VOL. 17

NO. 3

CHEMOTHERAPYIN VITROACTIVITY467

OF N',N'-ANHYDROBIS(B-HYDROXYETHYL)BIGUANIDE HYDROCHLORIDEAGAINST PARAINFLUENZA
VIRUS TYPE 3
OSAMU KITAMOTODepartmentandKIYOTAKETOBITAof Medicine (Director : Prof. OSAMU KITAMOTO), the Institute

Medical Science, the Universityof Tokyo, Minatoku,ofTokyo, Japan(Received May 12, 1968)

INTRODUCTION
N', N'-anhydrobis(8-hydroxyethyl)biguanidehydrochloride (ABOB) is an antiviral substancewhich has been used widely because of its prophylactic and therapeutic effect against influenza since
MELANDERand others first reported its anti-influenza
viral activity in 19601,2).
The Research Committee of Antiviral Substances
in Japan (chairman Prof. OSAMU KITAMOTO)
conducted double blind test of this drug in theepidemic of influenza A2 and 58,4), as well as insmall epidemic of adenovirus (type 3) infection5),
and in the latter KITAMOTOet al. found the effectiveness of ABOB against adenovirus infection for
the first time.
Later, KAJI et a16) showed that this drug has
inhibitory effect against adenovirus type 3 and 8
when tested in vitro.
There have also been many works concerning theefficacy of this agent against measles') or mumps8).
In the following experiment, we studied theantiviral activity of ABOB against parainfluenza
virus type 3 in cell culture system.
MATERIALS AND METHODSvirus
A newly isolated strain of parainfluenza virus type3(hemadsorption virus type 1) was used in the study.
This was passed in primary cynomolgus monkey
kidney cell several times, and the stock virus fluidwith infectivity titer of 107.5 TCID50/0.2ml andhemagglutination titer of 1 : 128 was obtained.
The stock virus was stored at —70°C until required.
cells
Primary cynomolgus monkey kidney cells (CMK)
were purchased through a commercial source andwere grown in the medium containing EARLE'S
basal saline solution, lactalbumin hydrolysate (0.5%), sodium bicarbonate (0.11%), inactivated bovine
serum (2%), penicillin (200 units/ml) and strepto-mycin (200 mcg/ml). When 30 X 104 cells per a tube
were seeded, confluent monolayers were usuallyformed in 5 days of incubation at 37°C.
HEp-2 cells were grown in EAGLE'S minimal
essential medium, supplemented with 10% inactivated horse serum and containing 20 mg % sodium
bicarbonate, 200 unitsiml of penicillin and 200 mcgf
ml of streptomycin.
Primary CMK were maintained with Medium 199
without serum, and HEp-2 cells with Medium 199
with 1% inactivatedhorse serum.
These media
contained 84 mg% sodium bicarbonate and appropriatequantity of antibiotics.
chemicals
Sterile 10 w/v% stock solution of ABOB was madeup in double distilled water and stored at 4°C until
required.
Toxicity of the agent against
the cells wasexamined in the following way.
ABOB was dissolved in various concentrations
in maintenancemedium and was administered to the monolayer cellsheet of primary CMK or HEp-2. The cell cultures
were incubated at 37°C for 7 days changing themedia every other day and the cytopathic changes
were observed microscopicallyat the end of theincubation period. The result is shown in Table 1.
Table 1. Toxicity of ABOB against HEp-2
and MK cell sheet (incubation period :7 days)
cellconc. of ABOB-----(mg/m1)

HEp-2

DDNNNN

1.0. 0
5.0
2.5
1.0
0.50
D= damaged,

N = not

damaged'MEDDDNNNCHEMO「468『HERAPY

APR.1969

PrimaryCMKseemedtobelnoresusceptibletothe

forthewholeoftheincubationperiodunlessagentthanHEp‑2ce1LTheconcentrationofABOB

otherwise加entio簸ed.

usedintheexperimentwaso.5mg/mlexceptinthe

Virusinoculationwasperformedasfollows.

isotopestudy,inwhichcaseitwas1、omglml.

MonolayercellsheetwaswashedwithPBS,and

ActinolnycinDwas己giftfromtheDepartm、ent

O.2mlofthevimsfluidwasinoculatedperatube,

ofAntibiotics,NationalInstituteofHealthofJapan.

Afteronehour'sadsorptionat37℃,thecellsheetFivemglmlofsolutionwaspreparedinpureethyl

waswashedwellwithPBStoremovetheunadsorb.

alcoholandstoredat‑20℃。

edvirus.Thecultureswereincubatedwithmainte.

withnance題ed呈umat37℃,changingthemediumevery

frem

otherday.

Tritiateduridine(3Hlabeledat5pesitien,specificactivityof5.oci/mM)waspurchased

isotopestudy

DaiichiChemicalsCo.,Ltd.

WhencompletemonolayerofHEp‑2cellwas

i㎡ectivitytitration
Infectivitytitrationwasperformedby

formed,withcellpopulationof25×104peratube,

meansof辱theculturedcellsweretreatedwithactinomycinD
he狙adsorptiontechniqueusingguineapigredblood￠ellsuspension,becausecytopathicchanges茎)roduced

2mcglm1,foronehourandtheRNAsynthesisof

byparainfluenzavirustype3ineither茎)rimarythehostcellwascompletelyinhibited.Insucha

血onkeykidneyorHEp‑2cellareinsuMcientfor

condition,parainfluenzaviruscanmultiplywithinquantitativeassayofinfectivity.

thecytoplasmofthehostcellwithoutbeingin‑
hibitedbythisagent9)。

Serialten‑folddilutionsofthetestviruswere

Thevirusatamuユtiplicityof200TCID50/cellwas

madeinthemaintenancemediumandO.2mlofeach
dilutionwasinoculationto2tubecellcuユtures。

theninoculatedtogetherwithactinomycin.After

Four丘fthofamilliliterofmaintenancemediu燃

one‑houradsorptiontime,thecultureswerewashedwasaddedandthecultureswerei難cubatedat37。C

withPBS,andmaintenancemed量umwhichcontained

for5dayschangingthe血ediumeveryotherday.

actinomycinDandtritiateduridineattheconcen。

Attheendoftheincubation,culturefluidswere

trationoflmcCilmlwasadded.Unlabeleduridine

femovedandthecellsheetwaswashedwithphos‑

atthecencentrationoflomcglmlwasadministeredatthesametime.

phatebufferedsalinesolution(PBS).Onemilliliter

Twotubecultureswereremovedimmediately,ofO.5%suspensionofwashed,citratedguineapig

3,6andghoursaftertheendoftheadsorptio皿

redbloodcellswereaddedtoeachtube.Tubeswereplacedsemihorizonta11yfGr30minutedat4℃,

timerespectivelyandthesampleswereprocessed

thecultureswerewashedwellwithPBSandthe

forcountingtrichloroaceticacid‑precipitableradio騨

testswerereadmiαoscopicallyat10×10magni‑'fi
catiens.Whenmαethan1!40ftheareashowed

activityusingliquidscinti11ationcounterandthe

hemadsorption,theculturewasjudgedaspositive。

treated,parainfiuenzavirusinoculatedHEp‑2cells

uptakeoftritiateduridinebytheactinomycin

wasestimated,hemagglutinationtest
HemagglutinationtestwasperformedusingO.5%
suspensionofguineapigredb1◎

RESULTS
AゆEffectofABOBen]productionofinfecti▼e◎dceUandatroom

temperature・

parain猛uenzavirUsinHEp…2cellculture
WhenO,5瓢9/mlABOBwasadministered24hours

Serialtwoイ01ddilutionsofthetest伽idwere
madeinPBSandO。4田Ioftheredbloodcellsus‑

priortovirusinoculati◎nandwaspreSentforthe
wholeoftheincubationperiod,virusyieldat48and96

pensionwereaddedtoequalvolumeofeachdilutien。
Hemagglutinationpattemwasreadafter75minutes、

hoursfollowingin◎culatiσn◎f103・oTCID50remained

andtheτeciprocalofthehighestdilutionthat

lo3・5andlo5・5TcID50/mlrespectively,which'infective

showedpositivehemagglutinationwastakenas

virustiteratthesepointsinthecontrolcultures

hemagglutinationtiter.

were105・sand106・7TCID50/mlrespectively,andat▼ir囎imc"lationanddrugadmini5t罫atio勤48hourstherewas1.810guniゼsreductioninthe

Administrationofthedrugtothecellsheetwas

productionof・infectivevirusand1.210gunit,sdonebychangingtheculturemediumtothe憩ainte・

reductionat96hoursbyO.5mg/mlofABOB(Fig。1).

nancemediumcontainingapPropriateconcentration

B。EぼectofABOBonfreehemagglutininofABOB.Thedrugwasusuallyadnユinistered24

pru血ctionbypar血hourspriortovirusinoculationandwaspresent

CMKcellculture

加enzavi皿sinprimary,

CHEMO†HERAPY

VOL.17NO・3

Fl9・LI酬勧e鰍469。fABOB。翻1「匡3・3。
工lthibfセ⊃ryeffecセ6fABOBo擁freehema一陣 撫 剛ir噛pe3m蒔P鰍1。漁 聯 一2ceUs

鮒1蜘 轟P雇 蜘晦卿1麟fl題e職

繍:'鵬 躍.麟離
edv塗 ㎡。的己吻theincttbati。

町

perie己含書 O冒 ξ隣oABOBロ
ABOBytesin'eee㎡

ー ロ
凸一・もfb厩H。Sdαysp・r.

日1

ξ モ虚に脚塾。聰

。s;nご犠も㎡沁為

Fluidsfrom,theprimaryCMKcellcultures
inoculatedwith103TCID500ftheviruswere

04696144h。睦rso臼

蹄cめafion

harvesteda.t48hours'intervalandtheirhe瓶ag‑
glutinintiterswereestimated.Theresultisshow蓋C.Effe￠tofABORontritiated.uridineuptake

jnFig.2.Ifvirus‑cellsystemwasincubatedwith

byHEp‑2cellstreatedwithac血omycinDandFiヒト2.Inhibiteryd冷ecそofABOBon・覇[ee馳 噸 臨 η 醐 蟹eをi。
吻
四mi湘一伽nz。 伽3{ype3inpri磁ry
加onkeykぎ 己翻eycells

藍no￠ulatedwithparain且ue麓zavirus
Tritiateduridineuptakebyvirus‑hostcellsystem
whenthecellwaspretreatedwith2mcg/mlof
actinomycinDincreasedgraduallyandreachedthe
peakat9hours(Fig.4。uppertracing).
Ifthecellsweretreatedwith1.Omg/mlofABOB

HAfiler

for24hoursbeforevirusinoculationandthen

128similarlyprocessedwiththecontro1,uptakeof
uridinewasmoderatelyreduced(Fig.4.10wer64tracing).

32

解6τ㎞ 脚 督・廊㎡赫憲1篠
驚e面Tdt・e;隣bR麗A。f幽 慌蹴 鴨 舳3噛ぞ。e肛s84002h。群s。fl繊
o.5mg/mlofABOBfor144hours,titeroffree

144』a士1。
渦

02004896

難 郵讐

030の=8鷹︒串歪4100

hemagglutininproducedwasaround1:16,while
controlshowedashighas1:128.

oInhibition・offreehemagglutininproductionwas

36S'
hours遵

noticedevenwhenthedrugwasadministeredat
thesametimewithoreven24hoursaftervirus

UridineuptakebynormalHEp‑2cellswasnever

Snoculation,iftheculturesystemswereincubatedinf正uencedbypre‑treatmentofthecellswith1.O

wviththe・drugforapPropriatelylongperied(Fig.3).

mg/mlofABOBfor24hours(Fig。5).

470

CHEMOTHERAPY

Fig.5. Effectof ABOB
oninitiateduridine
incorporation
into HEp-2 cells

8000

0 6000
g T-1
L'S 4000o—a no drug
ABOB
1.0Vint36hours

8DISCUSSIONABOB in the concentration of 0.5'mg/m1 inhibitedthe production
both of infective
virus and freehemagglutinin in vitro without damaging the hostcell, although the degree of inhibition is not sogreat.
Thus, there was 1 to 2 log unit's reduction
in infective virus production and 1/4 to 1/8 reduction
in free hemagglutinin
production.
When RNA synthesis of the host cell is completely
inhibited by actinomycin
D, uptake of tritiateduridine by virus-host cell complex will reflect thedegree of viral RNA synthesis within the cytoplasm
because actinomycin D does not interfere
withmultiplication of parainfluenza viruses. Our experiment showed that 1.0 mg/ml of ABOB 'reduced theviral RNA synthesis by around 30%.
These results might make it probable, on condition
that ABOB does not inactivate the virus outside
the cell nor does it inhibit the virus adsorption toand penetration
into the host cellm, that ABOB
primarily suppresses
the synthesis of viral RNA
within the host cell, and, as the result, there is areduction in infectivevirus
production or freehemagglutinin
production as was observed in ourexperiment.
The exact mode of action of this agent, however,will remain to be resolved.
SUMMARY
Inhibitory activity of N',N'-anhydrobis(0-hydroxyethyl) biguanide hydrochloride ' (ABOB) againstparainfluenza virus type 3 was reported.
When 0.5
mg/ml of ABOB was administered 24 hours prior touvirus inoculation and was present -for the whole ofthe incubatiori period, there was 1 to 2 log unit's
reduction in infective virus production in HEp-2
cells and 1/4 to 1/8 reduction in free ,hemagglutinin,
production in primary cynomolgus monkey kindney
cells.
ABOB in the concentrationof 1.0 mcgiml sup-20000APR. f969

pressed tritiated uridine incorporation into RNA toactinomycin D treated, parainfluenza
virus infecteck
HEp-2 cells by around 30%.
REFERENCES1) MELANDER,B.. N',N'-anhydrobis(-hydroxyethyl) biguanide HCl (ABOB) in prophylaxis.
and suppressionof experimental
influenza.
Antibiot. Chemother. 10 : 34, 1960
2) SJOBERG,E.: Experiments on prophylaxis ansuppression of epidemic influenza with N',N'anhydrobis ($-hydroxyethyl)biguanide hydrochloride(ABOB).
A double blind study..
Antibiot. Med. Clin. Ther. 7 : 97, 1960
3) KITAMOT000.: Studies on antiviral substance,
especially ABOB. Jap. Med. J. (Nihon IX
Shimpo) No. 2031 : 13, 19634) KITAMOTO, 0.: Studies on antiviralagent
against influenza. III rd International Congressof Chemotherapy. Vol. 1. p. 897, Georg Thieme,Stuttgart,
1965
5) KITAMOTO, 0.. Therapeuticeffects of ABU.;
on pharyngoconjunctival
fever.
Jap. Med. J.
(Nihon Iji Shimpo) No. 2211: 14, 1966
6) KAJI, M., KAMIYA, S., TATEWAKI, E., NAGA—
FUCHI, Z. & FUJIWARA, N.. Effect of N',N'anhydrobis (R-hydroxyethyl)biguanide hydrochloride (ABOB) against adenovirus.
Chemotherapy 14 . 66, 1966
7) HOPKINS, E. J., PYE, A. M., SOLOMON,M. &SOLOMON,S.: Controlled trial of "Virugon" intreatment of measles.
Brit. Med. J. 2 : 1263,1961
8) CURWEN,M.P., EMOND,R.T.D. & MCKENDRICK,
G. D. W.: Controlled
trial of A.B.O.B. inmeasles, chicken-pox and mumps. Brit. Med,J. 1 : 236, 1963
9) WHEELOCK,E.F.: Intracellular site of Newcas
tle disease virus nucleic acid synthesis. Proc.,
Soc. Exp. Biol. Med. 114 : 56, 1963
10) WAGNER, R. R.: Chemical and biological approaches to the therapy of viral diseases. Am.
Rev. Resp. Dis. 88 : 404, 1963