2nd & 3rd generation PS-wohrle

8/7/2019 2nd & 3rd generation PS-wohrle

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Russian Chemical Bulletin, V ol. 47, No. 5, M ay, 199g 807

P h otod yn am ic th erap y o f can cer :secon d an d th ird gen era t ion s o f p h o tosen s i t i z er s

D . W i ~h rl e, a * A . H i r t h , a T . B o g d a h n - R a i , a G . S c h n u r p f e i l , a a n d M . S h o p o v a b

alnsti tute of Organic and M acrom olecula r Chemistry, Department of Biology an d Chem is try, Universi ty o f Brem en,Postfach 330440, D-28334, Bremen, German y. *Fax: (0421) 218 4935. E-mail: [email protected]

bInsti tute of Organic Chemis try, Bulgarian Ac ade my o f Sciences ,Sofia, Bulgaria

Photodynamic therapy of cancer (PDT) at early stages of various types of cancer is basedon the use of the phototoxicity of photosensitizers (PS) which appeaes upon their excitationby light in tumor tissues. The review concentrates mainly on the data for the second andthird generations of photosensitizers: the st.ructure of PS, their photophysical and photo-chemical properties, and some results of their in vitro and in vivo application. A carefullydesigned PS should exhibit the following properties: long-wave absorption, good singletoxygen quantum yield, art intramolecular polarity axis, and low in vivo (photooxidation)stability. Uncharged PS that are lipophilic, positively charged, and capable of binding withmonoclonal antibodies are discussed as an example.Key words: photosensitizees, naphthalocyanines, phthalocyanines, porphyrins; photody-namic therapy of cancer.

I n t r o d u c t i o nAs a phototherapeutic method, the phetodynamictherapy of cancer (PDT) is based on the use of the

phototoxicity of photosensitizers (PS), which appearsafter they are excited by light, with the aim of localdestruction of various cancer tumors at their early andmiddle stages. The PS used for this purpose are diversederivatives of porphyrin and related macrocycles. Themethod is applicable for long-term control of manyearly stages of canc er and may be used as a palliative forcancer at advanced stages (see Refs. 1--4 for reviews).The treatment of a patient includes several steps.I. First, a solutio n of a suitable PS (0.5~ 5.0 mg per1 kg body weight) is injected systematically (intra -veneously, intraperitoneally; for skin cancer, local ad-ministration is also possible), and then time is allowedfor clearance of serum a nd a ccum ulatio n of the photodrugin the tumor. After ~10--30 h, the concentration of thePS in the tumor is often higher than that in peritumoraltissues. The fluorescence of the PS ill the u ltra-viole t orvisible light region may be detected after the irradiationwith low light intensitie s of shorter wavelenght and canbe used as a diagnostic means to probe the localizationof the tumor2. Irradiation with an ap propriate dose of visible ornear infrared light (in the longest-wavelength absorption* Universitat Bremen, Fachbereich 2 (Biologie/Chemie).Instimt I~r Organische und Makromolekulare Chemic, Postfach330 440. D-28334 Bremen. Deutschland.

region of the PS) results in the in situ activation of thephotodrug and subsequent photoreactions with compo-nents of the tumor tissue. Cell disintegration is com-pleted within the first hours after photodynamic treat-ment. Necrosis and apoptosis are considered as the maincell death mechanism s. 53. Profound vascular and cellular effects occur within12 to 18 I1, causing a number of processes. After 2--3weeks, hemorrhagic necrosis of the tumor tissues takesplace.The photosensitizers should exhibit good phototoxicityand low dark toxicity. In summary, tile phototoxicity ofa PS is determined by the following main factors: itsphotophysical and photochemical properties, its struc-ture and charge, its intrac ellula r con cen trat ion, and itssubcellular localization.5 Negatively charged and verypolar PS are trapped vi a pinocytosis and are thought tobe accumulated mainly in cytoplasmic liposomes. Cat-ionic PS penetrate through hydrophobic cellular mem-branes due to the fact that the membranes are chargednegatively on the inside surface. The PS which aredelivered by liposomes and then transported with lowdensity lipoproteins (LDL) mainly use the receptor-mediated endocytoxic pathway. After a nu mbe r of steps,this pathway ends up in lysosomes. The intracellularlocalization of PS depends on the pathway of theirtrapping. For example, hematoporphyrin derivativesHpD/PII (see below) have been detected mainly inmembranes (in cellular, nuclear, and mitochondriatmembranes and in endoplasmic reticulum). Other, morelipophilic anionic PS are also generally localized inTranslated from Izvestiya Akadem ii Na uk. Seriya K himicheskaya. No. 5. pp. 836--845, May. 1998.

1066-5285/98/4705-0807 $20.00 9 1998 Plenum Publishing Corporation


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80 8 Russ . Ch em .Bul l . , Vol . 47, No. 5 , Ma y, 1998 W r h r l e e t a l .

T a b l e I . Photosensi t ize rs s tud ied for c l in ica l appl ica t ionsPhotosensi t ize r Producer

Company, inst i tu te Ci ty (country)AI p h th a lo c y a n in e s(Ph o to se n s )Zn n a p h th a lo c y a n in e sam - T H P CPo rp h y c e n e sM o n o a sp a r ty l ch lo r in e s( M A C E )Purpurins ,Ce t io p u rp u r inTe x a p h y r in s8 -Am in o le v u l in i c a c id8 -Am in o le v u l in i c a c id

Research Inst i tu te of OrganicSemiprodu c ts and DyesBulgar ian A cadem y of Sc iencesSc o t i a Ph a rm c e u tCy to p h a rm /p a r tn e rNip p o n Pe t ro c h e m ic a lPDT In c .Pharmacyc l isD U S AM e d a c G m b H

Moscow (Russia )Sofia (Bulgar ia )Gu i ld fo rd (Gre a t Br i t a in )Me n lo Pa rk , CA (USA)To k y o ( Ja p a n )Santa Barbara , CA (USA)Pa lo Al to , CA (USA)T o r o n t o ( C a n a d a )H a m b u r g ( G e r m a n y )

a In preparation for clinical trials.

m e m b r a n e s t r u c t u r e s , w h e r e a s h y d r o p h i l i c P S s e e m t oa c c u m u l a t e i n l y s o s o m e s . C e r t a i n c a t i o n i c P S a c c u m u -l a te p r e f e re n t i a l l y i n m i t o c h o n d r i a . H y d r o p h o b i c P Sd e l i v e r e d b y l i p o s o m e s o r L D L h a v e b e e n f o u n d i nm e m b r a n e s , m i t o c h o n d r i a , e n d o p l a s m i e a n d ro u g hr e t i c u l u m , a n d i n e n d o t h e l i u m ( i n s m a l l e r a m o u n t s ) .

T h e p r a c t i c a l u s e o f p h o t o s e n s i t i z e r s r e q u i r e s t h a tt h e i r m u t a g e n i c a n d c a r c i n o g e n i c p r o p e r t i e s b e ta k e ni n t o c o n s i d e r a t i o n . N o s t a t i s t ic a l l y s i g n i f i ca n t m u t a g e n i cp o t en t ia l w as f o u n d f o r H p D / P I ! o r s u lf o n a t ed a l u m i -m ~m p h t h a l o c y a n i n e s , i I t s h o u l d , h o w e v e r , b e t a k e ni n t o a c c o u n t t h a t o n l y s m a l l a m o u n t s o f P S a r e in j e c t e di n t o an o r g a n i s m . I t is a l s o . i n t e r e s t i n g t o n o t e t h a t t h er o le o f m e t a b o l i s m i n r e m o v a l o f t h e s e c o m p o u n d s i se i t h e r s m a l l o r i ts c o n t r i b u t i o n i s n o t d e t e c t e d a t a l l; l aP S a r e r e m o v e d f r o m t h e o r g a n i s m u n c h a n g e d .

C l i n i c a l application o f PD T and related fields of therapyF o r in v ivo a p p l i c a t i o n s o f P D T , p h o t o s e n s i t i z e r s

w i t h a b s o r p t i o n i n t h e w a v e l e n g t h r a n g e f r o m 6 0 0 t o8 50 n m a r e e m p l o y e d . I n th i s s p e c tr a l r e g io n , t h e m a x i -m u m p e n e t r a t i o n d e p t h o f l ig h t in t o a t i s s u e c a n b eo b t a i n e d ( u p t o 2 - - 3 c m u n d e r f a v o r a b l e c i r c u m s t a n c e s ) .T h e m a j o r i ty o f c l i n ic a l a p p l i ca t i o n s o f P D T h a ve b e e np e r f o r m e d u s i ng a m i x t u r e o f h e m a t o p o r p h y r i n d e r iv a -t i ve s ( H p D ) a s t h e p h o t o s e n s i t i z i n g a g e n t . P a r t i a l l y p u -r if ie d H p D n a m e d P h o t o f r i n I I (P I I ; A m e r i c a n C y a n a -m i d / L e d e r l e L a b o r a t o r i e s , U S A ) o r s i m i l a r c o m p o u n d s( P h o t o s a n , P h o t o h e m , P h o t o c a r c i n o m i n ) ar e e m p l o y e d .I n a n u m b e r o f c o u n tr i e s ( C a n a d a , U S A , N e t h e r l a n d s ,J a p a n ) , t h e s e c o m p o u n d s a r e e x t en s i v e l y u s e d i n c li n i c a la p p l i c a t i o n s a g a i n s t s o m e t u m o r s . B e c a u s e t h e s e c o m -p o u n d s e x h i b i t s o m e d i s a d v a n t a g e s ( i n p a r t i c u l a r , l i g h tw i t h a w a v e l e n g t h o f ~6 3 0 n m i s r e q u i r e d ) , q e w p h o t o -s e n s i t i z e r s w i t h a b s o r p t i o n s h i f t e d t o w a r d t h e r e d r e g i o n

( 6 7 5 t o 8 0 0 n m r a n g e ) h a v e b e e n d e v e l o p e d . S o m e o ft h e s e P S a r e u n d e r c l i n i c a l p h a s e I / I I t r i a l s ( -[ a b l e l ) .

F o r tr e a t m e n t o f c u t a n e o u s l e s i o n s , P D T t r e a t m e n ti s a s ea s y as i l l u m i n a t i n g a n a r e a o f t h e s k i n . F o rt r e a t m e n t o f i n t e r n a l c a n c e r s , t h e l i g h t is d e l i v e r e d v iao p t i ca l f ib e rs . In m o d e m p h o t o d y n a m i c t r e a t m e n t , l a -s e r s a r e u s e d a s t h e l i g h t s o u r c e b e c a u s e o f t h e i r a b i l i t yt o c r e a t e a sm a l l c o h e r e n t l i g h t b e a m , w h i c h c a n b et r a n s f e r re d e f f i c i e n t ly t o a d i s t a n c e t h r o u g h v a r i o u s f i -b e r s 2 0 0 t o 4 0 0 p. m i n d i a m e t e r . S i n c e t h e l i g h t d e l i v e r ym e t h o d a n d l i g ht d o s e i ll P D T a r e d e t e r m i n e d b y t h ek i n d a n d s iz e o f t h e t u m o r , m u c h e x p e r i e n c e i s r e q u i r e df o r a s u cc e s s fu l t r e a t m e n t . G a s a n d s o l i d - s t a t e l a s er s a sw e l l a s dy e l a se rs p u m p e d b y A r l a s e r a n d N d - Y A Gl a s e r a r e u s e d m o s t c o m m o n l y . I n t h e f u tu r e , s e m i c o n -d u c t o r l a s er s t h a t a r e l es s e x p e n s i v e a n d m o r e e a s y toh a n d l e w i l l a c q u i r e g r e a te r i m p o r t a n c e . T h e t o t a l d o s e o fl i g ht d e l i v e r e d to n e o p l a s t i c a r e a s i s u s u a l l y v a r ie d f r o n t8 0 t o 6 0 0 J c m - 2 i n o r d e r t o r e d u c e t h e e f f e c ts o fh y p e r t h e r m i a _T h e a p p l i c a ti o n o f P S f or t h e i n a c t iv a t i o n o f v i ~ s e si n b l o o d i s c u r r e n t l y t h e s u b j e c t o f i n t e n s e i n t e r e s t .E x c e l l e n t v ir u s k il l i n g i s a c h i e v e d w i t h P S s u c h a sb e n z o p o r p h y r i n s a n d s u l fo n a t e d p h t h a l o c y a n i n e s , w h o s ea b s o r p t i o n i s r e d - s h i f t e d r e l a t i v e t o h e m o g l o b i n . T h et r e a t m e n t o f d i s e a s e s c a u s e d b y c y t o m e g a l o v i r u s , h e r p e ss i m p l e x v i ru s ( H S V ) , a n d h u m a n i m m u n o d i f i c i e n c y v i -r u s ( H I V ) i s u n d e r i n v e s t i g a t i o n . 6

A d m i n i s t r a t i o n o f ~ 5 - a m i n o l e v u li n i c a c i d ( ~5 -A L A ,N H 2 C H 2 C O C H 2 C H 2 C O O H ) i n d u c e s t he b i o sy n t h es i so f p h o to s e n s i ti z i n g c o n c e n t r a t i o n s o f e n d o g e n o u s p r o t o -p o r p h y r i n I X . D u e t o t h e a d v a n t a g e s o f t h i s m e t h o d ,s u c h a s t i s s u e s p e c i f i c i t y , r a p i d r e m o v a l , a n d p o s s i b i l i t yo f to p i c a l a d m i n i s t r a t i o n , ~ 5- A LA is s u b j e c t t o e x t e n s i v ep r e c li n i ca l a nd c l i ni c a l s t u d y b y p h o t o d y n a m i c t h e r a p ya s a p o ss i b l e r e p l a c e m e n t f o r t h e u s u a l P S ( M e d a cG m b H , H a m b u r g , G e r m a n y ) . 7


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P h o t o d y l~ a m i c t h e r a p y o f c a n c e r Russ . Chem. Bul l ., Vo l . 47 , No . 5 , May , 1998 809

P h o t o s e n s i t i z e r s i n P D TP h o t o s e u s i t i z e r s u s e d i n t h e r a p y s h o u l d e x h i b i t t h e

f o l l o w i n g p r o p e r t i e s :( I ) h i g h q u a n t u m y i e l d o f t h e t r i p le t s t at e (q0-r > 0 .4)co m bin ed w i th h igh t r ip le t l i f e t ime (XT > 100 ItS) be -c a u s e p h o t o c h e m i c a l r e a c t i o n s p r e d o m i n a n t l y o c c u r i nt h e e x c i t e d t r i p l e t s t a t e ;( 2 ) a b s o r p t i o n i n t h e l o n g - w a v e r e g io u ( k > 6 3 0 n m ,p r e f e r a b l y k > 6 8 0 n m ) w i t h h i g h e x t i n c t i o n c o e f f i -c i e n t s ( e > 5 0 0 0 0 L m o l - j c m - I ) i s r e q u i r e d t o i n -c r e a s e t h e p e n e t r a t i o n d e p t h o f li g h t i n to t h e t i s su e a n dt h e n u m b e r o f p h o t o n s a b s o r b e d , s i n c e li g h t f l ux i n t h et i ss u e d e c r e a s e s e x p o n e n t i a l l y w i t h d i s t a n c e ; t a

(3 ) l imi ted in v ivo s t a b i l i t y f o r r a p i d r e m o v a l f r o mt h e t u m o r a n d p e r i t u m o r a l t i s su e s;

( 4 ) s e le c t iv e t r a n s p o r t o f t h e P S t o t h e t u m o r i no r d e r t o r e d u c e s i d e e f f e c t s i n p e r i t u m o r a l t i s s u e s ;

( 5 ) l o w d a r k t o x i c i t y o f t h e P S a n d t h e i r d e g r a d a t i o np r o d u c t s ;(6) avai labi l i ty .I n f o r m a t i o n o n t h e P S u s e d f o r P D T i s s h o w n i nT a b l e s 1 a n d 2 . T h e s t r u c t u r a l f o r m u l a s o f c e r t a i n P S a r ep r e s e n t e d b e l o w .T h e p r i nc ip a l c a u s e o f p h o t o d a m a g e i n P D T i n v ol v est h e f o l lo w i n g p r o ce s s es . 5 T h e e x c i t a t i o n o f a P S o c c u r s

v ia a n i n t e r s y s t e m c r o s s i n g f r o m t h e e x c i t e d s i n g l e t s t a t e( I P S * ) t o t h e t r ip l e t s t a t e ( ~ P S * ) ( E q . ( 1 ) ) . T h e s o - c a l l e dt y p e 1 r e a c t i o n s c o r r e s p o n d t o r a d i c a l r e a c t i o n s w i t hp h o t o i n d u c e d e l e c t r o n t r a n s f e r ( r e d o x r e a c t i o n s ) a n do c c u r e i t h e r a s r e d u c t i v e q u e n c h i n g b y b i o m o l e c u l e s( E q . ( 2 ) ) o r a s o x i d a t i v e q u e n c h i n g b y O 2 ( E q . ( 3 ) ) .T y p e 1 I r e a c t io n s i n v o l v e p h o t o i n d u c e d e n e r g y t r a n s f e rf r o m t h e t r i p l e t s t a t e o f t h e P S ( 3 p s * ) t o t r ip l e t o x y g e n( 3 0 2 , 3 ~ - ) w i t h f o r m a t i o n o f si n g l e t o x y g e n ( t O 2 , l a g )f o l l o w e d b y o x i d a t i o n ( E q . ( 4 ) ) . I t is g e n e r a l l y a g r e e d

Me(HO)CH~ N HN ~ C H ( O H ) M e

" . - (C O z H C O 2 H

Hematoporphyrin

M e O 2 ~ / /

C 0 2 H C 0 2 M eBenzoporphyrin(monoac~d)

Etiopurpurin(used as a S n-complex)

R R

R R N~ ' N

AIm, Sirv, Ge~v / \Phthalocyanine (Pc ]

"CO2H

RNaphthalocyanine (Nc)

CO2HChlorine e6

RI

R Texaphyrin

OH

HO

OH

m-THPC

Porphycene


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8 1 0 Ru ss .C h e m .Bu l l . , Vo l . 4 7 , No . 5 , Ma y , 1 9 98 W r h r l e e t a l .

Table 2 . S o m e p h o t o p h y s i c a l p r o p e r t i e s o f f i rs t - a n d s e c o n d -g e n e r a t i o n p h o t o s e n s i t i z e r sP h o t o s e n s i t i z e r ~ ) 'm a x ~PT a "Pa b/ L m o l - I c m - I / n mHe ma topo rphy r in c 3500 630 0 .83 0 .65P h o t o f r i n I I ( P I I ) c - 3 0 0 0 ~ 6 3 0 - 0 .2Zn ph th a locy an ine c 150000 675 0 .6 0 .59AI ph th a loc yan ine- 105000 675 0 .38 0 .38te t rasu l fon ic ac id /Z n p h t h a l o c y a n i n e - 1 1 50 0 0 6 7 2 0 .5 6 0 .5 2te t rasu l fon ie ac id dZn nap htha lo cyan ine ~ 160000 764 0 .45Ben zopo rphyr in r | 18000 685 0 .60Bac te r ioch lor ine r t 50000 785 0 .32 0 .32Zn e t iopu rpur in c -700 00 ~690 0 .83 0 .57Porp hyce ne r 52000 630 0 .42 0 .30a Q u a n t u m y i e l d o f t r i p l e t s t at e .b Q u a n t u m y i e l d o f I O 2 .r T h e p a r a m e t e r s w e r e d e t e r m i n e d i n a n o r g a n i c s o l v e n t .a T h e p a r a m e t e r s w e r e d e t e r m i n e d i n w a t e r .

t h a t s i n g l e t o x y g e n i s t h e k e y a g e n t i n c e l l u l a r d a m a g e f lI n a d d i t io n , p h o t o t h e r m a l e f f e c ts d u e t o n o n r a d i a t i n gd e a c t i v a t i o n o f e x c i t e d s t a t e s a l s o l e a d s t o p h o t o s e n s i -t i z e d c e l l d e a t h ( E q . ( 5 ) ) . z ,9

P S + h v ~ I p s * --~ 3 p s * ( I )3 p S * + R S H o r R N R ' 2 --~

P S ' - + ( R S H ) o x o r ( R N R ' 2 ) o x ( 2 )3 p S * + 0 2 -- ~ P S " + + O 2 " -

- -* s u b s e q u e n t r e a c t i o n s O f c e l l u la r t a r g e t s ( 3 )3p s* + 30 2 - -~ PS + 102 --~

- ~ s u b s e q u e n t o x i d a t i o n o f c e l l u l a r t a r g e t s ( 4 )1PS* o r 3pS * ~ PS + Q - -> ce l l dea th ( 5 )T h e p h o t o s e n s i t i z e r s e m p l o y e d i n P D T o r i n v e s ti -

g a t e d f o r t h is p u r p o s e c a n b e d i v i d e d i n t o t h r e e g e n e r a -t i o n s :

- - F i r s t g e n e ra t io n : H p D , P I I ( m i x t u r e s o f P S ) ; n os e l e c ti v e a c c u m u l a t i o n i n t h e t u m o r .- - S e c o n d g e n e ra t io n : s t r u c t u r a l l y d i s t i n c t c o m p o t , n d s ,w h i c h i n m o s t c a s e s d i s p la y l o n g - w a v e a b s o r p t i o n ; n os e l e c ti v e a c c u m u l a t i o n i n t h e t u m o r .

- - Th i rd g e n e ra t io n : s e c o n d - g e n e r a t i o n P S b o u n d t oc a r ri e r s f o r s e l e c t iv e a c c u m u l a t i o n i n t h e t u m o r .

T h e f i r s t g e n e r a t i o n o f p h o t o s e n si t iz e r sH e m a t o p o r p h y r i n d e r iv a t iv e s ( H p D , P I I , P h o t o h e m ,

e tc .) b e l o n g t o t h e f i rs t g e n e r a t i o n o f P S a n d a r e m o s t l yu s e d i n c l i n i c a l a p p l i c a t i o n s . T h e y a r e o b t a i n e d b y t r e a t -m e , i t o f h e m a t o p o r p h y r i n w i t h 5 % H 2 S O 4 i,1 a c e t i c a c i da t r o o m t e m p e r a t u r e , s I n o r d e r t o p r e p a r e s o l u t i o n s f o ri n j e c t io n , H p D i s t r e a t e d w i t h a n a q u e o u s b a s e a n d t h e nn e u t ra l iz e d . H e m a t o p o r p h y r i n d e r iv a t iv e s ( H p D ) c o n si st

o f a m i x tu r e o f s e ve r al m o n o m e r i c a n d o l i g o m e r i c c o m -p o u n d s . 4 ,1 ~ T h e m o n o m e r i c f r a c t i o n i s r e m o v e d u s i n gH P L C o r G P C . T h e H p D p h o t o s e n s it i z e r an d i ts c o m -m e r c i a l v a r ia n t s e x h i b it f o u r m a i n d i s a d v a n t a g e s .1. T h e l o n g e s t - w a v e a b s o r p t i o n b a n d a p p e a r s a t6 3 0 n m ( w i t h a lo w e x t i n c t io n c o e f f i c i e n t ) ; t h e b i o l o g i -c a l e f f e c t s a f t e r ir r a d i a t i o n a t t h i s w a v e l e n g t h o c c u r o n l yt o a t is s u e d e p t h o f ~ 5 m m ( S c h e m e I , T a b l e 2 ).

2 . N o n s e l e c t i v e a c c u m u l a t i o n i n t h e t u m o r r e s u l t s i nd i s t ri b u t io n o f P S b e t w e e n t h e t u m o r a n d t h e s k i n , w i t ht u m o r / s k i n r a ti os o f ~ 2 9 I . T h e a m o u n t o f s e n s i t i z e rs i nv a r i o u s o r g a n s d e c r e a s e s i n t h e s e r i e s : l i v e r > u r i n a r yb l a d d e r > p a n c r e a s , k i d n e y , s p l e e n > s t o m a c h , b o n e ,l u ng , h e a r t > s k in > m u s c l e > > b r a i n . O n l y - 0 . 1 - - 3 % o ft h e i n j e ct e d P S a m o u n t is f o u n d i n t h e t u m o r t i ss u e .

3 . T h e s e n s i t i z e r i s r e t a i n e d i n c u t a n e o u s t i s s u e s fo r2 - - 3 m o n t h s ( a f te r 75 d ay s , 6 1 % o f P I I i n t h e s p l e e n a n d3 0 % i n t h e l u n g is s t il l p r e s e n t ) , w h i c h r e q u i r e s t h a t t h ep a t i e n t a v o i d s b r i g h t l i g h t .

4 . T h e c o m p l e x a n d d i f f ic u l t l y s e p a r a b l e m i x t u r e o fc o m p o u n d s d o e s n o t a ll o w o n e t o u s e o n l y i ts a c t i v ec o m p o n e n t s .

S c h e m e 1

SensitizerDccrea~ in stability

Inc~ ea ,e in absoq~ t ion ooeff ic icn tfpo t' rl < 104 to > 10 5 L to o l - i C l l l - i

P o t p h y d mI !550 600

Pht Iraloc'yani neP o r p h y c e ~ e s N a p h t h a l e c y a n i ~ , , s

C h l o r i n e st- 41 l! i i i

6 5 0 7 0 0 7 5 0 8 0 0 ~ / n mI R e ~ i ~s u i t a b l e f o rP D T [

A b s o r p t i on r ~ J o n o f p l ~ t o s e r ~ t i z c r s

TissueX / n m 5 5 0 5 9 0 6 2 0 6 8 0 8 0 0T r a n s m i s s i o n ( % )through the 15- ram t i s sue 10 -5 10 -4 10 -3 10 -2 10 - I

T h e s e c o n d g e n e r a t io n o f p h o t o s e n s i t i z e r sP h o t o p h y s i e a l an d p h o t o c h e m i c a l p r o p e r t i e s . T h e s e c -

o n d g e n e r a t i o n o f P S , w h i c h a r e i n s o m e c a s e s t t se d inp h a s e I o r p h a s e I I c l i n i c a l t r i a l s , h a s a b s o r p t i o n m a i n l yf r o m 67 5 to 8 00 n m L i g h t w i t h t h i s w a v e l e n g t h p e n -e t rd t e s in t o t i ~ u e s t o a d e p t h o f u p t o 2 - - 3 c m ( s e eS c h e m e I , T a b l e 2 ). T h e i n t e r p r e t a t i o n o f d o s e - - r e s p o n s e4 1 1r e l a ti o n s h i p s f o r t h e s e P S ( p t a t h a l o c y a n i n e s , , n a p h -13a bt h a l o c y a n i n e s , I z b c u z o p o r p h y r i ~ l s , ' . c h l o r i n e s , 4 p u r p u -


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6/10

S 1 2 R u .~ . C h e m . B u l l . . V o l . 4 7 . N o 5, Ma y , 1 0 9 8 Wiihrle e t a l

R +

O C 6 H + 3O C 6 H 13

a 1

B u tB u t

O P ' / =

R 1 - - ~ R IR; N ~ . % . , . : ~ . N

II l N - - Z n - - N I I l~ i N ~ N

R : ~ R ~R2 ;~ma~, (D M F } q~.,~C O O H 7 4 9 0 4 9O H 7 5 1 0 . 2 1

R +

N J ~

R e R 3 k , ,, = ~ ( D M F ) ' P aH O P y 7 1 6 0 . 4 3O P y O P y 7 1 6 0 3 9

tMe

dicarbonitr i les and 6 - t e r t - b u t y l - 2 , 3 - n a p h t h a l e n e d i -carbonitrile.tS'~ l'h e mixture of po rphyrazines with dif-ferent pyridyloxy substitttents was separated by the usualflash chromatography and co nv er te d into positively

charged PS by methylation with CHsl. I n v i t r o experi-ments with 10 IJM solutio ns of the PS con tai ni ng 5. I() ~Hel.,a cells in 10% aqueous fetal bovine serttm (FBSIshow. after incuba tion for 18 h, that the uptake of PSincreases with increasing number of positively chargedcenters and is 3%, 4 2%, and 5.4% for three, four, andeight positive charges, respectively . 9 However, cellphotot oxicity is highest for the PS with three positivecharges (shown in Fig. I as an e xample), because thesin,let oxygen quantum yield (qo.,~ = 0.64) is highest (forothe r PS, % < 0.49).Hydrophobie photosensitizers. Thc photosensitizerswhich are too hydrophobic to be water soluble, such asunsubstituted phthalocyanines, naphthalocyanines, ortheir derivatives with alkyl, aryl, ether, thioether, amino ,aminoalkyl, and am id , subs tituen ts, have to be dissolvedin a suitable delivery system before injection. Un ilame llatliposomes, e .g . , those based on dipalmitoyl phosphati-dylcholine (DPPC), or oil emulsions like Cremopholwere used as deli',ery systems. IlolLI9 This met hod pro-vides an easy way to make good PS available for PI)T.Even strongly aggregating naphthalocyanines can be dis-solved in monomcri c form in a queous DPPC. p Thehydrophobic PS arc located in the monomeric state tnthe lipophilic part of the double layers of liposomes(-50--100 nm in diamcter). After injection, the PS arepreferably transferred to low-density lip.proteins (LDI.)in the blood serum. The pla.~ma LDL consists of a corewith hydrophobic lipids surrounded by polar liptds andapoproteins (22 nm in diameter). One LDL can incor-porate more than 150 porphyrin molecules in the lipidcore due to hydrophobic interactions. The targeted de-livery of LDL carrying PS to the tumor is achieved dueto the uniquc features of receptor-mediated preferentialendocy'tosis of LDL by malignant cells9 Hence, a largeamount of LDL.-as.sodatcd i'S c a n b e transported. In v ivo

~ U . Ntichelsen, T Ni~hi~aka, I Okura. and D W~ihrle.unpublished resttlts

i8Oi +~,, 60 9

i \_ 40 i ' , 2%" I ',E i--. "\20 ', \

20 40 60 80 100C/p.mol L-t

i80 t ', b~" t ", \_ ~ 6 0 i ' - --d " -. , 2

? 40! '\ ": ',1 "" E ' 1 o .. +m 2 0 r . , ., " - .. . . . - . . _ +

t " i - - I

o 50 1 0 0 150 2 0 0 250 300 tirr/S)F i l~ . I . t h o t o t o x i c i t y o f Z n p o q ~ h ~ . r n z in c > , w i t h t l lr c t - t I ) :u + d f o , l r ! . ~ I - - ) q h ' ,e c h a r g c s i n t h . l a c e l l , ; { m c . t ~ : l t t o n o f [ ( } I . d l lO l o f | ) ~ ,

~ i t h 5 ' I t) " | t c L : l c c H s in 1 0 ~ F I 3 S I r . c o n c c n t m t i o : ~ d e p e n d e n c e ( i l h l m t n a t i o l l I ~ V ,, : l S 0 m W c m 2 | , b . d e p c n d c n c c c mt r r . + d l a t i o + l t l t l l C l ~ ( ) I l I \ V t ' + l l - . + )


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Photodynamic therapy of cancer R u s s . C h e m . B u l l . , V o l . 4 7 , N o . 5, M a y , 1 9 9 8 813

[ZnNcl/~tg per 1 g of tissue:

1.4 -;< -.n ....... c? - " - - < L L [ ..... : ) 2

1.0 \, -...c" ~

0 . 6 1 - a - - \ ~~'"-A.3 . . . . . . . . . . . . . . . . ~ _ ~

0 . 2 1 /H0 ~ . . . . . . . . . . . . . . . . . . . . . . . . . ,2 0 4 0 6 0 d h

Hg. 2. Pharmacokinetics of unsubstituted Zn naphthalocyaninein black mice C57 with Lewis lung carcinoma (size -5 mm)transplanted into a right leg (0.25 mg of PS in DPPC liposomeper I kg body weight, intraperitoneal injection). Curves- 1,tumor; 2, liver; 3 , skin.ki/min-1

0.16 iE0.12]p[0.08 '~

Jd

0.04 iI

O~

o

r 9

TAP Pc NcFig. 3. Photostability of various unsubstituted and substitutedZn H tetraazaporphyrin chelates (I0 -5 tool L-I) measured inDMF upon light illumination at k = 350--850 nm and apower of 20 mW cm -2 (n is the number of linearly annelatedbenzene rings; TAP is tetraazaporphyrin; Pc is phthalocyanine;Nc is naphthalocyanine). The measurements were carried outin the air.

pharmacokinetic experiments with mice bearing Lewislung carcinoma 16 h after administration of Zn- na ph-thaloc yanine s dissolved in l iposomes show a PStum or/ sk in ratio of ~4 : 1 (Fig. 2). lz L D L can also beused as a delivery system for PS.

Stability o f p h o t o s e n s i t i z e r s . Attention should begiven to the in v ivo stability of photosensitizers. Asmentioned above, HpD/PII, which have low selectivityfor accumulation in the tumor, are retained in cutane-otis tissues for 2 to 3 months. Nonselective Second-generation PS, such as sulfonated AJ phthalocyanines,can stay for several weeks in tissues others than thettnn or ta (skin), caus ing light sensitivity of the patient.On lhe other hand, if photobleaching or decompositioqof the PS occur be|bre in v ivo reactions with biomolecules;ire initiated, no tissue damage is incurred. Therefore,the following requirements ,'ire imposed on PS. The total

d / r a m~ l f 920 [ .... 8

1612 ,- / 7 ~o--~Qf--U~ 9 4

~ - o -'(2"- ~ - ~ ,~ .~ -~ ~ 2

r3 6 9 12 15 18 21Time after PDT/days

Fig. 4. Comparison of the growth rate of Lewis lung carcinoma(d is the mean tumor diameter) transplanted into a left leg ofblack mice C57 and treated with Zn naphthalocyanines withvarious substituents R (0.25 nag of Zn naphthalocyaninesdissolved in DPPC liposomes per 1 kg body weight): R -NHCOPh (/), NHCOCsHll (2), NHCOMe (3), H (4).NHCOC6H4OMe (5), NH 2 (03, NI-ICOCIIH23 (7); control(without PS) (8). The illumination dose was 450 J cm -2, 20 hafter intraperitoneal injection of PS.

in v ivo stability should not exceed 2-- 3 days, sincesecond generation PS reach maximum tumor/other tis-sue ratios in one day. The photostability should be lessthan 2--3 days. On the one hand, it should allow i n v i v ophotoreactions. On the other hand, however, self-shield-ing of PS with high extinction coefficients is known tooccur. Hence, decreased photostability increases thetherapeutic depth of laser light penetration into thetissue, because PS molecules in the upper cellular layersphotobleach during illu minatio n faster than those indeeper layers. Therefore, the absorption of the alreadydead cells and photoblea ched PS molecules allows deeperlight penetration during photody namic treatment.

The photostabil i ty of various Zn complexes oftetraazaporphyrins in DMF upon irradiation in the airwas determined. The measured decrease in intensity (A)of the longest-wave absorption (Q band) of the complex(and hence its concentration) obeys first-order kinetics:In(A0/Ar) = k i t . 2an It is seen that the photostabilitydecreases with an increase in the number of annelatedbenzene rings at tetra azap orph yrins (Fig. 3). z~Tetraazaporphyrins are too stable for the use i~ PDT. Itwas shown for zinc naphth aloc yan ines that the skin isalmost completel y cleared after ~72 h (see Fig. 2), whichmakes long-term skin sensitization unlikely. Photody-namic in v ivo experiments with mice bearing Lewis lungcarcinoma treated with very low amounts of differentlysubstituted zinc naphtha locyanine s can cause a pro-nounced delay in tumor growth depending on the sub-stituent (Fig. 4). 12 A reli able increase in the averagesurvival time of the mice was achieved.

C o m p a r i s o n o f t h e f i rs t a n d s e c o n d g e n e r a t i o n s o fPS, Figure 5 compares the effect of photodynamictreatment for different generations of PS (change in the


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814 Rus,v .Chem.B ul l . , l , b l . 47 , No . 5 , M ay , 1998 WOhrle et al .

d / I 1 " 1 m2 0 :1 6 ~1 2

2-2, . / J9 ~ , / It " J " ~ o . . . . . . 0 . . . . . 0 . . . . . 0/ , / - . J ~ ; 3

/ / " / -

3 6 9 12 15 t8 21Time after PDT/days

F i g . 5. Comparison of the growth rate of B16 pigmentedmelanoma (d is the mean tumor diameter) in mice underexposure to light (the dose was 360 J cm-2) in the region ofphotosensitizer absorption: /, HPD (5 mg per 1 kg of bodyweight, illumination at L = 630 nm); 2, ZnPc (0.3 mg per I kgof body weight, illuminationat ~. = 673 nm); 3, ZnNc (R = H)(0.3 mg per 1 kg of body weight, illuminationat ~. = 756 nm);4, NHCOPh (0.5 mg per I kg of body weight, illumination at~. = 774 nm).

main tumor diameter of B I6 pigmented melanoma),zlThis skin cancer is quite common in some countries,e.g. , in Australia, due to the increase in UV radiation onthe Earth. At the end of the observation period (21 daysafter PDT), the PDT effect was absent for the HpD PSand for ZnPc (as well as in control groups without PS).Substituted zinc naphth alocyan ines are the only knownPS that are active against pigmented melanoma. Of zincnapht haloc yanin es, the derivative with R = N HCO Ph ismost active and is now under preclinical study.

T h i r d g e n e r a ti o n o f p h o t o s e n s i t i ze r sIn addition to the characteristics of the second-

generation PS, photosensitizers of the third generationshould have properties providin g selective delivery of PSto the tumor tissue, e.g. , by conjugation to biomoleculessuch as monoclonal antibodies (mAB). Malignant tumorceils have cell surface antigens which are different fromthose of normal cells. Monoctonal antibodies directedtoward tumor cell antigens are able to couple to photo-sensitizers. The mAB--PS conjugate will bind specifi-cally to the tumor tissue and sensitize its photokilliugwithout damaging normal tissues, z-za This difference inaffinity to tu mor and n ormal tissues is favorable forimmunotherapy by mAB--P S conjugates. A large amou ntof research has been done in the last years on the use ofantibodies in the diagnosis and tre atm ent of cancer, z2bVarious toxic agents have been conjugated to antibodies,including radioisotopes, micro bial toxins, and PS. zzeAfter coupling of PS molecules to the antibody, it isimportant to keep the binding affinity of the immuno-globulins toward tumor-associa ted antigens.

Three strategies for binding PS are presently beingstudied: (I) direct binding of PS to the con sta nt part ofmAB (disadvantages: bin ding often also occurs to vari-able parts of mAB; accumulation of too small amountsof PS); (2) binding of PS to pol ymer s ( e .g . , dextran) andcovalent linking of the polym er to mAB (di::advantages:bindi ng often also occurs to variable parts of mAB;shielding of the mAB by the polymer); (3) multiplicativeeffect, which uses polyphasic tumo r therapy to ac cumu -late 106--)08 PS molecules (assuming 104--105 antigeubinding sites)} 3

Let us demonstrate some recent results of the latterstrategy, z4 To create a multi plica tive system, the accu-

. _ . 1 C e l l( 1 ) / / s u r f a c eA n t i g e n ~ \V /B M 7 I ~ ' 0B i o t i n ~

S c h e m e 3(2) ( 3 )

b ~ S ~ ~ 1 ~

+ S A + b P s + S A ._

( 4 ) + . , .

hv I c ~ o t o x i ~ I


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Photodynamic therapy of cancer Ru.~s .Chera .BufL . VoL 47 , No . 5 . M ay , 199 8 8t5

mulating properties o f the a vidin -bioti n system wereused for targeted delivery of PS specifically onto labeledtumo r cells. Sch eme 3 shows multiple bind ing of avidinand biotinylated PS to biotinylated mAB linked to thesurface antigens of the tum or cell: z3 (I) cells with tumo rmarker are treated with biotinylated mAB; (2) after theexcess mAB is removed, avidin or streptavidin is added(binding of avidin to biotin linked to mA B); (3) after theexcess avidin is removed, biotinylated PS (bPS) areadded (binding to various free sites of the mAB--avid inconjugate); (4) multiple a ddition o f avidin and then bPSprovides a high accumulation of PS on the surface oftumor cells. The experimental results presented belowdemonstr ate the results of this method.

Several biotinylated bPS were prepared by the reac-tion of sulfochlorides of Zn phthalocyanines withbiotinylated cadaverin.

bPS R 1b P S - t ZbPS-2 ZbPS-3 Z

a 5

N ~ N N 1

R3R2 R3 R4 R5H X X XZ X X XH Z Y Y

Ox=-o-C -~O O

HN~NHO H H

O OAt first, the binding of bPS to streptavidin-modified

mAB was studied by a modified enzyme-linked immuno-sorbent assay method (ELISA). Perforated plates werecoated with the Btust-Mucin Antigen (BMA) and thentreated with the biotinylated antibody BM7. Afterwards,they were incu bated wit h strept avidin (10 -8 g) for 15 rain.The bPS (5 9 10-7--5 9 10 -12 g) was a dded afte r that. Aftereach step, the treated plates were washed with pH 7.2buffer sohttions. In order to estimate the number of non-biotinylated antibody binding sites, a test with thebiotinylatcd peroxidase--o-phenylenediamine system wascarried out. It was found that, depending on thekind of bPS. the avidin binding sites were saturated by

Tab l e 3. Comparison of characteristics of first- (I), second-( I I ), and thi rd-generation ( I I ! ) photosensi ti ze rsCompared Effectparameter I I I 111Synthesis

Relatively cheap, Yes (+)* In most Noavailable cases (+)Structural No Yes (+) Yes (+)homogeneityProperties in solution

Long-wave No Yes (+) Yes (+)absorptionHigh extinction No Yes (+) Yes (+)coefficientHigh photosensitizing Yes (+) Yes (+) Yes (+)activityHigh stability:photooxidative Yes Yes and Yes andno (+) no (+)in the dark Yes Yes (+) Yes (4-)and no and ?

Properties in vitro andin vivoRapid accumulation Yes (+) Yes (+) 9in tumorSelective accumulation No No Yes (+)in tumorPhotodynamic Yes (+) Yes (+) Yes (?)activityStability in vivo Yes Yes and ?no (+)Low dark No (+) In most cases ?toxicity no (+)Wide spectrum Yes (+) Yes (+) ?of indicationsReduction of '~ Yes (+) '~metastases and ?

* The (+) sign indicates a positive effect.

5 . 10- 8g of bPS-I and only 5-1 0-[ ~ of bPS-2 orbPS-3. Thus, excellent binding of bPS was achieved.

After that, the dark toxicity and phototoxicity of thebPS were studied. Breast carcinoma cells (106 T47dcells) were treated with 4" 10 -6 g of bPS. No darktoxicity was detected. Un de r irradiation with only5 J cm -2 dose, the cells were photodyna mically de-stroyed: the cell lysis was -80 % with bP S-I or bPS-2and ~60% with bPS-3. Finally, steps (1)--(3) (see Scheme3) were carried out with two cell lines. Breast carcinomacells (106 T47d cells with BMA) and colon carcinomacells (SW 1222 cells with out BMA) were tre ated withbiotinylate d antibo dy BM7. After washing, 10 -8 g ofstreptavidin was added, and after one more washing,4- 10 -6 g of bPS-I was added. Irradiation with a5 J cm -2 dose resulte d in ~90% lysis of T47d cells and~45% lysis of SW1222 cells. These first experimentsindicate specific binding and phototoxicity of the bPS.Further work along this direction is in progress.


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816 Russ . Ch em. Bul l . , Vo l . 47 , N o . 5 , Ma y , 1998 WiShrle e t a l .

Table 3 summarizes the prese nt situation with differ-ent generations of photoseusitizers for PDT. The secondgeneration satisfies the conditions required for PDTbetter than the first generation. PS of the third genera-lion are still in the initial stages of research.

The future of PDT depends greatly on financial sup-port and easier approval procedures for clirtical testing,since a large number of suitable PS are available now.

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