Phytomedicine Vol. 1/1994, pp. 77-106

© 1994 by Gustav FischerVerlag, Stuttgart· Jena . New York Original Papers

SP-303, an Antiviral Oligomeric Proanthocyanidinfrom the Latex of Croton lechleri (Sangre de Drago).

R. UBILLAS1, S.D. JOLAD1, R.C. BRUENING1, M. R. KERNAN1,S.R. KING1, D.F. SESIN1, M. BARRETI1, C. A. STODDART1,1. FLASTER1, J. KU01, F. AYALA1, E. MEZA1, M. CASTANEL1,D. McMEEKIN1, E. ROZHON1, M.S. TEMPESTA1,D. BARNARD2, J. HUFFMAN2, D. SMEE2, R. SIDWELL2,K. SOIKE3,A. BRAZIER4,S. SAFRIN5,R. ORLAND06, P.T.M. KENNy6,N. BEROVA7\ K. NAKANISHI7.

1 Shaman Pharmaceuticals,213 East Grand Avenue, South San Francisco, CA 94080-4812, U.S.A.2 Institute for Antiviral Research, Department of Animal,Dairy and VeterinaryScience, Utah State Univer-

sity,Logan, UT, 84322 U.S.A.3 Tulane Regional Primate center, 18703 Three Rivers Road, Covington, LA, 70433 U.S.A.4 Southern Research Institute, Birmingham, AL, U.S.A.S San Francisco, General Hospital, InfectiousDiseases, Building 80, Ward 84, 998 Potrero Avenue, San

Francisco,CA 94110 U.S.A.6 Suntory Institute for Bioorganic Research (SUNBOR), Shimamoto-cho,Mishima-gun, Osaka 618, JA­

PAN7 Department of Chemistry, Columbia University, New York, NY 10027, U.S.A.

t on leave of absence from the BulgarianAcademy of Science, Sofia,Bulgaria.

Summary

SP-303, a large proanthocyanidin oligomer isolated from the latex of the plant species Crotonlechleri (Euphorbiaceae) has demonstrated broad activity against a variety of DNA and RNA vi­ruses. In cell culture, SP-3D3 exhibits potent activity against isolates and laboratory strains of res­piratory syncytial virus (RSV), influenza A virus (FLU-A) and parainfluenza virus (PIV). Parallelassays of SP-3D3 and ribavirin showed comparable activity against these viruses. SP-303 also ex­hibits significant inhibitory activity against herpesvirus (HSV) types 1 and 2, including herpesvi­ruses resistant to acyclovir and foscarnet. Inhibition was also observed against hepatitis A and Bviruses. The antiviral mechanism of SP-303 seems to derive from its direct binding to componentsof the viral envelope, resulting in inhibition of viral attachment and penetration of the plasmamembrane. Antiviral effects of SP-303 were measured by three distinct methods: CPE, MTT andprecursor uptake/incorporation. Cytotoxicity endpoints were markedly greater than the respec­tive antiviral endpoints. SP-303 exhibited activity in RSV-infected cotton rats and African greenmonkeys, PIV-3-infected cotton rats, HSV-2 infected mice and guinea pigs and FLU-A-infectedmice. The most successful routes of SP-303 administration for producing efficacy were: topicalapplication to HSV-2- genital lesions in mice and guinea pigs, aerosol inhalation to FLU-A-infect­ed mice and PIV-3-infected cotton rats, and oral dosage to RSV-infected cotton rats. A variety oftoxicological evaluations demonstrated the safety of SP-303, particularly orally, which was pre­dictable, since condensed tannins are a common dietary component. It is notable that the larger

78 R. Ubillas et al.

proanthocyanidins as a class have high antiviral activity, whereas most of the monomers are in­active. Clinical trials are ongoing to evaluate SP-303 as a therapeutic antiviral agent.

Key words: Croton lechleri (sangre de drago), oligomeric procyanidin SP-303, antiviral activity.

I. Ethnobotanical Background

The sub ject of this study, SP-303, is an oligomeric proan­thocyanidin derived from Croton lechleri, one of a variety ofspecies of tre ss within the Croton genus in the family Euph or­biaceae. This family is widely known for many medicinalpurposes (Von Reis and Lipp, 1982; McRae et aI., 1988). Ared, viscous latex is produced by several species within thesection Cyclostigma Griseb . of this genus. These species arewidely distr ibuted throughout Central and South America,ranging from Mexico to Paraguay and southern Brazil.

This red latex is one of the most common traditional me­dicines in all of Lat in Amer ica. Its diversity of ethnomedicaluses is summarized below. The latex is most commonly re­ferred to as "Sangre de Drago" (" dragons' blood" ) and"Sangre de Grado" Spanish names appl ied to the plant bythe Europeans upon the ir arrival in the New World fivecenturies ago. The origin of the name "Sangre de Drago"comes from a number of species in the botanical genus Dra­caena in the family Agavaceae. Stems of several of the 60species in thi s genus produce a red resin (giving rise to thename "dragons' blood" ) that has been used in varnishes.One particular species, Dracaena cinnabari Balf., wa s pro­babl y known in antiquity. A second species, Dracaena dra­co (L.), the dragons' blood tree, had been used as a woundhealing agent in Spain, the Canary Island s and other re­gions of Europe.

Many vernaculars for "Sangre de Drago" exist in man yspecific indigenous langu ages. In the language of the Unt­suri shuar, a species of th is genus bearing red latex is refer­red to as "Uruchnum" (Bennett, 1990). Among the Qu i­chua Indians of Amazonian Ecuador, it is "Lan huiqui "(King, pers. obs.). The Cofan Indians call it "Masujuain"(Ceron, 198 7); the Waorani Indians, "Conewe" (King,per s. obs.), In Mexico and Guatemala, one 0 the red latex­bearing species, Croton draco Cham & Schltdl., has at least18 different indigenous names including such variations of"dragons' blood" as "Lora Sangre" and "Sangre de Perro" ,as well as a number of non-Spanish derived names such as"Chucum" in the Lacondon language of the region of Chia­pas, or "Xixte" in the langu age of the Huasteca in San LuisPotosi, Th is is only a partial listing of the indigenousnames. Clearly, this plant has had widespread and diverseuse for healing among ind igenous groups throughout Cen­tral and South Amer ica, including the countries of Mexico,Guatemala, Panama, Colombia, Ecuador, Peru, Bolivia,Paraguay and Brazil.

The most well-known and widespread red latex-bearing

Croton species of the northwest Amazon, and a primar ysource for SP-303 is Croton lechleri Miill. , Arg. Thi s spe­cies is distributed in the countries of Colombia, Ecuador, .Peru and Bolivia. A closely related species that may be asynonym, C. draconoides Miill., Arg., is recorded in Peru,and the type specimen was collected in Brazil. There are,however, multiple other species that have yielded SP-303,ranging from Mexico to Paraguay and southern Brazil.Those species include C. draco, Cham & Schltd!., especial­ly wide-spread in Cent ra l America, C. urucurana, Baill. ,well-known in Paraguay and southern Brazil, C. sordidus,Benth ., of the Andean region, and C. gossypifolius Vah!.,common also in the Andean region . Add itional speciesthat yield red latex in a number of countries in Cent raland South Amer ica include C. ferrugineus, c. panamensisS. C. Klotzsch, C. palanostigma Mart. & Baill., and C.salutaris. Th is sub-group of the genus Croton is in need ofrevision because of the many synonym names for the spe­cies and the need for taxonomic clarificat ion for th is me­dicinally important group of New World plant s. The tax o­nomy and systematics of these species is currently underinspection and revision by Dr. Grady Webster (Webster,1993) and a doctoral candidate at the University of Cali­forn ia, Davis.

Ethn om edical Uses. The source for SP-303, "Sangre deDrago" of Central and South America, is one of the mostwidely found, known and used plant medicinal sourc es inthe entire hemisphere. It has been used for centuries andcontinues to be used by a wide diversity of people, especial­ly in Colombia, Ecuador, Peru, Bolivia, Paragua y, Mexicoand Cenral America. Users include nat ive, urban and mes­tizo populati ons, as well as urban healers; it is now increas­ingly being sold in urb an health food sto res. Thi s plantmed icine is extensively sold in Peru and Ecuador (Me za1994, in press). Its most common usages ar e: internally forcoughs, flu, "problems with lungs " , diarrhea, and for"stomach ulcers"; and topically as a wound healing agentfor cut s, open sores, herpes infections, for the gums aftertooth extraction and for oral open sores in and on themouth. In some urban areas, it is appli ed topically after sur­gery for enhanced wound healing. Th e general dosage forinternal use is 5-10 drops of the red latex in warm or coldwater, milk or in alcohol, 1-3 times a day for 5 days. Th isregimen is often repeated for a period of up to 3 weeks. It isappli ed directly to open wounds to stop blood flow andpromote healing. Diluted with water, it is applied as a vagi­nal wash for excessive vaginal bleed ing. No side-effectshave been reported after internal use.

In urban regions of many Andean countries and in Cen­tral America, the latex is currently being sold under at least20 different labels by a variety of "health food manufac­turers". These manufacturers sell small quantities of the li­quid in bottles for hepatitis, diabetes, ulcers, cancer preven­tion, tonsillitis, as an anti-inflammatory, and to enhancefertility and weight loss. It is also recommended for hem­orrhoids, acne and as vaginal wash. The predominant usesamong the indigenous populations are limited to topicalwound healing and internally for coughs, colds, diarrheaand stomach problems.

In summary, the Croton species that form a widespreadcomplex known regionally as "Sangre de Drago", plays acritical role in the local and urban traditional medicine ofnumerous countries throughout Latin America. Historical­ly, scientific interest in the wound-healing and antiinfectiveproperties of "Sangre de Drago" is well documented in theliterature (Bettolo and Scarpati, 1979; Persinos, 1972; Per­sinos-Perdue et al., 1979; Kitazawa et al., 1980; Vaisberg etal., 1979; Vlietinck, 1987), and general scientific interestcontinues to this day (Cai et al., 1991; Lewis et al., 1992;Tempesta, 1993).

Distribution, Density, and Harvesting of the SF-303Source Species. Over the past four years, Shaman Pharma­ceuticals has committed itself to the long-term sustainableharvesting and management of SP-303 source species. Wehave conducted 14 distinct studies in four different coun­tries on ecology, distribution, density, growth habits, mar­keting, agroforestry, management and collection methods.We have worked closely with indigenous peoples and scien­tists to develop sustainable harvesting methods that willhelp protect and manage this group of plants species. Ourconcerns are both to sustain the source for our lead antivi­ral products and to assist local populations in continuing tohave direct and easy access to the plant material as a sourceof regional traditional medicine.

In numerous examples, uncontrolled clear-cutting has de­stroyed the genetic diversity of a number of species through­out the tropics, and particularly in Amazonia. Specificallythe cases of rosewood oil (Aniba rosaeodora) and Oje (Fi­cus anthelmintica) have shown that intense market de­mands can cause dramatic ecological consequences bythreatening the gene pool and diversity of economically im­portant species. Since its beginning, Shaman Pharmaceuti­cals had committed itself to pay very close attention to thedetail of sustainable supply at every stage of the drug devel­opment process. Recent work by Cragg et al. (1993), dis­cussing the evolution of taxol and the supply crises that sur­rounded its source species, has further emphasized the im­portance of working on the long-term supply aspects of anypotential natural product for drug development. In ouropinion, a protocol incorporating all steps needed to con­serve and manage a target species should become an integralpart of any preclinical data package.

Our distribution studies have shown an extremely wide-

SP-303 79

spread occurrence and high natural density of the SP-303 ­bearing Croton species from Mexico to Paraguay. A densi­ty of 3 to 10 species per hectare seems to be common, espe­cially along riparian habitats throughout the northwestAmazon. These specific studies will be published elsewherein collaboration with the primary scientists who conductedthe research. These research data have been provided to lo­cal government ministries and conservation organizationsas well as directly to indigenous groups working on themanagement of this species.

Ecological habitat and distribution vary considerably.Most of the C. lechleri grows in the lowland northwesternAmazonian forest region from 100-600 meters in elevation.Related species occur from 700-2500 meters along theeastern and western slopes of the Andean regions of Ecua­dor, Colombia and Peru. Other Croton species in Mexicoand Central America are more adapted to moist tropicaland in some cases sub-tropical forest habitats in their mois­ture regime. The species in Paraguay and southern Brazil areadapted to the drier cerrado type of habitats with a muchlower level of annual precipitation.

The species C. lechleri and several related species exhibitclassic pioneer species ecological profiles. They are one ofthe three most successful emergent species appearing incleared fields and on roadsides throughout much of theEcuadorian and Peruvian forest regions between 600 and2500 meters.

The most common harvesting method for C. lechleri hashistorically been the slashing of bark, which initiates theslow release of up to several liters of latex from a standingtree of up to 6 years of age with an average diameter atbreast height (DBH) of 25 centimeters. Traditional harvestpractices utilize small quantities (less than 100 m!) of mate­rial for personal and family use. However, the increasingmarket for this material over the past decades has led to amore common harvesting practice of felling of the tree andsubsequent scoring of the bark at intervals of 6-12 inchesaround the circumference. This method yields up to 5-61i­ters of latex per tree depending upon the age and season ofthe harvest (i.e., latex yield is greater during the rainy sea­son).

Sustainability. Because this tree of the Euphorbiaceae fa­mily produces latex continuously, we originally focused onmethods for sustainably "tapping" the latex as it is donefor the harvest of natural rubber. We therefore commis­sioned a number of studies by experts on the morphologyon the genus of Hevea and Croton. While rubber has beensuccesfully tapped both in the wild and in plantations, thelaticifers of the red latex-bearing Croton species are distinctfrom those of the Hevea species. Rubber trees contain mainlatex "canals" in which the latex restores and flows contin­uously. In contrast, Croton laticifers contain many tubulesthat must be opened individually to release the latex inside(Rudall, pers. comm.). It is therefore not feasible to "tap"Croton tree species like Hevea rubber trees. However, the

80 R. Ubi lias et at.

rapid gro wth of the trees and high yield of SP-303 (- 1 % )provid es an excellent opportunity for managing these spe­cies in agro forestry systems where they only require theminimal management of high yieldi ng mixed-species secon­dar y forests.

Conservation and Management. M aintaining the integri­ty of th e tropical rainforest ecosystems requires integratingthe needs of th e people who live in a nd around these en vi­ronments. Long-term conse rva t ion management must beinterwoven with the cultural va lues and needs in reg ion swhere source species occur. Studies have shown that we canmanage and maintain the genet ic diversity of these speciesby cre ating sustainable harvest and management systems incollaboration with forest-dwell ing people.

We recognize this as an ecological and business imperative tomaintain operational and long-term supply and sound ecologicalmanagement for the benefit of local inhabitants. We have enteredinto agreements with indigenous federations such as the ConsejoAguaruna y Huamb isa of the northern Peruvian Amazon to sup­port and develop reforesration and management programs for thistree species. The Consejo Aguaruna y Huambisa provided us withan agreement from their annual congreso which included the sig­natures of 137 delegates representing some 30 000 individuals ofthese tribal groups in the norrhern Peruvian Amazon (King, 1994 ).This is an ongoing process requiring monitoring and reporrs fromShaman Pharmaceuticals to the various groups with which wework. The specifics on price, purchase and many other details havebeen stipulared by the various indigenous groups with which weare collaborating and are being monitored at the federation levelby organizations such as AIDESEP (the National Peruvian Indig­enous Peoples Organization of the Amazonian Region). We havealso initiated similar programs with the Cofan Indians as well asother indigenous groups in the rainforest regions of several An­dean countries. We are actively working with these and other indi­viduals to create management plans and conduct reforestationactivities that greatly exceed the amount of harvested material.The governments of the countries in which we are conductingthese activities require routine, long-term management plans toallow us to proceed with the collection and export of raw materi­als for this drug development program. We have helped create alarge body of information on the conservat ion and management ofthis wide-spread and highly important medicinal plant specieswhere very little baseline information existed previously. To pro­mote these business practices, we are also supporring school pro­grams and encouraging local students to understand the conceptsof reforestat ion and plant uses that are basic to their ethnobotani­cal and ethnomedical needs. We have been supporring communityseminars in conjunction with the Ministry of Agriculture to informfarmers of the imporrance of this tree and the value of applyingeven minimal management to it. We have also helped organizingseminars to train indigenous federation representat ives to furtherwork with their indigenous colleagues to maintain this imporrantresource for the future.

II. Isolation and Characterization of SP-303

Botanical class characteristics. Proanthocyanidins andproanthocyanidin polymers are found as naturally occur­r ing phenolic substances in a wide variety of plants, parti-

cul arl y those with a woody habit of growth. The generalchemical stru ctu re of monom eric and polymeric proan­thocyanidins is shown on p. 88 (Harborn e, 1988 ). Thepolym er consists of linear chains of 5,7,3',4'-tet rahydroxy(catechin or epicatechin) o r 5,6,3',4',5'-pentahy droxyflavan-3-ol (gallocatec hin or ga lloep icatechin) units linkedtogether through either C-4 to C-6 and /or C-4 to C-8.

The monomer unit of th e polymer chai n ma y be based oneither of two sterochem istries of th e C rin g, des ignated cisan d termed epicatechin or trans and named catechin, andon eith er of two B ring oxidation patterns, 3',4'-dihydroxy­ph enyl (designated as a procyanidin unit) or 3',4',5'-trihy­droxyphenyl (designated as a p rodelphinidin unit). There­for e the polymer chains which are based on four differentmonomeric units create a w ide variety of polymeric pro­anthocya nidins and a large number of possible isomers.

Proanthocyanidins (syn. leucoanthocyanins, or "con­densed tannins" ) ar e a class o f co mpounds that yieldcya nidin and or delphinidin [anthoc yan idins] on treatmentwith acid. They encompass all flavan-Ssol monomers,dimers and higher olig omers. Condensed proanthocya­nidins are the dimers and higher oligome rs of the variousflavan-Svols, with polymeric proanthocyanidins consistingof fla van-3-ol polym ers. Procyanidins are a subclass of pro­anthocyanidins, consisting of various flavan- Svols contain­ing only the B ring oxidation pattern of 3 ', 4 '-dihydroxy­ph en yl. Prodelphinidins ar e also a sublcass of proanthocya­nidins, co mposed solel y of B ring oxidation as 3',4',5'­trihydroxyph enyI. Leucoanthocyan id ins encompass all fla­va n-3 ,4-diol monomers as well as flavan-t-ols, and are an­other subclass of proanthocyanidins. Further examples ofsubclass members have been published by Harborne(1988), who gives the range of oxidation patterns found inth e pr oanthocyanidin class.

Fractionation and isolation. The lat ex of Croton lechleriwas found to have significant in vitro antiviral activity ininfluenza (FLU-A) and respi ra tory syncy tia l virus (RSY)screens and was initially subjected to a bioassay-guidedfract ion at ion over Sephadex to remove inac tive compo­nents, and concentrate bio act ivity. This evol ved int o a sea l­eable isolation scheme using th e seven-step process des­cri bed below. The product turned out to be the most bioac­t ive component in the latex, a red-brown proanthocyanidinoligomer, termed SP-303. Flavonoids in general and smallproanthocyanidins ha ve been reported to exhibit antiviralpr op erti es (Beladi et al. , 19 72; Kaul et a l., 1985; Takechi eta l., 1985; Selway, 1986; Vanden Berghe et aI., 1986; Nona­ka et aI. , 1989; Hudson, 1990 ; N onaka et aI., 1990). Infact , significa nt clinical effo rts involving a flavan and achalcone were undertak en to trea t rh inovirus infections,although neither were successful (Selway, 1986). Before webegan our work, there had been no published data on th ed irect antiviral effects of proanthocyanidin polymers, al­th ough they are currently the subject of considerable in­ter est as evidenced by patents and patent applications

(Itsuo et aI., 1989; Cariel and Jean, 1990; Nonaka et aI.,1990; Tempesta, 1993).

After addition of 3 parts isopropanol to the latex (Step 1),the supernatant of the resulting biphasic mixture was furth­er extracted with butanol (Step 2). The residual aqueoussolution was evaporated to dryness, dissolved in methanol,and precipitated with ethyl acetate (Step 3). The filtered su­pernatant was then purified by a combination of ionexchange chromatography and size exclusion chromato­graphy. Using water as the eluent, alkaloids (primarily tas­pine) present in the material (Persionos-Perdue et aI., 1979)were removed from the Step 3 intermediate by cationexchange chromatography on CM-Sephadex C-50. Furtherenrichment of the proanthocyanidin polymer-containingfraction was achieved by connecting a Sephadex G-50 col­umn to the outlet of the CM-Sephadex C-50 column. Withwater as the eluent, the proanthocyanidin oligomer ad­sorbed to the G-50 column while some of the more polarlow molecular weight compounds eluted. The bioactiveproduct was then eluted with 15 % aqueous acetone(Step 4). Fractionation of the Step 4 intermediate on Toyo­pearl HW-40S and elution with 20 % aqueous acetone re­moved most of the low molecular weight proanthocya­nidins. Further elution with 40 % aqueous acetone gave anintermediate of desired bioactivity (StepS). This inter­mediate was finally purified on Sephadex LH-20. Elutionwith 90 % aqueous ethanol followed by 20 % aqueousacetone removed remaining monomeric, dimeric, trimericand tetrameric proanthocyanidins, while elution with40-50 % aqueous acetone yielded a fraction comprisingmedium-sized oligomers, designated as SP-303 (Step 6).

The use of three successive gel permeation columns al­lowed rigorous control of the molecular weight averageand distribution of the final product. The moisture contentand grain size of the final product were controlled by dis­solving a given amount of the Step 6 material in twice theamount of water and evaporating the resulting syrup undervacuum for a specific time and temperature, then grindingand sifting the resulting solid material through U.S. Stand­ard Testing Sieves. The overall yield of SP-303 by thismethod was -1 %; this process has been recently improvedto allow larger batches to be prepared increasing the yieldto 12-15 % (wt./v of latex).

An HPLC procedure employing a highly cross-linked po­lystyrenedivinylbenzene gel permeation column was usedas an in-process control method for evaluating the purity ofmaterial produced in each of the above chromatographicsteps. The same procedure was used to evaluate the purityand molecular weight distribution of the final drug sub­stance. This procedure is described in more detail below.

One method that has been used to determine the number­averaged molecular weight of a proanthocyanidin is gelpermation chromatography (GPC) of the acetylated poly­mers (Williams et aI., 1983). The acetylations are normallycarried out by reaction of the polymers with acetic anhy-

SP-303 81

dride in pyridine. Gel permeation chromatograms are ob­tained using tetrahydrofuran as the mobile phase and poly­styrene-divinylbenzene as the stationary phase. Unfortun­ately, because of the limited solubility of SP-303 in pyri­dine, this method could not be used for SP-303. Direct in­jection of SP-303 resulted in adsorption of the materialonto the GPC column. However, by adding water to themobile phase to give 95 % aqueous tetrahydrofuran, it wasfound that all Croton lechleri proanthocyanidins, includingSP-303, eluted from the column with> 99 % recovery. Foraccurate molecular weight determinations by GPC, thestandards must have similar chemical properties to the ana­lyte. The ideal standards are monomers, oligomers andpolymers of the same structure type as the analyte. Polysty­rene standards (Polymer Laboratories) and proanthocya­nidin oligomers obtained from C. lechleri (Cai et aI., 1991)were used to prepare the calibration curves shown in Fig­ure 1 using a Polymer Laboratories PL Gel 500 column. Toincrease the resolution, this analysis was repeated usingtwo PL Gel Mixed E columns connected in series. The PLGel Mixed E column has a 3 micron particle size, with anefficiency of 50000 plates/m, equivalent to 3 columns ofthe same length containing 10 micron particles.

It is not surprising that the use of two types of polymerstandards resulted in different molecular weight predic­tions. It was assumed that the calibration curve based uponthe proanthocyanidin standards would give a more accu­rate estimate of the true molecular weight distribution inSP-303; the data were extrapolated as shown to cover theelution range observed for SP-303. The molecular weightrange and distribution of SP-303 as determined by thismethod is 1500-3300, corresponding to between 5 and 11flavanoid units, with a number-averaged molecular weightfor SP-303 of -2100 daltons and an average chain length of7 flavanoid units. Analysis of the gel permeation chroma­togram of SP-303 as shown in Figure 2 was performedusing a photo diode array detector. The UV spectra ob­tained at 20 % of peak upslope, peak apex and 20 % ofpeak downslope were overlayed and plotted (normalized)from 215-365nm and 250-365nm, as depicted in Figure3. These data clearly show the homogeneity of SP-303.

Chromatography

General. All preparative chromatography was conductedusing FMI pumps and UV/VIS detectors set at 450 nm(linear). Flow rates were 25 -50 rnl/rnin. Moisture contentsof SP-303 were determined using a Karl-Fischer device(Methrohm). Thin-layer chromatography was conductedon HPTLC plates (Merck) developed with EtOAc-HzO­formic acid - acetic acid (80 :20: 3: 2) and visualized byspraying with 1 % vanillin in 20 % ethanolic HCI.

Isolation. Isopropanol was added to the cold latex (41) inthe volume ratio of 3 : 1. After mixing, the material wasstored at 5°C for at least 15 hr, the clear red supernatant

82 R. Ubillas et al.

---e- polystyrene standards --i3- pro cyanidins

PL Gel Calibration

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was siphoned off from the residue , centri fuged, and thesupernatant evaporated to dryness under reduced pressureon a rotary evaporator. Thi s material (970 g) was parti­tioned between water (61) and butanol (3.6 1). The aqu eouslayer was removed, extr acted twice more with n-butanol,and evaporated under reduced pressure. The dried water­soluble fraction (700 g) was dissolved in methanol (21);ethyl acetate (121) was add ed, the mixture was thoroughlymixed, then stored at 5°C for the least 15 hr. The clear redsupernatant was siphoned off from the residue, filtered ,and evaporated to dryness to give the Step 3 intermedia te(390g).

The Step 3 intermediate (450 g) was dried in a vacuumoven at 35°C for at least 24 hr. It was then dissolved in wa­ter (1 1) and chromatographed on a system consisting of aCM-Sephadex C-50 colum n (20 x 100 cm) with a precol­umn (9 x 50 em) and a Sephadex G-50 column (20 x25 ern). The elution was initiated with water (20 I) throughthe ion exch ange columns and , after elution of the first pinkband, onto the Sephadex G-50 column. After disconnectionof the ion exchange columns the desired Step 4 intermediate(210 g) was eluted from the Sephad ex G-50 column with15% aqueous acetone (5 1) The Sephadex G-50 column wasregener at ed by eluting with water (20 I); the one-time use ofa precolumn allowed the larger ion exchange column to lastfor -20 runs .

The Step 4 intermediate (150 g) was chromatographed onan Toyopearl HW-40S column (10 x 50cm) using 20 %

aqueous acetone (16 1) as the eluent. After change to 40 %aque ous aceton e, the Step 5 intermediate (57 g) was elutedas a bro ad peak (8 1) and evaporated to dryness on a rotaryevaporato r. The Toyopearl column was regenerated by elu­ting with 60 % aq. acetone (41) followed by 20 % aq. acet­one (31).

The Step 5 intermediate (57 g) was chromatographed ona Sephadex LH-20 column with 90 % aq . ethanol (10 I) fol­lowed by 20 % (15 1), 40 % (5 1) and 50 % (4 1) aq. aceton eas eluents. The SP-303 eluted with 40 % aq. and 50 % aq.aceton e. The column was regenerated by washing with60 % aq. acetone (5 1) and 90 % aq. ethanol (5 1) . Th e finalproduct, SP-303 (35 g), was evaporated to dryness on a ro­tary evap orator and dr ied in a vacuum oven for - 24 hr at35°C. The overall yield of SP-303 from the latex is 1 % (4 1give 40 g).

Adjustment of Moisture Content and Grain Size. A sam­ple of SP-303 (100 g) was dissolved in water (200 ml) andevaporated to the consistency of a coarse, free-flowing sandwith an average moisture content of 15 %. Thi s materialwas ground directl y through a U.S. Standard Testing SieveNo . 45 : The ground substance was then sifted using a me­chanical sifting mach ine through a No.60 sieve to give auniformly sized product (300 ± 50 micron) .

Gel Permeation Chromatography. Gel permeation chro­matography was conducted on a Perkin Elmer LC-AnalystHPLC system with a PE LC-235 photo diode array detec­tor, column PL Gel 500 (7.5 x 300mm), mobile phase 95 %

SP-303 83

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5.IiIIlt 1C!SIIrI INlm .. lt hilrsts,st..,I

JltOm •••••• "-Ql~ 1I)11C't.l ; , rt rCl 11~:n: tS t!l1Axllhtll hl re 111S:n :tS t!rl

' Lll 7 _ 1 11~1iatt ••• ''' ' ~M ••• I0\n-e1 .. •. . 11 Dlhl\JtIl ••••••• l

lanat , . . .. 1 fcnat 11U• . . . lUll SIXl

II. art.( .... "l~ I: n HitLII: II_ Pwca-c. ~

1 7.111 ~ ~. 11 IQ:I .lKIXl llXUOX

114.l!a lou l WtIl. II IOU I ~ l :t :

o 5

Time (min)

10 15 Fig. 2. Gel. Permeation Chromatogramof SP-303.

ag. THF, flow rate 1 mllmin; diode arra y detector set at 280nm, Analysis of molecular weight range and distribution ofSP-303 was conducted using two PL Mixed E columns(7.5 x 300 mm) connected in series; mobile phase 95 % aq.THF; flow rate 1 milmin; detector 280 nm. Chromatogra­phic fractions were diluted 1 : 9 with THF containing 0.1 %BHT before analysis. SP-303 and dried intermediates weredissolved in 95% THF containing 0.1 % BHT (1 mg/ml) be­fore analysis.

Spectroscopy

Mass Spectrometry: Mass spectra were acquired on thefirst two sectors of a lEOl HX/HX110A tandem four sec­tor mass spectrometer, which was operated at -10 kV accel­erating potential. Spectra are averaged profile data of 3scans, which were acquired from 300 to 5000 m/z at a ratethat would scan from 1 to 6000 m/z in one minute as re­corded by a lEOl DA5000 data system. Ions were pro­duced by FAB using a lEOl FABgun operated at 6 kV in aconventional FAB ion source. A 3: 1 mixture of dithiothrei­tol and dithioer ythritol was used as the FAB matrix. Allmeasurements were performed on negatively charged ions.Calibration was performed using CsIlglycerol. Mass spec­tra were also provided by a Kratos MS50 mass spectrome­ter equipped with a Xe FAB source. The negative ion FABexperiment was performed with a mass resolution (M/LlM)of 1000, using CsI as calibrant.

NMR: lH and BC NMR spectra were obtained on a

400 MHz Varian instrument in D20 operating at 400 and100 MHz, respectively.

IR: Infrared spectra were obtained on a Perkin-Elmer1605 FT-IR instrument.

CD: Circular dichroism spectra were acquired on alASCO 702 instrument in both H20 and phosphate buf­fer.

Physical Properties of SP-303

Appearance: dark red-brown powder, light brown fluffysolid after lyophilization.

Solubility: Soluble in H20 and 5 % dextrose up to 50 mg/ml.

Melting point: > 500 "C (decomp.)Elemental analyses: %C 56.1-56.7, %H 4.6-4.9, %N <

0.5, %CI < 0.5, %S < 0.9Specific rotation: [a] D 0.0 0 (H20 , conc.: 0.22, 3.33 &

15.5. g/100 ml).Circular Dichroism: Amax =299 to 305 nm, M:: -1.02 to ­

1.10; Amax =255.5 to 256. 7nm, .1£: +0.452 to +0.658;(cone. 9.76 x 10-4 to 9.76 X 10-6 M in 0.1 M phosphate buf­fer @pH 6.7, respectively); Amax = 212.5 nm, Ll£: -2.815;Amax =236 nm, Ll£: +2.774 ; (conc. 385 x 10-6 M in H20 ).

UV-VIS: Amax: 205 , 275 and 460nm (H20 , Abs. 1.07,1.65, 0.25 @conc. 0.0055, 0.2 and 1.0 mg/ml).

FT-IR: 3380, 1690, 1610, 1570, 1450, 1343, 1200,1143, 1100, 1065, 1033, 825 and 725 ern" (KBr press,cone 2 mg SP-0303/200 mg KBr).

84 R. Ubillas et ai.

below, the structure of SP-303 has been assigned as a het­erogeneous, oligomeric pro anthocyanidin (condensed tan­nin) having an average chain length of seven units (rangingto 11), the monomers of which are the four related catechinand gallocatechin stereoisomers. This results in an averagemolecular weight of -2100 a.m.u. A very small amount ofa sub-unit with an anthocyanin character is also present ba­sed on UV-VIS spectral dat a, but its precise location withinthe individual chains remains to be determ ined.

The experimental range of cis/trans (catechin/epicate­chin) stereochemistries was shown to cover from 1: 2 to

2 : 1, employing l3C-NMR, CD and specific rotation data.The absolute stereochemistries of the monomers deter­mined from CD spectral data are consistent with the 2S ster­eochemistry of the monomers [(+l-catechin, (+)-gallocate­chin, H -epicatechin and (-)-gallepicatechin]. The oligomerunits are formed by linkages of monomers through 4-6 andlor 4-6 and/or 4-8 C-A ring positions, but the exact link­age pattern could not be determ ined spectroscopically.

Spectral Data: The UV dat a of SP-303 are closely relatedin appearance to those of the known proanthocyanidin s.However, the visible dat a are clearly different. The knownpro anthocyanidins are colorless (A 205, 240 and 275 nm)having no absorption in the visible range, while SP-303 is acolored compound with UV absorption in H20 at Amax 205(Abs. 1.07@ 5.5mg/ml ) and 275 (Abs. 1.65 @ 0.2mg/ml )

nm virtually identical to the known proanthocyanidins, butwith additional weak visible absorpt ion at 460 (Abs. 0.25@ 1.0 mg/ml) nm. Structurally, the visible data of SP-303suggest the presence of a flavylium moiety within the pro­anthocyanidin polymer, accounting for the observed redcolor. This is consistent with the visible spectroscopic datareported (Markham, 1982) for the closely related red-col­ored anthocyanins, which contain the flavylium/quinonemoiety (A 460-560 nm). Also, the visible absorption at A460 nm is ~ 500 times weaker in absorbence than the ultra­violet absorption at Amax 205 nm; this indicates that thechromophore is present at trace levels « 1 %) . Thus, whilecontributing the characteristic color, the f1avylium moietyrepresents a very minor portion of the overall structure ofSP-303.

The FT-IR data show a strong, broad band for OH stret­ching at v 3379; C=C stretching band s appropriate forphenyl units at v 1690, 1610 and 1570, phenolic C-OHstretching vibration at v 1200 , C-OH stretching bands at v1143 , 1102 , 1065, 1033, and a band at em" 826 consis­tent with a 1,2,3,5-tetra substituted benzene ring. The dataare consistent with a polyphenol structure for SP-303.

The l3C-NMR data of SP-303 are indicative of the pro­anthocyanidin class of polymers. This can be seen when thel3C NMR spectrum of SP-303 is compared to various pu­blished 13C NMR spectra of proanth ocyanidins (Harborneand Mabry, 1982; Haslam, 1989). In particular, the l3CNMR chemical shift data of SP-303 are consistent with anoligomer composed of both procyanid in [3',4'-dihydroxy-

366

w ,H

330 350 366 ..

om

om

...........--- - - - - - - -1

~-="""- - - 1

274 .3 290 310

•. u ' • "Cll i t " L" aJ. 1 I

250

274 .3 290 300 310 320 330 340 350

HII

215 230

250 260

II

file (ri l l Display Opt ions Inst".,enl s lash Status fmlVSI

Fig. 3. Diode Array Spectra of SP-303. Spectra taken at PeakApex, 20 % Upslope, and 20 % Downslope.

\H --

H --

.,\

HIe (rlit Display ~tions Inslnooents lesks Stat llS ImlVSl

IH-NMR: 15 in D20 @ 400 MHz: 2.2-3.0, 3.2-5.4,5.5-7.3 (all pekas very broad, ref. to HOD at 15 4.63).

13C-NMR: 15 in D20 @ 100 MHz: 28.7, 36.4, 38.2 , 66.7,72.6, 73.7, 76.6, 78.8, 82.9, 96.5, 97.6, 107.4, 109.2 ,115.5 , 116.8 , 120.0, 121.1, 13104, 133.9, 146.2, 15504,157.6 (all peaks broad, ref. to 1,4-dioxane @ 15 67.6).

Evidence for Structural Assignment. Extensive effortsemploying a wide range of anal ytical techniques have beendirected at determining the structure of SP-303. This workhas succeeded in identifying the types of monomeric unitspresent in the polymer, the stereochemical ratio of compo ­nent monomers as well as their absolute stereochemistries,and the range and distribution of chain lengths within theoligomer. Based on the inform ation obtained as described

phenyl] (C-1' = 133.9 ppm, C-2' = 115.5ppm, C-3' & C-4'= 146.2 ppm, C-5' = 116.8 ppm, C-6' = 120.0 ppm) andprodelphinidin [3' ,4',5'-trihydroxyphenyl] (C-1'131.4 ppm, C-2' & C-6' = 109.2 ppm, C-3' & C-5' = 146.2ppm, C-4' = 133.9 ppm) B-ring moieties with the individu­al flavanol C-ring units possessing both the 2,3-trans-3,4­trans [similar to catechin/gallocatechin; C-2 = 82.9 ppm, C­3 = 72.6 ppm, C-4 = 38.2 ppm] and 2,3-cis3,4-trans [simi­lar to epicatechin/galloepicatechin; C-2 = 76.6 ppm, C-3 =73.7ppm., C-4 = 36.4ppm] stereochemistries when com­pared to literature NMR assignments of related comp­ounds (Haslam, 1989). The A-ring 13C-NMR data of SP­303 (C-4a = -102 ppm (buried), C-5 = 157.6ppm, C-6 =97.6 ppm, C-7 = 157.6 ppm, C-8 = 96.5 ppm, C-8a =155.4 ppm) closely match published phloroglucinol (5,7­dihydroxy)-substitution NMR data (Harborne, 1988).However, the 13C-NMR data do not allow for unambi­guous assignment of 4-6 and/or 4-8 linkages between theindividual units within the SP-303 polymer due to peakbroadening and peak overlap in the 100-110 ppm regionof the 13C-NMR spectrum (the chemical shifts of C-6 andC-8 when C-substituted lie between 106-108 ppm). Theterminal unit is consistent with the published epicate­chin/galloepicatechin (C-2 = 78.8 ppm, C-3 = 66.7 ppm, C­4 =28.7ppm) 13C NMR data for related compounds.

The multiplicities [13C NMR: s in DzO @ 100 MHz:28.7, t; 36.4, d; 38.2, d; 66.7, d; 72.6, d; 73.7, d; 76.6, d;78.8, d; 82.9, d; 96.5, d; 97.6, d; 107.4, d; 109.2, d; 115.5,d; 116.8, d; 120,0, d; 121.1, d; 131.4, s; 133.9, s; 146.2, s;155.4, s; 157.6, s (all peaks broad, ref. to l,4-dioxane @ 867.6)], as determined by the Distortionless Enhancementthrough Polarization Transfer (DEPT) spectra, are furtherverification of the 13C-NMR assignments based on chemi­cal shifts as described above. The absence of small peaks inthe range 8 100-115 in the DEPT, when compared to thenormal broad-band decoupled spectrum, is consistent withtheir assignment as quaternary carbons buried under theprotonated carbons in this region of the normal 13C-NMRspectrum.

An Inverse-Gated Decoupled 13C-NMR experiment, per­formed with SP-303, removes the nuclear Overhauser ef­fects (nOe) normally observed on individual lC peaks, andthus allows for an accurate integration of the 13C-NMRspectrum. According to previous research, this informationallows measurement of the relative ratios of cis and transstereochemistries of the C-ring monomeric units throughdetermination of the integrated area for the C-2, C-3 peaksof catechin (lit. ()83.4, 73.1; obs. ()82.9, 73.7) and epicate­chin (lit. 8 76.6, 72.6; obs. () 76.6, 72.6). Due to overlap,only the 8 82.9 C-2 signal of catechin and the 8 76.6 signalfor C-2 of epicatechin are useful in determining the ratio,which is -2:1 in catechin!epicatechin. The method is alsouseful in determining the ratio of the terminal monomericunit to the rest of the units in the oligomer by looking at theratio of the C-2 signal of the terminal unit to all the other

SP-303 85

C-2 signals in the compound. These signals are readily di­stinguished both by chemical shift and by multiplicity asdetermined by DEPT (doublet at 8 36.4,38.2 vs. a triplet at()28.7). Using the relative areas of these signals, the ratio ofterminal unit/internal units becomes -1:6, which is consi­stent with an heptamer being the average chain length ofthe polymer. In summary, integration of selected 13C-signalsdetermined SP-303 to be a proanthocyanidin polymer withthe relative stereochemical ratio of approximately 2:1 cate­chin!epicatechin, as well as an average length of sevenunits.

The lH-NMR spectrum is notable for the extremebroadness of the resonances, which is consistent with theoligomeric nature of SP-303. The three very broad lH-peakscan be assigned on the basis of chemical shifts as follows: ()2.2-3.0, -CHz adjacent to an sp2 carbon, H-4 for the ter­minal unit; ()3.2-5.4, -CH adjacent to two sp2 carbons forall internal H-4's & -CH-O, H-2 for all units; 8 5.5-7.3,aromatic -CH for all units. The observed line-broadeningprecludes the application of 2-D NMR experiements suchas COSY, NOESY,]-Resolved, etc.; these were tried withoutsuccess under a variety of conditions on SP-303 in DzO,MeOH, DMSO, aetone, pyridine, and combinations there­of, at temperatures ranging from 20 to 80°C. Raising thetemperature, as well as lowering the viscosity of the NMRsolvent did little to sharpen the lH-NMR spectra. This be­havior is consistent with many polymeric materials.

A slightly better situation was found in a two-dimensio­nal heteronuclear lH_13C one-bond experiment, He­teronuclear Multiple Quantum Correlation (HMQC), inDzO. The spectra were poorly resolved because of thebroadness of both the parent lH_ and 13C-NMR spectra.However, the data did confirm the proton assignments ma­de above on the basis of lH chemical shifts. For example,the cross-correlation peaks observed confirmed the protonassignments of () 5.5-7.3 as aromatic protons, attached to

carbons ranging from () 96-121. This verified the proton!carbon assignments, and also is completely consistent withthe structural features of the C-ring, as well as the A- and B­rings found within the oligomer.

The mass spectrometry of SP-303 proved to be challen­ging, and many methods and instruments were involved inthe attempt to get significant results. After failing to obtainspectra using CIMS (Chemical Ionization), FDMS (FieldDesorption), SIMS (Secondary Ionization) TOFMS (Timeof Flight) and a variety of FABMS (Fast Atom Bombard­ment) techniques, and after talking with experts in the areaof proanthocyanidin mass spectrometry (Prof. Barofsky,pers. comm.), we decided to use negative ion FABMS utili­zing a matrix consisting of a 5:1 mixture of dithiothreitoland dithioerythritol known as "magic bullet". The spectra(Figs. 4, 5) were obtained under many different conditionsbut were consistently too weak to perform high resolutionand many MS/MS measurements. However, the spectra didallow for confirmation of a gross structure of the proantho-

86 R. Ubillas et al.

913138131371313613135131340a

0ll2 . 7

0

13

0

£I613c .9457 . 9

• 91}" 93~ .4 761.8-llJl. J. ~ j .l J. j j. ...I. 1 .. "'" ... ..I. .. ..6.

" . .. . .. ...~

~ aI ,

2

8

tlUC

'" 6"0C:l

.D

'"tl> 4'c;tl

0:::

18

3

Fig. 4. Negative ion fast atom bombardment mass spectrum (FABMS) of SP-303 in magic bullet mat rix from 300 to 1000 mlz.

2S-,--- - - - - - - ------------------------- -y

213

tlUC

'""0 l Sc:l

.D<'l

tl>'c;tl

0:::

1213 . 1 1594.6 1813 . 7

209 .9

Fig. 5. Negative ion fast atom bombardment mass spectrum (FABMS) of SP-303 in magic bullet matrix from 1000 to 4000 miz,

cyanidin oligomer made up of catechinlepicatechin andgallocatechinlepigallocatechin units. Examinatio n of theFABMS spectrum from 1000 -4000 m/z (Figure 5) revealsclusters of odd-numbered peaks corresponding to M-Hpeaks for a pentamer (5 x approx. 300 mol. wt. of mono­mer) at m/z 1505 (reI. abund 15), a hexamer (m/z 1809, reI.abund. 14), heptamer (m/z 2097, reI. abund. 19) an octa­mer (m/z 2401, reI. abund. 12), a nonamer (m/z 2689, reI.

abund. 8), a decamer (m/z 2993, reI. abund. 7) and an un­decamer (m/z 3297, reI. abu nd. 5). The even-numberedpeaks seen in Figures 4 and 5 at m/z 1200, 916, 762, 610and 458 are apparent fragments of larger parent(s). Also,each peak corresponding to an individual unit, for examplethe M-H peak at mlz 1809 for a hexamer, is really a clusterof peaks each differing by 16 a.m.u , or oxygen (i.e., M-Hm/z 1777, 1793, 1809, 1825) as shown in Figure 6. This is

SP-303 87

30 179 .17

1809 .1825 jliU 1777.20

182117ue<::l

"0 20c::l

I..0

~ ! Iii<::l

u

1\;;- 15

"<=J i"176 .22 I Ii

iU

Ili f! I'0:: Hl

!~

~5 I I ~1~ I i I I I I . I I -=r==;:---

1750 1760 17713 1780 1790 1800 1810 1820 18313 18 <:0

t'V Z

Fig. 6. Enlargement of the negative ion fast atom bombardment mass spectrum (FABMS) of SP-3D3 in magic bullet matrix form.

consistent with the hexamer being made up of varying ra­tios of B-ring oxidations (procyanidinlprodelphinidin),with the prodelphinidin occurring as the dominant oxida­tion pattern (i.e., 1778 a.m.u. corresponds to 3:3 procya­nidinlprodelphinidin units, 1794 a.rn.u, corresponds to 2:4procyanidin/prodelphinidin units, 1810 a.m.u. correspondsto 1:5 procyanidinlprodelphinidin and 1826 a.m.u. corre­sponds to 0:6 procyanidinlprodelphinidin-entirely prodel­phinidin B-rings). If the assumption is made that the surfaceactivitity and ionization energies of the individual units inthe matrix are not substantially different, then the relativeabundances of each of the parent M-H ions given abovecan be used to estimate relative percent of each of thevarious pentamer-to-undecamer oligomers in SP-303. Theaverage ratio of procyanidin/prodelphinidin units deter­mined by this method reflects a predominance of prodel­phinidin B-ring oxidation in SP-303. The above negativeion FABMS data define a range of oligomers for SP-303 upto the undecamer, each made up of catechinlepicatechinand gallocatechinl epigallocatechin monomers. The rangeand average molecular weight distribution of SP-303, clear­ly a heterogenous oligomer, are in accord with those deter­mined from the 13C NMR data. Overall, the MS data are inagreement with the 13C-NMR data and the HPLC-GPCmolecular weight determination by substantiating an aver­age oligomer chain length of seven, and an average molecu­lar weight of approximately 2100 a.rn.u.

Elemental analyses were performed on samples of SP­303. dried at 35-40°C under high vacuum. To avoid thepossibility of decomposition, no attempt was made to re­move all the H20 (e.g., by using P20S under high vacuum

and high temperature conditions) so that typically, 3-7%H20 remained after drying or lyophilization. The analysesare consistent with the following average formula for SP­303: (C15H120 6.S)n . 8 H20 , which represents approxi­mately 7% moisture. The calculated %C (56.8%) and %H(4.6%) using the formula are consistent with the observedranges (%C 56.1-56.7; %H 4.6-4.9). It should be notedthat the average formula is in accord with the mass spectralobservation that SP-303 is made up of varying ratios ofprocyanidinlprodelphinidin units. This is reflected in theformula ClsH1206.S, indicating an average distribution ofprocyanidinlprodelphinidin units with C15H120 6 for an allprocyanidin oligomer and C1sH120 7 for an all prodelphi­nidin oligomer.

The relative stereochemistry of the internal units of SP­303 was determined from the 13C-NMR discussion aboveas consisting of -2: 1 catechin (2,3-trans-3,4-trans )/epicate­chin (2,3-cis-3,4-trans) stereochemistries, with the terminalunit having the epicatechin (2,3-cis) configuration. Whatremained was to examine the absolute stereochemistries ofthe units themselves. Virtually all of the natural occurringf1avanoids including proanthocyanidins have the same ab­solute configuration at C-2, apparently following a com­mon biosynthetic pathway conserved in widely differingplants. The two stereochemical forms commonly found area 2,3-trans isomer (2R,3S) as in (+ )-catechin, and a 2,3-cisisomer (2R,3R) as in (-)-epicatechin. Based on biosyntheticconsiderations, SP-303 consists of monomeric units madeup of (+)-catechin, (+ )-gallocatechin, (-)-epicatechin and (-)­galloepicatechin. To confirm this, two sets of experimentswere performed. The specific rotation of SP-303 was mea-

IoJVv'

88 R. Ubillas et aI.

HOI

oJVv'

OH

OH

....6(HOH

HO

R = H, OHOH

techin, with (+)-gallocatechin and (-)-galloepicatechin pre­dominating. The sequence of the individual monomers witheach chain could not be determined. Also, the visible spec­tral data strongly indicate the presence of a f1avylium (antho­cyanidin) moiety, although the relat ively small fraction(<1% ) of this component has been an impediment to deter­mining its position within the oligomer. The average chainlength of SP-303 has been determined by 13C-NMR via inte­gration of the terminal unit/internal units as being a hepta­mer. Negative FABMShas also allowed estimation of the re-

sured in H20 over a range of concentrations, without anyrotation observed. By itself, this suggests that SP-303 isachiral or racemic. However, the 13C NMR shows an aver­age of 2:1 catechinlepicatechin (trans/cis) stereoisomers asdescribed previously. Using the specific rotation, the molefraction of the cis isomer X cis can be calculated by the fol­lowing formula: X cis = ([a] 578 + 320)/494 from the litera­ture, to give Xcis =(0 + 320)/ =0.65, which translates into a2:1 ratio of cis/trans (which is the same as a ratio of 1:2 ca­techinlepicatechin). When coupled with the 13C-NMR da­ta, the specific rotation data just described give an experi­mental range of X cis = 0.33-to-0.66 (±0.05). CD spectrawere obtained to confirm the chirality inherent in SP-303.The circular dichroism spectra confirm the chirality ofSP-303. In addition, the sign of the 280 nm circular dichro­ic bands is in accord with the C-2 stereochemistry assignedas 2R from literature precedent with other f1avanols, con­firming (+)-catechin, (+)-gallocatechin, (-)-epicatechin and(-)-galloepicatechin as the units making up the oligomerSP-303. Additionally, based on precedent, the positive CDat 236/212 nm indicates that the 4b stereochemistry[(-)-epicatechin & galloepicatechin or cis] is dominant overthe 4a stereochemistry [(+)-catechin &-gallocatechin ortrans]. Thus, the CD data support the interpretation of thespecific rotation data given above. In summary, the experi­mentally determined range of monomer stereochemistry ofSP-303 can be defined as Xcis = 0.33-0.66 of (-)-epicatechin& galloepicatechinl(+)-catechin & gallocatechin.

Structure Conclusions: The basic components of SP-303, aheterogeneous oligomer, have been established as (+)-ca­techin, (+)-gallocatechin, (-)-epicatechin and (-)-galloepica-

HO

OH

HO

R

h OH

...··UOH

R

h OH

...··UOH

OH

NR

h OH

...··UOH

OH

N =3-9R = H. OH

HO

OH

R =G =CatechinR = OH = Gallocatechin

HO

OH

R =H =EpicatechinR = OH = Epigallocatechin

lative proportions of oxidation within each of the chainlengths as well as their size range (up to Ll-mer). The sum ofthese data supports an average molecular weight of 21 00.

The relative stereochemistry is shown to range from 2:1to 1:2 ratios of catechin to epicatechin substructures, withthe terminal unit having the epicatechin structure. The ab­solute stereochemistry determined from CD is consistentwith the majority of naturally occurring flavanoids atC-2. Interestingly, the specific rotation of [a]578 = 0

0

isconsistent with the CD data, confirming the chiral natureof SP-303.

III. Antiviral Studies

Although a variety of plant derived drugs are used suc­cessfully in the clinic today, with the exception of podo­phyllotoxin, no other plant-derived antiviral agent is ap­proved by the FDA for commercial use (Rozhon et aI.,1994). The philosophy of Shaman Pharmaceuticals is toisolate and evaluate pharmacologically active componentsin medicinal plants that have ethnotherapeutic indicationfor certain diseases. Initial efforts in this area resulted in thediscovery of SP-303, a naturally occurring proanthocya­nidin oligomer (average molecular weight 2,100 amu). Thelarge body of virological preclinical data obtained onSP-303 is presented below. Some of the data in the reporthave been published previously (Soike et aI., 1992; Gilbertet aI., 1993; Wyde et aI., 1993a & 1993b).

Materials and MethodsViruses and cells. Table 1 lists the sources of RNA and

DNA viruses and cell lines used in this study. Antiviral ac­tivities presented here as 50% inhibitory endpoint or 50%effective dose (EDso) are in ug/ml.

Antiviral activity in cell culture. The antiviral activities ofSP-303 and known antiviral control compounds were de­termined using the viral cytopathic effect (CPE) assay, theplaque neutralization assay, the hemagglutination-inhibi­tion (HAl) assay, and/or the MTT antiviral assay: (i) ViralCPE assay. This assay has previously been described (Wydeet aI., 1993b). (ii) Plaque neutralization assay. The proce­dure used for the plaque reduction assay was adapted fromthat of Rozhon et al. (1993). Briefly, test compound wasmixed with virus and incubated 1 hr at 22°C before addi­tion to a cellular monolayer (60 mm diameter). Viral ad­sorption was performed at 3rc for 90 min before the viralinoculum-test compound mixture was aspirated and themonolayers washed with phosphate buffered saline. Anoverlay medium consisting of 0.76% carboxymethyl cellu­lose, MEM cell culture medium and 2% fetal bovine serum(56°C, 30 min) was added to the cells and incubation wascontinued for 6 days at 3rc. Plaques were visualized by

SP-303 89

staining with crystal violet (0.5% crystal violet, 10% for­malin: methanol, 1:1). (iii) HAl assay (for influenza virus Aonly): This assay was performed using a protocol similar tothat described by Dowdle et al. (1979). Briefly, cells thathad been treated with test agent were infected with FLU-Ain 96-well round bottom microtiter plates and the cells we­re incubated at 37°C. At the end of the day 5 of incubation,an equal volume of 1% washed chicken erythrocytes (Bio­Whittaker) was added to the culture medium. The platesthen were incubated at 4°C for 1 hr and the individualwells scored for hemadsorption activity. (iv) MTT antiviralassay. This assay was used exclusively for evaluating the ac­tivity of antiviral agents against hepatitis A virus (HAV). Asdescribed in the following section, MIT is usually used toassess adverse effects of potential antiviral agents in unin­fected cells by measuring cellular viability. However, withHAV-infected cells, MIT was also used as an indirect me­thod to measure the inhibitory activity of antiviral agents;the reagents and protocols used for both assays are identi­cal. Finally, the assay employed to test the activity ofSP-303 against hepatitis B virus (HBV) has been described(Korba and Milman, 1991; Korba and Gerin, 1992).

Selectivity index (SI). An SI (50% cytotoxic concentra­tion +50% antiviral concentration) for each virus testedwith SP-303 or known antiviral control compound in cellculture was calculated using the 50% cytotoxic endpointthat was determined in uninfected cells in an assay run inparallel with the antiviral assay. The cytotoxic measure ofantiviral agents was determined either by visual inspectionor by the MTT assay (Mossman, 1983). In either case, the50% cytotoxic endpoint, 50% inhibitory dose (ID so) is gi­ven in ug/rnl, For the MTT assay, a 96-well plate containingthe appropriate cells was incubated with different concen­trations of antiviral compound. At the end of the incuba­tion period, the effect of test compound on cellular viabilitywas evaluated by adding 25 ug/ml MTT (5 mg/ml stock inPBS) to each well and incubating for 3 hr at 37°C. The cul­ture medium-MTT mixture was aspirated and the MTTproduct, formazan, was solubilized with addition of 0.1 ml0.04 N HCI in isopropanol. Adsorption at 570 nm was de­termined for each well using a mikrotiter plate-readingspectrophotometer. A dose-response curve was generatedand the concentration of test compound giving 50% ab­sorption compared to the untreated control represented theICso• In addition, uptake and incorporation of radiola­belled biochemical precursors was used as an independentmeasure of the potential adverse effects of SP-303 in unin­fected cells.

Virucidal test: RSV (Long) and SP-303 (0-1000 ug/ml)were mixed and incubated for 1 hr at 37 "C, subjected to se­rial tenfold dilution, and titered for infectivity in HEp-2cells using the viral CPE assay.

90 R. Ubillas et al.

Table l. Listing of Viruses and Cell Lines Used to Test SP-303 and Their Sources.

Virus Source Cells Source Cell Origin

RespiratorySyncytial Virus HEp -2 ATCC hum. epid. carcinoma

MA-I04 Bio Whittaker Af. green monkey kid.RSV A (Long) ATCCRSV A (Tracy) Baylor U.!RSVA(WydelHouston/88) Baylor U.RSV B (46791) Baylor U.RSV B (47063) Baylor U.RSV B (18537) Baylor U.

Influenza Virus A MD CK ATCC canine kidneyA54 9 ATCC hum. lung carcinoma

FLU A Taiwan(HI Nl) Baylor U.FLU A NWS/33(HI Nl) ATCCFLU A Japan/305157(H2N2) ATCCFLU A Port Chal. 1/73(H3N3) ATCCFLU A Leningrad/86(H3N2) Baylor U.

Influenza Virus B MDCK ATCC canine kidneyFLU B USSR/I00/83 Baylor U.FLU B Yamagata/16/88 Baylor U.Hong Kong 5172 WHO!

Herpes Simplex Virus human foreskinType 1 HFF Clonetics fibroblast

MA-104 Bio Whittaker Af. green monkey kid.MDCK ATCC canine kidney

HSV·l 10186 (TK+) Well Res!HSY·l KOS (TK+) Well ResHSY· l 10166 (TK+) Well ResHSY· l McKrae 555A A.(TK+) NesburneHSV-l ACGr4 (TK) S. SachsHSY·l SC16-R5C1 (TK-) S. SachsHSY·1 BWVL 10168 (TK) Well Res

Herpes Simplex Virus human foreskinType 2 HFF Clonetics fibroblast

MA-104 Bio Whittaker Af. green monkey kid.MDCK ATCC canine kidney

HSY·2 MS ATCCHSY·2 £ 194 (TK+) M. FialaHSY·2 10167 (TK+) Well ResHSY·2 10165 (TK+) Well ResHSY·2 10634 (TK+) Well ResHSY·2 Cl 1252 (TK') S. SachsHSY·2 Cl1182 (TK') S. SachsHSY-2 10161 (TK-) Well Res

Human Cytomegalo-virus (AD-169) ATCC MRC-5 ATCC human lung

Table 1 (continued).

Parainflu enza Virus LLCHEp-2

PIV-l Baylor U.PIY-3 Baylor U.PIY-3 C243 SRP

Measles Virus Baylor U. Yero

Adenovirus-S Baylor U A549

Hepatitis B Virus George-'town U. 2.2.15

Herpes Z oster Utah!(Varicella) Virus State U. HFF

MRC-5

ATCCATCC

ATCC

ATCC

CloneticsATCC

SP-303 91

Rhesus monkey kid.hum. epid. carcinoma

Af. green monk. kid.

hum. lung carcinoma

HBYDNA transfectedhuman livercells

human foreskinfibroblastshuman lung

! Abbreviations used in this table include: WHO, World Health Organization; Well Res, Wellcome Research Laboratories; SRI, Sou­thern Research Institute; Baylor U., Baylor University; Utah State U., Utah State University, Antiviral Research Institute; GeorgetownU., Georgetown University.

2 Sells et aI., 1987.

Mechanism of action studies

Mechanistic studies with SP-303 comprised time-of-addi­tion of test compound relati ve to the viral infectious cycle,effect of test compound on attach ment and penetration ofvirus, and effect of test compound on vira l uncoating: (i)Time- of-addition studies: Test compound was added tocells at various intervals either before or after infection. De­pending on the study, test compo und was present through­out the incubation period follow ing its addition, or thecompound was aspirated after a defined period of incuba­tion . Viral replication was assessed by plaquing, except inone study with HSY-1 in which inhibition of viral DNAsynt hesis was the end point (Shipman et aI., 1976; Smee etaI., 1983). (ii) Effect on viral attachm ent: The method em­ployed was a modification of th at of Taylor and Cooper(1989). Briefly, [35S] methi on ine labeled puri fied virus wasincubated with cells for 1 hr in the presence of test com­pound befor e washin g the cells and determining cell-as­sociated radioactivity by liqu id scint illation spectrophoto­metry. (iii) Effect on vira l penetrat ion through the plasmamembrane: Virus was adsorbed to cells at 4°C before ad­din g of test compound and ra ising the incubation tempera­ture to 37° C. After 1 hr incub ation , unadsorbed viru s wa saspir ated and an y rem aining virus was neutralized by theaddition of neutralizing virus antibody. The antiserum wasrem oved and th e resulting virus was quantitat ed by pla­qu ing. (iv) Effect on viral unco at ing (HSY-1 only): The me­th odology used was that of Feldman et al. (1981) and Har­menberg et al. (1991). Briefly, cells that were pretreatedwith cycloheximide (200 ug/ml ) were infected with [3H]thymidine labeled HSY-1 and incubated for 4 hr at 3rc.Infected cells were subsequently washed, lysed with NP-40,and the cytoplasmic and nuclear components were sepa-

rated by centrifugation at 10,000 x g for 30 min. Radio­activity associated with each fraction was determined byliquid scintillation spectrophotometry. Since herpesviru suncoats at the nuclear membrane, radioacti vity associatedwith nuclei would indicate th at uncoatin g occurred nor­mally.

Determination of the affinity constant for SP-303 bin­dings to RS V. Methodologies using Scatchard analyses fordetermining affinity con stants for molecules binding to vi­ral part icles have been described (Bennett, 1978; Fox et aI.,1991 ). Briefly, [3H] SP-303 (sp. act. 1llCi/llmol) at concen­tr ati ons ranging from 0.1-50 ug/rnl and 12.5 ug/ml of un­labeled SP-303 were added to centrifuge tubes that con­tained purified RSV. The contents of the tubes were mixedand incubated at room temp erature for 40 min. Viralbound SP-303 was separated from unbound SP-303 by sub­jecting the tubes to two successive rounds of ultracentrifu­gatio n at 150,000 x g for 1 hr at 4°C. Th e amount of SP­303 bound to virus was determ ined by liqu id scintillationcounting. A Scatchard plot and the binding constant, Kd,

were dete rmined using the software program, K· cat™(Biometa llics, Inc. , Princeton , Nj), for ligand binding.

Evaluation of SP-303 in virus-infected animals. Studies inanima ls were performed as recommended in the Guide forthe Care and Use of Laboratory An imals (NIH Publication85-23). Relevant experimental details, including concen­trat ions of test agents and regimen s, are provided for all vi­rus/animals models in Table 2. (i) RSY-infected cotton ratsand parainfluenza virus -3: Infect ions of cotton rats andtreatm ents with antiviral test compounds are described byWyde er al. (1993a ). (ii) FLU A-infected mice: Descriptionsof FLU-infected mouse models have been described pre­viously (Grunert et aI., 1965; Schulman, 1968; Walker etal., 1976; Hayden, 1986; Wyde et aI., 1986). (iii) HSV 1- &

92 R. Ubillas et al.

Tabl e 2 . Summary of Dosage Regimens and Efficac y Obtained in SP-30 3-Treated, Virus-infected Animals.

Range of Do-2

Anim al Num. of Dosage sage Efficacy Control Evidence for SP- AdverseM ode l Studies Levels Routes Regimen! Endpoints Cmpds. 30 3 Efficacy Effects

RSv/Cotton 24 0.3-270 IP, PO, +24 hr viral titers ribavirin IP: 1-10 mg/kg/day wgt.loss atRat SPA (3 da ys) in lung PO: 1-10 rug/kg/day do ses above

(Wyde er aI., 1993 a ) efficac ySPA: 1.3 -10 do sesmg/kg/day (Gilbertet aI., 199 3 )

RV/Af. green 7 0.2-2 70 IV, PO -4 hr to +24 viral shed none IV, PO : AV C virus nonemonkey hr from throat shed curve versus

(10 days) and lungs time , mean peak viraltiters, du ration ofsheddingRefer to Tabl e 8(Soike et aI., 1992)

FLUN 10 0.3 -270 IP, PO , -4 hr to +14 oximetry, ribavirin, SPA: 85% sur vivor s decrease inM ouse SPA hr vira l titers amanta- compared to 0% in body temp.

(to 8 da ys) in lung, lun g dine pla cebo at 9 with SPAhistology, mg/kg/day route;pneumonitis, decrease insurvivors, wgt. at 21 6bod y temp mglkg/day

with SPAFLUN 1 1-10 IP -48 bod y temp., ribavirin , none slightFerrett (3 da ys) turbinate & amanta- pyrogenic

lung virus dine effecttiters, lunghistology,nasophar y .virus shed,pla sma viralant ibo dy

PARAFLU-3/ 2 1-10 IP +24hr vira l lung none vira l lung titer: 87- Refer toCotton Rat (3 da ys) titer 93% reduction RSV/Cotton

(Wyde er aI., 1993a) Rat sectionabove

HSV-l / 5 10-30% TOP ohr to + 6 cut ane ous ACV none noneGuinea Pig creams or hr viral lesions ,(Dorsal) ointments (8 days) vius shed,

sur vivorsHSV-2/ 3 IP, PO : 30- IP, PO, -2 hr to 6 hr external ACV, Lesion score reduct- wgt decreaseMouse 270; TOP (8 days ) genita l DHPG ion (AVC : lesion ver- by IP routeVaginal TOP: 5-10 % lesion s, sus time curve) at 30

creams or viru s shed, mg/kg/day by IP;ointments survi vors also for PO at 90

mg/kg/day & TOPwith 10% cream

HSV-2/ 3 10-15% TOP -48 hr to 6 externa l ACV Lesion score reduct- noneGuineaPig creams or hr genita l ion (AVC: lesion ver-

Vaginal ointments (8 da ys) lesions , sus time curve) withviru s shed , DMSO ba se cream;survivors also virus shed

decre ased 1-2 logloTCID 50 & increase insurvivor s (60% SP-303 versus 8% forplacebo) .

I Do sage levels are given in mg/kg/day.2 Abbreviations used for dosage routes: IP, intraperitoneal; PO , or al; IV, intravenous; TOP, topical; SPA, small particle aerosol.3 A minus sign (-) indicates a prophylactic regimen. For example, -48 hr indicates treatment with SP-303 was begun 48 hr before infec-

tion. A plus sign (+) indicates a therapeutic regimen. 0 hr indicates treatment was initiated at the time of infection. The number of daysin parenthesis ( ) indicates the length of the dosage regimen.

SP-303 93

Table 3. Antiviral Activity of SP-303 and Ribavirin Against Selected Common Respiratory Viruses.

SP-303 Ribavirin

Anti- Cyto- Anti- Cyto-viral toxicity viral toxicity

Virus N EDso1 IDso2 SP EDso

1 m.,2 SP

(A)RSVA 84 13.6:!:3.5 299:!:101 22 13.4:!:4.0 >1000:!:0 >75RSVB 35 6.7:!:0.7 158:!:30 24 8.0:!:2.3 >1000:!:0 >125FLUA 116 7.0:!:1.6 32l:!:105 46 7.3:!:1.9 > 751±128 >103FWB 6' 13.7:!:9.8 27l:!:103 20 5.8±1.2 > 847:!:153 >146PIV-1 38 3.0:!:1.5 129:!:32 43 10.7:!:1.3 >1000:!:0 > 93PIV-3 69 50.0:!:18 169:!:29 3.4 15.5:!:5.3 >1000:!:0 > 65

(B)Adcno-S 110 Not act! ' 182 1000 >1000 > 1Measles 110 Not act !' 24 32 >1000 > 31

I EDso is the 50% antiviral endpoint compared to the control and is expressed in ug/ml; values shown represent the arithmetic meanvalue of the EDso values for all viruses in the group. The assay used is the viral cytopathic effect assay and the endpoint was determi­ned visually.

2 IDso represents the 50% cytotoxic endpoint compared to the control and is expressed in mg/ml; values shown represent the arithme­tic mean value of the IDso values for all cytotoxic assays that were performed. The cytotoxic endpoint was determined either with MITor visually.

3 Selective Index: ICso + ECso'

4 RSV-A viruses tested includes: 4 Long isolates, 2 Tracy isolates, and 2 Wyde isolates. (Most viral isolates in this group were originatedfrom Influenza Research Center, Baylor University).

5 RSV-B viruses tested include isolates: 18537,46791, and 47063 (Baylor University).6 FLU-A viruses tested includes isolates: NWS133 (HI Nl ), japan/305157 (H2N2), Port Chalmersll /73 (H3N2), Leningradl86 (H3N2),

Shanghai, and Taiwan/86 (H1N1) (6 isolates).7 FLU B strains include: Hong Kon g/SIn, USSR/100/8 3 (3 isolates), and Yamagatall 6/88 (2 isolates).8 Three unnamed isolates.9 Isolates tested include: strain C243 and 5 unnamed isolates.10 Unnamed isolate.11 Not active.

2-infected mice an d guinea pigs: The HSV-murine and gui­nea pig models for evaluating antiviral agents are describedin detail in numerous publication s by Kern (1984, 1988,1990). (iv) RSV-infected Afr ican green monkeys: TheRSV/African green monkey model ha s been previously de­scribed Kakuk et al. (19 93) .

Statistical tests. Statist ical tests used to evaluate cell­based results included student's t-test ; for an imal studies,the Fisher exact test, ANOVA, and th e Mann-Whitney Utest were used .

Results of SP-303 antiviral activity studies

Respiratory Viruses. Table 3 presents the antiviral acti vi­ties (EDso's) of SP-303, determined by CPE assay and re­presented as the ar ithmetic mean of th e EDso values for allviral isolates or lab strains within a group, as well as the se­lective indices of the molecule against 6 groups of commonrespiratory viruses are presented in Table 4. The individualisolates comprising each viral group are indicated in the le-

gend to the table. The 50% antiviral endpoints for SP-303ranged from a low of 3.0 ug/rnl with parainfluenza virustype 1 (PIV-1) to a high of 50 ug/rnl for parainfluenza virustype 3 (PIV-3) . With the single exception of the PIV-3gr oup " , SP-303 exhibited potent anti viral activity againsta ll the resp iratory viruses that were tested. SI values withSP-303 ranged from 3.4 to 46 with th e mean value being26.4 (Table 3 ). For comparative purposes, the acti vities ofribavir in against the same viral gro ups are also shown.Note that the average antiviral endpoints of ribavirin ap ­proximate that of SP-303 for all vira l gro ups except PIV-3(Ta ble 4 ). Compared to SP-303, ribavirin exterted less ad­verse effect s on cells with an average 51 of> 101 (Table 3 ).SP-303 was inact ive against adenoviru s-5 and measlesviru s, whereas ribavirin exhibited activity against measlesviru s with an ECso of 32 ug/ml (Ta ble 3 ). Plaque reduction

.. Of 6 PIV-3 isolates tested, two were essentially inactive withant iviral endpoints of 93 and 94 ug/ml. However, the remainingthree isolates had endpoints of 5,8, and 1611g/ml.

94 R. Ubill as et a l.

Table 4. Antiviral Activity of SP-303 Against Respirator y Syncytial Virus! Using the Plaque Reduction Assay.

Antiviral .' Cyto toxicity" Selective"PFU Inoculum! EDso IDso Index

48136236366406730

3.33.03.53.04.84.6

83.283.24 1.683.241. 683.2

25281228

918

Ave. SI '"S.E.20", 7.6

1 Different amounts of vira l inocula (RSV,Long Strain; ATCC VR-26) were mixed with dilutions of SP-303 and incubated at room tem­perature for 1 hr before being added to HEp-2 cellular monolayers. After a 90 min adsorpt ion period, the SP-303/virus mixtur e wasaspirated from the cells, the monolayer washed, an overlay of carboxymethyl cellulose and MEM cell culture medium with 2% FBSwas added, and the monolayers were incubated for 6 days at 37°. Plaques were enumerated by staining with crystal violet.

2 Size of the viral inoculum in plaque forming units (PFU).3 Antiviral activity (EDsoin ug/rnl) is expressed at the 50% inhibitory concentration compared to the non-treated, infected contro ls.4 Cytotoxi city (IDso in lug/ml), detemined by MTT, is expressed as the 50% inhibitory concentration compared to the non-treated con­

trols.s Selectivity index =ID so + EDso

TableS. Effect of SP-303 on Uptake and Incorporat ion of [3H] Labeled Precursors.

Average50% Inhibitory Endpoints]Uptake Incorporation

Leu101

Thy122

Urid104

Leu64

Thy65

Urid73

] 50% inhibitory endpoints are expressed in ug/rnl, The period of incubation of SP-303 with HEp-2 cells was 18 hours. Abbreviations:Leu, leucine; Thy, thymidine; Urid, uridine.

analyses using SP-303 and RSV confirmed the antivi ralactivity of SP-303 observed in the cytopathic effect assays(Table 4 ). Six individual assays employing different quanti­t ies of vira l inocul a resulted in antiviral endpo ints ran gingfrom 3.3-4.8 Ilg/ml and SI va lues, determined by MIT,ranging fro m 9-28 w ith a mean value of 20 .

In addition to visua l and MIT evalu at ion s to assess th epotential adve rse effects of SP-3 03, a third study was doneto measure the effect the compound has on cellula r uptak eand incorporation of ami no acid (leucine ) and nucleic aci d(uridine and thymidine) precursors (Table 6). When diffe­rent concentrations of SP-3 03 were incubated with uninfec­ted cells in the presence of ra diolabeled precusors, the con­centrations th at inhibited 50% uptake and incorpora tionof these precursors were well above the 50% antivira l end ­points. Thus, the fact that th e antiviral endpoints of SP-30 3(Tables 4 & 5 ) were consistently less than those determinedin cytotoxity assays lends co mpelling evide nce to supportth e assertion th at th e antivi ra l activity of SP-303 ind epen­dent of th e cytotoxic effect o f the molecul e.

Herp esuiruses. The inhibitory activities of SP-303, deter-

mined by CPE assay, against either th ymidine kinase plus(TK+) or thymidine kinase minus (TK) herpes simplex virustypes 1 and 2 , are shown in Table 6. In addition, in Table 6,the antiviral activites of SP-303 against acyclovir resistant(ACVS)/fosca rnet resistant (FOS') herpes viruses, tha t wedetermined using th e plaque reduct ion assay, are shown.SP-303 was inactive against one strain of herpe s zoster (va­ricella ) virus which was test ed .

Usin g th e antivira l CPE assay (Ta ble 7), th e EDso va luesfor SP-303 agains t H SV-l , TK + of TK- st ra ins, range froma low of 3 ug/rnl to no activity at the high est concentrationtested. The average ECso for viruses in th e HSV-1 group is35.6 ug/ml, compared to 20.5 ug/ml for the HSV-2group, sugges ting that SP-30 3 has slightly greater potencyaga inst th e HSV-2 serotype. However, th e range of ECsovalues o bserve d for both serotypic groups was simi lar. Inhi­bition of H SV-1 strain 10168 and H SV-2 strains CI 11 82and CI 1252 suggests the po ssib ility th at SP-303, unl ikeacyclov ir, is act ive against TK- H SV strai ns. In reg ard to

po ssible adve rse effects of SP-303 on cel ls, the molecul eexerts a grea ter cytotoxic effect than acyclovir. W ith the ex-

SP-303 95

Table 6. Antiviral Activities of SP-303 Against Herpes viruses Determined from the Viral CPE Assay.

SP-303 Acyclovir

Anti- Cyro- Anti- Cyto-viral toxicity viral toxicity

Virus Strain (TK)l EDs02 ID

s03 SI4 EDs02 IDs03 SI4

HSY-l McKrae (+ ) 3 10.9 3.6 < 1 >1000 >1000HSY-l KOS (+) 57 75 1.3 < 1 >1000 >1000HSY-l 10166 (+) Not Acr! 47.6 < 1 >1000 > 625HSY-l 10186 (+) 54.4 75 1.4 1.6 >1000 > 625HSY-l ACGr4 H Not Acr' 56.2 19.2 >1000 > 52HSY-1 SC16(R5C1) H 58 75 1.3 36.9 >1000 > 27HSY-1 10168 (_)6 Not Act? 75 18.4 >1000 > 54HSY-1 10168 (-f 5.6 15.1 2.7 7.8 >1000 >128

HSY-2 10167 (+) 54.6 121 2.2 < 1 >1000 >1000HSY-2 10165 (+) 17.5 17.8 1 3.9 562 144HSY-2 10634 (+) 15.7 75 4.8 8.2 >1000 > 122HSY-2 E-194 (+) 2.3 8.2 3.6 6.3 >1000 > 159HSY-2 C[1182 H 10.1 75 7.4 52.4 >1000 > 19HSY-2 C[ 1252 H 22.8 75 3.3 124 >1000 > 19

I TK refers to a functional viral thymidine kinase (+) or a non-functional viral thymidine kinase (-) mutant.2 Antiviral activity (EDso in ug/rnl) is expressed at the 50% inhibitory concentration compared to the non-treated, infected controls.3 Cytotoxicity (IDso in ug/rnl) is expressed as the 50% inhibitory concentration compared to the non-treated controls.4 Selectivity index =ID so + EDso5 Not active at the highest concentration that was tested.6 Virus tested in vera cells.7 Virus tested in MA-I04 cells.

Table 7. Antiviral Activities of SP-303 Against ACY'/FOS' (A) and ACY'/FOS' (B) Herpes viruses Determined by the Plaque ReductionAssay' .

Antiviral Activity (EDso)1 -

HSYType Isolate SP-303 Acyclovir Foscarnet

(A)HSY-1 8901 08 3.2 2.5 10Not Typed? 920014 1.1 3.3 68HSY-2 890650 0.8 38.0 34HSY-2 890480 2.0 64.0 34HSY-2 910040 1.2 64.0 10Not Typed? 890490 1.1 70.0 32HSY-2 890600 1.9 108.0 27Not Typed- 900160 1.1 130.0 28

(B)HSY-2 900180 0.9 0.9 260HSY-2 90395 1.4 1.0 119HSY-2 920025 1.1 1.4 130HSY-2 920020 2.1 1.0 130HSY-2 910580 1.7 0.4 115HSY-2 890546 1.4 2.1 110HSY-2 920023 1.1 1.3 119HSY-2 90157 1.2 1.0 110

I Antiviral activity (EDso in ug/ml) is expressed at the 50% inhibitory concentration compared to the non-treated, infected controls. Al­though cytotoxici ty data are not shown in this study, SP-303 was not toxic at 100 ug/ml, the highest concentration tested. SP-303 waspresent only during the viral attachment phase, whereas ACY and FOS were present throughout the assay.

2 The immunotype of isolate is not known.• Safrin et al., 1993.

96 R. Ubillas et al.

ception of a single assay, IDso endpoints with acyclovirwere not obtained at 1000 ug/rnl (Table 6).

When 5P-303 was tested against ACV'/F05' andACV'/F05r (Table 7) H5V type 1 and 2 viruses using theplaque reduction assay, considerabl y greater potencie s wereobtained than those obtained from the CPE assay (Table6). As seen in Table 7, 5P-303 is highly active against theACV' and ACVs viruses listed in this Table are 1.6 and 1.5ug/ml, respectively. These values compare with 60 and 1.1ug/rnl EDso's for ACV' and ACVs viruses, respectively, andwith EDso's of 15.8 and 36.6 ug/rnl for F05S and F05' vi­ruses, respectively. Thus, as shown with the plaque reduc­tion assay, 5P-303 exhibits highly potent activity against 8ACV' and 8 F05' strains of herp es viruses. Although theseACV' viruses have not been characterized for TK- or TK+genotype, the results of this study stro ngly imply the abilityof 5P-303 to inhibit TK-,Acvr H5V. The major implicationof this study is that 5P-303 exerts its antiviral effect by amechanism different than that of acyclovir and foscarnet,which are both known to inhibit replication of H5V DNA(Hovi, 1988; Mertz, 1990 ).

When 5P-303 was tested against hepatit is A virus (HAV)using MIT to define anti viral and cytotoxic endpoints, anEDsoof 5.0±0.0 ug/ml (n = 2) was obta ined with a ICsoof135 ug/rnl (51 = 27) . Against hepat itis B virus (HBV), SP­303 at 50 ug/ml resulted in approximately a 50 % reductionof intra cellular HBV genom ic and replicative intermediateDNAs. No toxicitiy was detected at 50 ug/ml using neutralred stain ing.

Antiviral mechanism of SP-303

Virucidal test: Assessment of R5V infectivity when the vi­rus was incubated for 1 hr with increasing concentrationsof 5P-303 indicated that 5P-303 is virucidal at concentra­tions of ~ 125 ug/ml: at this concentration, a decrease inviral titer of 1.8 10glO TCID so is observed. Since the viruci­dal concentrat ion is well above the inhibitory concentra­tions observed under conditions employed for anti viralassays, the virucidal effect of 5P-303 does not playa role inthe latter assay.

The following observat ions have been made regard ingthe time during the viral infectious cycle that SP-303 exert sits antiviral effect: (i) Time-of-addition stud ies performed inrelation to the viral cycle indicate that the optimal time to

produce an antiviral effect with SP-303 is early in the viralreplicat ive cycle: For example , the most potent anti-R5V ef­fects are observed when 5P-303 (6 ug/rnl) is added to cellsconcomitantly with R5V and as late as 2 hours post-infec­tion : The R5V titer is decreased between 1-2 loglo TCIDsowhen SP-303 is added between -2 hr to +2 hr post-infec­tion. In contrast, if 5P-303 is added to cells at 4 hours post­infection, virtually no anti-R5V effect is observed. Similarfindings were observed when viral DNA synthesis was the

endp oint in H5V-1-infected cells: When SP-303 (75 ug/ml ]was added to cells before infection und allowed to remainwith the cells, viral DNA synthesis was completel y inhibi­ted when measured at 18 hr. If added immediately after a1 hr viral adsorption period and allowed to remain on thecells, 5P-303 blocked HSV DNA synthesis by 30%, but ifadded later than 1 hr, there was no effect on viral DNA syn­thesis measured at 18 hr:o(i i) 5P-303 retain s its anti viral ef­fect when cells are pretreated with the molecule for onehour and washed before infection. Cells pretreated with5P-303 for 1 and then subjected to as many as 10 washe sprior to infection inhibit R5V plaque formation by 60 %.Strong adherence to the plasm a membrane of cells may ac­count for 5P-303 retaining some of its antiviral activity un­der these experimental conditions. These findings contrastwith those obtained for ribavirin : If ribavirin is added to

cells during the viral adsorption period and the monolayeris washed to remove comp ound , no antiviral effect is seen.Based on the fact that ribavirin is a nucleoside analoguewhich exert s its inhibitory effect during replication of theviral nucleic acid, this result is not unexpe cted and supportsa different antiviral mechanism of 5P-303 compared to thatof ribavirin. The result s of the above studies indicate that5P-303 exerts its anti viral effect by inhibiting an early viralmechanism such as adsorption and/or penetration throughthe plasma membrane.

In a mechanistic study using [3S5]-labeled R5V that wascoincubated with 5P-303 at 10.5 ug/ml, a 50% inhibiti onof viral attachment to cells at 60 min compared to untrea­ted controls was demonstrated. A second mechani stic stu­dy, indicat ing an alternative antiviral mechanism, showedthat 5P-303 (10.5 ug/ml) inhibits nearly 100% of R5V pe­netr ation through the plasma membrane when the com­pound is added during the adsorption phase. Thus, R5V ismore sensitive to the effect of 5P-303 in inhibiting penetra­tion than attachment. Confirmation of binding of 5P-303to virus particles was determined using purified R5V and[3H]-labeled 5P-303 , and Scatchard ana lysis gave a Kd

value for 5P-303 binding to R5V of 23±10 x 10-8 M (datanot shown). Although these studies clearly show that 5P­303 binds to R5V, and that this interaction likely accountsfor inhibition of viral attachment and penetration, thesestudies do not preclude the possibility that 5P-303 adher­ence to plasma membranes also plays a role in blocking vi­ral infectivity.

Finally, 5P-303 fails to induce production of interferonwhen incubated with L-92 9 cells (data not shown).

Efficacy of SP-303 in virus-infected animals

Table 2 provides details on the concentrations of 5P-303that were tested, routes of admin istration, experimental pa­rameters for measuring efficacy, and other relevant infor­mation on the in vivo evaluati ons of 5P-303.

RSV-infected cotton rats: In the cotton rat model, SP-303was administered at several different dosage levels by threeroutes of administration: oral, intraperitoneal, and smallparticle aerosol (Wyde et al., 1993a; Gilbert et al., 1993).

(i) Oral evaluations: In a series of 12 efficacy studies inRSV-infected cotton rats (some are published in Wyde et al.1993b), SP-303 was administered in three dosages per stu­dy ranging from 0.3 to 270 mg/kg and given either once,twice, or three times daily. The efficacy of SP-303 wasmeasured from RSV recovered from lungs by transpleurallavage. In some of these studies, a ribavirin-treated groupwas included to compare its activity with that of SP-303.Comparing groups of animals receiving different dosagelevels to placebo-treated animals within individual studies,statistically significant reductions in viral titers are seenwith 1 mg/kg doses in 3 studies; with 3.0 mg/kg in 4 stud­ies; and with 10 mg/kg in 3 other studies (data not shown).With the exception of a single study, no dose over 10 mg/kgresulted in statistically significant reductions in viral titersunder the conditions employed. The data from each studywere analyzed by two statistical methods: (i) ANOVA wasused across all dosage groups (in a single study) to deter­mine if the overall reduction at all dosages was significantcompared to the control; and (ii) by linear regression to de­tect a dose-response related reduction- in RSV lung titers.Using the criteria of both requiring a statistically significanteffect produced by SP-303 for at least one dosage groupand evidence for a positive dosage response, 6 studies pro­vide reasonable evidence that SP-303 is active in RSV-in­fected cotton rats under the experimental conditions, whileonly 4 studies were clear failures. In two other studies,there was a statistically significant decrease in RSV titers inone dosage group, but evidence for a dose-response waspoor. In summary, 67% (8/12) of the studies exhibitedevidence for efficacy at any dosage level. When correlatingefficacy with the number of dosage groups per study thatresulted in significant reductions in viral titer,S studies hadonly a single dosage which was statistically significant,while 2 studies had 2 dosages that were significant and 1study was significant at all 3 dosages. Analysis of dataresulting from these studies did not enable conclusions tobe drawn about a dose-response effect or the advantages(or disadvantages) of once versus multiple daily dosing.The findings of this series of studies are consistent with thesmall sample sizes (n = 4/group).

(ii) IP dosage route: When SP-303 was evaluated in theRSV/cotton rat model using the IP route of administration,the overall results of nine individual experiments confirmedthe ability of this compound to reduce RSV pulmonary ti­ters. (Some of these studies are published in Wyde et al.,1993a). When the data were pooled, a dose-response rela­tionship to 10 mg/kg of SP-303 was evident. At dosages of30 mg/kg, however, slight toxicity of the compound wasobserved, with weight losses occurring in infected and con­trol animals. In 3 experiments, SP-303 was compared to ri-

SP-303 97

bavirin given at 90 mg/kg, with the latter drug showing asomewhat superior ability to reduce viral titers.

(iii) Inhalation route ofdosage: Administration of SP-303by small particle aerosol to RSV-infected cotton rats resul­ted in significant reductions in pulmonary titers of RSVwhen given at doses 1.3-10 mg/kg/day (Gilbert et al.,1993). However, at doses of 18.7 mg/kg/day, the compoundwas somewhat toxic, resulting in weight loss in comparisonto control animals. The reductions in RSV titers obtainedwith all groups treated with SP-303 by the inhalation routewere less than those seen in the ribavirin-treated positivecontrols.

RSV-infected African green monkeys: Seven studies wereperformed in which SP-303 was tested for efficacy in RSV­infected African green monkeys, the results of which aresummarized in Table 8. Three of these studies involved in­travenous (IV) dosage of SP-303 while the remaining fourstudies employed oral gavage (PO) as a means of delivery.Some of these data were presented at the InternationalAntiviral Meeting in 1992 and subsequently published asan abstract (Soike et al., 1992). In all studies, monkeyswere infected with RSV intratracheally and intranasally.The ability of SP-303 to produce efficacy in RSV-infectedmonkeys was assessed using the following parameters:(i) mean area under the curve (AUC) of RSV titer versustime after inoculation; (ii) mean RSV titer at all time points;(iii) mean peak RSV titer at each dosage level (or for eachexperimental group); (iv) mean time to first RSV shedding;and (v) mean duration of RSV shedding. Except for symp­tomatic effects which were not quantitatively evaluated,AUC is the most important assessment as it measures theeffect of SP-303 on reduction of RSV titers throughout theexperiment.

(i) IV dosage route: With doses of 0.2, 0.5, or 1.0mg/kg/day given in divided doses starting 4 h prior to in­fection and continued for 7 days, statistically significant,dose-dependent reductions in throat swab titers of RSVwere obtained in SP-303 treated-groups compared to place­bo-treated animals (Table 8). Indeed, animals receiving thehigh dose of 1.0 mg/kg/day had a reduction 3 loglo TCIDsoin peak virus titer, which represents a substantial reductionin oropharyngeal shedding of RSV. Also, these animalsseemed to experience fewer symptoms of infection. At the1 mg/kg dosage, the mean time to initial RSV shedding wassignificantly delayed and the duration of RSV shedding wassignificantly reduced compared to control animals (datanot shown). As a follow up to this study, the same high doseof SP-303 (1 mg/kg/day) was compared in groups treatedeither 4 hr (-4 hr) prior to infection or at 24 (+24) hr and 48(+48) hr post-infection (Table 9). In the -4 hr group, statis­tically significant reductions in comparison to control ani­mals were again seen in throat swab titers of virus, reduc­tion in mean duration of oropharyngeal shedding of virus(not shown), and a delay in mean time to onset of viralshedding (not shown). For the +24 hr group, there was a

98 R. Ubillas et a l.

Table 8. Antiviral Activity of SP-303 in RSV-lnfected Africa Green Monkeys.

Experi-:'Specimen- ment Mean Peaks

Study No. Regimen! Assayed Groups AUe4 Virus Titer

£-042 IV; BID; -4 hr throat Placebo 26.2 4.80.2 mg/kg 15.1 3.70.5 mg/kg 14.2* 3.71.0 mg/kg 3.7 1.5*

£-015 IV; BID; all throat Placebo 24.6 4.5groups tested -4 hr 13.7* 3.3at 1 mglkg/ +24 hr 19.9 4.3day +48 hr 24.0 4.3

£-044 IV;TID throat Placebo 22.2 4.65 mg/kg(-4 hr) 26.5 4.95 mg/kg(+24 hr) 29.6 5.520 mg/kg(+24 h4) 30.4 6.1

£-043 PO; OlD; throat Placebo 24.0 4.2-4 hr 10 mglkg 15.9* 3.7

30 mg/kg 18.6 3.790 mg/kg 16.4 3.3

PO; TID; Placebo 26.8 5 .2-4 hr 30 rng/kg 17.4* 3 7

90 mg/kg 18.7* 3.3270 mg/kg 12.4* 2.5*

£-024 PO; BID; throat Placebo 30.6 4.8+24 hr 10 mg/kg 33.7 5.2

30 mg/kg 28.4 5.290 mg/kg 25.1 4.8

PO; BID; lung Placebo 29.2 5.8+24 hr 10 mg/kg 21.8* 3.5*

30 mg/kg 24.7 4.890 mg/kg 19.2* 4.2*

! Abbreviations used under regimen: IV, intravenously; PO, orally; OlD, one dosage per day; BID, two dosages per day; TID, threedosages per day; and -4 hr indicates prophylactic study in which test compound is given 4 hr before infecting monkeys.

2 Sites from which viral specimens obtained: throat swab or bronchoa lveolar lavage.J Listing of placebo and experimenta l groups. Dosage levels are given as daily mg/kg and -4 hr indicates prophylactic study in which test

compound is given 4 hr before infecting monkeys; +24 hr and +48 hr indicate a therapeutic study in which test compound is given at24 hr or 48 hr, respectively, after infection of monkeys.

4 AUe represents area under the RSV titer versus time of inoculation curve.S Mean Peak Titer refers to the maximum titer of virus recovered at any time averaged for all monkeys in that group .

* ANOVA analysis demonstrating statistical signifancecompared to placebo groups at p < 0.05 using least significant difference method .

statistically significant reduction in mean viral titers calcu­la ted across all dosage groups (not shown), but not in theother measured parameters, and for the +48 hr treatedgroup, there were no significant differences between treat­ment and control g roups for any of the measured parame­ters. In the final IV study, the effects of increased dosageswas evaluated (Table 10). In animals treated either at -4 hr,+24 hr, or +48 hr, none of the measured parameters gave

evidence for efficacy.(ii) PO evaluations: In the first study (Table 8), once-a­

day dosing and three t imes a day dosing were comparedusing a prophylactic regimen by initiating dosage 4 hr be-

fore infection (i.e, - 4 hr). In the once-a-day dosing study,on ly the 10 mg/kg group had significant RSV reductions bymean AUe. Mean duration of viral shedding compared to

control animals also was significant (not shown) . In thethree times a day dosing study, statistically significant re ­ductions in AUe as well as peak viral titer were obtained atall three dosage levels.

In two final oral studies, reduction of RSV titers fromboth throa t swabs and bronchoalveolar lavage (BAL) fluidswere evaluated in monkeys receiving 10, 30, and 90mg/kg/day of SP-303 (Table 8). The rationale for examiningRSV titers in BAL specimens is tha t RSV in the lower respi-

ratory tract of humans is of greater significance pathologi­cally than virus confined to the nasopharyngeal cavity. Al­though some efficacy was observed in throat swab speci­mens at the 30 and 90 mg/kg dosages in regard to meantime to first RSV shedding, respectively (not shown), no ef­fect was observed with AUC and mean peak RSV titers atthe 10-90 mg/kg range. In contrast, for BAL specimens, the10 and 90 mg/kg groups were statistically significant com­pared to placebo-treated animals in respect to two test pa­rameters: AUC and mean peak RSV titer. In fact, mean RSVtiters in BAL fluids were reduced by approximately 2 10glOTCIDso units. These results are particularly significant be­cause administration of SP-303 was begun 24 hours afterinfection. Thus, the compound demonstrated efficacy in anRSV infection in the lung which represents the most signifi­cant clinical scenario of RSV infection in humans.

For either IV or PO studies with African green monkeys,no generalized toxic effect of SP-303 was observed. Occa­sionally, in the IV studies a small amount of irritation wasobserved at the intravenous site of injection. In the few in­stances when symptomatology was scored, observers feltSP-303-dosed animals experienced a decrease in rhinorheaand sneezing and coughing.

FLU-A-infected mice and PlV-3-infected cotton rats (Ta­ble 2). As resported by Wyde et al. (1993a) and Gilbert etal. (1993), SP-303 was efficacious in PIV-3- and FLU-A- in­fected animals, respectively (Table 4). Among IP, PO, andinhalation routes of dosage used with SP-303 in FLU-A-in­fected mice, only the inhalation route resulted in statistical­ly significant efficacy. Evidence for efficacy in FLU-infectedanimals treated with SP-303 by small particle aerosol (SPA)was demonstrated by oximetry (i.e., % saturation of arteri­al 02), pulmonary FLU titers, survivors, pneumonitis, andhistology of lung tissue. In two studies published using PIV­3-infected cotton rats (Wyde et aI., 1993a), SP-303 admin­istered IP at 3 and 10 ug/rnl, reduced virus in the lung by

SP-303 99

90% compared to control animals. Efficacy with other ex­perimental parameters was not reported for this model.

HSV-I- or HSV-2- infected mice and guinea pigs (Table 2).Parameters were used to assess efficacy in the HSV/mouseand guinea pig models: AUC from viral lesion score versustime curve, virus shed from lesions, and the number of sur­viving animals at the end of the experiment. In the murine/and guinea pig/ vaginal models efficacy due to SP-303 wasdemonstrable by IP, PO, and TOP routes of administration.In mice, the AUC determined from vaginal lesion scoreswas statistically significant for the SP-303-treated groupscompared to placebo groups by all three routes (IP: 6.7 at30 mg/kg/day vs. 14.0 for the placebo; PO: 6.2 at 270mg/kg/day vs. 10.5 for the placebo; TOP: 5.4 with 10%cream vs. 9.3 for the placebo), but a dose-response effectwas not apparent. Fig. 7 compares the effect of topical SP­303 and acyclovir treatment on murine genital lesions. Asseen, acyclovir is approximately twice as active as SP-303based on AUC: 6.7 for SP-303 and 1.4 for acyclovir.Neither titers of HSV-2 shed nor percentage of survivors forthe SP-303-treated animals were significantly different thanplacebo groups. Again, acyclovir or DHPG (ganciclovir),used as positive controls, were efficacious by most meas­ures.

Topical application of SP-303 to viral lesions in the HSV­2/guinea pig vaginal model resulted in statistically signifi­cant efficacy in regard to virus shed in 2 of 3 studies. (In thestudy in which SP-303 failed, acyclovir also was a completefailure.) In one of these studies, the decrease in virus shedcorrelated with a concomitant reduction in AUC as well asan increase in the percentage of survivors compared to thecontrol with one of 3 topical vehicles that was tested (Table2). The results of this study suggest that the formulation ofthe topical vehicle may be significant since vehicle 1, unlikethe other two vehicles, contained 78% dimethyl sulfoxide(w:w) (Fig. 8).

--0-- Vehicle 14

~0o

(J) 3

c.2U)III 2

...J

CcaIII:E

0

2 4 6 8 10 12 14

........<>........ Vehicle 2

····0···· Vehicle 3

----6---- ACV (5%)

- - -EB- - - Placebo

Regimen: +6 hr, 15% SP-303in different ointments, BID, 8days.

'Statistically significant corn­pared to placebo @ p < 0.05.

1 6 PFU shed & % survivors alsosignificant'.

Days Post-I nfection

Fig. 7. Effect of Topical Treatment of SP-303 on HSV-2 Vaginal Infection of Mice.

1DD R. Ubillas et al.

However, SP-3D3 was not efficacious in dorsally infectedhairless or shaved guinea pigs when applied topically as a3D% cream or ointment. In contrast, ACV was nearly al­ways efficacious in this model by a least one of the aboveparameters. In summary, SP-303 is capable of producing ef­ficacy in the HSV-2/mouse and guinea pig vaginal models,but a dose-dependent effect correlating to any antiviral pa­rameter was not observed.

Discussion of Virology Test Results

The antiviral activity of SP-3D3 against a variety of DNAand RNA viruses in cell culture and in animal models of in­fection is presented below. In cell culture, SP-303 is inhibi­tory against a broad spectrum of respiratory viruses thatcomprise three genera in two viral families: the Paramyxo­viridae (PIV-I & RSV) and the Orthomyxoviridae (FLU-A& FLU B). Moreover, SP-3D3 inhibits viruses representingthree non-respiratory viral families: Herpesviridae (HSV-Iand HSV-2), Picornaviridae (hepatitis A virus), and Hepad­naviridae (hepatitis B virus). The antiviral potency ofSP-3D3 in the antiviral CPE assay is similar to that of riba­virin against RSV, whereas against HSV-I and HSV-2 in theCPE assay, SP-3D3 is not as broadly active against acyclo­vir" herpesviruses as acyclovir". Alternatively, as meas­ured by plaque reduction assay, SP-303 has the advantageover acyclovir- against acyclovir- herpesviruses, as SP-3D3is highly inhibitory to these viruses. Likewise, SP3D3 ex­hibits significant inhibitory effects against FOS' herpes vi­ruses. In animal models of viral infection, SP-3D3 exhibitsactivity in RSV-infected cotton rats and African green mon­keys, PIV-3-infected cotton rats, HSV-2-infected mice andguinea pigs, and FLU-A-infected mice.

With the exception of nucleoside analogues, which mayhave broad antiviral activities, and interferon, which inhi­bits a variety of viruses (Hovi, 1988; Hirsch & Kaplan,199D), the broad "interfamily" antiviral activtiy of SP3D3,which is not a nucleoside and does not appear to induce in­terferon, is unusual, if not unique. The results of studiespresented here indicate that SP-3D3 interacts with the viralparticle outside the cell to exert its antiviral effect. In fact, ifSP-3D3 is added to infected cells after RSV or HSV has pe­netrated the plasma membrane, no viral inhibition is detec­ted. Furthermore, a study using radiolabeled SP-3D3 de­monstrated that little or no SP-303 crosses the plasmamembrane (unpublished results). Thus, the antiviral effectof SP-3D3 is manifest through extracellular interaction withvirus. In the case of viruses having lipid envelopes (i.e, RSV,FLU, PIV, & HSV), SP-3D3 must interact with either the li­pid moieties comprising the envelope or the virion glyco­proteins that protrude from the envelope or both. It is wellknown that tannins strongly bind to basic polymers andproteins (Goldstein and Swain 1965), and this probablyplays a significant role in the antiviral effect of SP-3D3. Re-

gardless of the viral structural component to which SP-3D3binds, binding studies with RSV and [3H]-labeled SP-3D3suggest that SP-3D3 binds in a specific manner to virus: AKd of 2.3 x 10-8 M was determined from the Scatchard ana­lysis, which is similar to affinities of known antiviral li­gands that bind to specific receptors on the virion particle.For example, the WIN molecules interact with the non­enveloped capsids of human rhinoviruses with Kd values inthe 10-8 M range (Fox et al., 1991).

In regard to distinguishing lipid constituents from viralglycoproteins as targets for SP-3D3 binding, it can be ar­gued that, since the viral envelope is derived from the plas­ma membrane, SP-3D3 should be equally active against dif­ferent viruses replicating in the same cell. Clearly this is notthe case (Tables 3, 6, 7). In light of this argument, it is inter­esting that SP-3D3 causes a reduction in the intracellularDNAs of hepatitis B virus (HBV) since the antiviral assayfor HBV employs chronically infected hepatocytes. SinceHBV acquires its envelope intracellularly, rather than bybudding through the plasma membrane (Robinson, 199D),it would seem that SP-303 inhibits HBV indirectly by inter­acting with the plasma membrane of infected hepatocytes.The one nonenveloped virus against which SP-3D3 is inhi­bitory is hepatitis A virus (HAV). Although antiviral me­chanism of action studies were not performed with HAVaswere done with RSV, and to a lesser extent with HSV, thisresult suggests that SP-3D3 interacts with some polypeptidecomponent on the surface of the HAV capsid to result in in­hibition. In any event, without further study, it is not possi­ble to define the precise nature of SP-303 interaction withthe viral envelope or-capsid.

The finding that SP-3D3 inhibits RSV-infection of HEp-2cells when the cells are pretreated with SP-3D3 and thenwashed extensively before infection suggests an inhibitorymechanism of SP-3D3 different than that of binding direct­ly to the viral particle. Under these conditions, it is possiblethat SP-3D3 adheres to the plasma membrane to render thecellular receptors for viral attachment inaccessible to virus,and thereby blocks virus attachment. Although the resultsof the binding experiment with purified RSV and SP-3D3clearly demonstrate that SP-3D3 interacts with the viralparticle, none of the cell-based assays that resulted in inhi­bition of virus discriminate between SP-3D3 binding to thecell or to the virus. In fact, it cannot be discounted thatboth mechanims have a role. It should be emphasized thatthe effect of SP-303 to inhibit RSV binding to the plasmamembrane or penetration through the plasma membrane ifattachment has occurred is consistent with a large body ofdata obtained on picornaviruses treated with the capsid­binding WIN molecules (Fox, 1986; Smith, 1986; Pevear,1989). Finally, precedence exists for plant-derived antiviralagents that interact with viral particles to inhibit early viralevents such as attachment, penetration, and/or uncoating.The best-studied of these agents are flavanoid molecules

SP-303 101

3.-------------------,

'D--iJf-i:J----o--o-{}-Q--G *

----0---· Placebo

........<>.. ...... ACV (5%)

'Statistically significant corn­pared to placebo @ p < 0.05.

SP-303 (10%)

Regimen : +6 Hr, SP·30310% ointment BID, B days.(Ointment: Squibb cr. base)

--0--

*.......(). .

1:) •• •0-' -0- --0"0"0··-0

,.0'"

~

...0

2

2.5

0.5

1.5

Q)...oo

(J)

l:.S!IJlQ)...J

l:l'GQ)

:E

4 6 8 10 12 14 16

Days Post-Infection

Fig. 8. Effect of Topical Treatment of Sp·303 on HSV-2 Vaginal Infection of Guinea Pigs.

that interact with the capsids of picornaviruses (Ishitsuka etaI., 1982; Superti et al., 1989; Conti et aI., 1992).

Except for topical application of SP-303 to HSV-Ilesionson the backs of guinea pigs, SP-3D3 exhibited efficacy in allother animal models of viral infection that were tested (Ta­bles 9-11). The routes of SP-3D3 administration that weremost successful in producing efficacy were: topical applica­tion to HSV-2 genital lesions in mice and guinea pigs, aero­sol inhalation to FLUA-infected mice and PIV-3-infectedcotton rats , and oral dosage to RSV-cotton rats . It shouldbe noted that SP-303 was not always active by all the expe­rimental parameters used to measure efficacy in a singlestudy, and did not always demonstrate clear dose-depen­dent effects. Despite this, SP-3D3 was clearly efficacious inmost of the virus/animal models used for this report.

Given that SP-3D3 possesses antiviral activity in cell cul­ture that, with few exceptions, approximates that of positi­ve control antiviral compounds, then it is worth conside­ring why the level of efficacy achieved with SP-303 in vivowas not always equivalent to that of the control com­pounds, or in one model why SP-303 was completely inac­tive. The best system to examine this question is that of theHSV/animal models that tested acyclovir or DHPG (gan­ciclovir) in parallel with SP-3D3. (Although ribavirin wasoccasionally orally dosed in parallel with SP-3D3 in cottonrats, the optimal way to dose ribavirin to achieve efficacy isby inhalation.) When topically applied to the backs ofHSV-I infected guinea pigs, no evidence was obtained withSP-3D3 to support efficacy, whereas, ACV almost alwaysafforded efficacy in this model. Moreover, when SP-3D3was applied topically to HSV-2 vaginal lesions in mice, thecompound was uniformly less active than DHPG. Thereappear to be two possible major explanations that explainthese observed differences: (i) SP-3D3 is not reaching thesite of viral replication, and/or (ii) SP-303 is reaching thesite of replication but its extracellular antiviral mechanismis disadvantageous. In regard to (i), evidence obtained with

three different topical formulations of SP-303 implies thatefficacy may be enhanced if the compound can be deliveredto the site of viral replication in sufficiently inhibitoryquantities, (Fig. 3). On the other hand, in regard to (ii),even if more SP-303 is delivered to the site of HSV replica­tion, the extracellular antiviral mechanism of SP-303 maypreclude it from being as efficacious as acyclovir. If the ma­jority of the virus in the lesion is highly cell-associated (i.e.,little extracellular virus), then acyclovir clearly has the ad­vantage with its intracellular antiviral mechanism. In fact,acyclovir is highly efficacious in either the HSV-l or HSV-2model topical models.

When the SP-303 is given to HSV-2 vaginally infected miceby the IP and oral routes, the level of efficacy achieved,based on AUC (lesion score versus time curve), is 4.7- and2.8- fold less than that achieved with DHPG. The explana­tion for these differences in activity may relate to pharma­cokinetic differences between SP-3D3 and DHPG. To affordefficacy in this system, an antiviral molecule must get fromthe peritoneal cavity or the intestinal tract into the circula­tion and eventually to the site of viral replication. This pre­sumption precludes SP-303 acting as a general immune sti­mulant or as interferon. Both possibilites have been testedand excluded (unpublished data).

Toxicology

The toxicity of SP-3D3 has been investigated followingparenteral and oral administration in acute and multiple­dose studies in rats , mice, dogs and monkeys. The acute,single dose toxicity studies conducted for SP-3D3 are sum­marized in Table 10.

As noted in Table 9, no mortality was observed in any ofthe species following oral administration of SP-3D3 insingle doses up to 30D mg/kg. There were no meaningful

102 R. Ubillas et al.

Table 9. Acute, Single Dose Toxicity Studies of SP-303· .

Species

Mouse

Rat

Rat

Dog

Study LD.

MDR#402

MDR #401

MDR#406

ITR #126

Route Dose Range Number of Animals Results(mg/kg)

LV. 6.25-50.0 10/sex/dose LDso>50.0 mg/kg(x 4 doses SP-303)

LP. 10.0-100.0 10/sex/dose LDso>100.0 mg/kg(x 4 doses SP-303)

LV. 3.0-50.0 6/sex/dose LDso>50.0(x 4 doses SP-303) mglkg**

LP. 10.0-100.0 6/sex/dose LDso>lOO.O mg/kg(x 4 doses SP-303)

P.O. 30.0-300.0 6/sex/dose LDso>300.0 mg/kg(x 3 doses SP-303)

LV. 10.0-18.9 2 LDso>18.9 mg/kg

• In the mouse and rat studies, a single administration of SP-303 was followed by a 14-day observation period. In the dog study, dogswere observed for five days after drug administration.

** There were 5 of 12 (42%) animals that died in this study following intravenous administration. LDsois probably between SO and 100mglkg.

Table 10. Repeated-Dose Toxicit Studies: Oral Administration.

Species Study LD. Route Daily Dose Duratio Number of AnimalsRange n(mg/kg)

Mo use Shaman" LP. 2110/20 30 5 female/dose (x3 doses)P.O. (IP) days 5 females/dose (x3 doses)

10130/90(Po)

Rat Shaman" P.O. 0/100/300/ 5days 4/sex/dose (x 3 doses)a900

Rat MBR #04- P.O. 0/10/200/5 14 5/sex/dose (x 3 doses)07 00 days

Rat VIC #086 P.O. 0/50/200/5 30 12/sex/dose (x 3 doses)b00 days

Dog ITR #218 P.O. 100 5 days 2 males test1 male control

Monkey Pilot P.O. 100/300/90 5 days 2/dose (x3 doses)0

Monkey ITR #226 P.O. 0/10/50110 14 3/sex/dose (x 3 do5es)cITR #25 012001500 days

Monkey ITR #240 P.O. 0/301100/2 30 6/sex/dose (control, high00 days dose)

6/sex/dose (low, mid dose)

• Non-GLP studya 2/sex in controlsb Plus 5/sex in control and high dose groups for recoveryc 6/sex in controls

No ObservableEffect Level(NOEL)

N/A

N/A

500mg/kg/day

>200 and <500mg/kg/day

N/A

N/A

>100 and <200mg/kg/day

>30 and <100mglkg/day

changes seen in food consumption, body weights, clinical

signs or effects observed at necropsy.Following intravenous and intraperitoneal administra­

tion in mice, the lowest doses producing letha lity were 25mg/kg and 10 mg/kg respectively. In rats, mortality was ob­served only in the high dose group (50 mg/kg) following in­travenous administ ration, and no lethality was observed

following intraperitoneal administration at doses up to 100

mg/kg.In multiple dose studies, the toxicity of orally adminis­

tered SP-303 has been examined in mice, rats, dogs andmonkeys for treatment periods varying . from 5 to 30 days(Table 10). Five-day pilot studies followed by 14 and30-day GLP trials have been co nducted in bo th rats and

SP-303 103

Table 11. Repeated-DoseToxicity Studies: Intravenous Administration.

Species

Rat

Dog

Dog

Design

Study MDR #4030,5,20,35 mg/kg i.v. x 5 Days5/sex/dose

ITR #1240,5,20,35 mg/kg i.v x 5 Days1/sex/dose at 0, 5, 20 mg/kg2/sex/dose at 35a mglkg

2 dogs received 35 mg/kg boluswhich was changed to 30 mglkg in­fusion (over 15 min) due to rapidloss of consciousness after bolus

ITR #1610,2.5,5, 10 mg/kg i.v.( 15 min in­fusion) x 5 Days3/sex/dose at 0, 2.5, 5 mg/kg5/sex/dose at 10 mg/kg

One dog of each sex in high groupplaced into a 2- or 4-week recoveryperiod.

Findings

1. Mortality: none2. Clin signs: changes at the inj sites and adjacent tissues3. Chern: Mid/High-increased WBC, Segs, platelets; deer RBCs, Hb, Hct (not considered

biologically significant)4. Path: incr weight in liver (all), kidney (low/mid) (not considered biologically signifi­

cant)5. Histo: limited to the inj sites - hemorrhage, necrosis, inflammation, or thrombosis (not

considered biologically significant)6. NOEL: 5.0 mg/kg/day

1. Mortality: none2. Clin signs: following 35 mglkg bolus included ataxia, loss of consciousness, increased

salivation, bradycardia coughing and retching (recovered 30 min post dose); Sx notobserved in same animals following 30 mglkg infusion; deer weight & food con­sumption (high)

3. Chern: MidlHigh-increased alk phos, ALT, LDH and sorbitol dehydrogenase; throm­bocytopenia (max deer 81% - 89% to 24,000-51,000/1l1); prolonged aPTT & PTwithlow fibrinogen;

4. Path: (in MidlHigh) dark discoloration of mucosa in proximal sm intestine, enlargedmesenteric lymph nodes, pigment in the reticuloendothelial system, sinusoidal histio­cytosis, pigmented mononuclear cells in centrilobular veins, dilated lymphatics, cyto­plasmic vacuoles in splenic RE cells; hemorrhages in serosa of the gall bladder, endo­cardium, colon, urethra, urinary bladder and skeletal muscle; chronic lymphadenitis,enlarged vacuolated histiocytes in sinuses of mesenteric lymph nodes and thrombosisof the injection/infusion site

5. Histo: Rx-related changes observed in all animals; changes in low group were few andslight in severity

6. NOEL: 5.0 mg/kg/day

1. Mortality: none2. Clin signs: no significant findings3. Chern: Thrombocytopenia (high) - onset 15 minutes post- dose day 2 until study ter­

mination, max decr of 52%-87% (nadir 33,000-180,000/1l1); values returned to pre­treatment levels within 1 to 5 days of recovery

4. Histopath: dark discoloration of the proximal small intestine (all) with dose-relatedincrease in incidence; hepatic sinusoidal histiocytosis, cytoplasmic vacuoles in splenicsheathed capillaries, enlarged and vacuolated histiocytes in medulla of mesentericlymph nodes, focal thrombosis in lungs (high); pigmentation in hepatic sinusoidal cellsand in histiocytes of prox sm intest villi (mid/high); histiocytic foci in prox sm intestvilli, thrombosis at inj site (all); histologic changes in low-dose animals were slight ormild: in the recovery phase, changes reported in the reticuloendothelial system gene­rally either showed no improvement or, in some cases, were more evident

5. NOEL: 2.5 mg/kg/day

monkeys. In the 14-day trials in rats, groups of 5 male and5 female Sprague-Dawley rats were administered SP-3Q3by oral gavage at doses of 0,10,200, or 500 mg/kg/day for14 days. No treatment-related clinical signs were observedand no animals died during this study. There was no effectof SP-303 on any of the hematological, clotting or clinicalchemistry parameters monitored in this study. There wereno significant findings at gross necropsy, and no histo­pathologic changes were observed in the high-dose group.

Following this study, groups of 12 male and 12 femaleSprague-Dawley rats were administered SP-303 by gavageat doses of 0, 50, 200, or 500 mg/kg/day for 30 days. Fiveadditional rats of both sexes served as recovery animals and

were assigned to the high dose and control groups. At theend of the 3D-day dosing period, the recovery animals wereobserved for an additional 30 days before sacrifice. Onehigh-dose male was found dead on day 21 but the cause ofdeath was not determined.

Several high dose animals demonstrated rough haircoatduring the latter weeks of treatment but this was not appa­rent during the 3D-day recovery period. Sporadic reduc­tions in food intake possibly accompanied by marginaldecreases in body weight gains were seen at the high doselevel. This was not seen following cessation of treatment,except for an apparent spurious decrease in body weightgain for high dose females. Slight decreases in urinary spe-

104 R. Ubillas et al.

cific gravity were observed for high dose females and possi­bly high dose males at week 5. As this effect was slight andwas not seen at other times or accompanied by any otherindications of renal toxic ity, its biological significance is un­kno wn. No other urinalysis measurements, or hematologyor clinical chemistry parameters were altered by SP-303treatment .

A 14-day dose ranging study was conducted with SP-303administered orally to Rhesus monk eys. The study was car­ried out in 2 separate segments; the results of both segmentshave been combined for this summary. In the first segment ,3 monkeys of each sex were assigned to the control group(0 mg/kg), low-dose group (10 mg/kg/day), mid-dose level(200 rug/kg/day), and highdo se group (500 mg/kg/day). Inthe second segment, 3 monkey s of each sex were assignedto the control (0 mg/kg), low-dose gro up (50 mg/kg/day )and high-dose group (100 mg/kg/day). Except for the doselevels administered, the an imals in both segments weretreated essentially ident ically. There were no changes in he­matology, blood chemistry, clotti ng test, or IgG or IgE le­vels that were considered to be biologically meaningful.One female dosed with 500 mg/kg/day SP-303 had an in­creased eosinophil count on study day 15. Histopathologi­cal examination revealed eosinophilic infiltration in the he­matopoetic organs. The toxicological significance of thisfind ing is unknown. Pathological lesions possibly associa­ted with administration of SP303 were restr icted to animalsreceiving 200 or 500 mg/kg/day. They consisted of pigmen­ted histiocytes in the small and large intestines, a superfi­cial gastropathy, thymic lymphoid involution. In add ition ,diffuse pigmentation in hepatic sinusoidal cells was ob­served in the 500 mg/kg group. A marked decrease in meanthymic weights with thymic involution, was evident in ani­mals of both sexes in the 500 mg/kg group.

In the next study, groups of 6 male and 6 female Rhesusmonkeys were administered SP-303 by stomach tube at do­ses of 0, 30,100 , or 200 mg/kg/day for 30 days. At the endof the dosing period, 4 males and 4 females from each groupwere sacrificed and submitted to complete necro psies, while2 males and two 2 from the contro l and high-dose groupswere maintained for an additiona l 30-days recovery period .One low-dose male died from stra ngulation with its neckchain on day 29. There were no other death s during the stu­dy period. Body weights, water consumption, clinicalpath ology, ophthalmology and electrocardiography evalua­t ions did not show any evidence of treatment-related find­ings. The only clinical findings of note were decreased ap­petite in some animals in all groups and loose or liquidstoo ls, predominantly in the high-dose group. Histological­ly, an increased numb er of pigmented sinusoidal cells in theliver (high-dose group), greenish pigmented histiocytes in alllevels of the small intestin e, an increased pigmentation inthe histiocytes of the colon and cecum were noted (mid- andhigh-dose groups) were noted. In the recovery animals, allfindings had notably subsided or disappeared completely.

In addition to the oral studies, repeat dosing via the in­travenou s route has been conducted in rats and dogs, and issummarized in Table 11. As noted in the table, intravenousadministration of SP-303 was much better tolerated in ratsthan in dogs. Intravenous administra tion of SP-303 for 5days was associated with significant hematologi c toxicity(primarily consisting of thrombocytop enia) in dogs. In ad­dition, dogs exhibited a numb er of histopath ologic changesincluding pigmentation and vacuoliza tion of histiocytesthrough out the reticuloendothelial system. Administrationof a 35 mg/kg bolus injection to dogs precipitated an epi­sode of ataxia, bradycardia and loss of consciousness fromwhich the animals recovered after about 30 minutes.

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Address:

M.S. Tempesta, Shaman Pharm aceuticals, 213 East GrandAvenue, South San Francisco, CA 94080-4812, U.S.A.