CA2384474A1 - Compositions and methods for protecting against sexually transmitted diseases - Google Patents

Abstract

The present invention provides compositions, formulations and methods for preventing sexually transmitted diseases, such as Neisseria gonorrhoeae, human papillomavirus, HSV-2 and HIV, based on a mixture of relative amounts of kappa and lambda carrageenans in. The present invention also provides a method of preparing sulfated polysaccharides such as the carrageenan mixtures in a from that facilitates formulation for pharmaceutical use.

Description

CBIt 3.8-Od7 CaMFO~x'Tr4NS ANb lulETtiaDS POR PROTECnON AO~'ST
Sl~Xt7ALLY TRANSM~D DxSEA.SE9 BACKf3Rt)UND OF ?I~ 1N'~ENTION
I o 0 0 i1 'I~e caaageonan idly of polymers ors palyaacaharidnc obtained $om the red algae commonly lmowa as seaweed. Caaagaenana have been uaad extensively in the food, pbarmaccutical and cosmetics industries as luhrica~sts, ernttlsifiexs, and crabilizirtg and dispersing agears. Extensive pharmacological and toxicological studies hare been conduatad, Cattageenan ltas bast found to be na~rt-tea by anal, deal, anti fnhalatioa tauter of administrations even at extremely high doses, The cartageenans wrra therefore elaaei~ed as ''ge~ally recommended as safe" (GRAS by the F1~A. in I97Z'. Furtber extensive anal pnacolanetic studies conducted in pigs, rats, twice, gerbils, guinea pigs, ferrets, bam:tezs, dvga, aid monkeys ~" showed that the breakdown of chc carrageenans in the gasC~iatestittal tract taste minimal at best and that absorption was virtually non-existent SUMMARY Ql? TldE SON
~ 0 0 0 21 Ot~ aspect of thr present iavtation is directed to s mia~fiu~es of kappa and lambda ~eoa~.ln~" ~ ulat3o:as aontai~ning thtc mixtures and nsethoda of use. 'f he iui.ctuxa of tiaras apeaifie carrain the n~Iative amounts provides uz~acpectodly superior atttimicrobial acti~iry compared to other carregeenans, ?he caxxageenan mixtures of ~
present invention xnay ba easily formulaxd for admiaixtration to a human or snitaah (preferably via the vaginal route) using sa aqueaua solution. In'a preferxtd embodiment,the mixture is present in tbt foo~lation in an amount of about 3°l°
~by ~veEght. The formulations ors particularly useful is protection agair~at contraction of sexually tra~nitted diaeases I o 0 0 3 7 Anotlxar aspect of the present invention is directed to a raetitod of processing, ze$tua~ or stabilixiaE a sulfatod- polysacel~ide such as ca~ageerlari, and more plef~bly the caarag~nan ~ixtura of the sent ir~~ention (herminafcer "a IrJ7t cawageenan miJ.-ture").
The ~sthod ails mixing the sulfated-polysaccharide in anhydrous yr powdery form with at least one dry buffer salt, followed by hydration of tl~e canrageonan e.g., by the aciditivn of orator or another aqueous solution. The meB~od overcomes several disadvantage associated with current techniques for processing catrugeeaszts from seaweed, and facilitates finer processing into ~oncaulations suibblc for phairaaceutical applications.
IaESCRIPTION OF THI; bfiA'WINGS
Iaooal Fig.t is a graph showing long term activity ofa ICI7~. carrngecnan mixture, Mica were cb~alleaged. with a 95-100°Y° iut~actaons dose of SSV-2 at various time intervals afEGr application oftha KI74 canagoenan niixtttte. Tba Kl7v. carrageanate rxtixtutc rr!ta;na some ravel of activity against HSV 2 avers a#ter 2~4 hopes. The data suggest that a woxne~a could be protected even if oo>osideeablc time elagscd bohxreen u.sa of the IGlA.
rarrageenaa mixture and CBitttB.
~ooo~l Pig 2 is a graph of Soutlurrn Blot byrbxidizstion of ItT pC~~uoducta from RNA
extracted from the spleens of rhice. Lang 2 s=td 3 are positive ceratrols.
x,anGS 4 to 8 are from mica that w~erc pretreated with a I~17~. carrag~an mixtctre S minutes befbxe viral challenge, Lanes 9 to 14~ are frog mica in4culatsd vaginally nvitir 1~1V.
DBTAtLBD DESCRXP1IDN OF I'FI8 fiTV~NT~DI~Ir 100101 ~azzageanaa is the structural compattsnt of;saweari and is extracted as three main types, ~aatnsty iota, kappa and lambda. xhe mixture corltaitLS about 83 to about 99%
lrratbda-carrageeaarr sad about 1 to about 15 °~ kappa-cazr&geanan, preferably ft~om aboac 1 to about 10°.~a, and mare preferably aboett 3°!e kappa Ca'trageexlan. In Othe7C pzefeired embodia, the ~mi~cture ig substantially or entirely ~ree of dextrose, an iagredieu:t oornenonty found in carra~roenaos used ins the food industry. 'the nnixatre o~
these specific carragaenans in the ~lativa amQUnts provide uacxlrectedly superior axt~mi~ro6ial activity compared to ocher catragcattans. Although carcageenan has ~t cQatinuutn of rrrolocuiar weights, tree mixtures of the pnesertt invemiort have an average molecular weight of about Z x 105 daltaets with lines than about 1% of molecules having an average moleon'tar weight of 2 x lOs daltot~ (as dctarinined by gel pertxteation chromatography and light scntterln~. Tints tenique physical propariy imparts nonabsorbability to the final formulation wltiah in 'rum provides prolonged aati~microbial aetiv~ity.
IoOZiI 'I'he oarragccnan mixtures of the present ~nrantion may br easily formulated fez vaginal (and even rectal) administration using an aqueov~s solution.
Vaginal ad~Oistratioa is the preferred mode of administration, In ~neral, tba amonat of the canragccnan znixmre wih vary from about 2 w about ~4 °~b by weight Tn a prcftrrad ranboditnent, the mixtxue is present in the formulation in set amount Qf about 3% by weight The fomduiatioa may iusther carttain other active te,g., vaginally administrable) actxv~e agtmts andlor ic~trt ints, dependi~zg upon the iarend~ed nat. The ~bsrnulatians are parDoulaxty useful is prateadon ag~air~st sexually txansxnattcd diseases a.g., by inhibiting infection by ~V, HPY, $5"il-2 and rJe~ssaria goxorrltaeae.

ZI The sanxatg~nan mixtuxo of the present irxvention remains stable if exposed to fiteezittg, ambient, ox boiling crmpaxatinvs, is compatible with the hua~m traginal enviroametxt, and is sat Systemically absorbed not degraded td any absoibablt by~ptoductg det~mental to htunans.
t00131 The kappa and laatbda c~z'ageenatts are commercially avaiailabla. They caa be taixad and fotnaulated inn aocardattce with standard techniques. A prefcrzed matbod for fomnulatfng the ~ cazrageenan mixtures of the present invention ivvolvas the uss of dry buffer salts. T'he mekhod oxtails mixing s Solid buffer salt and the IfJ7~
ca~geenan mixture, in a weight ratio of from about 1:1 ~ about 10:1, The mixture of solid buffer salt at~d the c~c~cagoeaan mixture is then satubilixed in watax or in, an aqueous soluti~, oo make the sulfatadgolysaccharide formulation, the p13 of dte sulfated pvlysacchaxide farmu~latioct is rhea adjusted to be fibm about 6.0 tp about $.0, This is typically achieved by t'he addition of en acid, such as FiCI or a base, such as NaOH. ha general, the viscority of the a~:Ifated polysaccharide founulutlon is from about 20,000 to about 100,000 ~S, prefbsably &oxn about 30,000 to about 35,000 CPS.
~oo~~tl Solid buffer salts itrcludc solid alkaline metal salh of acetic acid, citric acid and phosphoric acid, nrherei~a the solid alicalirte foetal phoapltatc buffer includes solid mixture of tri-basic attd dl basic al1'ali Salts of phosphate, preferably in aphydxous form, wherein alkaline metal includes, but is not limited to potassitun and sadiarn. Any physiologically acceptable buffet can ho used i0oi5! To enhance the stability of ttro sulfated polysacaharids formulation, at least one preservatirne can be added to the fatmutaticm. datives iztclude aTlc~r1 esters of para-hydrouybenzoie acid, such as methyl parao~drbenzoatey pxopyl pataoxybeazante, hydaratoin derivatives. parabens, such as ranthyl parahe~a, propioniate salts, triclosah tricarbapilide, tea use oil, alcohals, fstmtsol, ~tesol acetate, hexachlorophene and quaternary aanuonium Salts, streh as benzolconjure, zinc and aluminum salts, sodium benzoate, benxyl aloQkrol, bcnzal3tonzum ch~orlds and chlombntanol.
=00161 The preservative can be pxtsent iti the propofions indicated i:a the various pharmacopoeias, and is particular is a weight ratio to sulfated polysaccharide of from about 80:1 to about 1,0:1, p~a~rably from X0;1 to about 15:1.
too171 The mmthocl may also be u$ed to fomtulate orre~na other negatively c>~argad sulfated polysaccharides provided that thby exhibit anti~miembial propartios and arc nan-absorbable to vtro. Without #ending to be.bout~d by any paxaoular theoxY of operation, it is 'beticved that Sulfated-poiysacchasides such as oarrageenans including the iCl7~ carragocnan r~xturaa, azs dry povyders that arc cattcemely bY~'~~pio whatw eycposed tA the atmosphere.
The uptalct of atmosphexie xnaisture rata flue dry ittgrediem causes eluting of the aueterial.
The probtern compounded wl~au the maroexfal is then introduced into ~e aqueous base solution, such that complete incorpoxatiaa of the sulfated polyeaccltar~ds into the aqueous Solution cantrot be obtained. It is also believed that by mixing sulfated-pol~rsaccha~cide and at least one solid bni~r salt together, the solid buyer satt absorbs the atmospheric znoiatwe shat the sulfated-polysaccharide would have absotbcd when eatposed to the atmoaphgre, and prevents cluzctping of the sul~ted-polyascoharidt. It is rather believed that the process Sesves to increaso the solubility of sulfasad~olya*ccharida is water, arid acbiawes stabiliution of tbei p8.
La Q l81 The follawht$ axarrZples are inbetuied to fxu~Qter illustrabs cemin ~e~Onced embodzmtentss of the invention and are riot fnttndtd en limit the itttrention in any way.
Exempla I . P'RpDUCTxDN Op 50Q LIx'15RS ofthe KID. cantageenat~ mixture ~40i91 In preparing the 1Cla. caa~a~geenan mtxturt~, (1) the :formulation ingrc~dimts should be weighed individually in a clean, dry weighing vessel;: (2) the ingr~dierst's "aeraaf.
weight, oat protyocvl wdght; should be reeoxded in the rttanufactuilng production Ing regardless of even slight va~iatian haturcen the rivo; (3J ony bulls ingredient container containing an artifacts) or r~oata should not ha used at~d the container should be rinsed, sealed, marked "~ON~'AMINATED" and rcmorrrd fra~u production arca~ (4) in process prodnct<ori batch ahpuld not be txanafezred ~ro:a one vessel to another before maauficharing is completed end formuletiou has passed quality control testing;
and (3J
pmd~ac~tion vessel should remain closod during raanufactuair~ to avoid loss of avatar due to evaporation, especially dut'sng any steps that require heating.
ID 0 ~ O1 Additionally, aarrageenan has proven to be stable in tba solid.
state and the productiat~ state under a rraxiety of adverse canditians, including Erecting ox autoclaving, for 24 months.
300~~,1 In the courSa of preparing the xC/7,. coon mixture from 100 mL
labaraaory size bat~ehas on to scalo-up of x5 and 30 liter Iabaxatary batches to Snalizing the maanfacsuting procedure of 50Q litx batches, it bccxxna difficult so al~tam batxhto-batch coneisten:ry of the desired formulation. ?hr present method surprisingly overcutrc these dif~autties and produced fans of the ~ carragecttan mires having comds~nr batch to-batch guality, EQLT>pMENT:
i00Z~7 Producfiion Vessel - IKA, IrMA 9I500AI'lF'f'~" is a water jacket prodaction vessel that allows fot rapid heating and cooling of solution Boring production.
1~C#~'1'S:
10023 theK/A.CatfBgCCnenmixture;
Phosphate buffer ss;lirie (PB5) [ca~inung: NaCI - 1x0 mmollL, KCl -- 2.'~
mmollL, Phosp>:ata buffer (potstaaium phosphate monobasia aad sodium phosphate dibasic) - 10 mmolJY, - (Sigcua Aldrich, Saint Louis MO);
p-Hydroxybenzoie methyl ester (Methyl parabcn)- (N'ipa Laboratos~es, Poptypridd, L1K);
~yd~oric Acid (1d4~ - Merck, Dnaaatadt, (3crmany;
Purified rwator- Clean Chemical Sweddn, 8orlange, Sweden.
(1). Weigh ingredients iu the following qnatnitias:
ING 1. -..~-"'..~~.,.,QUAD.
' Purred watcQ (3 484.Q
Fates .

~e ~ ~~~ ~- l i.0 ka Pho atst i~uffer 4.8 ~
saline B9 o.s ~

drc~chloric acid .5 kg 109/r (2). Carefully end thoroughly mix the dry ingiodieats~ the KI~ catraagemttaa mLxture and Phospkuate buf9fer ale (pBS) togcthtr~
(3). Inspect production vasstI to ensure Q~t mixing chamber is clean, dry and flea of artif~, and that the botitnn tralue is closed;
(4). Fill the pcoductlan vessel v~rith 100,01:. (Part ~ of purified water at~d begin stircir~g:
turbizi 500 rpm, emd anchor 20 rpm. water is added in 3 parts, 'fhc first part is enough to dissol~ the mttlzyl pxteben. The secoad part aids irt reducing the temptratuce, sufficiently dilutes the HGl ao acidio hydrolyses of canrageenan does not occur ~ahila maintaining low enough Salutien levtl so when adding the caaageenan/F8S
mixture the dalivezy siove caps be lowered into the mixing vessel such that it does not come into aot~taet with the base solution and is lower than the rr~sal access hotels so ~e excessive 'dusthtg' of tht mi~-ture is sot lost. The third part completes the final Couaeutratxam, (5). Continua stirring :ad add 0.51cg of mothyl parabea attd 0.5 kg of HCI.
Clare vessel access hatch aa,d ixcat vvatx 75~ to 85°G. Once mmpemturo is reached continue stirring for a mi~rir~um of 10 minutos to dissolve rnettryl paraben:
(f). Discolttinue heating and add 250.0 kg (.part 1I] of purified water. Cool solution to 25°
to 30~G. The addition of the ovaber will expedite tlta cooling process. 1"he solution needs to be cooled so that it is not produciung steam whets the next sddiboa of ingredients is made, Besides prevtotinyecraber loss whop the vessel is Qpen for ~e next addition, steam causes the caaage~arnll~HS mixture to clump end stick to the sievd that is uaed in the addition;
(7). Open crease hatch and begin the addition of cur:agsovan. I PBS muxiure slowly through a sieve with gentle shakier. lAddition takes approxir~rely 20 mirnytes.
Cainaide the addition of the mixture with increasing the atlxrtng speed to s maati:mrm speed of turbo 1200 rpm end anchor z0 rpm. T'he viscoairy of't~ solution, increases expona~ia~y with the addition of cazragsan3n. If the stirring speed is not sigaiRcaat, the camgeenan forms 'hydra-sealed' clumps, which never become dissolved And iacorporatad into the solution, thereby tendering the bstdh ux~acceptsble.
('Plydro-sealed' clumps a:C pockets of day ra~ageenan, which ere surtou~ed with an outside casting of aeatf hydrated caarago~an, which becomes impenetrable to caster due to aarrageenan'a ext~mtly latge molecular weight and ffexi'hIe structure.);
(8). Close aace9s hatoh and continue stirring at maximuta~ spend, turbin 1200 rpm and anctaox ~0 rpzn. Add 13.0 kg ptuifled water (Part Ill) and disvannevt the waterlfae, close value. Teat aolutiou rrr ~5" to 80°C by applying 52~/ heat; and (9). G'Iteck that all the values are alased and apply the vacuum to the vessel at a00 mbar, Stir selution at slightly reduced speed, turbin 1100 rpm.and anchor ZO ipm, under vacuum fox 1.5 hr at 75° m 80~C. The constant stinting of the solution, which is necessary for even distn'bittion and complete ineoxpotatian of ingredients, causes txressive air entrapment 'rhe vacuum pulls dais air vut of the solution;
(10). Turn heating OFF. stirring OPF, and vacuum Ok~. liemave Testing Sample Pram ~psodt~tion vessel and tsst for Control Test #1 Complete incorporation and overt distribution;
Control Test #I: Complete ittcarpoiation and event distxt-butian b toaZ41 Remove approximately 90 ~L o~ the inrproress mixtw~e (use a Large orlfllce Z00 E~. pipette tfp to aid in removing the carrageenan solutioon) and min: is 10 NI. of a 0.1s/.
methyl blue ~'S (I:1, isopropyl alcohol: d~-12U) is a 500 ~.L Fppertdorfftube.
The mixture in the tube should nppeat as an even blue color. This indicates that,the 1GI~, carraEeenaa mixture is avartly distributed wxthia the solution. Prepare a mxcmacope elide v~nith a 10 uL, of this m3xt~ue; cover with a conrar slip and view under lrnv magni$catloa (10~. The iCl~.
carragoanatr miaoture atlouId appear as !ar$e purple atcauds. This indicates tI~ the IC/?~, carzagaaaan mixn~xa x8 complotely incorporated snd the solution is "PASS". If the strands are blue or large blue cluaaps era viseble, tftea the KI7v, carrageenan mixture is not campletcly incorporated and stilndoa is "FAIL", Continue processing the solution under the conditionQ
of step ~9. Itecjreck aalutiozr at O.S hour intervals until solution is "PASS".
(11 ). Whim the solution is "PASS" far Cool Tact #1, test for fontrol Test #2, pii;
Corttxol Test #2: ply Ioozsl Tlxe tasdng samgle should be cooled to ?SQCt 2 (a range o~23aG to Z?°C) tbr nesting. The pl:I should ba 7.0 t 0.1 (a range of b.9 to 7.1). This irzdicatcs that the solutloaf's ply is umfornt and the sohxtion is "PASS". If the soluko~a is not ar'sthin the accgptabls pH
range (b.9 to 7.1 ) the solution is "FAIL". If the solution is "FAIL", the solution needs to be adjusted, as needed with either 10°Io HCl (to decrease the p~ or 1N
NaOI:YT (to uxrease tlae pH) is 25 m~, iacr~~raeat$ until the solation is "PASS". With each iuerementsl addition o~
either acid or base, thorough atim~g (atirriag and vacuum condiriora step #9, ac added. heat) is seeded to amture even distdbutian throughout batch before re-testing the pFl.
kachacl:
solution altar stixringlvacuurn far 0.5 hoax. Continua in this manor until aaluaan it "'PASS".
(1~), Wren the sciatica is "PASS" for Control Test #~, begin cooling the msaesare to 25aC
t 2~ (f3oC to 2'ToC). The stirring sped, which should be OFF st dais point, will need to be increased as the solution thiclrent upon eooling_ 'At start; turbix~ C~ and anchor zQrpm, ina~se turban 20 xgm / 15 mm and increase anchor 10 rpm / 30 mm, ending twith fi~in 1000 rpm and anchor d0 xlrm, It is preferred not to increase stiirirtg to rapidly; athacwise, air entrapz<xent may result, rf this should bsppen, apply the vacuumv 400 mbar until adlutioa is free of sir babbles;
(13). Remove Final Teatiag Sa~uple from the production vessel and rarest for Control Test #2 pH an8 for Control Teat #3, 'Viscosity.
Control Tast i~3; Viscosity IO o~ 61 The testing aunpte should be heated to 35aC t2a (a rye of 33~ to 37°C).
Te optimize pcrformanee, the visa~ity chonld be about 34,000 bo about 44,440 CP. Viscosity meas~u:aa~eats indicate that the solution's viscosity is urtifatm with tf~e FC
Rafe~rance sample ind GCS pxoductiaat batches utd the solution is 'PASS". if the solution is "FAIL" obtain testing samples from tha top sad the bottom of pcoduation vessel and conduct Coaaol Test #Z, pH and ~annol Test #3, Viscosity on each sample. If the solution is still 'FAIL", ccpcac step #9 and step #12 end ictest the solution fox Co~rol 'Test 3#, Yiscnsity_ Tf solution is '~A1L" sa Out of Speoi~cadaas Study shall be undertaken to detcxmine tha source of our of sptcificatto~x production.
t d 0 ~ ~ ] It area di$covered that sdjustiu~g viscosity rovith t]te addition of water ykklS an usdcaovirn percentlctrat~on to the final production batch reoaeriag the production batch naacceptsble, ' (14). 'VVhe~t the solution is "'PA9S° for Goatral Tests #1, #2,and #3 it is an acceptable prpduetion' batch which cart be prxessed far the iiuas control testing. Comet the ccaasfex tube ca~ntainin~ a alter bad to the bottom value of the production vessel end transfer the formuiaxion into storage containers. Retain a ?est Sample fbr Micmbiological Tes#ng~ before filling applicators.
L o d ~ s~ The fuel fbanulation prepares. in the proaesa discecssed , above hes the following cor~npoacnt~
Weight/Peroent: 500 L~tars offarmulation oncnt ~ocnt pttri~ed Waccr 4 9ti.8 _0 k lvtatlr. $W 0.
I
ben pBS: -NaGl 120 zruool/I, ICCI ~_7 mmollL

llHt~e d S3ltt ~QI~L

10~C1 $

the 15 3.
cairra~eenan kg nnixtelre [0D2 ton bas a 9 p of about ] 7.0 whir Tho , waS adlua Vital by Addis fotaxula ~IG1 solution and 1:
t ratio of K~Pb, and xTa~HHi~tS~~.

ExampIt 2. F~'ea of Carrageenaa on ,Fftv Infections ih vitro It7o301 GactatgCCnus hoe been shown to block HfV'.and other enveloped virnses by savexal Iaboratorxes including the laboratory of the Fh"S. several different t~~pes of forget calls surd strains of FIIV have been araployed in these studies. Generally, 509~o b~iooldng is observed at a fevc~ micro~nsin~L This is sitru'lar to other sulfated poIysacclrarides such as and dextrixr snlfats.
'l~an~rle 3. Iatrs vaginal veal iafecdon studaes - HSV-2lMouse I0031I Besides being the first and the priz~capal in v~vo system to evaluate fin potential of candidate xni~crabicides in bloaki~ag viral infection, the HSY
2lsnouae (BslWC~
aysteut is widely ntili~d by most investigatory groups engaged is the dev~elopmrnt of a micxobioide. An important difference between the system established by Phillips m~° and other ~ysurns is the utt~lization of viral dose rarAge comparison. The staadaxd viral challenge dose, Z00°r6 infection dose ox 10' gfu, usod by others for cwaluation of a micmbicide is rate limiting. The large m~jorny of the microbieides under development, as well as marry of dse 4T'C spermicidea will sl~vv a si~nificattt rate of protection against USV 2 iofeotion at this viral ahallBnge doses. However, i~hillips has utilized a vii c~os~tetitratirnn mothod that wilt enable evahtatfan at viral clrallcnge doses of IQ°,10°, acrd 1,000 x 140%inf~4on dose.
Iao321 Using this viral cballenga doss System, a comparison study was conducted to evaluate the eorapaxiaoa protection rates of a number of rnVcrobicides under deveiopmtrnt, 4TC sptamiaides .aid lubricants, and possible forurulatiotrs fox ~tso as a placd~o in the clinical trials to cval>rate efficacy of a microbicide. In addition to the KI7v, cat5tagmixture, comparative test formulations wtna: micmbicidea under develo~rmant such es Buffe:Gel'~
and No Fam'1, OTC spermicides: K 'Y Flus° ~ytrol III, and. ~dvsntsge 5~, OTC yaaipal lubriea:r~: Rtplensa and Kr'Y' relly'~'; and possibly placebo foanulations:
.1.SR~6 Carbopol' and 2.5% methyl odlIuiose.
100331 Test formulations fell into three categozies with respect to officuoy izr pmroecting mice flrona vaginal HSV z inferevn. At the vinI ahallc~e dose of 10' pfti, with the exception of K-7~ JeIIy, Carbopol sad methyl cellutose, art Fannulatioa$
prawidad s significant tevet of piotec#an against in#hctioa from T~SV-2, Hovvevrt, at thB
~~iral challenge dose of 1 Q ; with tire cxceptioh of tt:e ~. caxrageenaa r~aixt~u~re, art formulations only pQOVided a minimum level of protection. Tire l~, carregeottatr mix4m twas~ the only folmulativn still affording a level of protactian against vial infection at tire viral challenge dose of l d' pfu ~°.

too3W Thtreftita, it can be concluded that the i~sV Zlmouse system csa bs etnployrd as a tneaus by rwbich candidate microbieides can be evaluated assd compared under the~aarnr reefing conditions to identify potential effective microbinides, fixsmple 4._ Duration of actirrity - HSV-/Mouse L 0 0 ~ 5 ~ Came of tea criteria set ~oith by Uh?,AIDS (World Health Orgaai~stioa, ADDS
branch) for an ideal micr4bicide states 'it arontld be activ~a upon insertion and for a long padod of tithe, gii, iag a woman male flexibility in product use. ~dditio~lly, tho time course for iafectioa by sell-flee or r"e11,-assxista3 ~'V' en occur may hat be immediate. Th: I:ISV-.
?,/atous8 system can be employed to evaluate the duration of time drat a microbicide ~ronld retain activity. This is done by iutra-vagin.'tl applioati4tt of a te$t founulation, waiting a set period of tithe, and then claalleaging mine with a lotown doss of vin~.a.
"Datation of activity"
tosting was conducted uaittg Oynol T.Te (a 2°r6 N-9 containing OTG
sgcrmicide), BuffetGtI° (a law pH mierobicida tuuler devclapment) and the alt, carrag~nnn mixtvro, at five minutes and 1.5, 3, and 5 hours follow formulation application. Hy the 1'lrhour time point, Gynoi A° no loatger a~'orded any pmtectiaxr against infection and Hu~fezGel' had dropped. td being only 30% ef$ctivc. &uffexGel's efEcacy continued to drop aver tune and ha longss afforded attar protection by ~6 hours. In rnsrictd coxatraat, the ~/a. carrageetian mioturo repnaitted 90-t0o~~o effecsive in protecting against HSV 2 infection up tp LS htar and in :ame cxpazimaats up to ~ hr3. The I:CfA. caaageanatt mL~ture continued to retain so3nc level of activity for up to 24 hours. Sre fxg. 1.
Example 5. iatta rectal viral infection studies - HSV-2JMouse too36~ Ideallyr, a atierabioide fat was effective in protecting against izifectiorl by filV could be used rectaily as well as vaginally. Using an ir~t~n- xec~tai viral challenge moditkatian of the HS'lt-lmouae systara an avalaation of the efficacy and safely of a mi4robioida was explored.
tooay~ Pre.-tre$tment of me rectum with the L~J~ catragcenan m3xhua significantly reduct8 the auaabec of anialals that became infected fdllovving rectal ahalleage with IiSV 2, cotttpared to pre~atmant with P89 or taethylcellulose {an inert placebo)".
~tatt~pla 6. Erect ofthe Itlh. carrageenan mixture on vaginal flora to ca el It is important that the use of a microbicide dons not disrupt the batance of the natural vaginal flara_ In vitro studies indicated that carrageenan, did not enhance or inhibit the grQwr6 rate of .Lacto6acil)us aeidophdlus. the mosx common bacteria present in the vaginal IO

ffoxa. A study canduotad fn 35 wamGn pazticipatittg in a Pha3e I cliuicsi trial tbr tho vaginat safety of the ICIJ~. carrageenaa mixture showed no significaa~t change in vag~aal flora, as aLeasurad by the presence at abssncc o~bactezial vaginvsie~' 8xampla 7. I~VIMonat viral tranapoxt systzrn 100391 ~4lthough mica oenttot ba infected with HTV, its has been ihavvn that v~
active at inattivatad vlNa is in~tilied unto t'6e vagina of mice, virus can be subsequently defeated is the lymph lodes by the use of reverse transccriptase polymexase chain reartian (RT-pCR}a fividonaa has been pxeaented rbac dandritia cells played a mie in the upr,~ka of virus end subsequent traagpork to the ly~ztpb nodes. 'l7ais c4nchrsian is in agreement mith studies impiicatiu$ detulZitic cells in the initial stage of saxuai transmission of HTV~.
i0o~01 Results indic~xe that the KIa. ceaageenan mixture is efifcacions in preveatlng HIV from reaahin,g the lymph node, preaumsbiy by blocking HIV transport from the ~ragina_ t O o ~i 11 HIV transport using a mouse system and Aldritl~ol~ 2 israotivatod virets were nsed_ Thris is a standard method for iaxctivating IiIV that dOCS nut alter the rriral enrrelnpe.
The spleen cad the lymph nodes ware assayed fox the deter#an of ~ in order to establish the spleen as as alternate r~osfto~ty xite for HIV. The splaen(as opposed to the lynrtph nodes) allows for obtaining ralativety iargc~r amounts of RNA for pcxfaami~ag 12T-PGR
far thr detectiaa of ItIIV_ Txr add'stivn, rxtracdon of spleens is lass time consuming t3ian xemaval of the Iyrnph modes thoteby lmasening the probability of RNA tlagrs~ation.
too~~1 To detesxnine the cfficaa}' of the ItI~, carrageenan mi~cairo in pxovonting HIV
trora crosain$ the cerv~icaiwaginaI battier, mice vrrermandomized into three groups: 1) aon-traatad PAS eontrnl mine; Z) mica pro-treated with methyl cellulose (inert placebo); and 3) mica pretxeatod wide tht Y!?~, carxagaanan mixture. Results are shown can the Southern Blot in Fig. 2 and the table below.
'rreauuent .~_Itotalosier a ge lvt~G~iy1 CelluIosa -tne ~a Y~o ~ppollatnb raxrageenan ' m a to04~1 Dsia from saline aaa methylcetiulose (control) treated and mice treated with the ~,/~. 03T12g~TJ3xl t111Xtt1Te. ZitC (IHIa ~h,OW ~tt tltC ~. ~gee~ ~
$1~1~f~, C~M~y reduces five number ofpositiva ~.e., irifectedj animals. Mathylccllulasa Karl no effect.
IOD44I 1"1t8 data indicates that the ~J7i. carrageenan mi~cturc is affeativc is prevesiting HxY frame leavistg the vaginal vault.
Example $. Cell ~'taf~cldng/Mottse System o g 5 7 It has pre~niously been suggested that sexual a~ansmission of HIV
could be mediated by ~Y-fnfoeted lymphocytes or macrophages in semen thst cross the genital tract epithelium"~~, In ordex to test the hypatha~is t'bat moraonuclaar blood calls ttaffio from the vsginal vault thron~"h intact epithelia, donble<-vitally-sGtained activated mononuclear blood cells (mouse) were placed in the vagina of mice. pour hours later, animals were sacrificed and tissues were p~rapa~tcd for examination by fluorescence microscopy.
Numesnus double-staiaad ceps ~rrare of~aarvtd is d1e con~neetiva tissue beneath the vaginal epithelium and is the iliac lymph nncios~. To evaluate the effect that the K/7~, carrageenan nai~ctura may have on blocking this procosS, at~ir~ala ~nvwra pre.oreatcd vwith the test formulation priox to instillation of labeled cello.
a t " a ~

aimala ned r.aIls i 4 FteC2e tll~9V~d IltaprCphBge 0 's control macro a 40f4 ~

~~

~
~
g cot with ~c~tm p t trit kacppallalmbda earn muctu~

lOoa61 Number of double stained cells is the iliac Iymgh nodes e~ mica.
Appauximately 1_S million cells were examined in each animal, Tbc data show thatthe K/7~
caaageanan mixture sigt~if carttIy mdaced the ,umber of Galls that ware observed in the -Iymph nodes.
>rxample 9 lvlicrobicide affect on papillousaviius ~ao~9] 'Ihe YGI7,, aan~agea~an mixture is also effective on blocking bovitle papillomavirus (BPV~ foal formation in vitro (data oat shown). The xC/~
carrageertan mixtvze is efficacious in preventing papillomavirus ifrorn trausfozmir~ human vaginal explants in a xenogxsph system, x'hc STAID mouse xmograph sysnm tmploys cxplants of human vaginal 1z tissue rolled into ayli~Cical tubes that are gxafted subcutaneously on Irli~DISCID
(i~mmuaodo~cient) mice. The grafts are aliawad to heal fbr two areelrs, at which time o~,e end of the tuba is opened and a tsar ac~ound it instilled followed by HPV
chslla'xge. In experimonLS evaluating the KIJI. csrtagrenan mixture, In 14 out of 14 saline treated coritral axptsusts were transformed. xn contrast, anly 3 Attt of 17 ratplat~ts 2raated veith the ICIa.
cairogc4nau miytuze was trat~sfbzmed (darts not shanvn).
All patent arts non patent pablicstidns cited in this apecif cataon are indicative of the level of skill of those skilled in the arc to which Ibis invention pertains. All these publications are hcrain iacorporat~ed by reference to the snn~e axteat as if each individual publication or patent application was speoifically at~d individually indieatad as being incorporated hexeia by refatance.
10097 ?hoe skilled in tha art will rcco~niza, or be able to ascertain, using na snore than routine expernneratatlon, antnaoua equivalents to tha specific 3ubstnaces and pmaedures described herein. Such equivaleixts are eot~sl,daned to be within the scrape of this i~a~~entian.

tc.~c~cr,~laa..1 I. Fo~ovd and Drug Adminigtratian. G12AS (Generally recognized as safe} tbad iagredients:
Caxrageet~an. IAA Publications PB~?.2I 206, (1970.
Z. $enitz, R.F,, Abraham. ri" Cxolberg, L. & Goulstost, F. ~C.auagaanan: un ulctro~caic agent. Tox~ftol. Appi. PJtarr~racol. 22, 282 (I972).
3. Fox, M.R.B. & Jaaobs, R.M. MetuI.Xotis in $iologiaal Systems., pp. 214-7'.48 (Maieel Ticker, Inc.,I9$~.
4.LuscQmbe, D.K. 8t Nichalls, P.J. Acute and subpcutC oral toxicity of AI~t 24388, a pnri~ed sodium Ii~sulphanate in rats.Fd Cos~d. TaxfcoZ lT, 229 237 (1973).
5. Naesa; H. T'b~c effect of mic~cabial and animal prQteinases on peptide= and protein Hgnoseilphonic acid complexes in agar gal. Actn VaG Scarad. I2, 592-6Da, x971.
Ref 'I~pe: Aba~act b. Sa:nman, S. 6c ltoberts, 13.C.K. The erect of xinc sulrplements on plaama zinc and copper lavels snd floe reported symptoms is ltealtliy volunteers, Jklc~l J.
A~rtralia rd6, ~4b-249 (1987.
7. L_S.~A. 8aatt~r Effects Assessmelox~ of for Zinc (and Cbmpounds). B3~.AJ540-i-96-048.
1984. Wa$hiutgtoa, D~.C., tJS EnvixanmentmJ Prdtc~ction ~.gency, dfFce o~Researoh at~d lhwalopment. Ref Typt: Data File 8. Walden, 1,T. & Derttth, 17. FDA Nevv Release 12155. ,FIaA PuSI'akiv~ns y2lSS, (1972).
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Tasic.26, I0~ 867-$80 (19$8j.
1 x. Phillips, D.'I~. & ~'au, X. Mechanism of trophobtast infection by HIV.~tX,D$~t~,s Xsetn Red ~ovirusts 9, ld9T-1705 (1992).
12. baba, M. er al. pentogan polysulfaate, a sulfated aligosaecharide, is a potent and seleetxve anti-HTV age»t in vitro. An~viral Res 9, 335-343 (1988).
13. $aba, M. Qt al. Mechanism o~ inbibitoty effect of dextran sulfate and heparin an replicaaan of bsunau iazm~.snodeficiency virus in ~~itro. Proc Narl Aran! Sri 85, b132-G 126 (1988).
y 14. Pe~rce-Prstt, R 8t Phillips, D.M. S~die: of adhesion of lyrmphaoytic acllg; Iatglicatioms lox se7u~a1 t~nission of httmaa imravunodeficicncy virus. .8iol lteprad 48, 431.1.5 (1993).
15. Pearve-Platt, 1~.. & Phollips, D.M. SnIfated palyaaccha~rides itrlnbit lymphoaytQto-epifhelisl traxrsmission of HIV=I. .~i01 Reprod .54,173-182 (~99~.
16. Maguire, R.;4,., Zxcharopoulo:, V.R, &c Phillips. p.M. Carragsenati N9 Spatmicides for Prevexntng Prcgnn~ecy and. Sexually ?ransmitted xn~ectiams. Sir Tra>asm DDS
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Sabatiec, d.M.
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.,

Claims (14)

1. ~A composition comprising about 85 to about 99% lambda-carrageenan and about 1 to about 15% kappa-carrageenan.

2. ~The composition of claim 1 comprising about 1 to about 1 to 10% kappa carrageenan.

3. ~The composition of claim 1 comprising about 5% kappa carrageenan.

4. ~An aqueous formulation comprising an anti-microbially effective amount of the composition of claim 1.

5. ~The formulation of claim 4 comprising about 2 to about 4% by weight of the comprising of claim 1.

6. ~The formulation of claim 4 comprising about 3% by weight of the composition of claim 1.

7. A method of preparing the composition of claim 1 comprising (a) adding a solid buffer salt to the mixture, and (a) dissolving the carregeenan mixture and the solid buffer salt in an aqueous solution.

8. A method of protzating against contraction of a sexually transmitted disease, comprising administering to a human the composition of claim 1.

9. ~The method of claim 8 wherein. the human is a female.

10. The method of claim 8 wherein the administration is vaginal.

11. The method of claim 8 wherein the sexually transmitted disease is HIV.

12. The method of claim 8 wherein the sexually transmitted disease is HSV-2.

13. The method of claim 8 wherein the sexually transmitted disease is HPV.

14. The method of claim 8 wherein the sexually transmitted disease is Neisseria gonorrhoeae.