J. Org. Chem. 48:5203-5207, 
(1983)]. The hexane-soluble and carbon tetrachloride-soluble fractions 
appeared rich in lipophilic phorbol esters, as determined by TLC and 
.sup.1 H-NMR analysis. The material that partitioned into chloroform (3.7 
g) did not appear to contain long-chain alkyl esters of phorbol by .sup.1 
H-NMR, but was active in the anti-HIV assay. The latter sample was further 
separated by gel permeation through Sephadex LH-20 (4.times.140 cm) with 
methanol/dichloromethane (1:1) into seven fractions. Fractions two and 
three were combined (2.3 g) and subjected to preparative HPLC using a 
water/methanol step gradient elution on C-18 reversed-phase sorbent. The 
fraction that eluted with 70:30 methanol/water (95 mg) was further 
purified by C-18 HPLC (70:30 methanol/water) to provide 15 mg of a pure 
compound which showed striking activity in the anti-HIV screen. Throughout 
the isolation procedure all fractions were tested for anti-HIV activity as 
described hereinbelow. 
The pure, active compound was an optically active, white crystalline solid, 
mp 215.degree.-216.degree., [.alpha.].sub.D +62.6.degree. (c 0.9, MeOH). 
The molecular formula of C.sub.22 H.sub.30 O.sub.6 was established by 
high-resolution, fast-atom bombardment (FAB) mass spectrometry (observed 
m/z 391.2088 for [MH.sup.+ ], calculated m/z 391.2119 for C.sub.22 
H.sub.30 O.sub.6). These data, and precedents that many species of the 
family Euphorbiaceae produce phorbol diterpenes, suggested that the 
compound was a monoacetylated phorbol diterpene derivative. Characteristic 
ultraviolet absorbances (EtOH) at 210 (.epsilon.=8900) and 236 nm 
(.epsilon.=5900) and infrared bands (CHCl.sub.3) at 3300, 1725 and 1705 
cm.sup.-1 supported this conclusion. Homonuclear decoupling and COSY 
(correlation spectroscopy) [Bax et al.: J. Magn. Reson. 44:542-561, 
(1981)] analyses of the .sup.1 H-NMR spectra (500 MHz, CDCl.sub.3) 
revealed a 12-deoxyphorbol nucleus. The .sup.1 H and .sup.13 C NMR 
resonances (See Table 1 below) were found to match literature values for a 
compound known as prostratin [Cashmore et al.: Tetrahedron Lett. 
20:1737-1738, (1976) and Evans F. J.: Naturally Occurring Phorbol Esters 
(Evans F. J. ed.) Boca Raton, Fla.: CRC Press, 1986, pp. 171-215] whose 
structure (FIG. 1) had previously been established by X-ray 
crystallographic analysis [McCormick et al.: Tetrahedron Lett. 
20:1735-1736, 
TABLE 1 
______________________________________ 
NMR DATA FOR PROSTRATIN 
Carbon # .sup.13 C.sup.a 
.sup.1 H.sup.b 
______________________________________ 
1 161.2 7.50 (br s) 
2 140.0 
3 209.2 
4 73.8 
5 38.6 2.53 (2H, br s) 
6 132.8 
7 130.2 5.73 (d, J = 5.7) 
8 39.1 3.18 (dd, J = 5.7, 5.3) 
9 76.1 
10 55.1 3.48 (br s) 
11 32.4 2.13 (ddq, J = 11.6, 7.0, 6.4) 
12 31.8 1.70 (dd, J = 14.5, 11.6) 
2.01 (dd, J = 14.5, 7.0) 
13 63.6 
14 36.2 0.88 (d, J = 5.3) 
15 22.7 
16 23.2.sup.c 1.09 (3H, s).sup.d 
17 18.6.sup.c 1.02 (3H, s).sup.d 
18 15.3 0.98 (3H, d, J = 6.4) 
19 10.1 1.58 (3H, dd, J = 2.7, 1.3).sup.e 
20 68.3 3.79 (dd, J = 12.7, 3.6) 
3.86 (br d, J = 12.7) 
21 173.2 
22 21.3.sup.c 1.55 (3H, s) 
______________________________________ 
.sup.a Spectrum obtained at 50 MHz in CDCl.sub.3. Assignments were based 
on literature values for 12deoxyphorbols [Cashmore et al.: Tetrahedron 
Lett. 20:1737-1738 (1976) and Evans FJ: in Naturally Occuring Phorbol 
Esters (Evans FJ ed.) Boca Raton, FL: CRC Press, 1986, pp. 171-215]. 
.sup.b Spectrum obtained at 500 MHz in C.sub.6 D.sub.6. Coupling constant 
are reported in Hertz. Assignments were based on homonuclear decoupled an 
COSY analysis. 
.sup.c Assignments may be reversed. 
.sup.d Assignments may be reversed. 
.sup.e Long range allylic and homoallylic couplings. 
Screening for Anti-HIV-1 Activity 
The XTT-tetrazolium anti-HIV assay was performed on crude extracts, 
chromatographic fractions and pure compounds as previously described 
[Weislow et al.: J. Nat. Cancer Inst. 81:577-586, (1989) and Gustafson et 
al.: J. Nat. Cancer Inst. 81:1254-1258, (1989)] but with several 
modifications. The human T-lymphoblastic cell line CEM-SS [Nara et al.: 
AIDS Res. Hum. Retroviruses 3:283-302, (1987)] was used as the target cell 
line and virus infections were performed using the RF variant of HIV-1, 
(MOI=0.05). Concentrated pellets of CEM-SS cells were incubated with the 
free virus for 1 hour at 37.degree. C. The cells were then diluted and 
inoculated into round-bottomed 96-well microtiter plates (Corning) at a 
density of 5000 cells/well. Test compounds and the appropriate controls 
were then added to the wells, and the plates were incubated for 6 days. At 
the end of the incubation period cell growth parameters and HIV-1 
activities were determined. 
The relative numbers of viable cells in the cultures incubated with various 
concentrations of prostratin were estimated using the XTT-tetrazolium 
procedure; surviving cells metabolically reduce XTT to a colored formazan 
product which is measured colormetrically [Boyd M. R.: In AIDS Etiology, 
Diagnosis, Treatment and Prevention, (DeVita V. T. Jr, Hellman S., 
Rosenberg S. A., eds). Philadelphia: Lippincott, 1988, pp. 305-317; 
Weislow et al.: J. Nat. Cancer Inst. 81:577-586, (1989) and Gustafson et 
al.: J. Nat. Cancer Inst. 81:1254-1258, (1989)]. 
An interesting result was obtained (FIG. 2A), which clearly showed the 
importance of inclusion of the uninfected, drug-treated controls for 
proper interpretation of the XTT assay results. Viewed in isolation, the 
XTT assay data on the virus-infected cells would have indicated only a 
modest enhancement in cellular production of XTT-formazan production in 
the presence of 1.0-200 .mu.g/ml of prostratin. However, in comparing the 
effects of prostratin over the same concentration range on the uninfected 
cells, it was apparent that the agent had a marked growth-inhibitory, or 
cytostatic, effect on these control cultures. Therefore, what might have 
initially appeared to be only a modest antiviral effect of prostratin was 
actually quite profound. In the presence of a broad range of 
concentrations of prostratin the number of viable cells, as indicated by 
XTT-formazan production, was essentially identical in the uninfected cell 
cultures or in the cultures infected with HIV-1. 
Aliquots of supernatant fluid from the test wells of the tetrazolium assay 
were removed prior to the time of XTT addition for quantitation of 
residual infectious virus using the syncytium assay described by Nara et 
al. [Nara et al.: AIDS Res. Hum. Retroviruses 3:283:302, (1987)]. Data 
were expressed as syncytium-forming units (SFU/40 .mu.l. Other aliquots 
were diluted 1:100 with Triton X-100 and analyzed for the HIV core 
protein, p24, as previously described [Gustafson et al.: J. Nat. Cancer 
Inst. 81:1254-1258, (1989)]. Correlative assays measuring the effects of 
prostratin on p24 viral antigen production and syncytium formation as an 
estimate of infectious virions, shows similarly striking inhibitory 
effects of prostratin at concentrations as low as 1.0 .mu.g/ml (FIG. 2B). 
Trypan Blue Dye Exclusion Assay 
Trypan blue dye exclusion was employed for secondary evaluation of drug 
toxicity and antiviral protection over time. A 50 .mu.l aliquot of cells 
was removed from each test well and mixed with 100 .mu.l of 0.4% trypan 
blue. The percentage of viable cells was determined microscopically by 
direct hemocytometer counts. The assays indicated that cell viability was 
greater than 90 percent for both the infected and uninfected cultures 
treated with prostratin (FIG. 4). 
FIGS. 3 and 4 illustrate results of a series of experiments, each performed 
as described above, except that instead of using 6-day incubation, cell 
viability assays were performed after 0, 1, 2, 3, 4 and 5 days of 
incubation. The data shown in FIG. 3 were obtained using the 
XTT-tetrazolium assay, whereas the data in FIG. 4 are from a trypan blue 
dye exclusion assay. In both cases, the results are entirely consistent 
with the data and interpretations from FIG. 2, further confirm the 
cytostatic effect of prostratin on the CEM-SS target cells, and illustrate 
the impact of this effect on the assessment of the anti-HIV activity of 
prostratin. With either of the cell viability end-point assays, the 
anti-HIV activity of prostratin is strikingly apparent after three or more 
days of culture growth. 
Protein Kinase C Assay 
Binding of [.sup.3 H]phorbol 12,13-dibutyrate ([.sup.3 H]PDBu) to protein 
kinase C partially purified through the DEAE-chromatography step [Jeng et 
al.: Cancer Res. 46:1966-1971, (1986)] was assayed in the presence of 
phosphatidylserine as described [Sharkey et al.: Cancer Res. 45:19-24, 
(1985)] except that incubation was for 5 min at 37.degree. C. Competition 
of [.sup.3 H]PDBu binding by prostratin was determined in the presence of 
4 nM [.sup.3 H]PDBu. Binding of [.sup.3 H]PDBu to intact CEM-SS cells and 
competitive binding by prostratin were assayed in RPMI cell culture medium 
containing 0.1 mg/ml of bovine serum albumin, 25 mM Hepes, pH 7.4. 
Incubation was for 30 min at 37.degree. C., after which the cells were 
chilled, an aliquot removed to determine total counts, and bound counts 
determined by filtration. Non-specific binding was determined in the 
presence of 5 .mu.M non-radioactive PDBu. Activation of protein kinase C 
enzymatic activity by PDBu or prostratin was determined by the procedure 
of Nakadate et al. [Nakadate et al.: Biochem. Pharmacol. 37:1541-1545, 
(1988)] 
Because prostratin is a phorbol derivative, it was of interest to see if it 
would bind to and activate, or inhibit, protein kinase C [Blumberg P. M.: 
Cancer Res. 48:1-8, (1988) and Nishizuka Y.: Nature 308:693-698, (1984)]. 
Interestingly, in contrast to many other phorbol derivatives, prostratin 
reportedly is not a tumor promoter [Zayed et. al.: Experientia 
33:1554:1555, (1977)]. The K.sub.i for prostratin was 12.5.+-.0.4 nM 
(mean.+-.SEM; 3 experiments). For comparison, the K.sub.i value for 
12-deoxyphorbol 13-isobutyrate was 2.1.+-.0.1 nM (mean.+-.range; 2 
experiments) and the K.sub.d for PDBu was 0.59 nM. Prostratin, like PDBu, 
stimulated protein kinase C (PKC) activity in vitro; at a concentration of 
1000 nM prostratin, stimulation of PKC was 95% of that exhibited by a 
concentration of 100 nM PDBu. 
Binding affinities of phorbol esters in cells are typically lower than 
those obtained with reconstituted protein kinase C [Blumberg et al.: in 
Mechanism of Tumor Promotion, Vol. 3. Tumor Promotion and Cocarcinogenesis 
in vitro, (Slaga T. J., ed.) Boca Raton, Fla.: CRC Press, 1984, pp. 
143-184], presumably reflecting the role of cellular calcium and 
phospholipid composition on binding [Konig et al.: J. Cell. Biochem. 
7:255-265, (1985)]. In the CEM-SS cells, [.sup.3 H]PDBu was bound with an 
affinity of 4.9.+-.0.8 nM (mean.+-.range; 2 experiments) and a B.sub.max 
of 1.2.+-.0.1 pmol/mg protein (mean.+-.range; 2 experiments). Prostratin 
inhibited [.sup.3 H]PDBu binding to the CEM cells with a K.sub.i of 
210.+-.30 nM (mean.+-.range; 2 experiments). The usual binding protocol 
were cells omits serum, because of reportedly variable effects of serum on 
binding [Blumberg et al.: in Mechanism of Tumor Promotion, Vol. 3. Tumor 
Promotion and Cocarcinogenesis in vitro, (Slaga T. J., ed.) Boca Raton, 
Fla.: CRC Press, 1984, pp. 143-184]. In any case, a substantial effect 
when 10% fetal calf serum was included in the assays of biological 
response was not observed in the CEM cells on the K.sub.i for prostratin 
(K.sub.i =190 nM; one experiment). 
Well-characterized biological responses to the phorbol esters include 
inhibition of binding of epidermal growth factor and release of 
arachidonic acid metabolites [Dell'Aquila et al.: Cancer Res. 
48:3702-3708, (1988)]. It was confirmed that prostratin induces both of 
these responses in C3H1OT1/2 cells. 
C3H1OT1/2 cells were grown and [.sup.3 H] arachidonic acid metabolite 
release determined as described previously [Dell'Aquila et al.: Cancer 
Res. 48:3702-3708, (1988)]. .sup.125 I-epidermal growth factor binding by 
C3H10T1/2 cells was assayed as described [Dell'Aquila et al.: Cancer Res. 
48:3702-3708, (1988)] using a 1 hr pretreatment with phorbol ester and 
then a 1 hr co-incubation with the phorbol ester and the .sup.125 
I-epidermal growth factor. 
The half-maximally effective concentrations of prostratin were 220 nM and 
1100 nM, respectively. The corresponding values for PDBu, determined in 
parallel, were 10 and 24 nM, respectively. 
Since prostratin acted like a typical phorbol ester in vitro, both in PKC 
enzyme preparations and in intact cells, albeit with 20-45-fold lower 
potency than PDBu, the activity of other phorbol derivatives in the 
anti-HIV assay was examined. The phorbol esters PMA (phorbol 12-myristate 
13-acetate), at a concentration of 50 nM, and PDBu (500 nM) were similarly 
cytostatic and protective against HIV-1 under the above-described 
experimental conditions. 
Cell Morphology 
Low-power photomicrographs (FIG. 5) taken of CEM-SS cells after 6 days in 
culture under varying experimental conditions vividly revealed both the 
antiproliferative and the anti-HIV-1 effects of prostratin treatment. 
Panel A illustrates uninfected control CEM-SS cells which have not been 
treated with prostratin. The cells form a uniform pellet several hundred 
cell diameters thick on the bottom surface of the rounded well. In this 
culture configuration the cells are in very close proximity to one 
another; however, they are not physically attached and can be separated by 
gentle agitation. Infection of CEM cells by HIV-1 results in dramatic 
morphological alterations after 6 days (panel B); the effects include 
giant cell (syncytia) formation, cell lysis and production of large 
quantities of cellular debris. In contrast, the macroscopic morphology of 
cell pellets of uninfected CEM cells (panel C) and cells infected with 
HIV-1 (panel D), both treated with 10 .mu.g/ml prostratin, were virtually 
identical. 
Prostratin treatment caused a decrease in the size of the cell pellet 
relative to the control cells in both cultures. However, the size of the 
cell pellet and the percentage of viable cells in cultures infected with 
HIV-1 and uninfected cultures were similar after prostratin treatment. 
Thus, although prostratin inhibited cell proliferation, it also appeared 
to block totally the cytopathic effects of HIV-1 infection. 
Observations of cellular morphology at higher magnifications confirmed 
that, in the presence of 1 .mu.M prostratin, syncytium formation was 
completely abolished. 
All publications mentioned hereinabove are hereby incorporated by 
reference. 
While the foregoing invention has been described in some detail for 
purposes of clarity and understanding, it will be appreciated by one 
skilled in the art from a reading of this disclosure that various changes 
in form and detail can be made without departing from the true scope of 
the invention.