CA2093825C - Proanthocyanidin polymers having antiviral activity and methods of obtaining same - Google Patents

Abstract

The present invention provides for proanthocyanidin polymers with significant antiviral activity. The proanthocyanidin po-lymers can be chemically synthesized or can be isolated from a Croton or a Calophyllum plant species. The present invention en-compasses methods of using proanthocyanidin polymers in treating warm-blooded animals, including humans, infected with par-amyxovaridae such as respiratory syncytial virus, orthomyxovaridae such as influenza A, B and C, and herpes viruses such as Herpes Simplex virus.

Description

1. FIELD OF THE INVENTION
The present invention relates to the use of proanthocyanidin polymers, having 2 to 30 flavonoid units, in treating respiratory virus infections.
Additionally, it has been found that the use of the proanthocyanidin polymers having 6 to 30 flavonoid units is effective in treating virus infections in ~o general. The chemical characteristics of certain newly discovered proanthocyanidin polymers are also encompassed.

2. HAC1~GROOND OF THE INVENTION
2.1 ETRNO80TANICAL 08E8 OF EXTRACTS FROM
THE CROTON TREE AND FROM CALOBBYLLOM
INOPHYLUM
A number of different Croton tree species, including Croton salutaris, Croton gossypifolius, 20 Croton palanostima, Croton lechleri, Croton erythrochilus and Croton draconoides, found in South America, produce a red viscous latex sap called Sangre de Drago. It is most often utilized by infixed descent and native people of the Peruvian Amazon for flu and 25 diarrhea. It is taken internally for tonsillitis, throat infections, tuberculosis, peptic ulcers, intestinal disorders, rheumatism and to enhance fertility and is used by both adults and children. It is also used extensively to stop bleeding, herpes, and ~ for wound healing. The sap is placed directly on open wounds as an anti-infective and to accelerate the healing process. It is applied to the gums of PC'f/US91 /07579 ,w, c c , patients after tooth extractions. It is also utilized as a vaginal wash in cases of excessive bleeding.
It has been shown that Sangre de Drago from ' Croton draconoides and from Croton Iechleri contains San alkaloid identified as taspine, which exhibits ' anti-inflammatory activity, Persinos et al., 1979, J.
Pharm. Sci., 68:124. Taspine has also been shown to inhibit RNA-directed DNA polymerase activity in the myeloblastosis virus, Rauscher leukemia virus and Simian sarcoma virus (Sethi, 1977, Canadian J. Pharm.
Sci., 12:7).
Calophyllum inophylum is a tree ranging from India to East Africa to Polynesia. Seed oil is used in folk medicine as an antiparasitie in treatment of ~5scabies, ringworm and dermatosis as well as other uses such as for analgesia. In Indo-China the powdered resin is used for ulcers and wound healing. In Indonesia the bark is applied externally to treat swollen glands and internally as a diuretic. The sap 20is used as an emollient for chest pain as well as for tumors and swelling. Leaf extracts are used as a wash for inflamed eyes. The Cambodians use Leaf extracts in inhalations for treatment of vertigo and migraine.
The Samoans use the sap as an arrow poison.
2.2 PROANTHOCYANIDIN MONOMERS AND
POLYMERS !~?dD THEIR D8E8 Proanthocyanidin and proanthocyanidin polymers are found as colorless phenolic substances in a wide variety.of many plants, particularly those with a woody habit of growth (e.g., the Croton species and Calophyllum inophylum). The general chemical structure of a polymeric proanthocyanidin consists of linear chains of 5, 7, 3', 4' tetrahydroxy or 5, 7, i 3', 4', 5' pentahydroxy flavonoid 3-0l units linked WO 921U6b95 ~ PCT/US91/07b79 together through common C(4) - (6) and/or C(4) - C(8) bonds, as shown below.
i7il ~~ X019 ilt . ~ I lo, ~. o . . ~.-J
i °"
~ boll VI/~
I10~ ~d ' i 0 ~
~~OII
al Biosynthetic studies have indicated that proanthocyanidin polymers consist of monomer units of the type shown below, See Fletcher et al., 1977, ' J.C.S. Perkin, 1:1628.
nll ~°~II
Clf /~.,,, _()1I r,lf. ~~, %,1l I ~Q I ~;. .J ~~'~' I It) ~.. ..: a..~k, «. . ,,~.. s ' "~ .1 °. ~.,: - ~ II
l~~ ~. ~.,~i~~ ~.=.....II
ao ~ ~ ~ ; oll UII clc '~~)11 'U11 clc 2a II-11 17 !d--II
2h .R = pll I Ir IZ - UI 1 The monomer unit (generally termed '°leucoanthocyanidin'°) of the polymer chain may be ' 25 based on either of two stereochemistries of the C-ring, at the 2 and/or 4 position designated ,cas (called epicatechins) or trans (called catechin).
Therefore, the polymer chains are based on different 30 structural units, which create a wide variation of polymeric proanthocyanidins and-a large number of possible isomers (Hemingway et al.; 1982, J.C.S.
Perkin, 1:1217). C13 NMFt has been useful to identify the structures of polymeric proanthocyanidins and 35 recent work has elucidated the chemistry of di-, tri~-and tetra-meric proanthocyanidins. Larger polymers of the flavonoid 3-0l units are predominant in most plants, and are found with average molecular weights iaVO 92/06695 PCT/US91 /07679 ;...,, above 2,000 daltons, containing 5 or more units, Newman et al., 1987, Mag. Res. Chem., 25:118.
Proanthocyanidins have been reported to possess protein binding ability and a possible i biological role (Newman et al., 1987, PJag. Res. Chem., 25:118). Proanthocyanidin monomers and diners have been used in the treatment of diseases associated with increased capillary fragility and have also been shown to have anti-inflammatory effects in experimental ,0 animals (Beladi et al., 1977, .~lnn. N.Y. ~icad. Sci., 2.84:358). A procyanidin monomer was found to have antiviral activity against Herpes Simplex virus in an in vitro assay. (Beladi, et al., supra.) Beladi et al. tested the viricidal effect of a number of ~5 flavonoid monomers including apigenin, pelargonidin, quercetin and a proanthocyanidin monomer on Herpes Simplex virus. The proanthocyanidin monomer was found to be the most effective in virus-inactivating activity, followed by pelargonidin and quercetin.
20 Apigenin had only a slight effect. The effect of quercetin and the procyanidin monomer on the multiplication of Herpes Simplex virus in HEp-2 cells was also investigated. As concluded by Beladi et al., the monomeric flavonoids tested had viricidal 25 activity, but only a slight inhibitory effect on virus multiplication in the in vitro assays.
Prior to the present invention, there has been no disclosure regarding the use of 30 proanthocyanidins containing two or more glavonoid units for treating respiratory virus infections.
Additionally, there has been no disclosure regarding , the use of proanthocyanidins of six or more flavonoid units for treating virus infections in general. , 35 Eor the purpose of the present application, a proanthocyanidin polymer isolated from the Croton species is designated °°proanthocyanidin polymer A" and that isolated from Calophyllum inophy~um is designated 5 ~ ~ ~ ~ ~ ~ ~ p~/Ug91/07679 °'proanthocyanidin polymer B". I3owever, such designation is solely for simplicity of discussion and not necessarily intended to imply that the polymers are different classes of compounds. Both proanthocyanidin palymer A and proanthocyanidin polymer B are considered to be within the term proanthocyanidin polymer as described herein.
2.3 ItR8PIRATORX 8~tN03tTIAL AIRQB
Respiratory Syncytial Virus (RSV, belongs to the virus family Paramyxoviridae, genus Pneumovirus, and is responsible for causing lower respiratory tract infections such as bronchiolitis and pneumonia in the infant and child. The virus is considered to be in ~5 important agent of acute respiratory disease in children and it is expected that up to 50% of infants will suffer from RSV infections during their first winter. In young children with pulmonary or cardiac disease, up to a 37% mortality rate has been reported 20 due to RSV. Although RSV respiratory infection is -common in the young, the virus is found in respiratory secretions of infected persons of any age. Outbreaks among the elderly have been associated with serious and fatal illness, and RSV infection can be the source 25 of fever and pulmonary infiltrates in immunosuppressed adults (England et al., 1988, An.n. Int. Med.,1s203).
A vaccine of inactivated RSV has proven to be ineffective against RSV infection and leads to an 30 unusual immune and lung inflammation upon subsequent RSV infection. Therefore, recent research has - concentrated.on developing either potent live vaccines or an effective antiviral compound.
~ Ribavirin, (1°(~-D-ribofuranosyl-l, 2, 4-35 triazole 3-carboxamide), the current drug of choice, has been found to reduce the severity of illness and amount of virus shed in acute respiratory infections of RSV or influenza virus. Currently ribavirin is WO 92/06695 p~'/US9'/07679--.
used for the treatment of RSV infections of the respiratory tract in aerosol form. However, because of its toxicity, ribavirin is less desirable for use systemically. ~dditionally,,ribavirin has been faund to be teratogenic in animals. This has raised serious cancern for female health care personnel administering c i or expased to ribavirin (Gladu et al., 1989, J. Toxic Env. Health, 28:1). ' ~0 2.4 OTTTIS ?IEDIA AIID OTITIB ERTEatNA
Otitis media, inflammation of the middle ear, is second only to colds as the most common disease of early childhood. lMore than 60~ of all children will have an episode of otitis media by age ~5 six. The greatest risk for the development of acute otitis media lies in infections caused by respiratory syncytial viruses, rhinoviruses, influenza A viruses or adenoviruses (Henderson, et al., IV. Eng. J. Med., 1982, 1377; Ruuskanin, et al., Pediatr. Infect. Dis.
20 J., 1989, 94; Sanyai, et al., J. Pediatr., 1980, 11).
It has been found that prevention of the respiratory viral infection decreases the incidence of otitis media (Heikkinen, et al., AJDC, 445 (1991). Use of a flu vaccine has been found to be effective. However, 25 there are a number of reasons why this is an unsatisfactory approach: it is necessary to administer the vaccine annually, two doses must be given initially, there is a problem of continual antigenic variation of the influenza viruses and the resulting fluctuation in efficacy of the vaccine and the cost.
s irrFL~Er~z~ vla~usE$-.., .. , Influenza viruses are members of the ~rthomyxovlridae family and are distributed world-wide. Five pandemics have accurred in the 20th century, with the 1918 outbreak killing at least 21 million. In the United States, aver 500,000 deaths W~ 92/06695 pCT/US91/07679 have been attributed to influenza epidemics over the last twenty years. The outbreaks are seasonal, occurring almost every winter, and transmission is primarily through the respiratory raute. Amantadine has been shown to shorten the fever duration and respiratory symptoms by only about 50%, but causes some central nervous system side effects.
Rimantidine, as yet unlicensed, shows a similar effect, although it is not associated with the central ~0 nervous system side-effects seen with amantadine.
Primary influenza viral pneumonia, a common complication of influenza infections, has not been shown to be treatable by either rimantidine or amantadine. Prophylactically, influenza has been 'S controlled by the use of inactivated influenza virus vaccines with about 80% protective efficacy.
2.6 ~IERPEB BIMF'LEX ~TRU8 Herpes simplex virus (HSV) is a member of 20 the Herpitoviridae famil y, and is distributed world-wide. Transmission of the virus occurs via direct contact from person-to-person, with common entry of HSV-1 through the oral cavity arid infection of HSV-2 through the genital tract. The prevalence of HSV
antibody is inversely proportional to socioeconomic status, with close to 100% of HSV-positive adults in underdeveloped countries and 30-50% in developed countries. Acyclovir, .idoxuridine (IDU or 2-deoxy-5-lodouridine), trifluridine and vidarabine (adenine arabinoside, Ara-A) are effective in treatment of various HSV infections, with trifluridine and acyclovir the drugs of. choice. Vidarabine, the choice _ for ophthalmic, infections, is not effective topically against herpes labialis or other skin/genital infections. Acyclovir is not effective topically when used against recurrent genital infections. However, it is effective when used i.v. and p.o. Acyclovir is PCT/U591l07679 ,...,~
_ ., ; .
8 _ not recommended for routine use in treatment of recurring FiSV genital infections as ganglionic latency is still present and outbreaks occur after treatment is stopped.
3 0 ~~Y ~~ ~~~~r I~~i~T~o The present invention relates to a method of treating respiratory virus infections, comprising administering to a warm-blooded animal, a therapeutically effective amount of an antiviral agent comprising a proanthocyanidin polymer. The proanthocyanidin polymer preferably contains 2 to 3o flavonoid units, more preferably 2 to 15 flavonoid units, and most preferably 2 to 11 flavonoid units.
The flavonoid units include but are not limited to catechins, epicatechins, gallocatechins, galloepicatechins, flavanols, flavonols, flavandiols, leucocyanidins, anthocyanidins, or combinations thereof. The flavonoid units can be singly or doubly 2~ linked to each other. More specifically, the method of the invention can be used to treat respiratory infections induced by a respiratory syncytialyvirus as well as Parainfluenza virus 3, Influenza A, Influenza B virus and virus associated with otitis media and otitis externs. The proanthocyanidin polymer can be administered intravenously, intraperitoneally, subcutaneously, intramuscularly, orally, topically, or by inhalation.
The proanthocyanidin polymers are effective against most of the viruses implicated in otitis media. They are soluble in agueous solutions, are readily formulated for parenteral, oral, topical (i.e., into ear and nose) administration and thus are admirably suited for use in the prophylaxis or treatment of otitis media.
The proanthocyanidin polymers are similarly effective against the viruses implicated in otitis dV~ 92/06695 ~ ~ ~ ~ ~ 2 ~ pCT/US91/07679 I
r externs. Because such viruses are located outside of the tympanic membrane, the proanthocyanidin polymers F
can be topically administered into the ear in a suitable formulation so as to come directly into contact with the viruses.
The proanthocyanidins useful for treating respiratory virus infections have a structure selected t0 PCT/US91 /07679 ...~
-from I, Il,,and III, and their esters, or ethers or the corresponding oxonium salts (HO)o /QHj~

n I II
/HOIa III
Where a, b = 1 to 3, x = 0 or 1, n = 0 to 28, preferably 0 to 13, more preferably 0 to 9.
TYae proanthocyanidin polymer useful for °
treating a respiratory virus infection can comprise 2 to 30, preferably 2 to 15, most preferably 2 to 1.1 !V0 92/0b695 ~ ~ ~ ~ ~ ~ ~ PCTlUS91/07679 monomeric flavanoid units having structure IV, or esters, ethers or corresponding oxonium salts thereof.
1 (OFI)r O \
(OH).
(ON), IV
where x, y = 1 to 3, z = 1 or 2.
Another embodiment of the invention further relates to a method of treating virus infections comprising administering, to a warm-blooded animal, a therapeutically effective amount of antiviral agent 2~ comprising a proanthocyanidin polymer containing 6 to 30 flavonoid units, preferably 6 to l5 flavonoid units and more preferably 6 to 11 flavonoid units. The flavonoid units include but are not limited to catechins, epicatechins, gallocatechins, 25galloepicatechins, flavanols, flavonols, flavandiols, leucocyanidins, anthocyanidins, or combinations thereof. The flavonoid units can be singly or double linked to each other. The method can be used to treat virus infections which are caused by paramyxovaridae, gp orthomyxovaridae or herpes viruses. The proanthocyanidin polymer can be administered intravenously, intraperitoneally, subcutaneously, intramuscularly, orally, topically, or by inhalation.
' The present invention also relates to 35 proanthocyanidins useful for treating virus infections in general having a structure selected from I, II, and III, above, and esters, ethers and corresponding W~ 92106b95 Ff:T/iJ~91107679, 0", - 12 - . .
, oxonium salts thereof, where n = 4 to 28, preferably ~4 to 13, most preferably 4 to 9.
The present invention also relates to novel proanthocyanidins which are obtained from a Croton 5species and from a CaZyphyllu;m.~.inophylum species and their esters, ether and oxonium derivatives. Such proanthocyanidin can be isolated from the whole plant, the bark, the leaves, the roots or the latex. In a preferred embodiment, the proanthocyanidin can be l0abtained from Croton Iechleri and from Calophyllum inophylum. These novel proanthocyanidin polymers are characterized by IR, UV-visible, and/or '3C NMR
spectroscopy.
~5 ~4. BRIEF DESCRIPTION OF THE FIGURES
Figure 1 Fourier-transform infra-red spectrum. of proanthocyanidin polymer A isolated from the Croton tree.
Ficrure 2 'H Nuclear magnetic resonance spectrum of 20proanthocyanidin polymer A isolated from the Croton tree. Sample was in Dz0 at 400 MHz.
Figure :3 Broad-band decoupled t'C nuclear magnetic resonance spectrum of proanthocyanidin polymer A in Dz0 at loo MHz.
FiQUre 4 The t3C NMR spectra of proanthocyanidin polymer B isolated from Calophyllum inophyIum, obtained at 100 MHz in DzO.
Figure 5 Fourier-transform infra-red spectrum o~
30proanthocyanidin polymer B isolated from Calophyllum inoplylum.
Firnxre 6 The effect of proanthocyanidiw polymer A and Amantadine on lung consolidation in mice infected with influenza A (HIN1) virus (early treatment initiation). ' 35Fi9u~ re 7 The effect of proanthocyanidin polymer A and Amantadine on lung virus titers in mice infected with influenza A (HIN1) virus (early treatment initiation).

i WO 92/06695 ~ ~ ~ ~ ~ ~ a p~/Ug9q/07679 ' Fiaure 8 The effect of groanthocyanidin polymer A and Amantadine on arterial oxygen saturation (SaO~) in male mice infected with influenza A (HIN1) virus (early initiation).
SFigure 9 The effect of proanthocyanidin polymer A and Amantadine on~arterial oxygen saturation (Sao2) in female mice infected with influenza A (HTN1) virus (early treatment initiation).
Fiaure IO The effect of proanthocyanidin polymer A
~Oand Amantadine on lung consolidation in mice infected with influenza A (HIN1) virus (late treatment initiation).
Figure 11 The effect of proanthocyanidin polymer A on lung titers in mice infected with influenza A (HIN1) ~5virus (late treatment initiation).
Figure 12 The effect of proanthocyanidin polymer A
and Amantadine on arterial oxygen saturation (Sa02) in male mice infected with influenza A (HIN1) virus (late treatment initiation).
20Fiaure 13 The effect of proanthocyanidira polymer A
and Amantadine on arterial oxygen saturation (SaO~) in female mice infected with influenza A (HIN1) virus (late treatment initiation).
Fiaure 14 The effect of Ribavirin on lung virus 25titers in mice infected with influenza A (HIND virus.
Figure 15 The effect of intraperitoneal Ribavirin treatment on lung consolidation in influenza A (HIN1) virus infected mice.
30 Figure 16 The effect of intraperitoneal Ribavirin treatment on blood Sa~2% in influenza A (HIND virus infected mice.
Ficture 1? The effect of proanthocyanidin polymer A
and Ganciclovir on HSV-2 vaginal lesions in mice.
35Figure 18 The effect of proanthocyanidin polymer A in 5% and IO% topical formulations, compared to placebo and acyclovir, in a 5% topical formulation.

-' 14 5. DETAILED DESCRIPTION OF 'THE INVENTION
5..1 DBEFOL PROANTHOCYANIDZN POLY?iERB
Proanthocyanidin oligomers or polymers, useful for the present anti-viral methods are 'comprised of monomeric units of leucoanthocyanidins.
Leucoanthocyanidins are.generally monomeric flavonoids which include catechins, epicatechins, gallocatechins, galloepicatechins, flavanols, flavonols, and flavan-3,4-diols leucocyanidins and anthocyanidins. The ~oproanthocyanidin polymers useful for treating respiratory virus infections have 2 to 30 flavonoid units, preferably 2 to 15 flavonoid units, and~most preferably 2 to 11 flavonoid units. The proanthocyanidin polymers useful for treating virus ~5infections in general, including but not limited to, infections related to influenza viruses, parainfluenza viruses, viruses of the types known .as paramyxoviridae, orthomyxovaridae, and herpes viruses, have 5 to 30 flavonoid units, preferably 2 to f5 20flavonoid units, and most preferably 6 to 11 flavonoid units.
Proanthocyanidin polymers having a varying number of flavonoid units are known and have been reported, for example, in W.L. Mattic:e, et al.
?5Phytochemistry, ~,, p. 1309-1311 (1984); Z.
Czochanska, et al., J.C.S. Chem. Comm., 375 (1979);
W.T. Jones, et al., Photochemstry, ,~, p. 1407-1409 (1976); E. Haslam, Plant Polyphenols, p. 75 (1989).
~ Those polymers having the recited ranges of flavonoids units and described in these references are useful for the methods of the present invention.

WO 92/06635 2 0 ~ ~ $ ~ ~C PCfi/US91/07679 15 - s r 5.2 METHODS FOR OBTAIArIING USEFtTL
PRO!-1NTHOCYAATIDIN POLSCPdERB
x.2.1 I80LATION
Useful proanthocyanidin polymers are obtained from various plants including but not .limited to the classes Filices, Coniferae, Monocotyledoneae, and Dicotyledonae (J. B. Harborne, THE FLAVONOIDS, ADVANCES IN RESEARCH SINCE 1980 (1988)). They can be obtained using the, entire tree or plant, the bark, '0 stems, roots or latex.
These plant source materials are extracted with water and/or a water miscible solvent. The preferred solvents are alcohol of 1-3 carbon atoms or acetone. The aqueous extract is used directly or ~5 after solvent removal. If the solvent is removed, the solid residue is redissolved in a solvent, preferably a lower alcohol or acetone, and insoluble materials are discarded. The soluble fraction is subjected to gel filtration (e. g., over a Sephadex), reversed-phase 20 column chromatography (e. g., C-8), or gel-permeation chromatography (e. g., divinyl benzene cross-linked gels) such as PL-GEL or membranes (e. g., an Amicon membrane) using water or water and a water miscible solvent, with or without a buffer, as the mobile 25 phase. The water miscible solvent is preferably a 1-3 carbon alcohol, acetone or acetonitrile.
The useful proanthocyanidin polymers of this invention are the fractions detected by ultraviolet (uv). They have. their major UV absorption maxima 30 between about 200-350 nm, with peaks usually at 200-210 nm, and 275285 nm.
With regard to the novel proanthocyanidin polymer compositions which form part of the basis of this invention, such polymers can be obtained from the 35 Croton tree or Calophyllum species by using the above-described extraction method. The soluble fraction is subjected to gel filtration, reversed-phase column dV0 92/06695 PCT/US91/07679 ,",~
2og3~~'a _ is -chromatography, gel-permeation chromatography, or membranes. Using water or water and a water miscible solvent, with or without a buffer as the mobile phase and the relevant fractions containing the polymers are 5detected using uv at a range of about 200-350 nm.
According to a preferred embodiment of the present invention, a novel proanthocyanidin polymer can be prepared as follows: Latex obtained from a Croton tree (e. g., Croton 3echlera) or from 10Ca1ophyllum inophylum is used directly or is concentrated by the removal of water (e. g., lyophilized). It is extracted with water, a lower alcohol of about 1-3 carbons, or with acetone to give an aqueous soluble fraction. The aqueous-soluble ~5fraction of the lyophilized or otherwise concentrated material is subjected to partitioning by ethyl acetate/water solution and to gel filtration (e~.g., Sephadex) using water and/or water and alcohol and/or water and acetone, with or without a buffer as the 20mobile phase. The fractions containing a chromophoric band are detected by ultraviolet (uv) (Amax about 200-350 nm) and collected and concentrated and subjected to gel filtration again, if necessary, to yield the 25proanthocyanidin polymer. Alternatively, the aqueous-soluble fraction described above is lyophilized or concentrated or is used directly and is subjected to reversed-phase column chromatography (RP HPLC), using water and/or water and acetone and/or water and 30 alcohol as the mobile phase. The fractions containing a chromophoric band detected by uv (max about 200-350 nm) are collected and concentrated, and subjected to RP HPLC again, if necessary, to yield the proanthocyanidin polymer. Alternatively, the agueous-35soluble fraction, described above, is lyophilized, concentrated or is directly subjected to gel permeation chromatography (GPC), using water and/or water and alcohol and/or water and acetonitrile, with WO 92/06695 - ~ ~ ~ ~ ~ ~ P~'/11591/07679 or without a buffer, as the mobile phase. The fractions containing a chromophoric band detected by r r uv at Amax about 195-350 nm are collected and ' concentrated and subjected to GPC again, if necessary, 5to yield the proanthocyanidin polymer. See examples ' i in Sections 6.1 - 6.3, infra.
As demonstrated in Section 6.2 when chromatographed on a Perkin Elmer LC-62o using Polymer Labs PL-gel 5m 500 A, 300 x 7.5 mm and THF-water (95:5 1D v/v) system at a flow rate of 1 ml/min, proanthocyanidin A has a retention time of 7.2 min.
and proanthocyanidiri B has a retention time of 6.5 min. Typical spectra for proanthocyanidin polymers are shown in Figs. 1 and 5.
i5 5.2.2 SY~lTHE8I8 OF U8EF'UL PROAIJTHOCYANIDIP1 POT.YMER~ APID DERIVATIZTEB THEREOF
It is known that leucoanthocyanidins can be condensed in mildly alkaline or acidic solutions to 20form proanthocyanidins. As an example, L.Y. Foo and R.W. Hemingway, J. Chem. Soc., Chem. Commun., 85 (1984) were able to synthesize a trimer from flavanol monomers. J.A. Delcoun, et al., J. Chem. Soc. Perkan T'rans. I., 1711 (1983) teaches that under mild, low 25pH, conditions and an excess amount of (+)-catechin, condensation occurred to form higher and predominantly linear [4,8~-linked oligomers. Specifically, dimers, trimers and tetramers of the flavonoid analogues were synthesized and characterized by NbIR spectroscopy, By 3D use of such condensation reactions proanthocyanidin polymers of up to about 30 flavonoid units can be synthesized for use in the methods of the inventian.
As an alternative to the above synthesis, enzymatic synthesis can be employed to produce the useful 35 proanthocyanidin polymers. The novel proanthocyanidin polymer compositions of the present invention ~~ 92/~6695 lP~f/US91/07679 ....., specifically exemplified herein can also be synthesized by such reactions.
Esters of proanthocyanidin polymers can be prepared by standard methods of acylation such as by 5the reaction of the polymer with acid chlorides or acid anhydrides in the presence of base. Of particular utility is the preparation by reaction with acid anhydrides in pyridine as utilized for the acylation of certain other lower molecular weight l0flavonoids by Thompson, et al., JCS, 1387 (1972), Fletcher, et al., JCS, 1628 (1977) and Hemingway, JCS, 1299 (1982).
Ethers can be prepared by standard methods of etherification such as by reaction of the polymer l5with alkyl halides and alkyl tosylates in bases.
Methyl ethers are readily prepared by the use of diazomethane as utilized in the etherification~of certain other low molecular weight flavonoids by Thompson, JCS, 1387 (1972), Hemingway, JCS, 1299 20 (1982) .
The oxonium salts (sometimes referred to as pyrilium salts or anthocyanidins) may be prepared by the so-called E~ate-Smith reaction (Chemistry and Industry, 1953, 377), by warming with aqueous or 25alcoholic acids (see also, Thompson, et al., JCS, 1387 (1972) and references cited in liaslam, PLANT
POLYPHENOLS, 1989, p. 28, Cambridge Press). This reaction can be advantageously catalyzed by iron 30 (Porter et al., Phytochemistry, 25, 223 (1986)).
5.3 CxARB~CTERIZATI02~T OF NO~EL
PROANTFiOCYANIDITT POLYMERS
Novel proanthocyanidin polymers prepared according to the present invention have solubility in ~5methanol and water. The polymers are soluble in water and aqueous solution including alcohol solutions to a degree of at least about 10 mg/ml.

W() 92/06695 .. 2 0 9 ~ ~ ~ ~ Pt T:T/LJS9a/07679 The proanthocyanidin polymers have been analyzed by a number of methods to determine their molecular weight and various other chemical and physical features. Various stereoisomers of 5proanthocyanidin polymers have been obtained and are within the scope of the present invention.
Column chromatography has been used to isolate water soluble fractions containing novel proanthocyanidin polymers varying in average molecular 'Oweight from about 700 daltons to about 3000 daltons, which corresponds to 2-3 to 9-11 average flavonoid units, respectively.
~3C-NMR spectroscopy indicates that the proanthocyanidin polymers contain flavonoid moieties 'sin which the individual flavonoid ring units possess various stereochemistries.
W-visible spectroscopy is consistent with the possible presence of a flavylium moiety or moieties within certain of the proanthocyanidin 20 polymers .
The characterization of proanthocyanidin polymer A and proanthocyanidin polymer S is further discussed in the examples at Sections 6.3 and 6.5.
25Functional derivatives of these polymers can be made and may be useful as intermediate for the preparatian of useful proanthocyanidin polymers.
5. ~ PROPHYLACTIC AND °rHEIt7~PEUTTC I18E8 OF pIt0~~3dTHOCYANIDI_1~1 hOLYMERB
30 proanthocyanidin polymers have been shown to be active in vitro and in vivo against a wide variety of viruses, and can be advantageously used in prophylactic and therapeutic:applications against 35 diseases induced by such viruses.
The proanthocyanidin polymers can be used either alone or in combination with other antiviral or antimicrobial agents to prevent and/or treat diseases WO 92/06695 . . . PC~'/US91/07679 "~
-induced by or complicated with viral infections from viruses including, but not limited to>: paramyxovaridae such as respiratory syncytial virus, orthomyxovaridae such as influenza A, B and C, and herpes viruses such 5as Herpes Simplex virus. -Proanthocyanidin polymers have various advantages in the treatment of viral infections including, but not limited to:
1) a broad range of antiviral activity;
2) very low toxicity;
3) no teratogenicity; and 4) _ application to both systemic as well as localized (i.e., topical) applications.
5.5 RODTES OF ADMIHIBTRATI03~1 The proanthocyanidin polymers of the present invention can be administered for prophylactic and v therapeutic applications by a number of routes, including but not limited to: oral, injection 20including but not limited to, intravenous, intraperitoneal, subcutaneous, intramuscular, etc., by topical application such as to nasal, nasopharyngeal linings and into the ear, and by inhalation via aerosolization and application to respiratory tract 251inings, etc.
When used according to the present invention against viruses, effective dose.ranges of the proanthocyanidin polymers are about 5.0 - about 30 30 mg/kg if given orally.(P.O.), about 0.1 - about 10 mg/kg if given intraperitoneally (I.P.)~, and about S --about 30 mg/kgjday if given by aerosol. Yf given topically; the proanthocyanidin polymer is applied in a suitable vehicle at a concentration of about 5 to 35 about 15% .
When administered to warm-blooded animals, including humans, the proanthocyanidins may be W ~ 92/06695 ~ ~ ~ ~ ~ ~ ~ PC'f/tJS91/07679 2i -combined with water, an aqueous solution or any physiologically acceptable carrier or vehicle.
The following series of Examples are presented for purposes of illustration and not by way 5of limitation on the scope of the invention.

6. EXAF~FLE~t 6.1 IBOL1~TIOI1 OF ~1 Pi0'~Eh PROANTHOCY7~3ITOIN
POLYMER FROM 1A CRCP3bN .LECHLERI BPECIEE
~PROANTHOCY~sFdIDIN POIeYMI~R A
In one series of experiments, a novel proanthocyanidin.polymer was obtained as follows:
C: lschleri trees were tapped and felled near the ~rillage of San Pablo de Guyana on the Nanay River 100 kilometers from Tquitas, Peru. The latex l5was obtained over a period of 24 hours by scoring the trees.
The latex (11) obtained from the Croton lechleri trees was diluted with isopropanol in the 20ratio of 1 part latex to 3 parts isopropanol (31} and allowed to stand for 15 hours at l0°C. The. resultant isopropanol-diluted latex was centrifuged, and the insoluble material was removed by decantation, leaving the mother liquor (3.7 1). The mother liquor was 25concentrated to dryness by rotoevaporation at 33°C to give 24o g of a deep-red brown powder. This concentrated material (9~0 g) was subjected to gel filtration using Sephadex CM-50 with water (20 1) as the mobile phase. The early-eluting fractions 30containing a red pigment were detected at ~ 200-350 nm or by visual detection of a red band eluting through a transparent column. To isolate the proanthocyanidin polymer, the early-eluting fractions were collected and subjected to gel filtration chromatography and/or 35HPLC as described below.
The HPLC isolation was performed by injecting 5 dal of concentrated samples onto a Perkin-Elmer LC Analyst liquid chromatograph system equipped with an IC-200 autosampler, LC 600 pumps and a LC-235 diode array detector using a Waters Ultrahydrogel 500 GPC column with Burdick and Jackson HPLC-grade water Sat 0.8 ml/min at ambient temperature, with detection at ~ 280 and 195 nm. The proanthocyanidin polymer Was found to have a retention time of 4.6-5.6 minutes depending on sample concentration, column conditioning and temperature.
'0 The red-pigment containing fractions isolated from SephadexT"" CM-50 were combined and subjected to gel filtration chromatography over ToyopearlT"° HW-40S (2 . 5 L) , using water, 10%
acetone/water, 20o acetone/water, and finally 40%
~5acetone/water in a step gradient fashion. The late-eluting red fractions detected by uv-vis as Amax 340 nm, were combined and concentrated for further purification on HPLC as described above. The combined fractions isolated from HPLC were again subjected to 20ge1 filtration chromatography over Toyopearl HW-40S to yield a proanthocyanidin polymer designated proanthocyanidin polymer A.
In another series of experiments, a novel 25proanthocyanidin polymer was obtained as follows:
Four liters of cold crude latex from the C.
Iechleri (or from the entire macerated plant, the bark or the roots) were diluted with twelve liters of isopropanol, stirred and stored at 5°C for 15 hours.
30The residue was removed by filtration and the solution evaporated to dryness, in vacuo, yielding about 970 g.
of solids.
The solids were added, with stirring, to 6 1 of water and 3.6 1 of n-butanol. The aqueous phase 35was separated and concentrated to dryness giving 700 g of material which was added, with stirring, to 2 1 methanol, and then I2 I of ethyl acetate was added.
The solution was kept at 15° for 15 hrs and the solid 2a93~~;~

material removed by filtration and discarded. The 1 solution was concentrated to dryness yielding ;
approximately 390 g. of crude proanthocyanidin polymer A. '.
The proanthocyanidin polymer A fraction was concentrated by a combination of ration exchange, adsorption and size exclusion chromatography as follows: 450-600 g of the crude proanthocyanidin polymer A was passed through a CM-Sephadex C-50 pre-column using de-ionized water as the eluent. The orange and dark red fractions were collected and then chromatographed, using de-ionized water, over another Sephadex C-50 column equipped with a LJV detector set at 460 nm, and 1-2 1 fractions were collected. The ~5 initial pink band was discarded and the subsequent effluent passed through a column containing Sephadex G-5o until the broad red-brown band was eluted. 15%
aqueous acetone was passed through the G-50 column until the effluent was colorless. Fractions of the effluent were examined by HPLC as described in Section 6.2, and those fractions containing proanthocyanidin polymer A were combined and evaporated to dryness yielding about 210 g, of proanthocyanidin polymer A.
6.2 POZtIFTCATTO~d OF PROAPrTIiOCYANIDIId POLYMER A
About 150 g. of crude proanthocyanidin polymer A, obtained as described in Section 6.l above, was dissolved in 300 ml 20% aqueous acetone and was chromatographed over a mixed-made gel-permeation/absorption column (Toyopearl HW40S, a spherical methyl methacrylate polymer of 40 ~Sm particle size). 16 1 were collected, then the eluting solvent was changed to 40% aqueous acetone and 8 more liters were collected, then the elution was continued with 4 1 of 60% aqueous acetone. The fractions containing proanthocyanidin polymer A, as shown by - 24 - PGT/tJS91/07679I:~
HPLC (See Section 6.3), were combined and the solvent removed in vacuo yielding approximately 57 g. of solid.
Final purification was accomplished by.a combination of adsorption and size-exclusion chromatographya 50-75 g. of ttie above solid were dissolved in ~0% ethanol and introduced onto a column containing Sephadex LH-20 (a cross-linked dextran gel with hydroxypropyl groups attached by ether linkages to glucose units of the dextran chains). hlution was carried out using 10 1 of 90% aqueous ethanol, then 15 1 of 20% aqueous acetone, 5 1 of 40% aqueous acetone, 5 1 of 50% aqueous acetone and then 5 1 of s0% aqueous acetone. Fractions were collected at 2 1 intervals and assayed by HPLC. The fractions containing proanthocyanidin polymer A were combined and evaporated to dryness in vacuo at 35° giving 35 g. of pure proanthocyanidin polymer A.
For the HPLC, a 30 cm gel-permeation column for non-aqueous mobile phases was used in which the stationary phase is Polymer Laboratories PL-Gel 5m 500 (a divinylbenzene-polystyrene polymer, 5 ~a particles, 500. pore size). The HPLC system was equipped with a diode array detector that generates W
spectra, with the detector set at 280 nm.
Samples are dissolved in 95% aq THF, which is also the developing solvent. At a flow rate of 1 ml/min, the proanthocyanidin polymer A peak maximum has a retention time of 7.2 ~ 0.5 min.;
roanthoc anidin p y polymer B has a retention time of 6.5 ~ 0.5 min, indicating that proanthocyanidin polymer B
has a larger molecular weight than proanthocyanidin polymer A.

WO 92/06695 - 25 2 ~ ~ ~ ~ ~ ~ pC~'/US91/07f>79 6.3 CF~EMICAL IDENTIIt~IC:ATIO~t AIdD STRUCTURAL
FEATURES 0f THE PRIaAIdTHOCYAP1IDIBd POLYMER ~I, In the infrared, the proanthocyanidin polymer A of the invention, obtained as described in Section 6.1 above, shows a very broad intense peak ranging from 3550-2500 and ether peaks at 1612, 1449, 1348, 1202, 1144, 1107, 1068 and :L027 cm-1. See Figure 1. t The 1H NMR spectrum in Dz0 (400 Ml3z 24°C) i0 exhibited very broad peaks at d 7.1, 6.9, 6.1, 4.7 and 2.8 ppm. fee Figure 2.
Ultraviolet-visible spectral analysis in Hzo revealed broad peaks at ~ 202, 235, (shoulder), 275, 305 (shoulder), 460 and trailing greater than 600 nm.
Although the UV data of the present polymer closely resembles'those of other known proanthocyanidins, the visible data are clearly different. The known proanthocyanidin~monomers and polymers are colorless (~ 205, 240, 275 nm) and have no absorption in the visible range, whereas certain of the isolated novel proanthocyanidin polymer A of the present invention are colored and have a visible absorption at 460 nm.
The color of the proanthocyanidin polymer suggests the a5 presence of a flavylium moiety or moieties within the proanthocyanidin polymer. This is consistent with the visible spectroscopic data reported for closely related monomeric anthocyanins which contain the flavylium moiety (~ 460-560 nm).
The 13C NMR spectra of the proanthocyanidin polymer was obtained at 100 MHz in DZO. The experiments performed were broad-band decoupled. As shown in FIG. 3, the 13C NMR spectrum in DZO exhibited very broad peaks at 8 155(C-5, C-7, C-9), 145(C-3', C-3b 5' )', 143 (C-3' , C-~4' ) , 130 (C-1' , C'-1' °, C-4'') , 128 (C-1'), 121(C-6), 116(C-2', C-5'), 109(C-8, C-2'', C-6'*), 97(C-6), 82(C-2), 76(C-2), 73(C-3), and 38(C-4) ppm, ', WO 92/06695 PCT/US91/07679 for the 'prodelphinidin 8 ring. The 13C NMR data suggest some key points of structural differences between the novel antiviral proanthocyanidin polymers of the present invention and known proanthocyanidin polymers. The most significant difference is a substantially larger peak in the region of 109 ppm for the proanthocyanidin polymers of this invention when compared with 13C Mgt spectra of other proanthocyanidin polymers known in the literature 0 (compare with spectra in Czochanska et al., 1979, J.C.S. Chem. Comm., p. 375-77; in Harborne, J.H. and Mabry, T.J. ed., The Flavonoids: Advances in Research, Chapman and Hall, NY, 1982, pp. 51-132). The 13C NMR data of the polymer is indicative of 5 the proanthocyanidin class of polymers. In particular, the 13C NMR chemical shift data of the isolated proanthocyanidin polymer (C-6'=132 ppm, C-2'=115 pm, C-3' & C-4'=145 ppm, C-5'=116 ppm, C-6'-107 ppm, C-3' & C-5' =146 ppm, C-4'-133 ppm) 20 is consistent with a polymer composed of procyanidin H
ring moieties with the individual flavonol C ring units possessing both the 2,3-trans and 3,4-traps [similar to (+)-catechin; C-2=83 ppm, C-3=73 ppm, C-4=38ppm] and 2,3-cis-3,4-traps [similar to (-)-epicatechin; C-2=77 ppm, C-3=73 ppm, C-4=73 ppm]
stereochemistries. The present experiment data indicates that proanthocyanidin polymer A is comprised of catechin, epicatechin, gallocatechin and galloepicatechin. The HPLC data suggest that the average number of flavonoid units is about 7. The HPLC data also suggests that number of flavonoid units vary from 2 to 11.
The proanthocyanidin polymer A is soluble in methanol, water and aqueous solutions. The proanthocyanidin polymer A is soluble in water at concentrations of at least about 10 mg/ml. The polymer has lower solubility in normal saline and WO 92lO~bb95 2 p 9 ~ g ~ ~ P(°T/US91/07679 _ 27 other salt solutions. Mass spectral analysis indicate that proanthocyanidin polymer A has a molecular weight average of about 2,100 daltons.
5. ~ I&OL~TIOId OF A PROIs.NTHOC~1ANIDI~I POLYMER
~ROlsi CAT..C9F'HYh.Li7!! Il'dOPI~YLilPi (PROANTHOCYANIDIly1 POLYMER 81 A novel proanthocyanidin polymer according to the present invention can be isolated by a method similar to that described above in Section 6.1 from the entire macerated plant, the bark, the leaves, the roots or the latex of Calophyllum anophylum.
AccorDing to a preferred method, the proanthocyanidin polymer is obtained by the method described in Section 5~1, except, that water is used as the preferred extraction solvent.
~In one series of experiments, a novel proanthocyanidin polymer, designated proanthocyanidin polymer B, was obtained from Calophyllum inophylum latex.
2,849 g of the latex from Calophyllum anophylum was mixed with 12.4 1 of a 1:1 mixture of isopropanol and water, stirred and stored at room temperature for 35 hours. The residue was removed by filtration and the solution was evaporated to dryness, in vacuo, giving 133.5 g of solids.
The solids were added, with stirring, to 30 g of methanol. The solution was then filtered away from the solids, and.a 1:1 mixture of water and ethyl acetate was added. The water fraction was separated and n-butyl alcohol was added. The water fraction was separated from the alcohol fraction and concentrated to dryness yielding approximately 10.4 g of crude proanthocyanidin polymer B.
3.'> The crude proanthocyanidin polymer B was passed through a CM°50 Sephadex CC column using de-ionized water as the eluting solvent. A red band was - 2 8 - -..., collected and fractionated using a LH-20 CC column, eluted with 70% aqueous ethanol solution and 20%, 50%
and 70% aqueous acetone solutions t:0 give, proanthocyanidin polymer B. At lower concentrations, a solution of proanthocyanidin polymer B is essentially colorless. At higher concentrations, the solution of proanthocyanidin polymer B is tan.
6.5 CHEMICAh IDENTIFICATION AND STRUCTURAL
FEATURES OF THE PROANTHOCYANIDIN
POLYMER B
The '3C NMR spectrum of proanthocyanidin polymer B (Figure 4) confirms that the polymer is a member of the class of proanthocyanidin polymers and comprises principally catechin and epicatechin monomeric flavonoid units. Gel permeation chromatography (GPC) indicates that proanthocyanidin polymer B has a molecular weight that is larger than proanthocyanidin polymer A. Consistent with the GPC
20 data, HPLC-GPC retention time of proanthocyanidin polymer B is shorter than that for proanthocyanidin polymer A under the same conditions -- again indicating the larger size of proanthocyanidin polymer B. HPLC indicates that polymer B has a molecular 25 weight average of about 3000 daltons, corresponding to an average number of flavonoid units of about 10. The HPLC data also suggests that the number of flavonoid units vary from 5 to 16. Mass spectral analysis indicates that proanthocyanidin polymer A has a 30 molecular weight average of about 2100 daltons.
The Fourier-transform infra-red spectrum of .
proanthocyanidin polymer B is quite similar to that of _ proanthocyanidin polymer A (See Figure 5 and Figure 35 Likewise, the W-visible spectrum of proanthocyanidin polymer 8 is similar to that of i i ~ ~ ~ 3 ~ 2 ~ 1PCT/US91/07679 proanthocyanidin polymer A except for the absence of the peak at 460 nm.

7. SCREENING OF PRORN'f~iOCYANIDIN POLYkiER A
FOR ANTI~IRAI. ACTIVITY
In one series of experiments, proanthocyanidin polymer A was tested for antiviral i activity against the following viruses: Respiratory' syncytial virus subtype variants, A2-Tracey, A-Long, B-46791, B-47063 and B-18537; parainfluenza virus, type 3 (PIV-3); adenoviruses, type 5 and 7; influenza, A-Taiwan (HIND; A-Leningrad (H3N2); A-Japan; A-Port Chalmbers; A-NwS33; B-USSR; B-Tama; B-RF; measles - virus, and Edmonston strain.
All viruses were obtained from the Influenza Research Center, Baylor College of Medicine, Houston, Texas, with the exception of measles virus which was obtained from ATCC. For comparison, ribavirin was included in the screening assay.
The following procedure was used to assay for antiviral activity. Assays were performed in 96°
well tissue culture plates. All dilutions and tissue culture suspensions were prepared in minimal essential medium containing antibiotics penicillin and streptomycin and 2% fetal calf serum (2% FCS-MEM).
Test compounds (0.05 ml) were added in quadruplicate to wells of the test plates. containing a subconfluent monolayer of HEp2 cells (ca. 3 x 103 cells). The compounds were diluted using serial 2-fold dilutions usually starting with a final concentration of 1 mg/ml. Approximately 100 median tissue culture infectious doses (TCID~) of the appropriate test virus in 0.05 ml was added. Tissue control wells contained medium, without virus or antiviral compound, and antiviral control wells contained antiviral compound i without virus. Ribavirin was included in each assay as a positive antiviral control, except for the ~'O 92/06695 2o~~~z~
PCT/US91 /0'7679 _ 3 0 - --adenoviruses where Ribavirin fails to demonstrate ' antiviral activity. Back titrations of each test virus were also included ineach assay. All plates were incubated at 37°C in a 5% COZ incubator. Virus i control wells were observed daily. When these wells exhibited 80-100% cytopathic effect (CPE), all wells were observed for CPE. In addition to visual and macroscopic observance of CPE, inha.bztion of syncytial formation was used to confirm activity against RSV.
In each antiviral assay, a 50% minimal inhibitory concentration (EDso) was determined. The EDso was calculated by determining the median minimal concentration of compound tested in wells inhibiting CPE 50% compared to virus control wells. The actual calculation of the EDso value was done with the aid of the computer program, "Dose-effect analysis with microcomputers" of Chou et al., 1984, Adv. Enz.
ReguZ., 22:27-55.
The effect of each compound on the growth of uninfected tissue culture cells, seeded at low densities to allow rapid growth, was also evaluated.
From this assay, a 50% minimal toxic concentration (IDsa) was determined. The assay for cell toxicity or inhibition of cell growth involved the following procedure. Test compounds (0.1 ml/well) were serially diluted 2-ofld. To the appropriate wells were added 0.1 ml of human HeLa, A549 or HEp2 cells, 0.1 ml of mouse L929 cells, 0.1 m of monkey Vero cells or 0.1 ml of canine MDCK cells. Approximately 3 x 103 cells were added to each well. Control wells consisted of wells containing a range of cell concentration (e.g., 3 x 103 s cells, 1:2 dilution of this number, 1:4, 1:8 and 1:16) in medium without any antiviral compound. A vehicle control consisting of serial dilutions of whatever vehicle was used for a particular compound (e.g., 1~%
DMSO, 50% methanol) was also included in each assay in WO 92/06695 ~ ~ ~ ~ ~ ~ ~ i'CT/US91/07679 duplicate. All plates were incubated at 37°C in a 5%
COZ incubator. After control wells containing cells, but no test compound, reached confluency, 3-[4,5-Dimethylthiazol-2-yl]-2,5-Diphea~yltetrazolium Bromide (MTT) was added to all wells. Three hours later, acid alcohol (0.1 ml of 0.4 N HCl in isopropyl alcohol) was added to each well to solubilize any precipitate formed in each well. Plates were read on a plate reader (W MAX, Molecular Devices) to determine the '0 optical densities in each well. The median concentration of antiviral compound in the last wells causing a 50~ reduction in O.D. was determined and termed "IDjo°'. All wells in the assay were also observed microscopically for inhibition of cell ~5 growth. The results are shown in Table 1.

WO 92/0S695 PCT/US91/07679"6,, _ .

TABhE ~
Egfect of In Yitro Screening ~or Antivirai Activity with Proaathacyaaaidin polymer A
EDso ~p~g/ml) Virus Proanthocvanidin Polymer A Ribavirin Experiment 1 RSV 8.5 4 Parainfluenza .virus >94 .

15.6 Influenza A 88 15.0 Influenza B 125 18.0 Adenovirus >125 Rhinovirus >125 Measles >125 31.3 'S Experiment 2 RSV 17.2 24 Parainfluenza virus 38 .

. ;15.7 Influenza A 31 . 14.3 Influenza B 125 18.0 Adenovirus 5 >250 >lODO
Adenovirus 7 >250 >1000 Rhinovirus >125 225 Measles >125 31.3 Experiment 3 Parainfluenza virus 3 5 12 Influenza A 4 14 Adenovirus 7 >250 >1000 In the first experiment of Table d, the EDso for the proanthocyanidin polymer A was determined to be 8.5 ~,g/ml for RSV. This activity compares quite closely to,ribavirin with a EDso determined to be 4.0 yg/ml for RSV. The Ipso (an index of cell toxicity) of the proanthocyanidin polymer A was 94 ~rg/ml. In the second experiment of Table 1, the EDso for the proanthocyanidin polymer A was 17.2 ug/ml for RSV as compared to 24.2 for ribavirin, and the IDso was 250 ,ug/ml. In the third experiment of Table 1 the EDso for 20~~82~
WO 92/Q6695 PCT/US9i/07679 ..
the proanthocyanidin polymer A was 12 ~g/ml and the Ipso was greater than 250 ~cg/ml, The results of antiviral activity of the proanthocyanidin polymer A compared to ribavirin in each assay was determined by calculating the drug Selective Index (ST) , defined as the ratio of ID~/EDso~
The Selective Index of the proanthocyanidin polymer A
for RSV was 11 and 14.5 and 21 in the three experiments.
Virus rating (VR) was obtained by averaging the sum of the CPE values (0 for no CPE; 4 for 100%
CPE) assigned to treated, infected monolayers at each compound concentratian. This average is subtracted from the average of the sum of the CPE values in an 'S equal number of virus control wells. A further adjustment was made to reflect any observed cytotoxicity. Values for VR were assigned as follows:
VR greater than 1, defined as definite anti-viral activity; a VR of 0.5-0.9, as moderate to questionable activity; a VR of 0.1-0.5, as slight activity. The slight activity could be attributable to cytotoxicity or eytopathic effects of the compound per se, The VR
of the proanthocyanidin polymer A against RSV was 1.5, indicating definite anti-viral activity in these in .5,itro assays. Also, the proanthocyanidin polymer A
demonstrated substantial anti-viral activity against parainfluenza and influenza A viruses in all three experiments.
In another series of experiments, polymer A
was tested for antiviral activity against RSV, types A
and B, influenza types A (Flu-A) and B (Flu-B), and parainfluenza types (PIV-1)~and 3 (PIV-3).
The cell lines used were Human HeLa, A549 and HEp-2 cells, mouse L929 cells, monkey Vero cells, canine MDCIC cells.
The effect of the proanthocyanidin polymer A
on the viability and growth of cells was determined by wa 9xioss9s PCT/IJ~91/0'7679....,., measurement of mitochondrial respiration and expressed as a 50% minimal toxic concentration {IDso). In this assay, as in the. ED50 assay, the p:roanthocyanidin polymer A was assayed in serial 2-:fold dilutions in quadruplicate across 96-well micro~titre plates.
Again, wells were seeded at the low density of 3x103 cells per well to allow for rapid growth, Vehicle controls which consisted of serial dilutions of vehicle (which, in the case of proanthocyanidin polymer A, was water)' were included in each assay in duplicate. Hlanks which consisted of media alone and media plus drug were also included. All plates were incubated at 37°C in a 5% C02 incubator until the tip concentration of cells in control wells reached confluency, usually 3 days. All wells in each assay were observed microscopically for cyctotoxicity. Then 0.05 ml of MTT (3-[4,5-dimethythiazole-2-yl)-2,5- .
diaphenyltetrazolium bromide)(5 mg/ml in PBS) was added to all wells and the incubation continued.
Three hours later acid alcohol {0.05 ml of 2d HC1 in isopropyl alcohol) was added to each well. The optical densities (0.D.) of each well were read at 490 nm using a plate reader (W MAX, Molecular Devices).
The percent viability of cells at each concentration of drug was calculated by dividing the mean of the O.D.'s of the drug treated wells {minus the mean of the drug blank) by the mean of the o.D.'s of control wells (minus the mean of the blank), and multiplying by 100. The effect of the vehicle on cell viability was similarly calculated. A dose response curve for the toxicity of proanthocyanidin polymer A was generated and the IDso determined.
The following procedure was used to assay for antiviral activity. Assays were performed in 96-well tissue culture plates. All dilutions and tissue culture suspensions were prepared in minimal essential medium containing antibiotics penicillin and Wa 92/06695 PCT/US91/07679 streptomycin and 2% fetal calf serum (2% FCS-MEM).
Test compounds (0.05 ml) were added in quadruplicate to wells of the test plates containing a subconfluent monolayer of HEp2 cells (ca. 3 x 103 cells). The 5 compounds were diluted using serial 2-fold dilutions usually starting with a final concentration of 1 mg/ml. Approximately 100 median tissue culture infectious doses (TCID~o) of the appropriate test virus in 0.05 ml was added. Tissue control wells contained 10 medium, without virus or antiviral compound, and antiviral control wells contained antiviral compound without virus. Ribavirin was included in each assay as a positive antiviral control, except for the adenoviruses where Ribavirin fails to demonstrate 15 aritiviral activity, Back titrations of each test virus were also included in each assay. A1,1 plates were incubated at 37'C in a 5% CO~ incubator. Virus control wells were observed daily. 'then these wells exhibited 80-100% cytopathic effect (CPE), all wells 2~ were observed for CPE. In addition to visual and microscopic observance of CPE, inhibition of syncytia formation was used to confirm activity against RSV.
In each antiviral assay, a 50% minimal inhibitory concentration (EDso) was determined. The EDso was calculated by determining the median minimal concentration of compound tested in wells inhibiting CPE 50% comparbd to virus control wells. The actual calculation of the EDso value was done with the aid of 3D the computer program, °'Dose-effect analysis with microcomputers°' of Chou et al., 1984, Adv. Enz.
Regul., 22:27-55.
The selective index for proanthocyanidin polymer A was then calculated as the ratio of the IDso over the EDso.
The results are shown in Tables 2-6. The results indicate that proanthocyanidin polymer A is as WO 92/06695 PC.'T/(JS91/0?679 ,...
~6 ~pg3~'~'~
effective as ribavirin for inhibiting growth of RSV-A
and S, plv-~,, pIV-3, FLU-A and FLU-B .in v.ftro. The selective index values indicate low cytotoxicity for proanthocyanidin polymer 1~.

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W a ~ po WO 92/06695 PC:f/L]S91/07679 .w, Fractions of proanthocyanidin polymer A have been further purified by HPLC using a GPC column and standards. Specifically, two fractions have been isolated: one fraction having a molecular weight average of 700 daltons (corresponding to 2 to 3 flavonoid monomeric units) and the second fraction having a molecular weight average of 3000 daltons (corresponding to 9 to 11 flavonoid monomeric units).
The two fractions show essentially.the same chemical shifts in the '3C and 'H NMR spectra as the unfractionated proanthocyanidin polymer A.
In vitro experiments indicate both fractions are active against RSV. Th.e fraction having an average of 2 to 3 flavonoid units has an an vitro RSV
antiviral activity of EDso=10 ~g/ml, while the second fraction having an average of 9-11 flanonoid units has an EDso=90 ~g/ml.
8. EFFECTIVENESS OF )jROAtdTFiOCYANIDIN
POLYMER ~. FOR TAERTMENT OF RgA
In vivo and in vitro assays indicate that the novel proanthocyanidin polymer A of the invention is a useful therapeutic agent for the treatment of RSV
infections. The present invention provides a safer therapeutic treatment for RSV infections without the toxic effects associated with Ribavirin, the currently approved composition for treatment of such infections.
Hispid Cotton Rats were innoculated with RSV
A2 intranasally on day 1 and given an intraperitoneal dose of 0.1-30.0 mg/kg of the proanthocyanidin polymer prepared in distilled water on day 2, 3 and 4.
Control animals received an equal volume of distilled water. In one experiment, as a comparison, a dose of 10-90 mg/kg of Ribavirin was used. All animals were killed on day 5. Lungs were removed and washed via transpleural lavage. The fluid collected was assessed for RSV titer in a microtiter assay using HEp2 cells.

WO 92/0G695 ~ PCf/US91/07679 Assays were performed in 96-well tissue culture plates. All dilutions and tissue culture suspensions were prepared in minimal essential medium containing antibiotics and 5% fetal calf serum (5%
FCS-MENt) . Lung lavage fluisi was added in quadruplicate to wells of t2ae test plates containing a subconfluent monolayer of cells (ca. 3x103 cells).
Approximately 100 median tissue culture infectious doses (TCIDso) of the appropriate test virus in 0.05 ml was added. Tissue control wells contained medium, but no virus. All plates were incubated at 3~~C in a 5%
COz incubator. Virus control wells were observed daily. When these wells exhibited 80-100% CPE, all wells were observed for CPE. In addition to visual ~5 CPE, inhibition of syncytia formation is used to confirm activity against RSV. Lung RSV titer logo was calculated as described by Dubovi et al., 1983; 1984.
The results are shown in Tables 7-8.
As shown in Table ?, for each experiment, the proanthocyanidin polymer A demonstrated a dose-dependent decrease in RSV titer compared to controls.
This indicates that the proanthocyanidin polymer A has effective antiviral activity against RSV an wivo. As shown in Table 8, direct comparison of the antiviral effect of the procyanidin of p ymer A to ribavirin (intraperitoneal administration) demonstrates that the polymer composition of the invention is significantly more potewt that ribavirin, the drug currently used to treat RSV infections.
Hispid Cotton Rats were inoculated with RSV
A2 intranasally on day 1 and given an oral dose of 1.0-10.0 mgjkg of the proanthocyanidin polymer on days 2, 3 and 4. All animals were killed on day 5 and lung tissues were collected. Lungs were removed and washed via transpleural lavage. The fluid collected was assessed far RSV titer in a microtiter assay using WO 92/06695 PCTlUS9l/07G79 ~,pg3~~~
HEp2 cells as described above. Results are shown in Table 9.
As shown in Table 9, the proanthocyanidin polymer administered orally significantly lowered RSV
titers at 10 mg/kg compared to controls. This indicates that the proanthocyanidin polymer has effective antiviral activity when administered orally against RSV and is more active than ribavirin.
3'ARLE ?
Effect of Proanthocyanidin Polymer A Rgainst RBV Infection of Cotton Rmts, Intraperitcneal Dosing ~5 Treatment Lung p (Mg/Kgj titer, EXPERIMENT ~1 Control 3.98 ~ 0.14 -__ (9549) 0.1 3.?8 ~ 0.31 0.08 (6025) 36.9$ kill 0.3 3.21 ~ 0.05 0.05 (1621) 83.0% kill 1.0 3.28 ~ 0.65 0.04 (1905) 80.1$ kill EXPERIMENT
#2 Control 4.3 0.8 ____ (20,000) 1.0 3.1 0.5 0.08 (1258) 94% kill 3.0 3.4 0:5 0.05 (2,511)87% kill 10.0 3.6 0.3 0.06 (3,981)80% kill i WQ 92/06595 ~ ~ ~ ~ ~ P~/Ug91/07679 I
Treatment Lung p (Mg(kg) titer, r i EXPERIMENT ~3 j , Control 3.98 ~ 0.14 ____ s (9,549) i 3.0 4.00 ~ 0.02 n.s.
(10,000) 0% kill 10.0 3.88 ~ 0.3 n.s.
(7,585) 20.6% kill 30.0 3.70 ~ 0.28 n.s.
(5,011) 47.5% kill Control 5.22 0.27 --.--(165,958) 0.1 4.87 0.32 n.s..

(74,131) 55.3%kill 0.3 4.25 0.39 0.014 (17,782) 89.3%kill 1.0 4.27 0.5 0.046 (18,620) 88.8%kill 3.0 4.41 0.19 0.012 (25,703) 84.5%kill WO 92/06695 . pCTI~.JS91/07679 Treatment Lung p (Mg/kg) titer, EXPERIMENT ,~5 Control 4.5 ~ 0.2 ____ (31,622) 4.5 ~ 0.2 n.s.
(31,622) 0~ kill 30 4.0 ~ 0.3 0.03 (10,000) 68.4 kill 0 90 2.8 ~ 0.3 r0.0001 (631) 98% kill ,Lung titer represents: RSV titer log ~o/g lung (Mean ~ S.D.) WO 92106695 '~ ~ ~ ~ ~ 2 ,~ p~/US91 /Q7679 fAHLE 8 Effect of Proanthoeyanidin Polymer 3~ and Ribavirin Against ItF3V
Infection of Cotton Rats, Intra~or3toaealDoaing Proanthocyanidin Polymer (Mg/Kg) Lung p titer, Control 5.22 0.27 ---(165,958) 0.1 4.87 0.32 n.s.

(74,131) 55:33% Kill 0.3 4.25 0.39 0.014 (17,782) 89.29% Kill 151:0 4.27 0.5 0.046 (18,620) 88.78% Kill 3.0 4.41 0.19 0.012 (25,703) 84.51% Kill Ribavirin(Mg/Kg) Control 4.5 0.2 ---(31,622) 10 4.5 0.2 n.s.

(31,622) 0% Kill 2530 4.0 0.3 0.03 (lo,ooo) 68.4% Kil1 90 2.8 0.3 <0.0001 (631) 98% Kill ,Lung titer represents: RSV titer log ,o/g lung (Mean ~ S.D.) s a a ~'O 92106695 PGT/U591/07679 2093$2~~ ....
TABLE 9 , Effect of Oral Proanthocyanidin Polder m T~gainst Rse Infection of Cotton Rats Treatment Lung p (Mg/Kg) titer, EXPERIMENT ,~1 Control 4.45 ~ 0.50 __ (28,183) 1.0 3.62 ~ 0.42 0.045 (4168) 85.7% kill 3.0 4.22 ~ 0.23 n.s.
(16,595) 41.1% kill 10.0 3.95 ~ 0.48 n.s.
(8912) 68.4% kill ~5 EXPERIMENT ,~2 Lung p titer, Control 3.86 0.55 ___ (7.244) 1.0 3.60 0.01 n,s.

(3,981) 45.0% kill 3.0 3.?1 0.50 n.s.

(5,129) 29.2% kill 10.0 2.90 0.57 0.06 (794) 9.0% kill ~093~25 WO 92/06695 Pt,f/US91/07679 EXPERIMENT ~'3 Lung titer, p Control 3.90 0.3 (7,943) 1.0 3.90 0.8 n.s.

(7,943) 0.0% Kill 3.0 4.00 0.8 n.s.

(io,ooo) o.o% Kill 1o Zo.o 2.90 0.5 o.oi (794) 92% Kill Ribavirin 3.30 0.5 0.07 (40 mg/kg) (1,995) 80% Kill ,Lung titer log ~a/g represents lung RSV titer (Mean D.) S.

An additional series of experiments:
conducted as described above confirms the an vivo activity of polymer A against RSV.
Proanthocyanidin polymer A displayed a mean EDso value of I.52 ~ 0.62 mg/kg following 3-day i:p.
administration, and 3.6 ~ 1.66 mg/kg following 3-day p.o. administration. See Table 10. The reference antiviral agent, ribavirin, displayed EDso values of 40 .1 6, and > 90 mg/kg following i.p. and p.o.
administration, respectively. Proanthocyanidin polymer A at a dose of 10 mg/kg p.o, resulted in a 68-92% reduction in lung RSV titers following oral administration, and a 21-80% reduction following i.p.
administration. Ribavirin (40 mg/kg) resulted in a >90% reduction in lung titers following oral administration, and a 50% reduction following i.p.
administration.

WO 92/06(i95 PCT/U~S91107G79 ,..1 Effect of Proanthocyanidin Polymer !~
Against RS~ inf~ction of Hispid Cotton Rats:
3-Day Intraperitoneal Dosing ED~(mg/kg) i~roanthocyanidin Ex~~~' Polymer A Ribavirin 1 1.0 40 2 0.3 30 3 3.0 50 0.3 5 3.0 Mean + S.E.M. 1.52 * 0.62 ~~ + 5.a ,0 3-Day Oral Dosing ED~(mg/gg) Proanthocyanidin E~p.# Polymer A
Ribavirin 1 3.0 >90 75 2 1.0 >90 A 3.0 5 1.0 Mean ~ SEM 3.6 + 1 b6 >90 In vi~.ra experiments have also been performed to study the effect of proanthocyanidin polymer A upon course of respiratory syncytial virus infection in African green monkeys.
Prpanthocyanidin polymer A was weighed out in 25 mg aliquots and refrigerated as the dry powder until use. Glucose was prepared as a 0.5% solution and refrigerated. A 25 mg sample of proanthocyanidin polymer A was dissolved in 50 ml 0.5% glucose daily and the solution sterilized by filtration through a 0.22 ~m membrane filter. Subsequent dilutions were anade in 0:5% glucose from the 0.5 mg/ml solution at 1:2 and 1:5 to give concentrations of 0.25 mg/ml and 0.1 mg/ml respectively.
Groups of three monkeys were dosed with proanthocyanidin polymer A at 0.5 mg/kg, 0.25 mg/kg or 0.1 mg/kg by administering the drug at 1 ml per kg of WO 92/06b95 ~ ~ ~ ~ ~ ~ ~ p~/US91107b79 -49°
body weight by intravenous injection. The time of administration of the drug infusion to each monkey was approximately 1 minute. A control group of three monkeys received 1,m1 of the 0.5% glucose solution.
Four hours after the initial treatment with proanthocyanidin polymer A, each of the monkeys was infected with a 10v dilution of stock respiratory syncytial virus. The titer of the stock virus was 1x105 TCID~/ml. Each monkey received 1 ml of the 10'z virus dilution by intratracheal inoculation and 1 ml applied drop-wise to external notes. The total virus dose therefore was 2x103 TCIDso/monkey. Treatment with proanthocyanidin polymer A was administered again 8 hours later resulting in daily doses of proanthocyanidin polymer A of 1.0, 0.5, and 0.2 mg/kg with three monkeys per group. Twice daily treatment continued at 8 a.m. and 8 p.m. for a total of seven days.
To determine virus shedding throat swabs were taken daily each morning prior to treatment with proanthocyanidin polymer A using a dacron nasal-pharyngeal swab. The swab was placed in 1.0 ml of tissue culture (minimum essential medium with a 10%
fetal bovine serum and penicillin, streptomycin and fungizone). Titrations were performed on the throat swab solutions by preparation of ten-fold dilutions which were inoculated into duplicate wells of BSC-40 cells Brawn in 24-well plates. Following incubation at 37°C in a COz incubator the titer of each specimen was determined by microscopic examination far viral cytopathology and the titer expressed as the logo of the TCIDm per ml. At. the time of each morning specimen collection each monkey was examined for thin~rrhea and other respiratory symptoms including coughing, sneezing and signs of respiratory distress.
The animal attenclant would also note any coughing or sneezing during routine daily care of the monkeys.

PCT/US91/07679, , Fourteen days after virus inoculation each of the , monkeys was bled and the sera tested for antibody titers in a neutralization assay against approximately 100 TCIDso of respiratory syncytial 'virus.
The results of intravenous dosing with ~'' proanthocyanidin polymer A are summarized in Table 11.
Each of the three control monkeys bdecame infected with respiratory syncytial virus, shedding virus in the oropharynx for 7 to 10 days or longer. Virus titers reached five and six logs,o in two of the three control monkeys and 3.5 logo in the third. In the monkeys receiving proanthocyanidin polymer A at the highest dose of 1 mg/kg/day, the time of virus shedding and titers of virus shed were much reduced. One monkey shed virus for one day, a second for three days, and a third for four days. In one monkey, the maximum virus titer was two logs. The mid dose of 0.5 mg/kg/day was also seen to shorten the time of shedding in two monkeys with the third monkey excreting virus in the oropharynx for nine days. Virus titers were generally ao lower although maximum titers of four logs were detected in two of the three monkeys and three logs in the third. A minimal effect was seen at the lowest dose of 0.2 mg/kg/day. Virus shedding was seen far 25 six or seven days and a maximum titers of three logs,o were seen in two monkeys and five logs in the third monkey. Antibody titers to respiratory syncytial virus were detected in all monkeys at 14 clays with titers of 1:40 to 1:160.
30 Daily mean logo titers are presented for each treatment group (See Table 12). Mean titers were lower in each of the three treatment grqups when compared with the mean titers in the control group on each of the sampling days. The mean titers were 35 considerably reduced in the 1.0 mg/kg/day treatment group, and a doss related reduction is also seen in the other two groups.

Wa 92/06695 PCT/U591/07679 Clinical symptoms were more prevalent in the control monkeys than in the monkeys treated with proanthocyanidin polymer A (See Table 13). Rhinorrhea is the most easily observed symptom and was seen in each of the three controls. No signs of toxicity were observed during daily examinations of the monkeys at the time of sampling or during daily inspection during the course of the study.
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o .a o ~ .i .a ~ ,q WO 92/06695 ~ ~ ~ ~ ~ ~ ~ p~/Ug93/07679 In summary, proanthocyanidin polymer A was seen to have a definite beneficial effect on respiratory syncytial virus infection in African green monkeys. A dose of 1.O mg/kg given by intravenous injection twice daily resulted in shorter duration of virus shedding from the orophar;ynx, reduced titers of virus shed and reduced clinical symptoms. A dose of 0.5 mg/kg administered twice daily was also effective in reducing all three parameters of infection although 0 to a lesser degree than was observed at the higher dose.
g. EFFECTIVENE88 OF PROANTHOCYANIDIN
POLYHER A
FOR TREATMENT OF INFLUENZA A
Male and female BALB/c mice were used in in vivo tests to study the effectiveness of proanthocyanidin polymer A against Influenza A. 'The mice weighed 13-16 g and were obtained from Simonsen 20 Laboratories (Gilroy, CA). They were quarantined 24 hr prior to use, and maintained on Wayne Lab Blox and tap water. Once, the animals were infected, 0.006%
oxytetracycline (Pfizer, New York, NY) was added to the drinking water to control possible secondary 25 bacterial infections.
Influenza A/NWS/33 (H1N1) was obtained from K.W. Cochran, University of Michigan (Ann Arbor, MI).
A virus pool was prepared by infecting confluent monolayers of Marlin Darby canine kidney (MDCK) cells, 30 incubating them at 37°C in 5% Coz, and harvesting the cells at 3 to 5 days when the viral cytopathic effect was 90 to 100%. The virus stock was ampuled and stored at -80°C until used.
The proanthocyanidin polymer A was dissolved 35 in sterile water for injection (WFI) each day at the appropriate concentrations and used immediately. 1-Adamantanamine HCL (amantadine) was purchased from WO 92/06695 PCT/US91/07679 ,_,, Sigma (St. Louis, Mo) and was dissolved in sterile saline. 1-~-D-Ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) was purchased from ICN
Pharmaceuticals, Inc. (Costa Mesa, CA),and was dissolved in sterile saline.
An Ohmeda Blox 3740 pulse oximeter (Ohmeda, Louisville, OH) with ear probe ati:achment was used.
Each mouse lung was homogenized and varying dilutions assayed in triplicate for infectious virus in MDCK cells as described by Sidwell, RW, Huffanan JH, Call E.W., Alghamandan H., Cook P.D., Robins, R.K.
"Effect of Selenazofurin on Influenza A and H Virus Infections in Mice", ~Antiviral Res., 6:343-53 (1986).
Two experiments were ponducted as follows:
Experiment 1: Seventeen male and seventeen female mice were infected intranasally (i.n.) with an approximate 90% lethal dose of virus (0.06 ml). This viral inoculum was equivalent to an approximately~105 cell culture 50% infectious doses in MDCK cells.
Treatment with 30, 10 or 3 mg/kg/day of proanthocyanidin polymer A or 125 mg/kg/day of amantadine was begun 2 days pre-virus exposure and continued once daily for 8 days. Pulse oximeter readings of arterial oxygen saturation (SaOz) were determined on 5 male and 5 female mice daily through 10 days post-virus exposure and again on days l4 and 21. On infection days 2,4,6,8;10,14 and 21, 3 mice (2 male, l female or 2 female, 1 male) were killed and their lungs removed, graded for consolidation, and frozen for laser assay of virus titer. Consolidation was scored from 0 (normal)-to 4 (100% consolidation).
The same animals used for SaOZ determinations were held ~ .
for 21 days and deaths noted as they occurred. As toxicity controls, 3 male and 3 female sham-infected mice were treated concomitantly with each dosage level of test compound. These animals together with normal WO 92/06b95 ~ 9 ~ ~ ~ ~ PCT/US91/07679 untreated mice were weighed immediately prior to treatment and 18 hr after the final treatment.
Experiment 2: This experiment was identical to Experiment 1, except therapy began 4 hr post-virus exposure.
Statistical Evaluation: Increase in survivor number was evaluated using chi square analysis with Yate's correction. Mean survival time increases, virus titer and SaOi value differences were analyzed by t-test.
Lung consolidation scores were evaluated by ranked sum analysis. The Exstatix~' program run on a Macintosh II
computer was used to determine ~ standard errors (S.E.).
An overview of the results of these ,' experiments is shown in Tables 14 and 15. The overview of the two experiments shows mean data compiled from all time points in which assays were made. The toxicity controls for these experiments indicated the 30 mg/kg/day dose of proanthocyanidin polymer A was toxic to the mice.
Experiment 1 (Early treatment initiationl: The summary of Experiment 1 (Table 14) indicates significant influenza disease-inhibitory effects of the 30 and 10 mg/kg/day dosages of proanthocyanidin polymer A based on-the reduction of lung consolidation. The SaOi~ were significantly increased in the infected female mice receiving the 10 and 3 mg/kg/day doses.
Amant.adine was intended as a positive control in these experiments. Amantadine did not appear significantly effective in this experiment.
However, it has been subsequently learned that this particular influenza virus is resistant to Amantadine.
More detailed examination of these parameters can be seen .in Figures 6-9 for Experiment WO 92/x6695 PCT/US91/07679 .-.
_58w , ~1. Lung consolidation (Figure 6) was particularly inhibited on the early sampling days; the consolidation gradually increased in all groups through the remainder of the experiment, but never reached the level of the virus controls.
Lung virus titers (Figure l0) were not reduced early in the study, but by .day 8 and 10, significant reductions were seen in mice treated with the 10 and 3 mg/kg/day dosages of proanthocyanidin polymer A.
The Sa02 data were separated for male and female mice, since 5 animals of each sex were used for these determinations. Essentially no inhibition of Sa02 decline was seen in male mice treated with either compound (Figure 1~). The female mice appeared to respond better, however (Figure 13), with the 10 and 3 mg/kg/day doses of proanthocyanidin polymer A
inhibiting by approximately 10% of the Sa02 decline.' It should be noted that in this experiment, therapy ceased on day 6, and by the next day, the SaOZ in the 20 treated animals began to decrease at a rapid rate.
This suggests a need to continue therapy a few additional days.
Experiment 2 (hate treatment initiation): The summary results of this experiment are seen in Table 15. It should be noted that in this delayed treatment study arll the male toxicity control mice survived the high dose proanthocyanidin polymer A-treatment, although they lost considerable weight. These animals were slightly older than those used in Experiment 1, and weighed an average of 1 g more.
As seen in Experiment 1, no significant increase in survivors was seen in the infected, treated groups, although significant mean survival time increases were observed in female mice treated with 10 and 3 mg/kg/day of proanthocyanidin polymer A

WO 92/06695 ~ ~ ~ ~ ~ ~ p~'/US91107679 (Table 15). Overall mean lung scores were again ' reduced.
The alternate-day lung consolidation findings are shown in Figure 10.- Proanthocyanidin ;
polymer A therapy again caused inhibition of consolidation, although to a lesser extent than seen using earlier-initiated therapy.
Lung virus titers (Figure 11) were only marginally reduced by proanthocyanidin polymer A
therapy.
The SaOz data for male and female mice are summarized in Figures 12 and 13. As was observed in Experiment 1, the female mice tended to respond better to proanthocyanidin polymer A therapy than the males, with somewhat variable, but dose-responsive inhibition of Sa02 decline. We conclude this material has a moderate effect against the murine influenza infection. v In view of amantadine's inactivity, an experiment was run using a 75 mg/kg/day dose of ribavirin, which is known to be highly active against the influenza virus infections (Sidwell, R.W., Huffman, J.H., Hare, G.P., Allen, L.B., Witkowski, J.T., Robins, R., "Broad-Spectrum Antiviral Activity of Virazole: 1-a-D-ribofuranosyl°1,2,4-triazole-3-carboxamide", Science, 177:705-6 (1972)). The drug was given i.p, twice daily for 5 days beginning 4 hr post-virus inoculation. The same disease parameters as used in the present experiments were employed with the ribavirin study.
The overall results of this ribavirin experiment are shown in Table 16. The drug was not lethally toxic, although it caused moderate host weight loss. A11 infected, ribavirin-treated mice survived the infection. Lung consolidation and lung virus titers were significantly reduced, and the mean SaOZ decline was inhibited. The lung consolidation, W~ 92/U6695 PCT/US91/U7679 20~3~'~~ -60-virus titer, and Sao2 data are shown in more detail in ' Figures 14-16.
Thus, in this experiment, ribavirin exerted significant influenza inhibition.
Proanthocyanidin polymer .~ when given to influenza A (F31N1)-infected mice i.p. once daily for 8 days, exerted moderate inhibition of the infection.
The material was most efficacious when treatment was initiated 2 days pre-virus exposure, although significant inhibition was also seen when therapy was ~~ delayed until 4 hr post-virus exposure. The toxicity controls did not survive a dose of about 30 mg/kg/day.
Disease-inhibiting effects were primarily limited to IO mg/kg/day. Amantadine was ineffective against this influenza virus strain. Ribavirin was inhibitory to '' the virus infection. It is concluded that proanthocyanidin polymer A has anti-influenza virus activity.

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_ 6q _ , 10. EFFECTI~ENEES OF PROANTHOCYADIIDIN
FOLYMER d~
FOR TREATMENT OF >?ARp,.INFLtIENZA ~IRt78 Twelve African green monkeys without antibody to parainfluenzavirus, type 3, (PIV-3) were used in an experiment to determine antiviral activity of proanthocyanidin polymer A. The monkeys were divided randomly into four groups of three monkeys each. The respective groups received proanthocyanidin polymer A at doses of lo, 3.3, or 1.0 mg/kg/day with the drug dissolved in 0.5% glucose. A control group of three monkeys received 0.5% glucose without the drug. each of the monkeys received treatment by intravenous bolus injection as divided doses twice t5 daily at 8 a.m. for 7 days. The injections were made into the saphenous vein of sedated monkeys. See Table 17.
Treatment was initiated four hours before virus inoculation. Four hours later, a 10-2 dilution of PIV-3 was prepared and inoculated as a 1 ml volume intratracheally and a 1 ml volume placed on the external nares. The titer of the inoculated virus was 103'5 TCIDsa per ml.
Throat swabs were taken daily.and placed in 1 ml of tissue culture medium. Fluid was expressed from the swab and the tissue culture fluid was titrated for virus. Tenfold dilutions of the throat swab specimens were prepared and inoculated into duplicate wells of 24 well tissue culture plates seeded with Vero cells. Titers were determined by microscopic examination of the cultures for viral cytopathology. The monkeys were observed daily for clinical symptoms including rhinorrhea, sneezing and Coughing. At 14 and 21 days post-infection, blood was drawn for detection of antibody to 1~IV-3 using a serum neutralization assay.

Each of the 12 monkeys became infected with PIV-3 with the majority shedding virus within 24 hours after virus inoculation. Virus shedding continued in all monkeys through the eighth day post-infection with some monkeys shedding virus through day 9 and 10. See Table 18.
The mean virus titers for each group are shown in Table 19. Proanthocyanidin polymer A at 10 mg/kg/day appear to reduce the mean titers on all of the days post-infection by one log or better. The 3.3 mg/kg/day dose was seen to have a slightly lesser effect than the 10 mg/kg/day dose but again all titers were less than the controls. No appreciable effect was seen at 1.0 mg/kg/day.
Symptoms of respiratory infection were more common in the control monkeys than in any one of the three proanthocyanidin polymer A treated groups.
During the 11 days of observation with 3 monkeys per group, 33 total symptom days are possible. In the group receiving proanthocyanidin polymer A at 10 mg/kg/day only 7 of those 33 days were observed to have monkeys with symptoms. The 3.3 mg/kg/day dose resulted in only 2 days and the 1.0 mg/kg/dose resulted in 7 days. The principal symptom observed was rhinorrhea (See Table 20).
Antibody titers to PIV-3 at 14 and 21 days after virus inoculation are reported. All monkeys were observed to develop antibody with what appeared to be slightly higher titers in the treated monkeys when compared with the untreated controls. See Table 21.
Thus, proanthocyanidin polymer A was seen to have a beneficial effect an the course of parainfluenzavirus, type 3, in African green monkeys treated with 10 and 3.3 mg/kg/day with proanthocyanidin polymer A. Proanthocyanidin polymer A was seen to reduce the titers of virus shed in the W~ 92/06695 ..~.
65 _ ~~~~~~~hroats of infected monkeys and may have reduced the symptoms seen with this infection.
Table 17. Treatment Groups of ?~gr:Lnan Graen pgon)caya selected to Evaluate th~ ~tiviral Activity of Proanthoeyan.idaaa Polymer ear Against Parairaflu~azavirus, Typ~ 3 Btonke~ l~u.~bersea height ()sg)Trentaa~eut pith Proanthoc~amidamm polymer a 3Ø 5 sg~kg, b.i.d., i.~.

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a 1.8 5 ~g/l~g, b.i.d., i.v.

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~ .w W~O 92/06695 PCT/US9t/07679 Table 20. Total Days in ~lhich Mon~Ceys showed l~ymptoms to Parainfluenzavirus, Typ~ 3 Infection ra-~s~ent s Da~Is/~otal Control, tilucose 13/33 P. Pol.A, 10 asg/lag/dap 7/33 P. Pol.7e, 3.3 s~g/kg/dag ~ 33 P. pol.~, 1.0 a~g/i:g/dap '7/33 Table 29.. Antibody Titers to Parainflu~nzavirus,' Type 3, in African Gr~en Maakeys Infected with th~ virus to and Treated kith Proanthocyanidin ~olym~r ~°
~6oake~ Antibods Titer at nmg.a p.a.
Tseataent equsber 1A age 31 Daa?s Control L337 laAO 1:160 6.51c 10346 1:a0 1a~0 glucose, 1.a. x3A5 1:80 11160 h.Polyaes i~, L299 1:160 1:320 10 sg/kg/dap, 80341 1:160 1:320 i.~. E.300 lalAO 1a3a0 '~.~olg~er 3!, DC1AA lsso laso 3.3 sg/8cg/dag, L30a 1:160 1a3a0 1r 1t1!3 la 1G0 1:110 ~. PolTser A, L3D1 1:160 Ia3a0 1.0 a~g/kg/daip 80347 1:80 1:160 1.r. ~34a la4o 1:18o lo.t EFFECTTVENESB OF PROA~ITEOCYANIDIN
POLYMER A FOR TREATMENT OF HERPES
SIMPLE7C yIR08 TYPE 2 y3IGINITIB
In vivo experiments were performed to study the effectiveness of proanthocyanidin polymer .~ in comparison with ganciclovir against herpes simplex virus type 2. 6anciclovir is known to be effective against herpes simplex virus type 2.
Tn one series of experiments, both compositions were formulated for intraperitoneal injection by dissplving in physiological saline.

WO 92/afi695 PCT/US91 /0679, ~gg3~~5 Swiss Webster female mice (Simonsen Labs, Gilroy, CA) weighing approximately 20 grams each at the start of the experiment were infected intravaginally with herpes simplex virus type 2 (HSV-5 2), E194 strain. This was accomplished in a 3-step process. First, the vagina of each mouse was swabbed for 5 seconds with a cotton tip applicator dipped in 0.1. N NaOH. This treatment irritates the vaginal area so that the infection takes better. Approximately 1 '0 hour later each vagina was dry swabbed for 5 seconds.
Then an applicator dipped iri virus medium was used to swab each mouse for 20 seconds. The swabs were gently and slowly twisted back and forth during the time they were in place.
,5 Six hours after virus infection, intraperitoneal (i.p.) injections were administered using proanthocyanidin polymer A, ganciclovir, or placebo. Treatments were also given i.p. twice daily on days l through 7 after virus challenge. The daily 20 dose of each compound was 30 mg/kg/day, with 15 mg/kg given at each injection.. Since on day 0 of the infection only one dose was given, the daily dose for that day was 15 mg/kg/day.
Groups of 5 mice were sham-infected using 25 the process described above for virus infection, except that no virus was present for the final step.
These mice were treated the same way and at the same times as above.. Mice were checked daily for survival, and weights were recorded before the first (day 0) and 30 after the last (day 8) treatment.
Lesion scores in infected mice were determined daily on days 3-14 of the infection. A
i score of 1+ indicates redness immediately around the vagina. 2+ indicates spread of the lesion toward the anus. 3+ indicates a lesion (usually with swelling) from the vagina to the anus. There.are variations to this since same mice may have a vaginal lesion plus a 1V~ 92/06695 ~ ~ ~ ~ ~ ~ ~ PC'f/US91/07679 _ 71 lesion on the tail. Because many of the mice go on to die, the lesion score near the time of death is carried through to the end of the a4 days. If this were not done, lesion scores in the placebo group would appear to go down as the most affected mice die off. Some animals developed hind limb paralysis (and later died). This condition did not add to the lesion score.
Deaths were recorded daily for 21 days. The mean day of death calculation took into account only mice that died. Vaginal virus titers were made by titration of virus obtained from vaginal swabs 3 days after virus inoculation. These titrations were conducted in Vero cells in 96-well plates.
~5 Calculation of virus titer was made by the 50~
endpoint dilution method of Reed, L.J. and Pleunch, Pi., Am. J. Hyg., 27493-498 (1938).
Statistical interpretations of survival (Fisher exact test), mean day to death (Student's t test) and virus titer (Student's t test) were made by two-tailed analyses.
Table 22 below shows the average lesion scores with standard deviations and statistical analyses for the experiment.

W~ 92/0b695 p~'/U~91/07679 .~

Table 22. Effect of Intraperitoneal Proantbocyani~din polymer A Tr~atment on 8erpea 8impl~x 'Virus Type a (88~-2) ~aginitis in Rice.
~s~ra ~ t.esion scorn Proanth. l~ol. aanciclovi;c ~

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3 a.a~o.a' 0:1~0.a o.3to.3 a A.ato.3 ~.at~.a** I.atl.o 5 O.?t0.!** O.atO.a** 1.9~1.a 5 l.3tl.a** O.atO.a*** 3.ltl.a 1.531.3** O.atO.a*** 3.111.5 i0 8 i.831.5* 0.3t0.a*** 3.01.5 a.atl.a o.ato.3*** 3.atl.a to a.3tl.a 0.530.5*** 3.a11.3 11 a:311.5 0.5$-.a*** 3.a11.3 1a a.a$l.a 0.5it0.a*** 3.a11.3 13 1.831.5* 0.510.5*** 3.a31.a la l.8t1.5* 0.510.5*** 3.131.a Grand llvg. 1.510.8*~ O.atO.a*** a.511.0 (nags s-la) alftes visas cP~sllenge.
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p <0.05, °' g>c0.01-' p10.001 (two-tailed Student's t t~at).
Proanthocyanidin polymer A showed a statistically 2p significant decrease in average lesion score compared to the placebo control. Ganciclovir was also effective. Figure 17 gives a visual impression of the degree of lesion inhibition exhibited by 30 mg proanthocyanidin polymer A per kg relative to the placebo control and ganciclovir. Table 23 shows survival of the treated mice.

PCd'/US91 /07179 Table 23. ~fgect of Intraperitoneal proanthocyanidin Polymer A Treatment on Survival and Vaginal ~irus Titer in i~4ice infected Intravagina:Lly with ~ierpes simplex 'virus Type 2 (IiSV-2 ) , Rurvivors/ Dfean lDa~ ~liseas Titan Ccaaoound Doa~ ~(otal l~) to Death lDav 3) ~irus-Infected Pro. Pol. ~ 30 x/10(40) 9.513.2 2.911.1 GanciClovis 30 10/10(100)** >21 1.7t1.0*
Placebo - 3/20(15) 8.212.9 3.811.2 i7ninfected- 'Poaicitv Controls lost Wit. ~a~i7e~
Pso. Pol.~l 3D 5/5(100) >21 +2.8 Ganciciovir 30 5/5(I00) >21 +1.0 Placebo - 5/5(100) >21 X2.5 'Log'o50% cell vulture infeetious dose per ~l.
°Deteriined on day 8. Represents the diffesenee in gra~rs beteoeen dap 9 and day 8 weights. 'p<0.002, t~ao-tailed lPisbes osact test (survival) as two-tailed Student's t test (aemn day to death and virus titer parameters.) ~5 Proanthocyanidin polymer A showed an increased survival (40x survival compared to 15~ in the placebo group). The ganciclovir group had 100% survival.
Table 23 also shows that proanthocyanidin polymer A
treated mice had less virus than the placebo control, 20 but the results were not statistically significant.
Ganciclovir caused a significant reduction in virus titer.
Results in toxicity control mice showed proanthocyanidin polymer A was well tolerated and 25 caused no adverse effects at the doses used.
Ganciclovir~treated mice gained less weight than placebo controls.
Thus, proanthocyanidin polymer A
administered intraperitoneally at a dose of 30 mg/kg/day had, moderate antiviral activity in this model. There was no apparent toxicity of the proanthocyanidin polymer A formulation to mice.
Another series of preliminary experiments, ~5. in which the proanthocyanidin polymer A composition is administered orally, showed some anti-FiSV-2 activity but only at the highest dose administered, i.e., 270 mg/kg/day.

In another series of experiments, the proanthocyanidin polymer A composition was formulated for topical application against HSV-2. The efficacy of proanthocyanidin polymer A is compared with ganciclovir and acyclovir. Both ganciclovir powdre and acyclovir cream were obtained commercially.
Squibb cream base #8, which served as a vehicle and as t0 the topical placebo control, was also commercially obtained.
The methods of infecting the mice, setting toxicity control and the parameters measured to evaluate the infection were the same as described above for the intraperitoneal tests. Topical treatments on the day of infection were given at +6 hours. They were then given twice daily for 7 more days.
Treatment for 7.5 days with proanthocyanidin polymer A at two doses resulted in a statistically significant reduction in lesion scores of the period of evaluation for the l0% dose. This is presented graphically in Figure 18 and in Table 24. The 5% dose also reduced lesion development relative to the placebo control, but the results were not statistically significant. Acyclovir cream nearly completely prevented lesion formation in infected animals. More mice treated wtih proanthocyanidin polymer A at 5% and 10% doses survived the infection that placebo controls (Table 25) and vaginal virus titers were reduced at the 10% dose. In addition, the numbers of mice without lesions on day 14 were greater than in the placebo group. None of the uninfected toxicity control mice lost weight or died as a result of treatment with any of the cream formulations.
(Table 25).

~~93~~5 WO 92/46695 pCT/US91/07579 It can be concluded that topically applied proanthocyanidin polymer A as a 1e)% cream was active.
Only three mice developed vaginal lesions, but the lesions all progressed to maximum severity. The proanthocyanidin polymer A cream 1'ormulations were somewhat dry to the touch, since t:he composition hydrated and absorbed same of the water from the Squibb cream base. This may have affected the performatnce of the proanthocyanidin polymer A cream.
Partial hydration of the material in water prior to mixing it with the cream base may provide a more aqueous and bioavailable product for topical application.
Table 2~. effect of topieal Proanthocyaaidin Polym~r A Treatment on herpes gimplex Qirus Type 2 (gg~_2) vaginitis in Mice.
Averaa Lesion Score Day' P. A P. Pol ~ A l Pol. 5% A 0%

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Results from iri vi ~tro tests far RSV
antiviral activity, performed under the same procedures as the tests with proanthocyanidin palymer A, indicate that proanthocyanidin polymer ~ is effective and has an EDso of 6 ~ag/ml.
The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the.invention. Any equivalent embodiments are intended to be within the scope of this invention.
Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Claims (18)

WHAT IS CLAIMED IS:

1. A proanthocyanidin polymer composition, characterized by:
(a) the capability of dissolving in water and/or aqueous solution;
(b) the capability of exerting a pronounced antiviral effect as demonstrated in in vitro assays for antiviral activity against respiratory syncytial virus, types A and B; parainfluenza virus, types 1 and 3; influenza virus, types A and B; and as demonstrated in in vivo tests for antiviral activity against respiratory syncytial virus;
influenza virus, type A; parainfluenza virus, type 3; and herpes simplex virus, type 2;
(c) comprising a proanthocyanidin polymer having a structure comprising flavonoid units selected from the group consisting of catechins, epicatechins, gallocatechins, galloepicatechins and combinations thereof;
and (d) 13C NMR spectra having peak positions at .delta. 154.2, 145.1, 143.7, 132.8, 131.2, 130.3, 120.9-118.6 (series of broad peaks), 116.1, 115.4, 114.3, 108.0, 106.3, 96.6, 95.3, 81.8, 77.6, 75.3, 72.6, 1.5, 65.6, 37.1, 35.3, and 27.7.

2. The proanthocyanidin polymer composition of claim 1 having an average of about 2 to about 11 flavonoid units.

3. The proanthocyanidin polymer composition of claim 1 having an average of about 7 flavonoid.

4. The proanthocyanidin polymer composition of claim 1 wherein the 13C NMR spectrum having peak positions at .delta.

155, 145, 130, 128, 121, 116, 109, 97, 82, 76, 73 and 38.

5. The proanthocyanidin polymer composition of claim 1 further characterized by:
(a) infrared spectral analysis of intense peaks ranging from 3350-2500 and other peaks at 1612, 1449, 1348, 1202, 1144, 1107, 1068 and 1207 cm-1;
(b) an ultraviolet spectra having broad peaks at wavelength 202, 235 (shoulder) and 275, and 305 (shoulder) nm; and (c) visible spectral absorption at wavelength of about 460 nm.

6. The proanthocyanidin polymer composition of claim wherein the infrared spectra has peaks at 3379, 1891, 1611, 1518, 1448, 1343, 1200, 1143, 1102, 1054, 1032, 825 and 725 cm-1.

7. The proanthocyanidin polymer composition of claim 1 obtained from a Croton species.

8. The proanthocyanidin polymer composition of claim 7 obtained from Croton lechleri.

9. An ester, ether or oxonium derivative of a proanthocyanidin polymer, in which the polymer is characterized by:
(a) the capability of dissolving in water and/or aqueous solution;
(b) the capability of exerting a pronounced antiviral effect as demonstrated in in vitro assays for antiviral activity against respiratory syncytial virus, types A and B; parainfluenza virus, types 1 and 3; influenza virus, types A and B; and as demonstrated in in vivo tests for antiviral activity against respiratory syncytial virus;
influenza virus, type A; parainfluenza virus, type 3; and herpes simplex virus, type 2;
(c) having a structure comprising flavonoid units selected from the group consisting of catechins, epicatechins, gallocatechins, galloepicatechins and combinations thereof; and (d) 13C NMR spectra having peak positions at .delta. 154.2, 145.1, 143.7, 132.8, 131.2, 130.3, 120.9-118.6 (series of broad peaks), 116.1, 115.4, 114.3, 108.0, 106.3, 96.6, 95.3, 81.8, 77.6, 75.3, 72.6, 71.5, 65.6, 37.1, 35.3, and 27.7.

10. A proanthocyanidin polymer composition characterized by:
(a) the capability of dissolving in water and/or an aqueous solution;
(b) the capability of exerting an antiviral effect as demonstrated in in vitro assays for antiviral activity against respiratory syncytial virus, types A and B;
parainfluenza virus, types 1 and 3; influenza virus, types A and B; and as demonstrated in in vivo tests for antiviral activity against respiratory syncytial virus; influenza virus, type A; parainfluenza virus, type 3; and herpes simplex virus, type 2;
(c) comprising a proanthocyanidin polymer having a structure comprising flavonoid units selected from the group consisting of catechins and epicatechins; and (d) a 13C NMR spectrum having peak positions at .delta. 155, 145, 134, 121, 118, 116 (shoulder), 97, 76, 73, and 38.

11. The proanthocyanidin polymer composition of claim 9 which is obtained from Calophyllum inophylum.

12. An ester, ether or oxonium derivative of a proanthocyanidin polymer, in which the polymer is characterized by:
(a) the capability of dissolving in water and/or aqueous solution;
(b) the capability of exerting an antiviral effect as demonstrated in in vitro assays for antiviral activity against respiratory syncytial virus, types A and B;
parainfluenza virus, types 1 and 3; influenza virus, types A and B; and as demonstrated in in vivo tests for antiviral activity against respiratory syncytial virus; influenza.
virus, type A; parainfluenza virus, type 3; and herpes simplex virus, type 2;
(c) comprising a proanthocyanidin polymer having a structure comprising flavonoid units selected from the group consisting of catechins and epicatechins; and (d) a 13C NMR spectrum having peak positions at .delta. 155, 145, 130, 128, 121, 116, 109, 97, 82, 76, 73 and 38.

13. A proanthocyanidin polymer composition obtained from a Croton tree by a method which comprises:
(a) extracting the whole plant, the bark, the stems, the roots or the latex of the Croton tree with water or a water miscible solvent selected from a lower alcohol of about 1-3 carbons or acetone or combination thereof to obtain an aqueous soluble fraction;
(b) subjecting the aqueous soluble fraction to gel filtration, ultrafiltration or chromatography using aqueous and/or organic solvents; or combination thereof and (c) collecting the fraction detectable by ultra violet spectroscopy having a .lambda. maximum at about 200-350 nm, wherein the collected fraction is characterized by having 13C NMR spectra having peak positions at .delta. 154.2, 145.1, 143.7, 132.8, 131.2, 130.3, 120.9-118.6 (series of broad peaks), 116.1, 115.4, 114.3, 108.0, 106.3, 96.6, 95.3, 81.8, 77.6, 75.3, 72.6, 71.5, 65.6, 37.1, 35.3, and 27.7.

14. The composition according to claim 13, in which the Croton tree is Croton lechleri.

15. The composition according to claim 13 wherein the 13C NMR spectrum having peak positions at .delta. 155, 145, 130, 128, 121, 116, 109, 97, 82, 76, 73 and 38.

16. The composition according to claim 13 further characterized by:
(a) infrared spectrum having intense peaks ranging from 3350-2500 and other peaks at 1612, 1449, 1348, 1202, 1144, 1107, 1068 and 1207 cm-1;
(b) an ultraviolet spectra having broad peaks at wavelength 202, 235 (shoulder) and 275, and 305 (shoulder);
and (c) visible spectral absorption at wavelength 460 nm.

17. A proanthocyanidin polymer composition obtained from Calophyllum inophylum by a method which comprises:
(a) extracting the whole plant, the bark, the stems, the roots or the latex of the Calophyllum inophylum tree with water or a water miscible solvent selected from a lower alcohol of about 1-3 carbons or acetone or combination thereof to obtain an aqueous soluble fraction;

(b) subjecting the aqueous soluble fraction to gel filtration, ultrafiltration or chromatography using aqueous and/or organic solvents; or combination thereof and (c) collecting the fraction detectable by ultra violet spectroscopy having a .lambda. maximum at about 200-350 nm, wherein the collected fraction is characterized by having 13C NMR spectra having peak positions at .delta. 154.2, 145.1, 143.7, 132.8, 131.2, 130.3, 120.9-118.6 (series of broad peaks), 116.1, 115.4, 114.3, 108.0, 106.3, 96.6, 95.3, 81.8, 77.6, 75.3, 72.6, 71.5, 65.6, 37.1, 35.3, and 27.7.

18. The composition according to claim 17 wherein the 13C NMR spectrum having peak positions at .delta. 155, 145, 130, 128, 121, 116, 109, 97, 82, 76, 73 and 38.