CA2202467A1 - Use of anthocyanidin and derivatives for treatment of retroviral infections - Google Patents

Abstract

The invention relates to the use of an anthocyanidin or an anthocyanidin derivative of general formula (I) wherein R1, R2, R3 and R6 independently of each other is H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R4 is OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R5 is H, OH, and Y is a counterion or a salt, prodrug or complex thereof for the preparation of a pharmaceutical composition for the prevention and/or treatment of retroviral infections in mammals, as well as to novel anthocyanidin derivatives of general formula (I) and methods for preparation of said compounds and novel pharmaceutical compositions.

Description

Wo 96/11692 1~ 5 USE OF ANTHOCrANIDIN AND DERIVATIVES FOR TREATMENT OF RETROVIRAL 11~
The present invention relates to the use of an anthocyanidin or an anthocyanidin derivative of the general formula I or a pharr~r-~ut~ y acceptable salt, prodrug or complex thereof 5 for the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease caused by a retro-virus in a mammal ; n~ i n~ a primate such as a human. The composition may be useful in the prevention of infection by HIV, the treatment of infection by HIV and/or the treatment 10 of the resulting acquired immune deficiency :~yl~dL~ (AIDS) and in the treatment of other retrovirus-related diseases.
BACKGROUND OF THE INVENTION
A retrovirus designated human i r~-~P~iciency virus (HIV) is the causative agent of the complex disease termed Acquired 15 Immune Deficiency Virus (AIDS), and is a member of the lenti-virus family of retroviruses. M.A. Gonda, F. Wong-Staal, R.C.
Gallo, '~Sequence Homology and Morphological Similarity of HTLV III and Visna Virus, A Pathogenic Lentivirus", Science a27, 173 (1985); P. Sonigo, N. Alizon et al., "Nucleotide 20 Sequence of the Visna Lentivirus. Rela~ionship to the AIDS
Virus~, Cell 42, 369 (1985) . The complex disease AIDS in-cludes progressive destruction of the immune system and degeneration of the central and peripheral nervous systems.
The HIV virus was previously known or referred to as LAV, 25 HTLV-III or ARV.
Other retrovirus-related diseases are described in John M.
Coffin, "Retroviridae and Their Replication", pp. 1437-1500 in "Virology", second edition, ed. ~y B.N. Fields, D.M. Knipe et al., Raven Press, Ltd. New York, 1990, see e.g. Table I.
.
30 Anthocyanins are the most important group of water-soluble plant pigments visible to the human eye. As the anthocyanins seem to have non-toxic effects on the human being, their possible pharmaceutical use has been further~investigated.

wos61116s2 }~l,.. J.

r~l2T~Tr.~n v~ L OF ~E3E Li~ U..
The present ~nventicD. ~cr~nQ~q that anthocyanidi3 a~d antho-cyanidin derivatives ae .~on-cytotoxic ~.... r.~ Lions can ex} ibit artiYira.} ef r~c~s i~ HIV ;~f~'ct~ cells.
5 The present i~ve~tio~ relates to the u e of an anthocya2idi~
or an anthocya~ idisl der_vat~ve of the general formula I
~OH
R, wherein R~, R2, R3 and R6 i~ r 1 Iy 0~ ea~h other are H, OH~ Cl-6-alkoxy, ar. -o-glycosyl grDup, an -O-gly~osyl group which is lD suoseituted wit~ one or nore a~yl group~, or an -O-glycr~syl moiety _ 5i nlJ ae ~ east two glycosyl grsups and ac least one acy} group ~. .. "_-~ so that at least o~e acyl group i3 locaeed ~etween ewo cLycosyl group~, R4 i5 OEi, alkoxy, an -O-glycosyl group, an -O-glyco~yl group 15 which is ~11herir11t~i wit~ one cr o~e acyl groups, or an -O-glycosyl moieey ~c~ T 5 at leasC two glycosyl groups and at least one acyl gro~n .. 5~.r~J..d SO eh~t at least one acyl group is loca~ed }~e~wee!! ~wo glycosyl sroups, Rs i3 H, OE~, and 2C Y is a co~t~Lion, or a prodrug or co~Dlæ~ :hereo~ for the pr~ra~r1r~r~ of a rh ~ r~l~ri ~1 ~7~: n ~or the prevention and~or treat-RECTIFIED SHEET (RULE 91) ISA/EP

ment of a disease caused by a retrovirus i~ a mammal includ-ing a primate such as a human.
In particular, the invention relates to the use of a ~ ~u-,d wherein at least one of R3, R4, and R6 is an -O-glycosyl 5 group, an -O-glycosyl group which is substituted with at least one acyl group, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is_ located between two glycosyl groups. The -O-glycosyl moiety may comprise at lO least two glycosyl groups and at least one acyl group arranged alternate with one glycosyl f ollowed by one acyl group; an acyl group may also be located at the very end of the moiety.
A presently preferred embodiment of the invention is the use 1~ of the ~ petanin wherein, with reference to formula I, Rl is OCH3, R2 i s 0}~, ~
R3 is 6-O- (4-0-B-p-cuul.,c,L~yl-~-~-rhamnopyranosyl) -~-D-gluco-pyranosyl, 20 R4 is ,B-D-glu~uLJy~
Rs is H, and R6 ~is OH
Other presently pr~ferred embodiments are the use of the individual anthocyanins outlined in Tables I and II below, 2~ e.g in compositions wherein the relatiYe quantities of the various anthocyanins are as outl~ned in Table I or Table II

_ WO 96/11692 . r~ ,C 'c ~

TA;3~E I
Structures and relative proportions (9~) of the i~dividual antho-cyanins in the first purified Vaccinium myrtillus sam~le (sample II) with reference to formula I, R4 is OH, Rs is H and R6 is OH
COMPOUND ~I R2 R3 Proportions ( 9f ) 1. nPlnhi~;rl;n-3-~,llAr-~q;r',~ 0~ OP, -O-galactosyl 10.6 10 2. n~lrh,n;~';n-3-glucoside O~l OP. -O-glucosyl 10 . 7 3. Cyanidin-3-5~1=rtrqi~ 0~} ~3 -O-galactosyl 6.8 4, rl lrh;n;~in-3- ~ =
Ar~h;nrq;~ o~ o}~ -O-arahinosy1 10.6 5 . Cyanidin- 3 glucoside OX P; -O-gluc osyl 8 .

6 . Petunidin- 3 -rJ~l q~ -rq; ~. ~ OC~3 OX -O-galactosyl 4 . 6 20 7. Cyanidin-3-arabinoside OE~ P; -O-arabinosyl 8. Petunidin-3- -:
:~r=h;nAqi~" OCP.3 O}~ -O-glucosyi 15.2 9. Peonidin-3-5~1Artnq;rlP OC1~3 J~ -O-galactosyl 1.2 10. Petunidin-3- ` -==
:lr~h;n~lq;~. OCP.3 ~ -O-arabinosyl 2.9 11. Peonldin-3-glucoside OCH3 H -O-glucosyl 30 12. Malvidin-3-5~ nq;~l OC}~3 OCP3 -O-galactosyl 10.3 13. Malvidin-3- ~ :
glucoside _ OCP.3 oc~3 -O-glucosyl 14.1 14. Peonidin-3-ArFih;n~q;~ OCP.3 ~ -o-araoinosyl 0.9 1S. Malvidin-3-arahinoside OCP.3 oc~3 -O-arahinosyl 4.0 ~ Pigment ~ and 8 together 4 0 ~ Pigment 11 and li together WO96111692 I~,1,.. J._. lo.

TA;3LE I I
Structures and relative proportions ~ ~ ) of the individual antho-cyanins in the second purified V~rr;n~ 7 myrtillus sample (sample III) with reference to formula I, R4 is OH, Rs is H
5 and R6 is OH
COMPO7~D Rl R2 R3 Proportions (~) n~l rhi ni rli n -3 _ 10 r~lRrtnqis~. OX OX -O-galactosyl 6.4 2. n,31rhini,1in-3- = ~
glucoside ox ox -O-glucosyl 7 . 4 3. Cyanidin-;-galactoside ox x -O-galactosyl 20.2 15 4. T) lrhini~r-3-RrRhinnqltl~' OX OX -O-arahinosyl 11.0 5 . Cyanidin- 3 -glucoside ox x -O-glucosyl 22 . 8 6 . Petunidin- 3 -~7-1~rtnq;~ OCX3 OX -O-galactosyl 1.6 7 . Cyanidin- 3 -RrRhinnqit;o OX X -O-arabinosy 8. Petunidin-3-RrRhi nnqi ~ OCX3 OX --o--glucosyl ll . 4 25 9. Peonidin-3- ~:
~7=ORrtnqi~l OCX3 X -a-galactosyl 1.2 . Petunidin-3-RrRhinnqi~ OCX3 OX -O-ara_inosy ll. Peonidin-3- ~= -glucoside OCX3 X -O-glucosyl ~t 12. Malvidin-3- =~
rRlRrtncjrl OCX3 OCX3 -O-galactosyl 3.5 . Malvidin-3- :=~
glucoside OCX3 OCX3 -O-glucosyl 7 . 4 3 5 14 . Peonidin- 3 - : :
arahinoside OCX3 X -O-arahinosyl trace . 7~alvidin- 3 -RrRhi nnqi S OCX3 OCX3 -O-arahinosyl 2 .1 4 0 ~ Plgment 7 and 8 together ~ Pigment l0, ll and 12 together ~ =~
The compound or mixture of compounds may be further def ined as an anthocyanidin or an anthocyanidin derivative; which, when dissolved in DMSO at a r~nm.on7-ration so that the final ~5 concentration of DMSO does not~ exceed 0.2~6 v/v DMSO, and tested as described in section 2 . 3, does not have a cytotoxic effect on the growth of uninfected SupTl cells resulting in a decrease in ODs80 of more than l096 as a result of incubation with the anthocyanidin or the anthocyanidin derivative, and 50 when tested as described in section 3 has an antiviral effect on the growth of Molt 3 IIIB cells infected with HIV-l WO 96111692 ~ .a IOJ

defined as a decrease in syncytia formation of more than lO96 as a result of 48 hours of incubation with the anthocyanidin or the anthocyanidin derivative.
It is contemplated that the anthocyanidin or anthocyanidin 5 derivatives and pharmaceutically acceptable salts thereof inhibits the reverse transcriptase or HIV integrase encoded by huTnan ;mmllnn~Pfirjency virus tHIV) type l (HIV-l) and type 2 (HIV-2) . The exact, -h~ni P~ of action i8 yet unknown, but if desired it can be further analysed by PCR of the various 10 intermP~l; =tPq in the replication cycle in order to ascertain at which stage the replication is inhibited by the antho-cyanidin or a~thocyanidin derivatives.
Based upon the disclosure of ~ the present invention, the person skilled in the art will be able to test the compounds 15 of formula I as (~ 1 iner1 above without using inventive skill.
Substances which are rr~n~i ~Pred useful may then be tested for cytotoxic ef f ects in other appropriate cell systems such as di f f erent f ibrobl as t s ( e . g . HeLa ) or other uninf ect ed T - ce l l lines, uninfected T-cell (e.g. cell lines from ATCC), and in 20 primary human lymphocytes from blood donors, e.g. enriched for CD4+ cells by means of Dynabeads~. Furthermore, toxicity tests may be performed such as single dose toxicity tests, e.g. ~Dso ~i.e. the dosage at which half of the expPr~i- t~l animals die). In addition to the LDso value in rodents it is 25 desirable to determine the highest tolerated dose and/or lowest lethal dose for other species, e.g. dog and rabbit. If the in ~ritro test results are- promising and the LDso is high, clinical experiments using humans may be approved takinq into consideration that at present no treatment exists of -AIDS.
30 The person skilled in the art would by use of methods described in standard textbooks, guideli~es and regulations as well as common general knowledge within the field be able to select the exact dosage regimen~ to be implem.ented for any selected compound using merely routine experimentation proce-3 5 dures . _ WO 96/11692 P~'~

During the process, the person skilled in the art may decide not to rt~nt; nllp studying all the initially selected com-pounds, or it may be decided to synthesize and test new com-pounds in view of the initial toxicity and biological results 5 obtained.
Experi~ents in ~ ~y~ e88 to further characterize general biolo~;rAl ef~ect~ and antiviral p~ v~e ies of anthocyanidins or anthocyanidin derivatives To further study the effect of anthocyanidins in relation to lO antiviral potont;Als of these compounds and to get a mecha-nistic understanding of how these compounds interfere with the re~lication of retroviruses such as HIV, a number of different experiments have been initiated or designed.
The experiments can be divided into groups according to the l~ goals of the studies:
I . Cytotoxic ef f~cts i) In tissue culture studies ii) ~n vivo studies in mice II. In vitro studies with defined enzymatic systems i) Reverse transcriptase (RT) ii) Integrase (I~) III. Antiviral effects Qf the compounds measured in tissue culture studies i) Testing the effect of the compounds on the labora-tory strain of ~IV in different cell lines ii) Analysis of the progress of retroYiral replication in the presence of compounds at r=nnr~ntrationS

WO96/11692 PCrlNO95/00185 giving substantial inhibition of cytQpathr~g~n; r effects and virus rrrrlllrt;r~,n iii) Testing the effect of the ~ ullds on HIV iso-lated from patients in priwary cell lines 5 Studies in qrou~ I i~ includes characterization of effects of the compounds on cell growth of a number of different estab-lished cell lines like the CD4+ human cell lines with lympho-cytic phenotypes (Jurkat, CME, X-9, Molt-3, all from ATCC~, the monocytic cell line U937 (also from ATCC), and a CD4+
lO HeLa (fibroblast) cell line with the ability to permit repli-cation of the laboratory strain of HIV when exposed to this virus. The studies also include the human epithelial cell lines SGHTL-34 (derived from gl. thyroidea) and 293 (derived from kidney) obtained from Professor Johan R. Lillehaug, lS Department of biochemistry and molecular biology, University of Bergen, Norway) and which cannot be infected with HIV due to the absence of CD4 receptors. Peripheral human lymphocytes are also i nrl ll~P~ in these studies . These cells are isolated from normal healthy blood donors, isolated by standard J,ym-20 phoprep methods (Nycodens), incubated with the test com-pounds, stimulated with phytr,~m~rglutinin or cytokines and tested for their ability to incorporate radioactive thymidine .
The aim of these studies is to determine what doses of the 25 test compounds human cells can tolerate without affectïng the growth potential of these cells. Furthermore, these studies will be P~n~P~ to include long terin effects on the cells of low r~nr,Qntrations of the test compounds. At doses where growth is affected, the aim is to study the ~ ni r_q of 30 growth inhibition. To get a general idea of how these com-pounds interact with cells at toxic or semitoxic doses, the cells are f irst characterized af ter treatment with test compounds using electron microscopy. Based on the results of those studies, different biochemical studies will be: designed W096/11692 p~ ,,.,~, to further elucidate the -h ~ni r~ behind the cytotoxic effects .
- Using these tissue culture systems, phar~-~r~kin~ric proper-ties of the ~u~,uuullds will be studied, the goal being to evaluate the efficiency of uptake as well as the stability of 5 the _ ~,uu--ds in human cells.
The main goal of the group I ii ) studies is to determine ~D50 in mice. As part of these studies, it is also desired to eva-luate the clearance of the different compounds by analyzing urine samples f rom the treated animals .
10 The group II i) ana ii) studies include a number of different tests designed to find how the test ~ul~uullds interact with different activities of IN and RT like substrate interac-tions, template interactions and protein-protein interac-tions.
15 III i) Based on the results from the group I i) studies, the effect of the test compounds at doses not affecting cell growth on syncytia formation and virus production will be studied in the same cell lines . The aim is to f ind if there are cell line specificities with respect to the antiviral 20 activities of the compounds studied.
At doses giving an inhibition of viral production, the group III ii) studies will be conducted. These experiments involve extraction of viral components from infected cells after treatment with the test ~ uullds. The analysis of the ex-25 traots include different types of PCR analysis of viralnucleic acids (RNA and DNA) to determine at what stage of the replication cycle inhibition occurs. These studies will be complemented with analysis of viral proteins in the extracts.
For the protein analysis, the viral proteins will be meta-30 bolically labelled during infection and treatment, precipi-tated with specific antisera and/or ~ntiho~ies, and analyzed by SDS-PAGE and autoradiography.

WO 96/11692 F~,1,..J. _ .

Since the laboratory strains of HIV do not efficiently infect CD4+ cells from healthy individuals, virus isolated from HIV-positive individuals will be used to infect freshly isolated CD4+ cells from blood donors. The cells will be tested with 5 test compounds at co};Lcentrations not inf luencing the stabi-lity of these cells to be stimulated by phytohemagglutinin or cytokines .
Exam~les of "C1 6 alkoxy" are methoxy, ethoxy, propoxy, iso-propoxy, n-butoxy, isobutoxy, tert.butoxy, pentoxy and hex-1 0 oxy .
In one embodiment of -he invention the alkoxy is selected fra~n the group consisting of methoxy, ethoxy, ~ropoxy' iso-propoxy, and butoxy, such as Rl, R2, R3, and/or R4 beiIlg methoxy . In a presently pref erred embodiment of the inven-15 tion, the anthocyanin or the anthocyanin derivative isderived ~rom an anthocyanidin selected from the group con-sisting of pelargonidin, apigeninidin, and aur~n~in;~in.
.
In ce~tain emhn~ q of the_invention at least one of R
and R2 is ~, whereas in other embodiments at least one of Rl 20 and R~ is CH. In a presently preferred ~Tnho~ , the antho-cyanin or the anthocyanin derivative is derived f rom an anthocyanidin selectea from the group consistlng of cyanidin, delpninidin, luteolinidin, trlcetinidin, 6-hydroxy-cyanidin, 6 - hydroxy - ~e 7 nh; n i din, ~-methyl - cyanidln, and pul r hc. 7 l; (1; n 25 In still other embodiments, at least one of Rl and R2 is alkoxy . It is presentl y pref erred that in this embodiment the anthocyanin or the anthocyanin derivative is derived from an anthocyanidin selectea from the group co~sisting of -peonidin, petunidin, malvidin, -osinidin, europinidin, hirsutidin, and 3 o capensinidin .
The glycosyloxv~ may ~be selected f rom the group conslsting of mono-, di-, t~l-, oligo-, polysaccharides, and deri~atives thereof In particula-, the glycosyloxy may be substituted _ CA 02202467 l997-04-ll Wo 96/l 1692 1 ~l,. .~J.

with one or more acyl groups, or the glycosyl may comprise at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups.
In particular, the acyl group may be selected from the group consisting of acyl groups derived from aromatic and aliphatic acyl groups, such as the group consisting of 4-coumaric acid, caffei~c acid, ferulic acid, sinapic acid, 4-llydLo~y~ zvic acid, gallic acid, acetic acid, oxalic acid, malonic acid, malic acid, maleic acid, and succinic acid.
In one embodiment of the invention, the glycosyl group is a group derived from a ~s~rrl~ride selected from the group consisting of gIucose, galactose, rhamnose, arabinose, xylose, and glucuronic acid.
In another ~mhrrli t, the glycosyl group is a group derived from a ~li c~rr7l~ride selected from the group consisting of 1 , 2 - gluco sy lglucos ide ( s ophorose ), 1 , 3 - g luc osylglucos ide (laminariobiose), 1 , 6-glucosylglucoside (gentiobiose), 1 , 2-xylosylgalactoside (lathyrose), 1,2-rhamnosylglucoside (neo-hesperidose), 1~6-rhamnosylglucoside (rutinose), 1,2-xylosyl-glucoside (sambubiose), 1,6-arabinosylglucoside, and 1,6-rham. nosylgalactoside .
In a third ~m~o~li , the glycosyl group is a group derived from a trisaccharide selected from the group consisting of 1, 2-glucosyl-1, 6-glucosylglucoside~ 1, 2-glucosyl-1, 6-rhamno-sylglucoside, 1, 2-xylosyl-1, 6-glucosylglucoside, and 1, 2-xylosyl-1, 6-glucosylgalactoside.
Although it is contemplated that the compositions may be useful for the. treatment of all retrovirus-related diseases, the invention in particular relates to the use of a compound for the prevention or treatment of infection by a retroviruS
such as infection by Human Il ndeficiency Virus (~IV) =

wo 96/11692 }~l,..J 5 C
;2 and/or ror prevent- on or treatment of Accuired Immune Deti-ciency Syndrome (AIDS ) .
Some or the, u " c ,ithin the generai ~ormula I are known, see e 5. "The Flavor.olds~i, ea. J. B. TT=rhnrno, T.J. Mabry and 5 X. Mabr-~, Cha?man & :-.ail, 1975, "The Flavonoids. Advances in Research", ed. J.B. -:= ~nrrP a=nd T.J. Mabry, Chapman & Hall, 1982, "The Flavonoids Advances in ~esearch since 1950", ed.
J.B ~rhnr-o, Chapman & Hall, 1g88, "The Flavonoids.
Advances in Researc.- since 1986", ed. J.B. ~;lrhnrno, Chapman & Hall, 1994 and -e~e~e-ces in Chemical Abstract, Vol. 119 to 123 under the Generai Subj ect Index entry Anthocyanins .
Howeve-, the invent~ on ~ n a ,~urther aspect relates to novel an~hocyanin der_~.rat_-~es o~ the general ~ormula I
~1 ~OH
R6 ~O~R, R/~ - R3 ~nero r ~
15 R,, R" R3 and ~6 ~ onAont-ly Of each other are ~, OH, alkoxy, an -O-glycosv~ g_oup, an ~a-glycosyl grou? which is subst~~ ~ted wit:. ore c_ mo-e acyl groups, or an -O-glycosyl moietv comp-:si-.g at :east t~o glycosyl groups and at least one ac~ l group ar~~r.ceà so that at least one acyl group is 2 0 located betweer two _ ~cosvl groups, R4 is CH, alkoxy, an. -~-gLycosvl grou~, an -O-giycosvl group which is subst~-_ ec ~i_- one or one acyl groups, or an -O-glycosyl moiet~ co~ p-:si-g at~least two glycosyl groups and at leas~ one ac~ l a-- ? ar-an~ed so that at least one acyl 25 grou? is loc~ted be~-~een. two glycosyl groups, àuDa 1 1 1 U I t SHEET
ISA/EP

CA 02202467 l997-04-ll Wo 96/11692 1~l,. ,J: c c R5 is H, OH, and Y is a counterion, or a prodrug or complex thereof with the exception of the compounds i~n~r~ above.
Furthermore, the invention relates to a method for the prepa-ration of a novel anthocyanidin or an anthocyanidin deriva-tive of the general formula I as defined above, the method comprising isolation and purification of the anthocyanidin or an anthocyanidin derivative essentially by the method out-lined in Example 1. The man skilled in the art will be aware that in isolation and purif ication of known or novel antho-cyanidin and anthocyanidin derivatives, the method described in Example 1 may be amended as d~uLu~.iate e.g. by use of other extraction procedures and cl.l~ to~raphic techniques.
Alternatively, the , .~--ds which are to be used according tû the invention or novel compounds according to the inven-tion may be synthesized e.g. as described in Iacobucci. G.A.
and Sweeny, J. G. (1983), "The chemistry of anthocyanins, anthocyanidins and related flavylium salts~, Tetrahedron, 39, 2Q pp. 3005-3038 or as described in Elhabiri, M. et al. (1995), "Anthocyanin chemical synthesis: an important access to natural and synthetic pigments", Polyphenols Actualites, No.
13, pp. 11-13. Chemical synthesis of the anthocyanidins and the anthocyanidin derivatives may give c-~Lu~.iate; ~' -q to stabilize the compounds.
In general, anthocyanins from blueberries are rather simple anthocyanins. Compared to other anthocyanins, in particular those acylated with aromatic acids like petanin (Sample I), they are more unstable and may therefore be less useful for pharmaceutical purposes. Thus, forms of anthocyanins involv-ing co-pigmentation of anthocyanins and intra- and inter-molecular association states of anthocyanins are within the scope of the present invention.

CA 02202467 l997-04-ll Wo 96/11692 P.~ a~

Each anthocyanin may exist on an extraordinary number of equilibrium f orms . Together with the variation of building blocks of each anthocyanin and the pns~;h;1ity of existing in several association states (;nrlll~l;n~ ~C~o~;~tion with metal 5 ions such as MgZ+, Fe2+, Fe3+ and Al3+, other rh~nn1 ir~ such as cinnamic acids and other :flavonoids, and polymeric mate-rial) this allows quite a number of structural mo-l;f;~t;ons whi ch may inf luence ef f ect s / activity ~
As a consequence of asymmetric centres, the compounds of the lO present invention can occur as mixtures of diastereomers, racemic mixtures and as individual i~n~nt;, rS All asymme-tric forms, individual isomers and combinations thereof are within the scope of the present invention.
Pharmaceutical compositions comprising mixtures of antho-15 cyanins derived f rom e . g . blueberries such as Myrtocyan~
(Vaccinium Inyrtillus anthocyanosides corresponding to 25~6 as anthocyanidines) as well as topical medicinal compositions cnnt~;n;n~ fruit juice or fermented fruit juice as described in C~ 1086651, a topical composition consisting of an ~ïsopro-20 panol extraction of mountain ash berries as described in US4 ,132, 782, alcoholic extracts of anthocyanosides described in FR 2456747, compositions comprising bilberry anthocyanidines, grape anthocy~n;~;n~ or elder~anthocy~n;~l;n~c described in GB 1, 589, 294 and anthocyanidin~ glycosides extracted from 25 bilberries, black currents and blackberries described in US
3, 546, 337 are known. However, these_compositions are based upon partially purified produc~ts from fruit or berries and, in addition to the anthocyanin, do also contain other com-pounds with a potential pharmaceutical activity such as 30 flavonoids. In contrast, the present invention is based upon much more purified anthocyanins.
A further aspect o~ the invention thus relates to a pharma-ceutical composition comprising an anthocyanidin or antho-cyanidin deriuative of the general f ormula I

Wo 96~11692 } ~~ S

wherei~
Rl, R2, R3 and R6 in~i~pon~ nrly of each other are }I, OX, alkoxy, an -O-glycosyi group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-g}ycosyl 5 -moiety compr sing a~ eas~ :wo glycosyl groups and at least one acyl group arranged so tha~ at least one acyl groUD is located between two giy~csyl groups, R4 is O}I, alkoxy, an -O-glycosyl group, an -O-glycosyl group whlch is substltute~ with one or o~e acyl groups, or an -O-10 glycosyl moiety comc-:sing at least two glycosyl groups and at least one acyl g-cc~p ar-anged so that at least one acyl group is locatea be~ween _wo glycosyl groups, Rs is H, O~i, and lS Y is a counterion, or a prodrug or comp ~ ex ~~Lereof with the exce~Dtion of the above I r: nno i comccsit- ons .
The cytoto:c c anà an~~ l effects in EIIV infected cells have been ~ in~a _~ one anthocyanin sample i-qol~t~ from 20 blue po~atoes (Solar.~ ~ ~ ercsum) (Sample I) and two antho-cyanin samDles iqol~r.~i '~om blue})er-ies (Vaccir~ium myrtillus (Samples II ana rI_~. sam~le I nnnt~inq one clean anthocyanin (called pe~ani~) cc~c- sing an aglycone, three m - r~h~ide moieties and one arc.~ 3c~ l group. Samples II and III both SVVVI11UI~ SHEET
_ 15A/~P

Wo96/11692 ~ . r contain a mixture of anthocyanins. Each anthocyanin in these mixtures are buiLt from only one aglycone and one - n::ar~h:q_ ride. Sample III cnnt~;n~ the same, however, a reduced number of anthocyanin compared to Sample II. Sample I which -n~t~inc 5 only one, rather complex anthoc~yanin, shows the best test results .
For sample II at ~nn-~ntrationS above 20 ~Lg/ml, an antiviral effect is observed (Figure 7). When studying the figure, it is noted that an antiviral effect can also be observed at a 10 concentration of 1 ~Lg/ml, however, this effect is not clearly reproduced at concentrations between l and 20 ~g/ml. In this respect, it should be emphasized that Sample III is a puri-f ied sample of Sample II which may include com~ounds with no antiviral ef f ect as well as compounds which do have the 15 desirea antiviral effect. This fact may explain that no dose-response curve can be obtained for this sample.
Further studies may be perf ormed with respect to various con-centrations of the compounds and mixtures of compounds accor-ding to the invention in order to titrate the exact concen-20 tration at which a cytotoxic effect or an antiviral effect isobtained.
A particular preierred ~mh~im~nt of the invention relates to a pharmaceutical ~composition comprising petanin in combina-tion with a pharmaceutically acceptable excipient.
25 Other preferred ~mhoriimontS are pharmaceutical compositions comprising a mixture of individual anthocyanins as outlined in Table I~or in Table II in ~ in~tion with a pharmaceuti-cally acceptable exci~ient. Also pharmaceutical compositions comprising a novel anthocyanin derivative in combination with 30 a pharmaceutica~ly acceptable excipient are within the con-cept of the present invention.
With respect to the counterion Y, it should be recognized that the particular counterion forming part of the salt of Wo 96/11692 P~

this invention is no~ of a critical nature, as long as it is compatible with the anthocyanidin or anthocyanidin derivative cation. The counterion is in particular a pharmacologically acceptable anion. The counterion may be organic as well as 5 inorganic in nature.
The term "pharmaceutically acceptable anion" as used herein refers to anions in the salts of the above formula which are subst~nti~lly non-toxic to living organisms. Typical pharma-ceutically acceptable anions include those derived from a 10 mineral or organic acid.
Examples of such inorganic acids are hydrochloric acid, lly~iLvbLvllic acid, hydroiodic acid,~ sulfuric acid, rhnqFhnric acid and the like, and examples of the organic acids are p-toluenesulfonic acid, methanesulfonic acid, oxalic~acid, p-15 bromophenylsulfonic acid, carbonic acid, succinic acid,citric acid, benzoic acid, acetic acid and the like.
Examples of the anions are sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, di-hydrogenphosphate, metaphosphate, pyrophosphate, chloride, 20 bromide, iodide, acé~ate, proprionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, pro-pionate, oxalate, malonate, succinate, ~suberate, sebacate, fumarate, ma1eate, butyne-1, ~-dioate, hexyne-1, 6-dioate, benzoate, chlorober,zoate, methylh=n~o~e, dinitrobenzoate, 25 hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulf onate, phenylacetate, phenylproprionate, phenyl-butyrate, citrate, lactate, ~y-hydroxybutyrate, glycollate, tartrate, meth~nl~qlll fonate, propanesulfonate, naphthalene-l-sulfonate, n~rhth~l ene-2-sulfonate, and mandelate anions, and 3 o the like . Pref erred anions are those derived f rom mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid and j th~n~qul~onic acid. _ =

W096~1 69 . S,l -o.
1~1 The compositions of :'-,e presen~ inven~_on are useful in the prevention or ~reatmen~ of infection by the human i '~
cienc~,r virus (XIV) and the trearment of consecuent p~thnl rri -cal conditions suc- as AIDS. 'rreating AIDS or preventing or 5 treati3g ~niect_on~,~y U.IV is defined as inrlllrinr, but not being limitea to, t-ea~ing a wlde range of states oi }~IV
infection: AIDS, ARC (AIDS related comolex), both symptomatic and asym~tomat_c, ana actual or potential exposure to EIIV.
For exam~le, the ccm~os;;ions oft this invention are useful in 10 treating infect:on_y ULV aite- s~qr~rt~ past ~UU~UL~:: to XIV by, e.g., hlood _-ansfusion, organ tr~n.qpl ~nt, t~ CU-y~
of body fluids, bites, arri~i~nt~l neeale stick, or ~U9UL-::
to patient blooa du-:-g surgery.
For these purcoses, ~.e rn"rnrun~q of the present i~ve~tioa lS may be aaministerea --ally, parenteraily (;ncll~in~ subcuta-neous ~ nj ect~ ons, ~ noas~, irtramuscular, intrasternal inject-on or in~usic- t~rrnir -,~q), by inhalation spray, or rectally, in dosacle u~it fnrm~ tirnq r~nr:lininr cor,vl~ntinn~l non-toxic pharmaceu~:_ally acceptable carriers, adjuvan~s and 20 vehicles.
Thus, in acco-aance wi _h t~e oresent ~ ~vention there ~ s fur-ther providea a met.c 'or the preven~ on and/or treatment ûf a disease caused hy a -etrovi-us, the method comprising admi-nister -g to a mamma; ~ neea thereof an ef iective amount of 25 an ant'r.ccyanir. der :a~ ve of ~ the general f ormula -RS
R~J\~R3 R~
whereir. .
.,uua 1 11 u 1 ~ SHEET
ISA/EP
-CA 02202467 1997-04-ll Wo 96/11692 R1, R2, R3 and R6 indepenaently of each other are H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least 5 one acyl grou~ arranged so that at~ least one acyl group is located between two glycosyl groups R4 is OH, alkoxy, an -O-glycbsyl group'~ an -O-glycosyl group which is substituted with one or one acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and 10 at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R~ is H, OH, and ==
Y is a counterion, 15 or a prodrug or complex thereof.
The treatment involves administering to a patient in need of such treatment a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically effective amount of a compound of the present invention, or a pharmaceutically 20 acceptable salt thereof.
These pharmaceutical compositions may be in the form of orally administrable suspensions or tablets; nasal sprays;
sterile injectable preparations, for example, as sterile injectable aqueous or oleaginous suspensions o~ supposito-25 ries. : ~
When administered orally as a suspension, these compositionsare prepared accordi~g=to technlques well-known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or:sodium alginate 3 o as a suspending agent, methylcellulose _as a viscosity enhan-cer, and sweeteners/flavouring agents known in the art. As immediate release tablets, these compositions may contain mi~L..~:~ y:~Lalline cellulose, dicalcium phosphate, starch, mag-nesium stearate and lactose and/or other excipients, binders, p~tPn~PrF~, ~;c;nte~rants, ~ ntc and lubricants known in the art.
5 When administered by nasal aerosol or in~1Ptinn~ these com-positions are prepared accQrding to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other sui-table preservatives, absorption promoters to enhance bio-lO availability, fluorocarbons, and/or other soll-hili~in~ or dispersing agents known in the~ art.
The injectable solutions or suspensions may be fnr~ tPd according to known art, using suitable non-toxic, parenteral-ly acceptable diluents or solvents, such as mannitol, l, 3 -15 but~n~ l, water, Ringer' s solution or isotonic sodium chlo-ride solution, or suitable dispersing or wetting and suspend-ing agents, such as sterile, bland, fixed oils, inf-lll~in~
synthetic mono- Qr diglycerides, and fatty acids, including oleic acid. - =
20 When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, syn-thetic glyceride esters or polyethylene glycols, which are solid at: ordinary temperature but liquidify and/or dissolve 25 in the rectal cavity to release the ~rug. =
Dosage levels of the order of 0 . 02 to 5 . 0 or lO_ 0 g per day are useful in the treatment or prevention of the above-indi-cated conditions, with oral doses two to f ive times higher .
For example, infectïon by HIV~ is effectively treated by the 30 administration of from l.0 tQ 50 mg of the compound per kg of body weight from one to four times per day. In one preferred regimen, dosages of 100-400 mg every six hour~s are admini-stered orally to each patient. It will be understooa, how-ever, that the specific dose level~ and frequency of dosage Wo96/11692 1 for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific ~ employed, the metabolic stability and length of ac-tion of that compound, the age, body weight, general health, 5 sex, diet, mode and time of administration, rate of excre-tion, drug combination, the severity of the= particular con-dition, and the host undergoing therapy.
The anthocyanidin or anthocyanidin derivatives may be useful either as compounds or mixtures of compounds, pharmaceuti-10 cally acceptable salts, pharmaceutical comE~osition ingredi-ents, either solely anthocyanidin or anthocyanidin deriva-tives or in combination with other anti-viral agents, immuno-modulators, antibiotics or vaccines. For example, the com-pounds of this inver,tion may be effectively administered, 15 whether at periods of pre-exposure and/or post-exposure, in combination with effective amounts of other antiviral agents, uulators, anti-infectives, or vaccines known to those of ordinary skill in the art.
LEGEND TO FIG~RES
20 Figure l shows the relationship between ce~l ~umber and staining by MTT.
Figure 2 shows the ef f ect Qf DMSO on cell growth and tkat 0.33~ DMSO can be used as a solvent for ~the compounds without affectlng cell growth.
25 Figure 3 shows the effect of petanin in different crnrPntra-tions dissolved in DMSO on the growth of SupTl cells measured after five days of incubation.
Figure 4 shows the effect of the first purified Vaccinium myrtillus sample (Sample II) in different rrnrPntr~tiQnS
30 dissolved in DMSO on the growth of SupTl cells measured after ~8 hours of incubation.

WO 96111692 ~_1/..

Figure 5 shows the effect of the second purified Vacr7ni1~m myrtillus sample (Sample III) in different rnnc~n~rations dissolved in DMSO on the growth of SupTl cells measured af ter five days of ;n~1lhat;nn.
5 Figure 6 shows the effect of petanin in different rnrl~P~tra-tions on the inhibition of formation of syncytia. The effect i8 shown as a percentage of ~the formation of syncytia in cells incubated with only DMSO.-Figure 7 shows the effect of the first purified Vaccinium 10 myrtillus sample (Sample II) in different r-nnn~ntrations on the inhibition of formation of syncytia. The effect is shown as a percentage of the f ormation of syncytia in cells incu-bated with only DMS0.
Figure 8 shows the ef f ect of the second purif ied vaccinium 15 myrtillus sample (Sample III) in different rnnr~ntrations on the inhibition of formation of syncytia. The effect is shown as a percentage of the formation of syncytia in cells incu-bated with only DMS0.
Figure 9 shows the high performance li~luid chromatography pro-20 files of ~the anthocyanin content of Solanum tuberosum durinr, the purification procedure. A, crude extract; B, after parti-tion against ethyl acetate and treatment with Amberlite XAD-7; C, af ter droplet - current chromatography; D, af ter Seph~dex LH-20 gel filtration. The different samples are monitored 2S simultaneously at two different spectral areas (i and ii).
Figure lO shows the structure: of petanin, which is the antho-cyanin isolated from Solanum tuberosum. = ~
Figure ll shows the anthocyanin content o the f irst purif ied Vaccinium myrtillus sample ~Sample II~ detected at 5~20 +
30 20 nm. The peaks are labelled according to the numbers given in Figure~12.

Wo 96/11692 Figure 12 shows a) the structures and b) the relative propor-tions (g6) of the individual anthocyanins in the first puri-fied Vaccinium myrtillus sample (Sample II) .
Figure 13 shows the anthocyanin content ~ of the second puri-fied Vaccinium myrtillus sample (Sample III) detected at 520 i 20 nm. The peaks are l~lh~ d according to the numbers given in Figure 14.
Figure 14 shows a) the structures and b) the relative propor-tions ( ~ ) of the individual anthocyanins in the second puri -lO fied Vaccinium myrtillus sample (Sample III) .

CA 02202467 l997-04-ll Wo 96/11692 EXAMPLES
TEST MEl~IODS
Det~3~;n ~t~n of cytotoxic and antiviral effects in lIIV
in~ected cells of ~ __ '~ or mixture~ of - __ all accord-5 ing to the in~e~tion 1. Cultivation of ce] ls The human CD4+ lymphocyte cell line Sup T1 derived from a Non-Hodgkin' s T-cell lymphoma patient (Smith et al. ~1984), Cancer Research 44, 5657) was a gift from Dr. J. Sodroski at 10 the Division of }Iuman Retroviruses, Dana Farber Cancer Insti-tute, Harvard Medical School, Boston, U.S.A., and was chosen for these studies due to its high content of CD4+ receptors and ability to f orm large syncytia f ollowin~ inf ection with HIV-1. The cells were cultivated as suspension cultures in 15 plastic flasks (NUNC, Copenhagen, Denmark - T25 flasks or T125 flasks) in RPMI 1640 medium (Bio Whittaker, Walkers-ville, MD, USA) supplemented with 59~ v/v fetal calf serum, 2 mM glutamine (both from Bio Whittaker) and ABAM (Cat.No. A
ggo~, Sigma Chem. Company, an O.lM antibiotic and antimyc~tic:
20 solution containing penicillin and fungizone) in 1 mM final co~centration and gentamicine (Bio Whittaker) to a f inal concentration of 50 I~g/ml at 37C and 59~ CO2 in an incubator (Assab ~ebo BioMed).
Counting of cell numbers was performed the same day the 25 experiments started using the Trypan blue exclusion method ~ Tissue Culture Chemicals, a catalogue from Sigma, 1994J and a Burker counting chamber ( "ASSISTENT", Germany) at a magni-fication of 400x. The ratio between the living and dead cells was at -least 95/5 in all experiments detL ~mi norl as described 30 in John Paul, "Cell ~ Tissue Culture", p. 368, Fifth Edition, Churchill ~ivingstone, 1975). Prior to the experiments the mediurn was half-changed in order to add new growth compo-nents. The cell density was adjusted to approximately 5 x WO 96/11692 25 r~
105 cells/ml and kept at this cnnr~ntration tllL~U~llUUL the experiment by countinr the cell number and adding new medium as d~L~Liate or, if n~r~cs~ry, by centrifugation of the cell suspension and r~CllcpQnci nn of the cell pellet in an 5 G~L~Liate amount of RPMI 1640 medium.
HIV virus producing Molt 3 IIIB cell line The cell line was est~hl; ch,-d by infecting Molt 3 cells (_merican Type Culture Collection, ATCC CRL 1552) with the HIV-1 strain HTLV IIIB obtained from Dr. W. A. Haseltine at 10 the Division of ~Iuman Retroviruses, Dana Farber Cancer Insti-tute, Harvard Medical School, Boston, lJ.S.A. The Molt 3 IIIB
cell line is rrnr~l~rinr virus particles constitutively.
The cells were cultivated as suspènsion cultures in plastic flasks (NUNC, ~-nponh~ren~ Denmark - T25 flasks or T125 15 flasks) in RPMI 1640 medium (Bio Whittaker, Walkersville, MD, USA) supplemented with 5% v/v fetal calf serum, 2 mM
glutamine (both from Bio Whittaker) and ABAM (Cat.No. A ggog, Sigma Chem . Company, an 0 . lM antibiotic and antimycotic solution rnnt~ininJ penicillin and fungizone) in 1 mM final 20 rnnr~ntration and gentamicine (Bio Whittaker) to a final rnnr.~ntration of 50 ~Lg/ml at 37C and 5% CO2 in an incubator (Assab ~ebo BioMed) .
2. Cvtotoxicitv of the com~ounds or mixture of coml~ounds tested 25 2.1. The MTT assay method for determining the number of viable cells The principle of this assay is based on the cleavage of the yellow tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Thiazolyl blue, Product No.
30 M 5655, Sigma Chemical Company) to form formazan crystal due to the dehydrogenase activity in the living cells ~(Mosman, T.
et al. J. Immunol. Methods, 65, 55). A standard curve for the Wo 96/11692 MTT assay wa6 est~hl;~ho~i (Fig. l) by rl;l~tin~ tiFIlly growing SupTl cells at known cell numbers in a standard medium (RPMI 1640) into a 96 wells tissue plate (NUNC) at a total volume of lO0 ,ILl followed by adding 50 Ill of MTT rea-gent (3 mgfml in phosphate buffer solution (PBS), pH 7.20) to each well. After addition of the MTT reagent, the plate was inr1lh~t~ at 37C and 5g~ CO2 for 3 hours in an ;nr11hator (Assab Kebo BioMed) . Then the cells were centrifuged at 2, 000 rpm (800 x g) for lO minutes in a centrifuge equipped with micro-titer plate holders (Beckman centrifuge, G3-6). After centrifugation lO0 ~Ll of ~supernatant was removed from the wells. For this purpose a multi r~n~l micro-pipette was used ~Finnpipettes, Finland). The pelleted cells were resuspended in lO0 I~l DMSO (dimethyl sulfoxide, Merck) and the plates were gently shaken by hand for about lO minutes at room temperature be~ore the absorption was read in an E~ISA reader (TiterekX Multiskan Plus MK II photometer~ er~uipped with a 580 nm light filter ~Flow ~aboratories, USA). The standard curve of the relationship between cell number and staining by MTT
is shown in Figure l with a ranging from lo3 cells/well to 5 x 104 cells/well corresponding to ODs80 = . Ol and ODS80 =
0.50, respectively. As shown in Figure l, within the amount of cells used, there is a linear rPl~t~nnRhip between the number of living cells and the intensity of staining between cell numbers o~ 20 . 000 and 60 . 000 . A new standard curve is est~hlic~d as appropriate e.g. when a new series of experi- _ ments are started by a hitherto unexperienced person_ The repro~ ri hi 1 i ty of the standard curve is good.
2.2. Determination of the effçct of DMSO on cell growth 3 o Since t~e water solubility of the compounds to be tested varies, the compound or mixture of compounds to be tèsted are dissolved in DMSO prior to addition to the cell cultures. The ef f ect of DMSO on the cell growth was theref ore tested . The cells were added to a 96 wells micro-titer plate; each well containing 1 x 104 cells in lO0 ILl of RPMI 1640 medium. To the suspension of cells was then addea DMSO at dif ferent Wo 96/11692 r~l,..J,_ rnnr,~ntrations ranging from 0 . Ol~ v/v DMSO to 1. 0~ v/v DMSO.
Following j nrl.h~t; on in an incubator (Assab Kebo BioMed) at 37C with 5~ C02, the amount of living cells as a function of the DMS0 rnnr~ntration was evaluated after l, 2, and 5 days 5 of incubation by applying the MTT assay as described above.
From the results of the experiments (Figure 2) it can be deducted that the maximum rnnr~ntration of DMS0 that could be used without affecting cell growth was 0.2~ v/v. Above that rnnr~ntration DMSO has a significant effect on the growth of 10 SupTl cells. At 0.2% v/v cnnr~ntr~tinn or lower of DMS0, practically no difference between cells with or without DMS0 could be observed. For this reason compounds to be tested in SupTl cultures in the presence of DMS0 have to be kept in solutions at rnnrl~ntrations so that the final rr~nr~ntration l5 of DMSO does not exceed 0 . 2~ v/v DMS0 .
2.3. Effect of test~ substances on the growth of uninfected SupTl cells For each substance to be tested or for each mixture of sub-stances to be tested, 3 parallel experiments were done.
20 Survival of cells were tested after l, 2, 5, and 7 days, respectively, after starting treatment of the celis with the substances. The cells were ~-;nt~;n~d in a g6 wells micro-titer plate. To each well l x lOg cells in lO0 ~l RPMI 1640 medium were added. To the suspension of cells was then added 25 lO l~l of the test substance in DMSO and RPMI 1640 medium in order to ensure that the final rnnr~ntration of DMSO did not exceed 0.2~6 v/v. As control, cells with or without DMSO were used. At the end of incubation at 37C and 5~6 CO2 for 3 hours in an incubator (Assab Kebo BioMed) ) with the compounds, 3 o survival of the cells was measured by adding the MTT reagent and the samples were processed as described above in section 2 .2 .
The results of the 3 extracts containing 3 different com-pounds or mixture of compounds dissolved in DMSO after five 35 days of incubation are shown in Figures 3-5.

CA 02202467 l997-04-ll 9~ )l 3. Testina compounQs or mixture of r .Lllds for antiviral ef f ects The screening of antiviral effect of different compounds or mixtures of compounds was based on measuring the ~orma~tion of 5 syncytia as the exact number of syncytia present after infec-tion of cells with HIV-1 can easily be counted by use of an inverse microscope and thereby an effect obtained by the compound or mixture of compounds added can be measured.
~IV-1 cnnt~;ninr supernatant from Molt 3 IIIB cell super-10 natant was prepared by centrifugation of the Molt 3 III3 cellculture at 1, 000 rpm in a 3~rkr-nn GS-6 centrifuge equipped with a GH- 3 . 7 rotor ~or 5 minutes . In order to standardize the supernatant with respect to the amount of virus, p24 Ag was measured using an ELISA based technique (Sundr~vist et al.
(1989), J. Medical Virology 29 :170-175) .
E:ach virus supernatant used in the experiment had a F24 Ag cnnrl~ntration of 1.5 - 2 ng/105 cells. ~ach T25 (~UNC~ flask was filled with 1 ~ 104 cells/ml in a total volume of 5 ml.
The test substances was added 3 0 minutes prior to the addi-20 tion of the virus cnnt~ininr supernatant and during t=his pre-incubation the flasks were kept at 37C and 59s C2 in an incubator (Assab Rebo BioMed) . After pr~inrllhation~ 500 ~11 of virus supernatant was added. The number of syncytia was counted after 24 and 48 hours o~ incubation at 37C and 596 CO
25 in- an incubator (Assab Kebo BioMed) (this time was found to be the standard tir~es for optimal syncytia formation for this cell line at the cnnron~ration of virus used).
For each test substance 2 flasks were used and the syncytia were counted by rol~ntinr the number of syncyt1a at 5= diffe-30 rent places on each flask in an inverse microscope (OlympusCR 2) using a magnifiri~tion of 10x, thus giving 10 indepen-dent countings for each test substance. The parallels ob-tained we~e within +- 10~6 for each experiment.

WO96111692 ~,1,..J S.lD~O.

The results of the 3 extracts cnnt~;nin~ 3 different com-pounds or mixture of c _ u-~ds are shown in Figures 6 - 8 . For each compound or mixture of ~ ~ ul~ds, the inhibition of formation of syncytia is shown as a percentage of the forma-5 tion of syncytia in untreated cells.
ExaMPLEs EXaYPLE 1 Isolation and purif;c~t;nn of the anthocy~ni~, pet~nin, from Sample I, blue potatoes (Solanum LUI~e~L _ L. ) and a mixture 10 of anthocy~nins from Samples II and III, b~ h~ ~ies (Vacci-nium nyrtil l us L . ) Sam~les Sample I: Tubers ~297 g) of Solanum tuberosum L. (anthocyanin pigmentation in skin and flesh) from cultivation at the Agri-15 cultural University of Norway, NLH-As, Norway, were collected in October 1994, cut with a pair of scissors and extracted for 3 hours (three times) with methanol cnnt~;nin~ O.1~ v/v rnnr~ntrated hydrochloric acid.
Sample II and SampIe III: Ripe berries of Vaccinium myrtillus 20 L. were collected in Asane near Bergen an the west coast of Norway in August 1992. The frozen berries (100 g) were extracted for 5 hours (twice) with 500 ml of methanol con-taining 0 . 059; V/V Cnn~-~ntrated hydrochloric acid.
For all three samples: The f iltered extracts were combined 25 and cnnc~ntrated under reduced pressure at 28C.
Procedure for ~urification of the sam~les For sample III, the lower layer of n-butanol-acetic acid-water (4:1:5, V/V) was used as mobile phase. A flow rate of CA 02202467 l997 - 04 - l l W096/11692 r~l/..J S.O~JO5 9 ml/hour was used tllLuuyllc~-lt the experiment. Some stationary phase (110 ml) was displaced prior to elution of the first drop of mobile phase. Then 150 fractions, each of 4 ml, were collected. Fractions 13-15 were collected and cnnr~nt~ated 5 under reduced pressure at 28C before the sample was sub-jected to gel filtration.
The r~nr~nt~ated solution (ca. 100 ml) was washed twice with 100 ml ethyl acetate, and the lower layer was further ~concen-trated under reduced pressure at 28C before it was passed through an 18 x 2 . 6 cm Amberlite~ XAD-7 column (an ion e~ul~dlly~ resin ~rom BD~ Chemicals Ltd. ) which had been washed in advance with distilled water. The XAD-7 column (with the adsorbed anthocyanins ) was washed with 2 1 of distilled water. To elute the anthocyanins, 300 ml each of 50 % aqueous 15 methanol and anhydrous methanol (both c~ nt~in;nr~ 0 .5% v/v CF3COO~I) were used successively.
Droplet counter-current chromatography (DCC~) was carried out using a Tokyo ~;k~k;k~; Eyela Model DCC-300 chromatograph ~itted with 300 glass capillaries ~40 cm x 2 mm i.d. ) .
20 For Sample I, the upper layer of n-butanol-acetic acid-water ~4:1:5, v/v) was used as mobile phase. A flow rate of 10 ml/hour was used throughout the experiment. Some statio-nary phase (100 ml) was displaced prior to elution of the ~irst drop o~ mobile phase . Then 45 fractions ,` each of 7 ml, 25 were collected. Fractions 12-35 were collected and concen-trated under reduced pressure at 28C before the sample was subjected to gel filtration.
For Sample II, the lower layer of n-butanol-acetic acid-water ~4:1:5, v/v) was used as mobile phase. A flow rate of 3~ 10 ml/hour were used throughout the experiment. Some statio-nary phase ~150 ml) was d~splaced prior to elution of the =
first drop of mobile phase. Then 160 fractions, each of 4 ml, were collected. Fractions 20-100 were co~lected and concen-CA 02202467 l997-04-ll WO96111692 T~ J7~ o~

trated under reduced pressùre at 28C before the sample was subjected to gel filtration.
For Sample III, the lower layer of n-butanol-acetic acid-water (4:1:5~ v/v) was used as mobile phase. A flow rate of 5 9 ml/hour was used tl~Juyl~ut the experiment. Some st~;nn~ry phase (110 ml) was displaced prior to elution of the first drop of mobile phase. Then 150 fractions, each of 4 ml, were collected. Fractions 13-15 were rnll !~cted and concentrated under reducea pressure~at 28C before the sample was subject-10 ed to gel filtration.
Gel filtration was performed on a 100 x 3 cm Sephadexe~ L~I-20 column using 4096 v/v aqueous methanol cnnt~ining 1~ v/v CF3COO~ as eluent. All the anthocyanin fractions belonging to each sample were put together and evaporated to dryness under 15 reduced pressure.
Monitorinq of fractions Thin-layer chromatography (TLC) analyses were performed on 0.1 mm cellulose layers (Schleicher and Schull, F1440) in the following solvent systems:
A. Formic acid-concentrated hydrochloric acid-water (5:1:5, v/v) B. n-Butanol-acetic acid-water (4:1:5, V/V, upper phase ) .
~igh performance liquid chromatography (XPLC) was carried out 25 using a slurry packed ODS-E~ypersil column 20 X O . 5 cm, 5 ~lm) .
Two solvents were used for elution ~A: formic acid-water (1:9, v/v) and B: formic-acid-water-methanol (1:4:5, v/v).
Several slightly different elution profiles were used: A
typical elution profile was composed of=~isocratic elution (90% v/v A, 10~6 B) over 4 min, linear gradient from lOgs v/v s to 10096 B over the next 17 min, followed by linear gradient ~o 96/11692 ~ Pcr~095100~85 from lO096 B to lO96 v/v B over l min. The flow rate was 1.5 ml min~l, and aliquots of lO ~l were injected. W/Vis absorption spectra were recorded using a photodiode array detector (HP
1050 , Hewlett-Packard), and spectral mea~u~ - q were made over the wavelength range 210-600 nm.
The relative ~uantities of the indiYidual anthocyanins in the purified Vaccillium myrtillus sample ~Sample II) were based on integration of the different peaks in the HPLC chromatogram ~Figure ll) of the purified sample. This chromatogram was lO recorded by measuring the absorption values on every second nm between 500 and 540 nm simultaneously, and do` not take into account the different molar absorption coefficients of the individual anthocyanins. ~ ~
In a similar manner, the relative quantities of the indi-15 vidual anthocyanins in the second purified Vaccinium myr-tillus sample (Sample III) were based on integration of the different peaks in the HPLC chromatogram (Figure 13) of the purified sample.
Contents of` Sample I
20 Figure 9 shows the high performance liquid chromatoyraphy pro~iles of the anthocyanin content of ~olanum tuberosum during the purification procedure. A, crude extract; B, after partition against ethyl acetate and treatment with Amberlite XAD- 7, C, after droplet-current chromatography, D, after 25 Sephadex L~-20 gel filtration. The different samples are monitored simultaneously at two different spectral areas ~i and ii ) .
Figure lO shows the structure of petanin, which is the antho-cyanin isolated from Solanum tuoerosum.
30 A sample of lO mg of petanin was tested for biological acti-vity as described in Example 2 . l .

Wo 96/1169~ F~~ 7 `7-0~7 33 ~ ~ :
Contents of Sam7,~1e II
Figure ll shows the anthocyanin content of the first purified Vaccinium myrtillus sample detected at 520 + 20 nm. The peaks are 1,7h~- 1 Pd according to the numbers given in Figure 12 .
5 Figure 12 shows the structures and b) the relative propor-tions ( ~ ) of the individual anthocyanins in the f irst puri -fied Vaccinium myrtillu~s sample.
A sample (600 mg) of this purified anthocyanin mixture of V;7Cr;n777m myrtillus was tested for biological activity as lO described in Example 2 . 2 .
Contents of Sam~le III
Figure 13 shows the anthocyanin content of the second puri-fied Vaccinium myrtillus sample detected at 520 + 20 nm. The peaks are labelled according to the numbers given in Figure 15 14.
Figure l~ shows a~ the structures and b) the relative propor-tions (9~) of the individual anthocyanins in the purified Vacc~nium myrtillus sample. =
A sample (16 mg) of this purified anthocyanin mixture of 20 Vaccinium myrtillus was tested for bioIogical activity as described in Example 2 . 3 .
EXaMP~E 2 Deternination of cytotoxic a7ld antiviral effects in lIIV in-fected cells in three different extracts ~nn7-:l;nin~
25 or mixtures of ~ according to the invention The cytotoxic effect and the antiviral effect in HIV infected cells of the three extracts obtained according to Example l were tested as desc-ibed in Test Methods, sections 2 and 3.

Wo 96/11692 r~ c Cytotoxic ef f ect of a compound or mixture of eompounds i~s defined here as the rnnr~ntration of the eompound or mixture of compo~nds which effects the growth rate of the eells tested . Here, a cytotoxic ef f ect of a compound or mixture of 5 m ~ is rrnci~lored present if a deerease in OD5~0 of more than lO~ is observed as a result of ineubation with the compound or mixture of ~ . With respect to the eytotoxie effeet, the results are shown in Pigures 3-5.
-An antiviral effect is here considered present if a decrease 10 in syncytia formation of more than lO96 is observed as aresult of =, nrllhaf i ~ with the compound or mixture of c~;l-pounds With respect to the antiviral ef f ect, the results are shown in Figures 6 - 8 wherein f or each compound or mixture of compounds the inhibition of formation of syneytia is shown as 15 a pereentage of the formation of syncytia in untreated cells.
In Figures 6-8 are shown the results after 24 hours and/or 48 hours. At 48 hours the same pattern is observed although the total amount of syncytia is higher. All three compounds or mixture of ~UIllyu~ s have a cIear inhibitory ef f ect on the 20 cytopathogenic effect of XIV although complete inhibition of syncytia formation cannot be obtained at the experimental conditions used.
Cytotoxic and antiviral ef fect of Sample At concentrations between 0.02 and 0.2 mg/ml, a cytotoxic 25 effect on cell growth rate is observed (Figure 3) .
At r~nc~ntrations above 1 ILg/ml, an antiviral effect ~is observed (Figure 6) .
-II Cytotoxic and antiviral effect of Sample II
At conron~rations above 40 llg/ml, a cytotoxic effect on cell 30 growth rate is observed ~Figure 4) .

wo 96/11692 . . r~l,..J

At cnnnontrations above 20 llg/ml, an antiviral effect is observed (Figure 7). When studying the figure, it is noted that an antiviral effect can also be observed at a rr1nr~ntra-tion of 1 llg/ml, however, this effect is not clearly repro-5 duced at c~ ntrations between 1 and 20 llg/ml.
III Cytotoxic and antiviral effect of Sample III
At c~n~ ~ntrations above 0, 03 mg/ml, a cytotoxic effect on cell growth rate is observed (Figure 5).
At cnnr~ntrations above O.l llg/ml, an antiviral effect is ob-lO served (Figure 8).

Claims (30)

36

1 The use of an anthocyanidin or an anthocyanidin derivative of the general formula I
wherein R1, R2, R3 and R6 independently of each other are H , OH , alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R4 is OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or one acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R5 is H, OH, and Y is a counterion, or a salt, prodrug or complex thereof for the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease caused by a retrovirus in a mammal including a primate such as a human.

2. The use according to claim 1, wherein the anthocyanidin or the anthocyanidin derivative, when dissolved in DMSO at a concentration so that the final concentration of DMSO does not exceed 0.2% v/v DMSO, a) when tested as described in section 2.3, the anthocyanidin or the anthocyanidin derivative does not have a cytotoxic effect on the growth of uninfected SupT1 cells resulting in a decrease in OD580 of more than 10% as a result of incubation with the anthocyanidin or the anthocyanidin derivative, and b) when tested as described in section 3, the anthocyanidin or the anthocyanidin derivative has an antiviral effect on the growth of Molt 3 IIIB cells infected with HIV-1 defined as a decrease in syncytia formation of more than 10% as a result of 48 hours of incubation with the anthocyanidin or the anthocyanidin derivative.

3. The use according to claim 1 or 2, wherein alkoxy is selected from the group consisting of methoxy, ethoxy, propoxy, isopropoxy, and butoxy.

4. The use according to claim 3, wherein R1, R2, R3, and/or R4 is methoxy.

5. The use according to any of claims 1-4, wherein at least one of R1 and R2 is H.

6. The use according to any of claims 1-4, wherein at least one of R1 and R2 is OH.

7. The use according to any of claims 1- 3, wherein at least one of R1 and R2 is alkoxy.

8. The use according to claim 5, wherein the anthocyanin or the anthocyanin derivative is derived from an anthocyanidin selected from the group consisting of pelargonidin, apigeninidin, and aurantinidin.

9. The use according to claim 6, wherein the anthocyanin or the anthocyanin derivative is derived from an anthocyanidin selected from the group consisting of cyanidin, delphinidin, luteolinidin, tricetinidin, 6-hydroxy-cyanidin, 6-hydroxy-delphinidin, 5-methyl-cyanidin, and pulchellidin.

10. The use according to claim 7, wherein the anthocyanin or the anthocyanin derivative is derived from an anthocyanidin selected from the group consisting of peonidin, petunidin, malvidin, rosinidin, europinidin, hirsutidin, and capensinidin.

11. The use according to any of the preceding claims, wherein the glycosyloxy is selected from the group consisting of mono-, di-, tri-, oligo-, polysaccharides, and derivatives thereof.

12. The use according to claim 11, wherein the glycosyl group is substituted with one or more acyl groups, or the glycosyl moiety comprises at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups.

13. The use according to claim 12, wherein the acyl group is selected from the group consisting of aromatic and aliphatic acyl groups.

14. The use according to claim 13, wherein the acyl group is selected from the group consisting of acyl groups derived from 4-coumaric acid, caffeic acid, ferulic acid, sinapic acid, 4-hydroxybenzoic acid, gallic acid, acetic acid, oxalic acid, malonic acid, malic acid, maleic acid, and succinic acid.

15. The use according to any one of claims 11-14, wherein the glycosyl group is a group derived from a monosaccharide selected from the group consisting of glucose, galactose, rhamnose, arabinose, xylose, and glucuronic acid.

16. The use according to any one of claims 11-14, wherein the glycosyl group is a group derived from a disaccharide selected from the group consisting of 1,2-glucosylglucoside (sophorose), 1,3-glucosylglucoside (laminariobiose), 1-6-glucosylglucoside (gentiobiose), 1,2-xylosylgalactoside (lathyrose), 1,2-rhamnosylglucoside (neohesperidose), 1-6-rhamnosylglucoside (rutinose), 1,2-xylosylglucoside (sambubiose), 1,6-arabinosylglucoside, and 1,6-rhamnosylgalactoside.

17. The use according to any one of claims 11-14, wherein the glycosyl group is a group derived from a trisaccharide selected from the group consisting of 1,2-glucosyl-1,6-gluco-sylglucoside, 1,2-glucosyl-1,6-rhamnosylglucoside, 1,2-xylo-syl-1,6-glucosylglucoside, and 1,2-xylosyl-1, 6-glucosylgalac-toside.

18. The use according to claim 1, wherein at least one of R3, R4, and R6 is an -O-glycosyl, an -O-glycosyl group which is substituted with at least one acyl group, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups

19. The use according to claim 1, wherein R1 i s OCH3, R2 is OH, R3 is 6-O- (4-O-E-p-coumaroyl-.alpha.-L-rhamnopyranosyl) -.beta.-D-gluco-pyranosyl, R4 is .beta.-D-glucopyranosyl, R5 is H, and R6 is OH.

20. The use according to claim 1, wherein the composition consists or the individual anthocyanins outlined in Table I, preferably in the relative quantities outlined in the table.

21. The use according to claim 1, wherein the composition consists of the individual anthocyanins outlined in Table II, preferably in the relative quantities outlined in the table.

22. The use according to any of the preceding claims for the prevention or treatment of infection by a retrovirus such as infection by Human Immunodeficiency Virus (HIV) and/or for prevention or treatment of Acquired Immune Deficiency Syndrome (AIDS).

23. A novel anthocyanin derivative of the general formula I
wherein R1, R2, R3 and R6 independently of each other are H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R4 is OH, alkoxy, an -O-glycosyl, an -O-glycosyl group which is substituted with one or one acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R5 is H, OH, and Y is a counterion, or a prodrug or complex thereof with the exception of the compounds mentioned in "The Flavonoids", ed. J. B. Harborne, T.J. Mabry and H. Mabry, Chapman & Hall, 1975, "The Flavonoids. Advances in Research", ed. J.B. Harborne and T.J.
Mabry, Chapman & Hall 1982, "The Flavonoids. Advances in Research since 1980", ed. J.B. Harborne, Chapman & Hall, 1988, "The Flavonoids. Advances in Research since 1986", ed.
J.B. Harborne, Chapman & Hall, 1994 and in references in Chemical Abstract, Vol. 119 and 123 under the General Subject Index entry Anthocyanins.

24. A pharmaceutical composition comprising an anthocyanidin or anthocyanidin derivative of the general formula I
wherein R1, R2, R3 and R6 independently of each other are H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R4 is OH, alkoxy, an -O-glycosyl, an -O-glycosyl group which is substituted with one or one acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R5 is H, OH, and Y is a counterion, or a prodrug or complex thereof with the exception of Myrto-CyanR
(Vaccinium myrtillus anthocyanosides corresponding to 25% as anthocyanidines), topical medicinal compositions containing fruit juice or fermented fruit juice as described in CA 1086651, a topical composition consisting of an isopropanol extraction of mountain ash berries as described in US
4,132,782, the alcoholic extracts of anthocyanosides described in FR 2456747, the compositions comprising bilberry anthocyanidines, grape anthocyanidines or elder anthocyanidines described in GB 1,589,294 and the anthocyanidin glycosides extracted from bilberries, black currents and blackberries described in US 3,546,337.

25. A pharmaceutical composition comprising petanin in combination with a pharmaceutically acceptable excipient.

26. A pharmaceutical composition comprising a mixture of individual anthocyanins as outlined in Table I in combination with a pharmaceutically acceptable excipient.

27. A pharmaceutical composition comprising a mixture of individual anthocyanins as outlined in Table II in combination with a pharmaceutically acceptable excipient.

28. A pharmaceutical composition comprising a novel anthocyanin derivative according to claim 23 in combination with a pharmaceutically acceptable excipient.

29. A method for the preparation of an anthocyanidin or an anthocyanidin derivative of the general formula I as defined in claim 23, the method comprising isolation and purification of the anthocyanidin or an anthocyanidin derivative by the methods outlined Example 1.
30. A method for the prevention and/or treatment of a disease caused by a retrovirus, the method comprising administering to a mammal in need thereof an effective amount of an anthocyanin derivative of the general formula where in R1, R2, R3 and R6 independently of each other are H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with ore or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R4 is OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or one acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R5 is H, OH, and Y is a counterion, or a prodrug or complex thereof.

of the anthocyanidin or an anthocyanidin derivative by the methods outlined in Example 1.

30. A method for the prevention and/or treatment of a disease caused by a retrovirus, the method comprising administering to a mammal in need thereof an effective amount of an anthocyanin derivative of the general formula I

wherein R1, R2, R3 and R6 independently of each other are H, OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or more acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R4 is OH, alkoxy, an -O-glycosyl group, an -O-glycosyl group which is substituted with one or one acyl groups, or an -O-glycosyl moiety comprising at least two glycosyl groups and at least one acyl group arranged so that at least one acyl group is located between two glycosyl groups, R5 is H, OH, and Y is a counterion, or a prodrug or complex thereof.