antiviral res 1995

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E L S E V I E R Antiviral Research 28 (1995) 343-357

Q A n t i v i r a l

R e s e a r c h

Ant iv i ra l ac t iv i ty o f se lec ted acyc l ic nuc leos ideana logues aga ins t human he rpesv i rus 6

D . R e y m e n a,*, L . N a e s e n sa , j . Ba l z a r i n i a , A . H O 12~b ,

H . D v o [ ' fi k o v f i b, E . D e C le r c q aRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 1 O,

B-3000 Leuven, Belgiumb Institute o f Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague,

Czech Republic

Received 2 May 1995; revised 12 July 1995; accepted 12 July 1995

A b s t r a c t

Human herpesvirus 6 (HHV-6) was examined in vitro for its sensitivity to a broad range o fnucleoside analogues, including acyclovir (ACV), ganciclovir (GCV), penciclovir (PCV), buci-clovir (BCV), brivud in (BVDU), the NT-isomer o f 6-deoxyganciclovir ($2242), foscarnet (phos-phonoformic acid, PFA), and several acyclic nucleoside phosphonate (ANP) analogues such as(S)-HPMPA, (S)-HPMPC, PMEA and PMEDAP. Antiviral efficacy was monitored microscopi-cally by the inhibitory effect of the compounds on HHV-6-induced cytopathic effect in humanT-lymphoblastoid HSB-2 cells. In addition, a newly developed immunofluo resc ence /flo w cyto-metric assay (FACS) was used to determine HHV-6-specific antigen expression. A close correla-tion was observed between the antiviral data obtained by the microscopic assay and the flowcytometric assay. Marked antiviral efficacy was noted for $2242, PFA and the ANP analogues(S)-HPMPA, (S)-HPMPC, (S)-cHPMPC, (S)-3-deaza-HPMPA, (S)-3-deaza-cHPMPA, (S)-HPMPG and (R)-HPMPG. Also, PMEA and PMEDAP proved highly active against HHV-6infection, whereas (S)-FPMPA and (R)-PMPDAP were inactive. ACV was only slightly protec-

tive against HHV-6, and no activity was found for GCV, PCV, BCV and BVDU. Overall, theefficacy of the nucleoside analogues against HHV-6 appeared to correlate with their efficacyagainst human cytomegalovirus (HCMV).

* Corresponding author. Tel.: +32 (16) 332176; Fax : +32 (16) 337340; E-mail: [email protected].

0166-3542/95/$09.50 © 1995 Elsevier Science B.V. All rights reservedSSDI 0166-3542(95)00058-5



34 4 D . R e y m e n e t a l . / A n t i v i r a l R e s e a r c h 2 8 ( 1 9 9 5 ) 3 4 3 - 3 5 7

K e y w o r d s :Human herpesvirus 6 (HHV-6); N ucleoside analogues; A cyclic nucleoside phosphonate analogues

1 . I n t r o d u c t i o n

H u m an h e rp esv i ru s 6 , o n e o f t h e m o s t r ecen t ly d i sco v ered m em b ers o f th e h e rp esv i ri -dae , was in i t ia l ly i so la ted f rom per ipheral b loo d lymp hoc y tes o f pat ien ts wi th theacq u i red im m u n e d e f i c i en cy sy n d ro m e (A ID S ) o r w i th v a r io u s ly m p h o p ro l i f e ra t iv ed iseases (Josephs e t a l . , 1986 ; Salahudd in e t a l . , 1986) . Al though HHV-6 is morpho log i-ca l ly s im i l a r t o o th e r h u m an h e rp esv i ru ses an d m ay sh a re p a r ti a l h o m o lo g y w i th h u m ancy to m eg a lo v i ru s (H C M V ) (E fs t a th io u e t a l. , 1 9 8 8) , m o lecu la r an aly s is an d im m u n o lo g i -ca l d a t a r ev ea led th at H H V-6 i s c l ea r ly d i s tin c t f ro m E p s te in -B a r r v i ru s (E B V ) , H C MV ,herpes s im plex v i rus types 1 and 2 , and v ar ice l la-zoster v i rus (Josephs e t a l. , 1986 ;

Salahudd in e t a l. , 1986) . Tw o gene t ica l ly d is t inct var ian ts (A and B ) o f HH V-6 ha vebeen descr ibed . In teres t ing ly, most s t ra ins that have been iso la ted f rom immunosup-pressed pat ien ts (par t icu lar ly t ransp lan t pat ien ts) belong to the B sub type (Dewhurs t ,1994) . These f ind ings suggest that th is sub type may be o f h igher c l in ical impor tancethan the A sub type, a l though the re la t ive p revalenc e o f the two sub types and thei rv i ro log ical effects in heal thy and in pat ien t popu la t ions rem ain i ssues that need to befu r ther exp lo red (Yalc in e t a l. , 1994) .

H H V-6 h as b een im p l i ca t ed in th e p a th o g en es i s o f a n u m b e r o f d iseases . I t h as b eencausal ly re la ted to exan thema sub i tum (roseo la in fan tum) (Yamanish i e t a l . , 1988) ,h e te ro p h i l e an tig en -n eg a t iv e in fec t io u s m o n o n u c leo s i s an d p o ss ib ly K ik u ch i -F u j im o to ' sd isease (Krue ger and Sand er, 1989 ; Salahu dd in e t a l ., 1993). A lso , react ivat ion o f

la ten t ly in fected cel l s may con tr ibu te to the p rogress ion o f ch ron ic fa t igue syndrome,cer ta in au to - immune d iseases (e .g . , sys temic lupus ery thematosus and S j~gren ' s syn-d ro m e) an d v a r io u s ly m p h o p ro l i f e ra t iv e d i so rd er s ( e.g ., n o n -H o d g k in ' s l y m p h o m as)(Kr ueg er and Sander, 1989 ; Salahudd in e t a l. , 1993) .

H H V-6 h as a l so b een p ro p o sed to b e a co fac to r in th e p ro g ressio n o f A ID S , s in ce th ece l l tro p i sm o f H H V-6 i s s im i l a r to th a t o f h u m an im m u n o d ef i c i en cy v i ru s ty p e 1( H I V- 1 ). C o i n f e c ti o n o f H I V- l - i n f e c t e d l y m p h o c y t e s b y H H V - 6 h a s b e e n d e m o n s t r at e dto increase the cy topath ic effect o f HIV-1 (A gut e t a l. , 1989 ; Lu sso e t a l. , 1989). T hes t im u la t in g e f f ec t o f H H V-6 o n H IV-1 r ep l i ca t io n m ay b e b ased o n t r an sac t iv a t io n o fHIV-1 LT R by HH V-6 -speci f ic DN A frag me nts (Enso l i e t a l. , 1989 ; Horvat e t a l ., 1989 ;T h o m p so n e t a l ., 1 9 9 4; W an g e t a l. , 1 9 9 4) . A l so , H H V -6 in fec t io n o f ly m p h o cy t i c ce ll s

has been shown to upregu la te CD4 express ion , render ing the cel l s more suscep t ib le toHIV-1 in fect ion (Lusso e t a l. , 1991) . Ho we ver, the c l in ical re levance o f these in v i t rod a ta r em ain s to b e e lu c id a ted . T h e h ig h l ev e l s o f H H V-6 th a t h av e b een d e tec t ed inA ID S p a t i en t s , m ay b e r e l a t ed to an ac t iv a t in g ro l e o f H H V-6 in th e p ro g ress io n o fA ID S , o r m ay resu l t f ro m an o p p o r tu n i s t i c H H V-6 in fec t io n in th e im m u n o su p p ressedpatients (Sp ira et al . , 1990; Co rbel l ino et al . , 1993).

T h e p resen t s tu d y w as a im ed a t ev a lu a t in g th e in v i t ro sen s i t i v i ty o f H H V-6 to an u m b er o f an t iv i ra l n u c leo s id e an a lo g u es , k n o w n to b e e f f ec t iv e in th e t rea tm en t o fin fec t io n s w i th o th e r h e rp esv i ru ses o r H IV. We h av e a l so ev a lu a ted th e an t i -H H V-6



D . R e y m e n e t a l. / A n t i v i r a l R e s e a r c h 2 8 ( 1 9 9 5 ) 3 4 3 - 3 5 7 345

ac t iv i ty o f sev e ra l acy c l i c n u c leo s id e p h o sp h o n a te (A N P ) an a lo g u es . T h ese a re n o v e lnucleo t ide analogues in which an a lky l s ide-chain con ta in ing a phosphonate g roup , i sl inked to a pur ine o r pyr imid ine base . Th is c lass o f b road-spect rum an t iv i ra l agen ts

exh ib i t po ten t and se lect ive act iv i ty no t on ly agains t herpes- and re t rov i ruses , bu t a lsoagains t adeno- , pox- , hepadna- and pap i l lomaviruses (De Clercq e t a l . , 1987 ; De Clercq ,1991, 1993; B alzarini et al. , 1993; N aesen s et al ., 1994). The antiviral activ it ies foun dw i th th ese co m p o u n d s w ere co m p ared w i th th e i r ac t iv i t i e s ag a in s t o th e r h u m an h e r-p esv i ru ses , e sp ec ia l ly H C MV.

2 . M a t e r i a l s a n d m e t h o d s

2.1 . Cel ls

H S B -2 ce l ls (A m er ican Ty p e C u l tu re C o l lec t io n N o . C C L 1 2 0 .1 ) , an im m atu reT- ly m p h o b las to id ce l l l in e , e s t ab li sh ed in 1 9 66 f ro m p er ip h e ra l b lo o d ce l l s (P B L ) o f ap a t i en t w i th acu te ly m p h o b las t i c l eu k em ia (A d am s , 1 9 6 8 ) w ere p ro p ag a ted in R P MI1 6 4 0 su p p lem en ted w i th 1 0 % fe t a l ca l f se ru m , L -g lu tam in e (2 m M ) , so d iu m b ica rb o n a te(0 .075%) and gen tamycin . Cel l cu l tu res were incubated a t 37°C in a CO2-con tro l ledincubato r.

2 .2 . Vi rus

T h e p ro to ty p e G S s tr a in o f H H V -6 (S a lah u d d in e t a l. , 1 9 8 6) w as k in d ly p ro v id ed b yDr. D.V.M. Ablash i (Nat ional Cancer Ins t i tu te , Nat ional Ins t i tu te o f Heal th , Bethesda,MD ., USA). H igh- t i tered v i rus s tocks( 1 0 4 C C ID so /m l , a s ca l cu la t ed acco rd in g to th em eth o d o f R eed an d M i in ch ) w ere p rep a red in H S B -2 ce l ls . T h ere fo re , ap p ro x im ate ly15 X 1 0 6 ce l ls w ere p e l l e t ed , i n fec t ed w i th H H V-6 in a 1 -m l v o lu m e an d in cu b a ted fo r1 -2 h a t 3 7 °C . T h e n , t h e in fec t ed ce l ls w ere r e su sp en d ed in m ed iu m a t a co n cen t ra t io nof 0 .75 )<1 0 6 ce l l s /m l . C e l l cu l tu res w ere su b cu l tiv a t ed ev e ry th ree to fo u r d ay s b ytw o - to th ree - fo ld d ilu t io n w i th f r e sh m ed iu m . Vi ra l g ro w th w as ex a m in e d m ic ro sco p i -ca l ly b y th e ap p ea ran ce o f cy to p a th ic e f f ec t (C P E ) an d a l so m o n i to red fo r an t ig enex p ress io n b y im m u n o f lu o re scen ce / f lo w cy to m et r i c an a ly si s (FA C S an a ly si s ; as d e -sc r ib ed b e lo w ) . Wh en C P E w as a t i t s m ax im u m ( i . e . , a t s ev en to t en d ay s a f t e rin fec tio n ) , fe t a l ca l f se ru m w as ad d ed to a f in a l co n cen t ra t io n o f 2 0 % . T h e in fec t ed ce ll sw ere th en th o ro u g h ly re su sp en d ed , d iv id ed in a l iqu o t s an d s to red a t -7 0 ° C .

2 . 3 . Co mp o u n d s

P h o sp h o n o fo rm ic ac id ( fo sca rn e t , P FA ) an d (R ) -9 - (3 ,4 -d ih y d ro x y b u ty l )g u an in e(bucic lov i r, B CV ) were ob ta ined f rom Astra L~ikemedel (SiSdert~i lje, Swe den) , 9 - (2 -hy-d r o x y e t h o x y m e t h y l ) g u a n i n e ( a c y c lo v i r, A C V ) w a s p u r c h as e d f r o m W e l l c o m e L a b o r a to -r i e s (R esea rch Tr i an g le P a rk , N C , U S A ) an d 9 - (1 ,3 -d ih y d ro x y -2 -p ro p o x y m eth y l )g u an in e(g an c ic lo v i r, G C V ) w as f ro m S y n tex (P a lo A l to, C A , U S A ) . 9 - [4 -h y d ro x y -3 - (h y d ro x y -



3 4 6 D. Reymen et al./Anticiral Research 28 (1995) 343-357

methyl)butyl]guanine (penciclovir, PCV) was obtained from Hoechst AG (Frankfurt amMain, Germany). (1R-l c , 2/3, 3o~)-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine[( - )BHCG] and 1-(2-deoxy-2-fluoro-/3-D-arabinofuranosyl)-5-iodocytosine (FIAC) were

obtained from Bristol-Myers Squibb (Wallingford, CT, USA). 2-Amino-7-[(1,3-dihy-droxy-2-propoxy)methyl]purine ($2242) was kindly provided by Dr. G. J~ihne (HoechstAG).

(E)-5-(2-Bromovinyl)-l-(/3-D-deoxyribofuranos-l-yl)uracil (brivudin, BVDU) and5-iodo-l-(/3-D-2-deoxyribofuranos-l-yl)uracil (idoxuridine, IDU) were synthesized byDr. P. Herdewijn (Rega Institute for Medical Research, Leuven, Belgium). l - / 3 - D -

Arabinofuranosyl (E)-5-(2-bromovinyl)uracil (sorivudine, BVaraU) was from YamasaShoyu (Choshi, Japan). The acyclic nucleoside phosphonates evaluated were: 9-(2-phos-phonylmethoxyethyl)adenine (PMEA) and its bis(pivaloyloxymethyl)-ester [bis(POM)-PMEA], 9-2(-phosphonylmethoxyethyl)guanine (PMEG), 8-aza-PMEG, 9-(2-phos-phonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), (S)-9-(3-hydroxy-2-phosphonyl-

methoxypropyl)adenine [(S)HPMPA], (S)-3-deaza-HPMPA, (S)-cyclic-HPMPA [(S)-cHMPA], [(S)-3-deaza-cyclic-HPMPA [(S)-3-deaza-cHPMPA], (S)-7-deaza-HPMPA[(S)-7-deaza-HPMPA], (S)-I -(3-hydroxy-2-phosphonylmethoxypropyl)c ytosine[cidofovir, (S)-HPMPC], (S)-cyclic-HPMPC [(S)-cHPMPC], (S)-9-(3-hydroxy-2-phos-phonylmethoxypropyl)guanine [(S)-HPMPG], (R)-9-(3-hydroxy-2-phosphonylmeth-oxypropyl)guanine [(R)-HPMG], (S)-cyclic-HPMPG [(S)-cHPMPG], (R)-cycl ic-HPMPG [(R)-cHPMPG], (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-di-aminopurine [(S)-HPMPDAP], (S)-8-aza-HPMPDAP, (R)-9-(2-phosphonylmeth-oxypropyl)-2,6 diaminopurine [(R)-PMPDAP] and (S)-9-(3-fluoro-2-phosphonylmeth-oxypropyl)adenine [(S)-FPMPA]. All the acyclic nucleoside phosphonate analogueswere synthesized by Dr. A. Hol~ and Dr. H. Dvo~kov~ (Academy of Sciences of the

Czech Republic, Prague, Czech Republic) according to the published procedures (Hol~et al., 1989; Hol~, 1993; Starrett et al., 1992; Dvo~kov~ et al., 1993; Dvo~kovfi andHol~, 1993; Jindfich et al., 1993; Balzarini et al., 1993), except for PMEA, bis(POM)-PMEA, (S)-HPMPC and (S)-cHPMPC which were kindly provided by Gilead Sciences(Foster City, CA, USA).

2 . 4 . A n t i - H H V- 6 a s s a y

The inhibitory effect of the compounds on HHV-6 replication in HSB-2 cells wasmonitored by the inhibition of virus-induced cytopathogenicity (CPE), which was basedon the appearance of large refractile cells in HSB-2 cells. Therefore, HSB-2 cells were

infected with HHV-6 at a multiplicity of infection (MOI) of 0.001. Immediately afterinfection, 100 /~1 of the infected cell suspension was transferred to the wells of aflat-bottomed microtiter plate containing 100 /xl of various concentrations o f the testcompounds. At day 3 and day 7, cell cultures were subcultivated by two-fold dilutionwith medium containing fresh compound. Microscopical evaluation of viral CPE wasperformed on day 10. Antiviral activity of the test compounds was expressed as ECs0,i.e., the concentration required to inhibit HHV-6-induced cytopathogenicity (CPE) inHSB-2 cells by 50%. The selectivity index was defined as the ratio of the cytostatic(CC50) to the antivirally effective (ECs0) concentration (FACS results).



D, Reymen et al./Antiviral Research 28 (1995) 343-357 347

2 . 5 . F l o w c y t o me t r i c a s s a y

A fte r m ic ro sco p ica l ev a lu a t io n , ce l l cu l tu res w ere fu r th e r an a ly zed b y an in d i r ec t

i m m u n o f l u o r e s c e n c e / f l o w c y t o m e t r i c a s s a y ( FA C S a n a l y s i s ) . T h e c e l l s w e r e c e n -t r ifu g ed , w ash ed tw ice w i th p h o sp h a te -b u ffe red sa lin e , an d a h ig h - t i t e red an t i -H H V -6p o ly c lo n a l an t i se ru m (k in d g i f t f ro m D r. G .R .F. K ru eg er, U n iv e r s i ty o f C o lo g n e ,G e rm a n y ) w as ad d ed . A f t e r 5 0 r a in in cu b a t io n a t 3 7°C , the ce l ls w ere w ash ed tw ice w i thP B S . T h en , t h e ce l l s w ere in cu b a ted w i th f lu o resce in i so th io cy an a te co n ju g a ted F (ab ' ) 2-f r ag m en t s o f rab b i t an t i -h u m an im m u n o g lo b u l in an t ib o d y [R aH -Ig -F (ab ' )2 -F IT C ] (P ro -san , Ghen t , Belg ium) fo r 50 min a t 37°C, washed once wi th PBS and f ixed in 0 .37%p ara fo rm ald eh y d e in P B S . T h e sam p les w ere th en an a ly zed w i th a f lu o rescen ce -ac t iv a t edce l l so rt e r (FA C S ta r, B ec to n D ick in so n , Mo u n ta in View, C A , U S A ) eq u ip p ed w i th ana rg o n - io n l a se r (S p ec t r ap h y s ics Mo d e l 1 6 4 ) a t 4 8 8 n m an d 2 5 0 m W l ig h t o u tp u t . T h eg reen f lu o rescen ce w as f i l t e red th ro u g h a 5 3 0 /3 0 b an d p ass ad so rp tio n f il te r. F o r each

sam p le , 5 0 0 0 to 1 0 ,0 0 0 ce ll s w ere an a ly zed b y a C O N S O RT 3 0 sy s tem (B ec to nDick inson) . The th resho ld o f posi t iv i ty fo r the g reen f luorescence was arb i t rar i ly se t ,based on the un in fected con tro l samples . Al l the data were expressed in a logf lu o rescen ce h i s to g ram fo rm ; a th ree -d im en s io n a l d i sp lay o f t he h i s to g ram s w as o b -t a in ed b y th e LY S Y S so f tw are p ro g ram (B ec to n D ick in so n ) . T h e E C s0 , t h a t i s t h eco n cen t ra t io n r eq u i red to su p p ress H H V-6 -sp ec i f i ed an t ig en ex p ress io n b y 5 0 % , w asca lcu la t ed f ro m th e co n ce n t ra t io n -d ep en d en t in h ib it io n cu rv es .

2 .6 . Cy tos ta t i c a s say

HSB-2 cel l s were t ransferred to the wel ls o f a f la t -bo t tomed micro t i ter p la te and

cu l tu red in th e p resen ce o f d i f f e ren t co n cen t ra t io n s o f t he t e s t co m p o u n d s . T h e cu l tu resw ere su b cu l t iv a t ed as m en t io n ed ab o v e . O n d a y 7 , t h e ce ll s w ere co u n ted w i th a C o u l t e rC o u n te r. C y to s t a t i c e f f ec t s o f t h e co m p o u n d s w ere ex p ressed as C C 5 o , o r co n cen t ra t io nreq u i red to in h ib it ce l l g ro w th b y 5 0 % .

3 . R e s u l t s

T h e an t i -H H V -6 ac t iv i ty o f d i f f e ren t n u c leo s id e an a lo g u es w as in v es t ig a t ed inH H V-6 - in fec ted H S B -2 T - ly m p h o b las to id ce l ls . A s sh o w n in Fig . 1 , i n fec t io n o f th ece l ls r e su l t ed in a c l ea r H H V-6 - in d u ced cy to p a th ic e f f ec t (C P E ) , i .e . , r e f rac t i l e b a llo o n -

in g ce l ls (p an e l B ) , a s c o m p ared to m o ck - in fec ted ce l ls (p an e l A ) . S u p p ress io n o f v i ra lC P E b y ( S ) - H P M P C a t a c on c e n t ra t io n o f 4 / z g / m l ( 1 4 / x M ) , is v is u a li se d in p an e l C .P o ten t ia l t o x ic i ty o f t h e co m p o u n d s w as a l so sco red m ic ro sco p ica l ly [p an el D ; (S ) -H P MP C a t a co n cen t ra tio n o f 1 0 0 /x g / m l (3 6 0 / zM)] . T h i s to x ic ity co u ld b e easi lyd i s t in g u i sh ed f ro m th e cy to p a th ic e f f ec t o f t h e v i ru s ( co m p are p an e l B w i th D ) . T h i sen ab led accu ra t e d e te rm in a t io n o f th e an t iv i ra l e f f i c i en cy o f th e t e s t co m p o u n d s , r e l a tiv eto thei r cy tos ta t ic effect .

In ad d i t io n , t h e e f f i cacy o f th e t e s t co m p o u n d s ag a in s t H H V -6 w as d e te rm in ed f ro mth e i r i n h ib i to ry e f f ec t o n H H V-6 - in d u ced an t ig en ex p ress io n in H H V-6 - in fec ted H S B -2



348 D, R e ym e n e t a l. / A n t iv i ra l R e s e a rc h 28 (1995) 343 -35 7

cells, as measured by a newly developed, highly sensitive indirect immunofluorescence/flow cytofluorographic method (FACS analysis). This technique specifically distin-guishes between the uninfected and the infected cell population and allows a reliable

determination of the percentage of HHV-6-infected cells in the whole cell population.With this method, concen tratio n-depe ndent inhibitio n curves were obtained, as exempli-fied for PFA, PMEDAP, (S)-HPMPC and acyclovir (ACV) in Fig. 2.

Fig. 1. Inhibitory effect of (S)-HPMPC against HHV-6-induced cytopathic effect (CPE) in HHV-6-infectedhuman T-lymphoblastoid HSB-2 cells. Panel A: mock-infected cells; Panel B: HHV-6-infected HSB-2 cells atday 7 post infection; panel C: HHV-6-infected cells treated with (S)-HPMPC at a concentration of 4/zg/ml;Panel D: mock-infected cells treated with (S)-HPMPC at a concentration of 100 tzg/ml.



D . R e y m e n e t al . / A n t i v i r a l R e s e a r c h 2 8 ( 1 9 9 5 ) 3 4 3 - 3 5 7 349

Fig. 1 (continued).

P FA w as a p o ten t in h ib i to r o f H H V-6 rep l i ca tio n w i th an E C s0 o f 5 .8 /x M, asm e a s u r e d b y t h e i n d i re c t i m m u n o f l u o r e s c e n c e / f l o w c y t o m e t r i c a s sa y ( Ta b l e 1; FA C S ).T h e se l ec t iv i ty in d ex o f P FA , o r r a t io o f cy to s t a t i c o v e r an t iv i r al ly e f f ec t iv e co n cen t ra -t ion , was 141. The se data are in ag reem ent wi th those pub l ished e lsew here (Agut e t a l .,1989a, 1991 ; S t re icher e t a l. , 1988) . Th erefo re , PFA was used as a referen ce com pou ndth ro u g h o u t th e su b seq u en t an t iv i ra l ac t iv i ty an d cy to to x ic i ty ex p er im en t s .

A cy c lo v i r (A C V ) h ad a r a th e r p o o r p ro tec t iv e e f f ec t w i th an E C s0 o f 2 7 0 /x M an d ase l ec t iv i ty in d ex o f o n ly 3 . H o w ev er, co m p o u n d s th a t a re s t ru c tu ra l ly r e l a t ed to A C V,n am e ly, g an c ic lo v i r (G C V ) , b u c ic lo v i r (B C V ) an d p e n c ic lo v i r (P C V ) d i sp lay ed n o



350 D. Reymen et al./Antiviral Research 28 (1995) 343-357

P F A

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CC

PMEDAP

I o . s

i 2.5

- - 5 12.5

- 6 2 . 5

c c

OCWcO

Z

WOuJ

Wrr

j ~ ( ~ - H PM PC

0 . 5

~ 5

1~ 5

VC

C C

A CV

80

4OO

VC

CC

LOG GREEN FLUORESCENCE

F i g . 2, I n h i b it o r y e ff e c t o f P E A , P M E D A P, ( S ) - H P M P C a n d a c y c l o v i r ( A C V ) a g a i n s t H H V - 6 - s p e c i fi c a n ti g e ne x p r e s s i o n in H H V- 6 - i n f e c t e d H S B - 2 c e l ls , a s d e te r m i n e d b y i n d i re c t i m m u n o f l u o r e s c e n c e / f l o w c y to m e t r i ca na lys i s (FAC S ) . C C = m oc k- in fe c te d c e ll c on t ro l ; VC = v i rus - in fe c te d c e l l c on t ro l .

an t i -HHV-6 ac t iv it y a t su b to x ic co n cen t r a ti o n s (Tab le 1 ). No r d id F IAC, BV DU an d i tsc o n g e n e rs B Va r a U a n d I D U . I n c o n tr a st ( - ) B H C G d i sp l a y ed m o d e r a te a n t i- H H V- 6act iv i ty wi th and ECso of 8 .4 /x M and a se lect iv i ty index of 4 . Marked an t iv i ra l ac t iv i tywas a l so n o t ed fo r t h e N7 - i so mer o f 6 -d eo x y -g an c i c lo v i r $ 2 2 4 2 (ECso: 0 .0 2 /x M,

select iv i ty index : 42) , w hich i s in agreem ent wi th the data repor ted by N eyts e t a l.(1994) .Sev era l acy c l i c n u c l eo s id e p h o sp h o n a tes d i sp l ay ed a mark ed in h ib i to ry e f f ec t o n

HHV-6 rep l icat ion . The h ighest an t iv i ra l ac t iv i ty was no ted wi th the HPMP der ivat ives ,thei r ECs0 values being (in increas ing order) : 1 .2 tzM [(S)-3 -deaza -HPM PA ], 3 .7 / xM[(S ) -3 -d eaza-cHPM PA] , 5 .6 / zM [ (S ) -cHPM PC] , 5 .6 / x M [ (S ) -HPM PG] , 6 .2 / x M[(R) -HPMPG] , 1 0 /x M [ (S ) -cHPMPG] an d 11 /x M [ (S ) -HPMPC] . Fo r t h ese co mp o u n d s ,the se lect iv i ty index was 10 -30 . A s t rong an t i -HH V-6 act iv i ty was a l so observ ed for thePM E d er iv a t iv es PM EA an d it s b i s (POM) es t e r p ro d ru g b is (POM )-PME A, P M EG an d



D. Reymen et al. / Antir, iral Research 28 (1995) 34 3-35 7

Table 1Antiviral efficacy of selected nucleoside analogues in HHV-6 infected HSB-2 cells

351

Compo und ~ ECso t' (/ xM) based on CCs~ ~ (/ tM ) Selectivity index d

CPE FACSPFA 8.4_+4.8 5.8_+2.3 818 _+316 141$2242 0.03 _+ 0.02 0.02 _+ 0.003 0.85 _+ 0.22 42PMEDAP 11 _+4.1 9.1 _+6.1 164 _+23 18PMEA 30 _+ 14.3 28 _+22.1 175 _+38 6bis -(POM)-PME A 0.56 _+ 0.08 0.53 _+ 0.1 1.9 _+ 1.4 4PMEG 1.7 ± 0.4 1.5 _+ 0.9 1.6 _+ 0.2 18-aza- PMEG > 25 > 25 45 _+ 1.6 -(S )- HP MPA 10 _+ 3.6 8.0 _+ 3.0 46 _+ 9 6(S)- cHPMPA 42 _+ 1.3 32 _+ 14.8 146 _+47 5(S)- 3-de aza -HPMPA 1.4 + 0.25 1.2 _+ 0.3 36 -+ 0.5 30(S)-3-deaza-cHPMPA 3.6 _+ 1.3 3,7 _+ 2.7 45 _+ 30 12(S) -7-d eaz a-HPMPA > 10 > 10 12 _+2 -

(S)-HPMPG 4.9 _+ 0.9 5.6 _+ 0.5 122 22(R) -HPMPG 5.6 5:0.01 6.2 -+ 0.2 87 14(S)-cHPMPG 6.9 -+ 1.3 10 -+ 4.1 153 -+ 32 15(R)- c-HP MPG > 25 > 25 57 + 40 -(S)- HPMPDAP 26 -+ 3.1 27 -+ 8.5 404 _+ 147 15(S)-8 -aza- HPMPDAP > 300 > 300 > 300 -(S)-HPMPC 14 _+6.6 11 _+10.6 141 _+16 13(S)-c-HPMPC 6.3 + 3.0 5.6 _+ 5.5 109 _+ 86 20(S) -FPMPA > 300 > 300 > 300 -(R)- PMPDA P > 300 > 300 > 300 -ACV 179 _+52 270 _+70 861 _+73 3GCV > 25 > 25 54 _+ 2.0 -BCV > 200 > 200 277 _+53 -

PCV > 400 > 400 ND -( - ) B H C G 7.4_+0.0 8.4_+0.4 37 _+8.0 4FIAC > 1 > 1 2.4_+2.7 -BVDU > 1 > 1 1.2_+ 0.8 -IDU > 1500 > 1500 > 1500 -BVaraU > 75 > 75 > 75 -

ND = not determined.For abbreviations: see section 2.3.

b Antiviral activity was expresse d as ECso, or com pound concen tration required to exert a 50% inhibition ofHHV-6-induced cytopathic effect (CPE), or a 50% inhibition of HHV-6-specific antigen expression, asdetermined by immu nofluo resce nce/f low cytometric analysis (FACS).c Cytostatic effect was expressed as CC5o, or compound concentration that reduced cell growth by 50%.d Selectivity index was defined as the ratio of CC50 over ECs0 (as determined by FACS analys is).

PMED AP, t he la t t e r hav ing an ECso o f 9 .1 / xM and a se l ec t i v i t y i ndex o f 18. In

c o n t r a s t , n o a n t i - H H V- 6 a c t i v i ty w a s o b s e r v e d f o r t h e P M P a n d F P M P d e r i v a t i v e s .

I n p a r a l l e l w i t h FA C S a n a l y s i s , m i c r o s c o p y w a s p e r f o r m e d t o d e t e r m i n e t h e in -

h i b i t o r y e f f e c t o f t h e t e s t c o m p o u n d s o n H H V- 6 - i n d u c e d c y t o p a t h o g e n i c i t y. T h e m i c r o -

s c o p i c a l d a t a s h o w e d a c l o s e c o r r e l a t i o n w i t h t h e r e s u l t s o b t a i n e d b y t h e i n d i r e c t

i m m u n o f l u o r e s c e n c e / f l o w c y t o m e t r i c m e t h o d ( Ta b l e 1) . A c o r r e l a t i o n c o e f f i c i e n t o f



352 D . R e y m e n e t a l . // A n t i v i r a l R e s e a r c h 2 8 ( 1 9 9 5 ) 3 4 3 - 3 5 7

1000

oOL)LLg -"o

oOw

10 0

- j

r = 0.972

10 • •

0.1

0.01 "~. . . . . . . . . . I . . . . . . . . I . . . . . . . .0.01 0.1 1 10 100 1000

EC50 asde te rmined by CPE (p.M)

Fig . 3 . Compar i son o f t he an t iv i ra l da t a ba sed on HHV-6-spec i f i c an t igen de t ec t i on , a s de t e rmined byi m m u n o f l u o r e s c e n c e / f l o w c y t o m e t r i c a n al y s i s ( FA C S ) , a n d o n m i c r o s c o p ic a l e v a l u a t io n o f H H V - 6 - i n d u c e dcy topa th i c e ffec t (CPE) . Da ta shown a re t he ECs~~ va lues ex t rac t ed f rom Tab le I , and fo r : PFA, co mp oun d$ 2 2 4 2 , A C V, P M E D A P, P M E A , ( S ) - H P M PA , ( S ) - H P M P C , ( S ) - H P M P D A P, ( S ) - 3 - d e a z a - H P M PA , ( S ) - 3 -d e a z a -c H P M PA , ( S ) - c H P M PA , ( S ) - H P M P G , ( R ) - H P M P G , ( S ) - c H P M P G a n d ( S )- c H P M P C .

1000 EA

•P M100 Oa PFA ~ •

"r') 10 ~ (S)'HPeMpDAP

"~' 1 Z (S)-HPMPG

• ~ • (R)'HPMP~G (S)-cHPMPCt~ $2242OLLI

0.010.01 0.1 I 10 100 1000

EC50 ag ainst HHV -6 (1~1)

F i g . 4 . C o m p a r i s o n o f t h e a n t i - H H V- 6 a c t i v i t y ( FA C S r e s u l t s ) o f s e v e r a l n u c l e o s i d e a n a l o g u e s a n d t h e i re ff i cacy aga ins t HC M V [da t a taken f rom S noeck e t a l . (1988) , Andre i e t a l . (1991) , Ba l za r in i e t a l. (1993) andNeyts e t a l . (1994)] .



D . R e y m e n e t a l . / A n t i v i r a l R e s e a r c h 2 8 ( 1 9 9 5 ) 3 4 3 - 3 5 7 353

0 . 9 7 2 w a s c a l c u l a t e d u p o n c o r r e l a t i o n a n a l y s i s o f th e ECs 0 v a l u e s o b t a i n e d b y t h e f l o wc y t o me t r i c me t h o d a n d t h o s e o b t a i n e d b y t h e mi c r o s c o p i c a s s a y ( F i g . 3 ) .

4 . D i s c u s s i o n

A m o n g t h e d i f f e r e n t c o m p o u n d s e x a m i n e d h e r e , P FA e m e r g e d a s t h e m o s t s e l e c t i v ea n t i - H HV - 6 a g e n t , w it h a n ECs 0 o f 5 .8 / z M ( a s me a s u r e d b y FAC S a n a l y s i s ) a n d as e l e c t i v i t y i n d e x o f 1 4 1 . Th i s r e s u l t i s i n a g r e e me n t w i t h t h e d a t a o b t a i n e d b y s e v e r a lo the r g roups (St re iche r e t a l . , 1988 ; Agu t e t a l . , 1989a , 1991) . Overa l l , PFA and i t sc l o s e l y r e l a t e d p y r o p h o s p h a t e a n a l o g u e p h o s p h o n o a c e t i c a c i d a p p e a r a s s t r o n g i n h i b i t o r so f H H V- 6 r e p li c a ti o n . T h e r e f o re , P FA c a n b e r e g a r d e d a s a r e fe r e n c e c o m p o u n d f o rHHV-6 d rug sens i t iv i ty de te rmina t ions . In con t ra s t , l i t e ra tu re da ta (St re iche r e t a l . , 1988 ;Ru ss le r e t al ., 1989 ; A gu t e t a l. , 1989a , 1991 ; B urns an d Sand fo rd , 1990 ; Di Lu ca e t a l. ,

1 9 9 0 ) o n t h e a n t i - HH V- 6 a c t i v i t y o f ACV a n d r e l a te d a c y c l i c n u c l e o s id e a n a l o g u e ss h o w l e s s c o n s i st e n c y. I t s h o u l d b e n o t e d t h a t t h e s e r e p o r t e d r e s u lt s we r e o b t a i n e d u n d e rd i f f e ren t expe r imen ta l cond i t ions , i . e . , wi th d i f f e ren t HHV-6 s t r a ins , in d i f f e ren t ce l lt y p e s a n d u s i n g v a r i o u s p r o c e d u r e s f o r v i r a l i n f e c t i o n .

Th e s e d i f f e r e n c e s i n s e n s i t i v i t y a p p e a r n o t t o b e r e l a t e d t o t h e HHV- 6 s t r a i n u s e ds i n c e Ag u t e t a l. ( 1 9 9 1 ) h a v e d e m o n s t r a t e d h o m o g e n e o u s s u s c e p t ib i l it y o f d i f f e r e n tH H V- 6 i s o l a t e s f r o m H H V- 6 s u b t y p e A o r B t o a n u m b e r o f a n t i v i r a l c o m p o u n d s i nv i t r o . Ho we v e r, t h e c e l l t y p e u s e d ma y b e a mo r e i mp o r t a n t f a c t o r i n t h e d i s c r e p a n c i e ss e e n i n th e a n t i v ir a l r e s u lt s . Da t a o n e f f i c a c y o f AC V a n d G CV a g a i n s t HH V - 6 h a v e i na l l p r e v i o u s c a s e s b e e n o b t a i n e d i n f r e s h h u ma n l y mp h o c y t e s (S t r e i c h e r e t a l. , 1 98 8;Ru ss le r e t a l ., 1989 ; A gu t e t a l ., 1989a , 1991 ; B urns and Sa ndfo rd , 1990) , the an t iv i r a l

c o n c e n t r a t i o n s r a n g i n g f r o m 2 t o 2 0 / x M f o r AC V a n d f r o m 1 t o 2 5 / x M f o r GC V. I nHSB- 2 c e l l s , we a n d o t h e r s ( S t r e i c h e r e t a l . , 1 9 8 8 ) o b s e r v e d o n l y mo d e r a t e a n t i - HHV- 6a c t i v i t y f o r AC V a n d l i tt le i f a n y a c t i v i ty f o r GC V.

Severa l au tho rs (St re iche r e t a l . , 1988 ; Burns and Sandfo rd . , 1990 ; Di Luca e t a l . ,1 9 9 0; G o m p e l s e t al ., 1 9 9 5 ) h a v e s u g g e s t e d th a t HH V- 6 l a c k s it s o w n p h o s p h o r y l a t i n ge n z y me s [ s u c h a s v i r a l t h y mi d i n e k i n a s e ( TK) ] . Th u s i t a p p e a r s t h a t , i n o r d e r t o e x e r tt h e i r a n t i v i r a l e f f e c t o n H H V- 6 , A C V a n d G C V m e r e l y d e p e n d o n t h e p h o s p h o r y l a t i n gc a p a c i t y o f t h e c e l ls , wh i c h ma y d i f f e r c o n s i d e r a b l y f r o m o n e c e l l ty p e t o - a n o t h e r. Th ei n a c ti v it y o f B V D U a n d r e la t e d a n a l o g u e s (i .e ., B Va r a U ) a g a i n st H H V- 6 , m a y b ee x p l a i n e d b y t h e a b s e n c e o f a v ir u s - e n c o d e d T K in t h e H H V- 6 - i n f e c t e d H S B - 2 c e l ls . I nc o n t r a s t to n u c l e o s i d e a n a l o g u e s wh i c h a r e s u b s t ra t e a n a l o g u e s o f th e DN A p o l y m e r a s e ,

PFA i n t e ra c t s w i t h t h e a c t iv e s i te o f th e e n z y m e a s a p r o d u c t a n a l o g u e o f p y r o p h o s p h a t e .I n t h i s c o n t e x t , t h e c o n s i s t e n c y i n t h e a n t i v i r a l d a t a f o r PFA i s mo s t p r o b a b l y r e l a t e d t ot h e f a c t t h a t n o p h o s p h o r y l a t i o n i s r e q u i r e d f o r PFA t o b e a c t i v e a g a i n s t t h e v i r a l DNAp o l y m e r a s e .

Th i s i s t h e f ir s t r e p o r t o n t h e a n t i - H HV - 6 a c t i v i ty o f t h e a c y c l i c n u c l e o s i d e p h o s p h o -n a t e ( ANP) a n a l o g u e s . I n t e r e s t i n g l y, s e v e r a l c o mp o u n d s b e l o n g i n g t o t h e HPMP s e r i e se m e r g e d a s p o t e n t a n d s e le c t iv e in h i b it o rs o f H H V - 6 : ( S ) - H P M P C , ( S ) - c H P M P C ,( S ) - H P M PA , ( S ) - 3 - d e a z a - H P M PA , ( S ) - 3 - d e a z a - c H P M PA , ( S ) - H P M P G , ( S ) - c H P M P Ga n d ( R ) - H PM PG , t h e i r ECs 0 v a l u e s r a n g i n g f r o m 1 t o 11 / x M a n d t h e i r s e l e c t i v it y i n d e x



35 4 D. Reymen et al. / An tiviral Resear ch 28 (1995) 343 -3 57

r a n g i n g f r o m 6 t o 30 . Th u s , t h e b r o a d - s p e c t r u m a n t i - h e r p e s v i ru s a c t i v it y o f th e H PM Pa n a l o g u e s e x t e n d s t o H H V- 6 .

M a r k e d a n t i - H H V - 6 e f f e c t w a s a l so n o t e d w i th th e P M E c o m p o u n d s P M E A a n d

PM ED AP ( E C s 0 : 2 8 / ~M a n d 9.1 / z M, r e s p e c t iv e l y ; s e l e c t iv i t y i n d ex : 6 a n d 1 8 ,r e s p e c t i v e l y ) , wh i c h h a v e p r e v i o u s l y b e e n s h o wn t o i n h i b i t r e t r o v i r u s e s a s we l l a she rpe svir use s (Ba lzar in i e t a l . , 1989, 1991; Nae sen s e t a l . , 1989, 1994) . In contra s t ,( S ) - F P M PA a n d ( R ) - P M P D A P , w h i c h a r e k n o w n t o b e a c ti v e a g a i n st re t ro v i ru s e s , b u tno t he rpesv i ruses (Ba lza r in i e t a l. , 1991a , 1993) , likewise f a i l ed to show e ff i cac y aga ins tH H V- 6 .

Th e a c t i v it y o f th e AN P a n a l o g u e s a g a i n s t H HV - 6 wa s c o m p a r e d w i th t h e i r a c t i v it ya g a i n s t HCMV ( F i g . 4 ) . To p l o t t h i s f i g u r e , d a t a f o r HHV- 6 we r e t a k e n f r o m Ta b l e 1( FA CS a n a l y si s ), wh e r e a s t h e HC M V d a t a we r e t a k e n f r o m t h e l i te r a t ur e ( Sn o e c k e t a l. ,1988; An drei e t a l. , 1991; Ba lzar in i e t a l . , 1993; N ey ts e t a l . , 1994). I t shou ld be takeni n t o a c c o u n t t h a t t h e a c t i v i t y a g a i n s t HHV- 6 a n d HCMV wa s d e t e r mi n e d a t d i f f e r e n t

o c c a s i o n s , i n d i f f e r e n t c e l l s a n d b y d i f f e r e n t me t h o d s . A b e t t e r c o r r e l a t i o n ma y h a v eb e e n f o u n d i f e x p e r i m e n t s w i t h b o t h v i r u s e s c o u l d h a v e b e e n d o n e i n th e s a m e c e l ls .N e v e r t h e le s s , i t a p p e a r s th a t t he D N A p o l y m e r a s e s o f H C M V a n d H H V- 6 , w h i c h h a v eb e e n s h o wn t o b e s t r u c t u r a l l y r e l a t e d , a l s o d i s p l a y a s i mi l a r s e n s i t i v i t y t o p o l y me r a s einh ib i to r s (Bapa t e t a l . , 1989) .

HHV- 6 s e e ms t o b e s e n s i t i v e t o t h o s e n u c l e o s i d e a n a l o g u e s , s u c h a s ( S ) - HPMPC a n do t h e r A NP a n a l o g u e s , t h a t f o r th e i r a n t i v ir a l a c t i v i t y d o n o t d e p e n d o n a c t i v a t io n b y t h ev i r a l TK ( Ne y t s e t a l. , 1 9 9 0 ) . I t i s g e n e r a l l y h y p o t h e s i z e d t h a t th e a n t i - HC M V a c t i v it y o f( S ) - H P M P C i s b a s e d o n in h i b it io n o f H C M V D N A p o l y m e r a s e b y th e a c t iv e m e t a b o li t e( S ) - H P M P C p p , w h i c h i s f o r m e d f r o m ( S ) - H P M P C b y c e ll u la r e n z y m e s ( H o e t a l. , 1 99 1;Su l l i v a n e t a l . , 1 9 9 3 ) . Fu r t h e r s t u d i e s a r e n e e d e d t o r e v e a l wh e t h e r ( S ) HPMPCp p , a n d

t h e ANP a n a l o g u e s i n g e n e r a l , a c t a g a i n s t HHV- 6 a c c o r d i n g t o t h e s a me me c h a n i s m a sf o r H C M V .

Se v e r a l a u t h o r s h a v e s u g g e s t e d , b a s e d o n i n v i t r o d a t a , t h a t HHV- 6 ma y e n h a n c e HI Vrep l i ca t ion and p lay a c ruc ia l ro le in the p rog ress ion o f AID S (Ag u t e t a l ., 1989 ; Enso l ie t a l . , 1989; Ho rv at e t a l. , 1989; L uss o e t a l . , 1989, 1991; Th om ps on e t a l . , 1994; W an ge t a l. , 1994) . The c l in ica l imp l ica t ions o f these in v i t ro f ind ings r e ma in to be c la r i f i ed(Sp i ra e t a l . , 1990) . Also , the c l in ica l ro le o f HHV-6 as an oppor tun i s t i c pa thogen inHI V - i n f e c t e d in d i v i d u a ls r e ma i n s u n c l e a r. Ne v e r t h e l e s s , c o m p o u n d s t h a t i n h i b it HH V - 6r e p l i c a t i o n ma y b e b e n e f i c i a l i n t h e t r e a t me n t o f AI DS p a t i e n t s , e i t h e r d i r e c t l y b ysuppress ing an oppor tun i s t i c HHV-6 in fec t ion , o r ind i rec t ly by inh ib i t ing the s t imu la t iono f HI V r e p l ic a t i o n b y HH V- 6 . I n t h is c o n t e x t , t h e d u a l a n ti v i ra l a c t i v i ty o f PM EA a n d

P M E D A P a g a in s t b o t h H I V a n d H H V - 6 m a k e s t h e se c o m p o u n d s o f p o te n t ia l ly g r e a tv a l u e i n t h e t r e a t me n t o f HI V- i n f e c t e d i n d i v i d u a l s .I n c o n c l u s i o n , f r o m t h e p r e s e n t s t u d i e s , s e v e r a l a c y c l i c n u c l e o s i d e p h o s p h o n a t e

a n a l o g u e s p r o v e d t o b e e f f i c i e n t a g a i n s t HH V- 6 . Th e h i g h e s t p o t e n c y a n d s e l e c t iv i t y wa se x h i b i t e d b y t h e HPMP c o n g e n e r s [ i . e . , ( S ) - HPMPC] , a n d t h e s e a g e n t s s h o u l d b e f u r t h e re x p l o r e d i n t h e i r o wn r i g h t f o r t h e t h e r a p y o f HHV- 6 i n f e c t i o n s . A l s o , c l i n i c a l s t u d i e ss h o u ld b e p e r f o r m e d t o a s s e ss t h e p o te n t ia l o f P M E A a n d P M E D A P i n t h e t re a t m e n t o fAI DS p a t i e n t s s u f f e r i n g f r o m c o n c u r r e n t i n f e c t i o n s b y h e r p e s v i r u s e s s u c h a s HHV- 6 .B i o c h e mi c a l s t u d i e s a r e u n d e r wa y t o d e l i n e a t e wh i c h e n z y me s a r e i n v o l v e d i n t h e



D. Reymen et al./ Antiviral Research 28 (1995) 343-357 35 5

a c t i v a t i o n a n d a n t i v ir a l a c t i o n o f th e A N P a n a l o g u e s . F u r t h e r m o r e , w e a r e p l a n n i n g t o

e v a l u a t e i n v it r o th e e f f i ca c y o f t he A N P a n a l o g u e s a g a i n s t d u a l i n f e c t i o n s b y H I V a n d

H H V - 6 .

Acknowledgemen ts

W e t h a n k C h r i s t i a n e C a l l e b a u t f o r f i ne e d it o r ia l h e lp . D i a n e R e y m e n i s a R e s e a r c h

A s s i s ta n t f r o m t h e B e lg i a n " N a t i o n a a l F o n d s v o o r W e t e n s c h a p p e l i j k O n d e r z o e k "

( N F W O ) . T h i s w o r k w a s s u p p o r t e d b y g r a n t s f r o m t h e B i o m e d i c a l R e s e a r ch P r o g r a m m e

o f t h e E u r o p e a n C o m m u n i t y , t he B e l g i an F o n d s f o r G e n e e s k u n d i g W e t e n s c h a p p e l i j k

O n d e r z o e k ( K r e d i e t N o . 3 . 0 1 8 0 .9 5 ) , t h e B e l g i a n G e c o n c e r t e e r d e O n d e r z o e k s a c t i e s

( K r e d i e t N o . 9 5 / 5 ) , a n d t h e G r a n t A g e n c y o f t h e C z e c h A c a d e m y o f S c i e n c e s ( N o .

4 5 5 4 0 7 ) .

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