Nucleotide Dimers as Anti Human Immunodeficiency Virus Agents

Chapter 11

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Nucleotide Dimers as Anti Human Immunodeficiency Virus Agents Elliot F. Hahn , Mariano Busso , Abdul M. Mian , and Lionel Resnick 1

2

3

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IVAX Corporation, 8800 NW 36th Street, Miami, F L 33178 Departments of Dermatology and Pathology, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, F L 33140 Department of Oncology, University of Miami Medical School, Miami, F L 33131 1

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3

A series of nucleotide homo and heterdimers were synthesized from nucleosides which include azidothymidine, 2', 3'-dideoxyadenosine and 2'3'-dideoxyinosine. The compounds were evaluated with respect to anti-HIV a c t i v i t y , cytotoxicity, i n v i t r o s t a b i l i t y and i n vivo distribution. On an equimolar basis, greater anti-HIV potency and enhanced cytotherapeutic indices were obtained with heterodimers relative to the corresponding monomers. In v i t r o s t a b i l i t y of the dimer phosphate linkage was found to be species dependent. After intravenous administration to rats, the distribution of 3'-azido-3'3'-deoxythymidilyl-(5'5')-2',3'-dideoxy;-5'-adenylic acid, 2-cyanoethyl ester (AZT-P(CyE)ddA) and AZT i n plasma and brain was similar. In 1981, the acquired immunodeficiency syndrome (AIDS) was reported as a new c l i n i c a l entity (1-3). An intensive research effort led to the i d e n t i f i c a t i o n of the e t i o l o g i c agent responsible for the disease. (4-7). The pathogen, human immunodeficiency virus (HIV), i s a non-oncogenic retrovirus closely associated with the l e n t i v i r u s family (8,9). The discovery of HIV led to the development of therapeutic strategies directed against the virus. The different stages i n the replicative cycle of HIV provide various targets at which a n t i v i r a l agents may intervene. 0097-6156/89/0401 -0156$06.00/0 ο 1989 A m e r i c a n C h e m i c a l Society

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


11.

HAHNETAK

157

Nucleotide Dinners

T h e s e have been t h e s u b j e c t o f a number o f r e v i e w s (10.11) and i n c l u d e : ( 1 ) t h e b i n d i n g s t a g e a t w h i c h CD4 r e c e p t o r d e c o y s a n d d e x t r a n s u l f a t e s a c t , (2) e n t r y i n t o t h e t a r g e t cell where drugs that block fusion or uncoating might intervene, ( 3 ) t r a n s c r i p t i o n o f RNA t o D N A , t h e l e v e l at which reverse t r a n s c r i p t a s e i n h i b i t o r s would f u n c t i o n , (4) i n t e g r a t i o n o f DNA a n d e x p r e s s i o n o f v i r a l genes, where "integrase" inhibitors or "anti-sense" construct would act, (5) viral p r o t e i n p r o d u c t i o n and assembly c o u l d be m o d i f i e d by p r o t e a s e i n h i b i t o r s and agents which affect, myristylation or glycosylation and f i n a l l y , (6) budding o f t h e v i r u s w h i c h may b e p r e v e n t e d b y interferons. The most e f f e c t i v e approach has i n v o l v e d t h e s y n t h e s i s o f i n h i b i t o r s o f r e v e r s e t r a n s c r i p t a s e , t h e u n i q u e enzyme a s s o c i a t e d w i t h t h e r e p l i c a t i o n o f HIV. The r a t i o n a l e for t h e u s e o f a n t i v i r a l a g e n t s i n HIV d i s e a s e i s predicated on t h e assumption t h a t r e p l i c a t i o n i s n e c e s s a r y f o r the development of progressive d i s e a s e . I n h i b i t i o n o f H I V may permit the regeneration or prevent additional deterioration o f t h e immune s y s t e m . These drugs would p r o v i d e the g r e a t e s t p o s s i b i l i t y of o b t a i n i n g an immediate c l i n i c a l i m p a c t on t h e c o u r s e o f t h e d i s e a s e . Since the discovery of reverse t r a n s c r i p t a s e occurred i n 1970 (12.13) . compounds t h a t inhibit t h e enzyme had been d e v e l o p e d and were a v a i l a b l e f o r e v a l u a t i o n a t the a d v e n t o f t h e AIDS c r i s i s . To d a t e , t h e most p o t e n t and selective anti-HIV compounds a r e a series of 2', 3 dideoxynucleoside analogs. These compounds a r e thought t o be s u c c e s s i v e l y p h o s p h o r y l a t e d by h o s t c e l l enzymes t o y i e l d 2 , 3 - d i d e o x y n u c l e o s i d e - 5 - t r i p h o s p h a t e s , which are analogs of 2 -deoxynucleoside-5 -triphosphates, the natural substrates for cellular DNA polymerases and reverse transcriptase. The 2 ,3 -dideoxynucleoside-5 triphosphates function as substrates for HIV reverse t r a n s c r i p t a s e a n d t e r m i n a t e v i r a l DNA c h a i n e l o n g a t i o n b y i n c o r p o r a t i o n i n t o t h e v i r a l genome ( 1 4 ) . Only 3'-azido2 ,3 -dideoxythymidine (AZT) h a s b e e n a p p r o v e d b y t h e FDA f o r t h e t r e a t m e n t o f HIV i n f e c t i o n . AZT was c h o s e n for c l i n i c a l e v a l u a t i o n on t h e b a s i s o f i t s s e l e c t i v e i n v i t r o antiviral e f f e c t a g a i n s t HIV. The c l i n i c a l t r i a l s with AZT have f o c u s e d on p a t i e n t s w i t h AIDS and AIDS-related complex (ARC). In t h e s e p a t i e n t s , AZT i n d u c e d clinical and l a b o r a t o r y improvements. AZT t h e r a p y a l s o exhibits a degree of success in reversing HIV induced dementia (15.16.17). C u r r e n t s t u d i e s aim t o d e t e r m i n e i f AZT is effective in preventing the development of AIDS in a s y m p t o m a t i c HIV s e r o p o s i t i v e individuals. AZT therapy was a s s o c i a t e d w i t h t o x i c i t i e s t h a t may l i m i t its use, which p r i m a r i l y i n v o l v e d bone marrow s u p p r e s s i o n i n the form of anemia and n e u t r o p e n i a ( 1 5 . 1 6 ) . Therefore, other s t r a t e g i e s w h i c h i n v o l v e c o m b i n a t i o n s o f a n t i HIV agents are also being pursued. The most p r o m i s i n g o f t h e 2 ' , 3 · 1

1

1

1

1

1

1

1

1

1


1

158

NUCLEOTIDE

ANALOGUES

dideoxynucleosides to be used individually or in c o m b i n a t i o n w i t h AZT a r e 2 · , 3 · - d i d e o x y c y t i d i n e (ddC), and 2·,3 -dideoxyadenosine (ddA). The u l t i m a t e success of t h e s e a g e n t s a g a i n s t HIV is determined in the clinical arena. Combinations of drugs which permit a reduction in i n d i v i d u a l d o s e s w o u l d be a means o f d e c r e a s i n g t o x i c i t y .


1

Another approach to combination therapy involves the use of either homo or heterodimers of specific dideoxynucleosides. We hypothesized that when the dideoxynucleosides are linked v i a a phosphate bridge the d i m e r i z e d compounds c o u l d p r o v i d e s u p e r i o r p h a r m a c o l o g i c a l effects for the following reasons 1) two different nucleosides are delivered to the c e l l simultaneously, 2) a masked phosphate i s p r e s e n t w i t h masking u n i t a l s o b e i n g active, 3) t h e a g e n t c o u l d f u n c t i o n a s a p r o d r u g , a n d 4) a c t i v i t y a s t h e i n t a c t d i m e r was p o s s i b l e . In general, the i n i t i a l phosphorylation of the nucleosides to yield mononucleotides is a l i m i t i n g step for the formation of these a c t i v e metabolites (18). However, AZT i s u n i q u e and the i n i t i a l phosphorylation i s not rate l i m i t i n g . If the nucleotide dimers cross the cell membrane (passive diffusion) and are hydrolysed intracellularly (data indicate that at least part of the dimer crosses the c e l l membrane and i s h y d r o l y s e d intracellularly manuscript i n preparation) , they would y i e l d 1 mole of mononucleotide and nucleoside. It is possible that the nucleotide p r o d u c e d w i l l be f u r t h e r anabolized to its triphosphate l e v e l r a t h e r than be a s u b s t r a t e f o r t h e p h o s p h a t a s e s and produce the nucleoside. Therefore, this process will eliminate the need for the initial obligatory phosphorylation step and may result in a superior therapeutic index. If the nucleotide dimers are hydrolysed before their uptake, the nucleotide phosphatases will convert the mononucleotide into the nucleoside. I n t h i s f a s h i o n , t h e d i m e r s may a c t a s " d e p o t forms" for t h e i r r e s p e c t i v e n u c l e o s i d e s a n d may y i e l d a favorable therapeutic index. We h a v e s y n t h e s i z e d a s e r i e s o f c o m p o u n d s w h i c h are homodimers or heterodimers of s p e c i f i c dideoxynucleosides and h a v e shown t h a t t h e s e a g e n t s p o s s e s s a c t i v i t y against HIV. The phosphate linked dimers of the d i d e o x y n u c l e o s i d e s have t h e g e n e r a l f o r m u l a shown i n F i g . 1 where R and R may b e A Z T , ddA o r d d l and R may b e hydrogen, cyanoethyl or e i t h e r a metal anion or organic anion salt. It should be noted t h a t R and R may b e d e r i v e d f r o m t h e same o r d i f f e r e n t d i d e o x y n u c l e o s i d e s to g e n e r a t e p h o s p h a t e - l i n k e d c o m p o u n d s t h a t a r e e i t h e r homo or hetero dimers. T h e d i m e r s may b e p r e p a r e d b y c o u p l i n g the nucleoside to be e s t e r i f i e d with a nucleoside 5 phosphate in the presence of a a r y l s u l f o n y l condensing agent and a base such as i m i d a z o l e (Fig. 2) . A general procedure for the synthesis of AZT-P-ddA, which is 2

3

1

2

1


11.

HAHNETAL.

159

Nucleotide Dimers

applicable for the synthesis of a l l nucleotide dimers is as follows: T h e n u c l e o s i d e - 5 - c y a n o e t h y l p h o s p h a t e was synthesized using a modification of the procedure described by Tener (19). Thus, the barium salt of cyanoethyl phosphate (5.0g) was converted to the pyridinium salt under anhydrous conditions and reacted with AZT (2.0g) in the presence of dicyclohexylcarbodiimide (6.18g) a t room t e m p e r a t u r e f o r 48 h o u r s . T h e r e a c t i o n was s t o p p e d b y a d d i t i o n o f w a t e r (10ml) and the product was purified by column chromatography to o b t a i n 2 . 0 7 g (69%) o f t h e d e s i r e d p r o d u c t . A solution of AZT 5 - c y a n o e t h y l p h o s p h a t e (600mg, 1.5mmol) and 2 , 3 dideoxyadenosine (352mg, 1.5mmol) was p r e p a r e d i n 60 m l of d i s t i l l e d pyridine. T o t h i s s o l u t i o n , 1 . 2 1 g (4mmol) of 2,4,6-triisopropylbenzenesulfonyl c h l o r i d e was a d d e d a n d s t i r r e d f o r 30 m i n , f o l l o w e d b y t h e a d d i t i o n o f 0 . 9 8 4 ml (12 mmol) o f N - m e t h y l - i m i d a z o l e . After stirring at room t e m p e r a t u r e f o r 18 h o u r s , t h e s o l v e n t was removed under vacuum and the residue was purified by column chromatography using s i l i c a g e l . The c y a n o e t h y l phosphate e s t e r w a s h y d r o l y z e d a t r o o m t e m p e r a t u r e w i t h 6 0 m l o f 15% ammonium h y d r o x i d e s o l u t i o n a n d t h e p r o d u c t was purified by f l a s h chromatography u s i n g s i l i c a g e l t o o b t a i n A Z T - P ddA i n 73% y i e l d .


1

1

1

1

To o b t a i n the c y a n o e t h y l phosphate e s t e r , the crude product prior to hydrolysis w i t h ammonium h y d r o x i d e was p u r i f i e d by chromatography. In initial studies, we determined the anti-HIV activity of the dimers using assays which measured i n h i b i t i o n of syncytium formation, reverse transcriptase p r o d u c t i o n a n d HIV a n t i g e n e x p r e s s i o n . Anti-HIV

Evaluation:

A syncytium i n h i b i t i o n assay that i s a safe, simple, rapid, quantitative and s e n s i t i v e screening system has been developed to detect p o t e n t i a l a n t i - H I V drugs. The assay has been standardized and validated for high capacity anti-HIV screening. Compounds c a n be i d e n t i f i e d and p r i o r i t i z e d b a s e d upon a n t i - H I V e f f e c t s . Potency is expressed as effective dose 50%(ED50), toxicity as i n h i b i t o r y d o s e 50% ( I D 5 0 ) a n d t h e c y t o t h e r a p e u t i c index as ID50/ED50. 1) I n h i b i t i o n o f s y n c y t i u m f o r m a t i o n . MT-2 c e l l s a r e used as targets because of their sensitivity to HIV i n f e c t i o n and the f o r m a t i o n of g i a n t syncytia that are quantifiable. The number and t h e t i m e n e c e s s a r y f o r the production of syncytia i s a function of the input virus inoculum. Target MT-2 c e l l s a r e exposed t o DEAE-Dextran ( 2 5 u g / m l , S i g m a ) f o r 20 m i n u t e s , w a s h e d , a n d i n f e c t e d w i t h


160

N U C L E O T I D E ANALOGUES

HIV-l(III-6) (MOI = 0 . 0 0 1 ) a t 37°C in humidified a i r c o n t a i n i n g 5% C 0 . A f t e r 9 6 h o u r s s y n c y t i a a r e c o u n t e d i n a microtiter configuration and compared t o controls. Uninfected and infected MT-2 c e l l s without exposure to drug and uninfected c e l l s exposed t o drugs a r e used as controls. A l l c u l t u r e s a r e performed i n t r i p l i c a t e on two sets of experiments.


2

To i n v e s t i g a t e t h e i n h i b i t o r y e f f e c t s o f t h e d r u g s on H I V - i n d u c e d s y n c y t i a f o r m a t i o n (20) , t h e n u c l e o t i d e d i m e r s were c o m p a r e d t o t h e i r monomers a n d t h e i r c o m b i n a t i o n s a t multiple concentrations ( F i g . 3) . AZT-P-ddA and AZTP(CyE)-ddA exerted the strongest protective e f f e c t against the development of HIV-induced syncytia. AZT-P-ddA and i t s cyanoethyl phosphate d e r i v a t i v e a t a concentration of 0.5um c o m p l e t e l y p r o t e c t e d M T - 2 c e l l s from t h e f o r m a t i o n of syncytia. AZT r e q u i r e d a c o n c e n t r a t i o n o f l u M , ddA, lOuM, and t h e c o m b i n a t i o n o f AZT + ddA r e q u i r e d 0.5uM t o achieve f u l l protection. No a n t i - H I V i n h i b i t o r y effects were seen a t c o n c e n t r a t i o n s below O.OluM.

2)

I n h i b i t o r y e f f e c t on r e v e r s e t r a n s c r i p t a s e p r o d u c t i o n .

Reverse transcriptase assays were performed as p r e v i o u s l y d e s c r i b e d ( 2 0 ) . When M T - 2 c e l l s w e r e i n f e c t e d by HIV, peak r e v e r s e t r a n s c r i p t a s e l e v e l s i n t h e c o n t r o l cultures without d r u g were g r e a t e r than 50,000 CPM/ml (uninfected control cultures gave background counts of 300cpm/ml) ( F i g . 4) . A significant inhibition o f HIV r e v e r s e t r a n s c r i p t a s e p r o d u c t i o n was o b s e r v e d i n a d o s e dependent manner when HIV-infected MT-2 c e l l s were cultured i n the presence of nucleosides and nucleotide dimers. AZT-P-ddA, AZT-P(CyE)-ddA and the combination of AZT + d d A , c o m p l e t e l y i n h i b i t e d t h e p r o d u c t s o f r e v e r s e t r a n s c r i p t a s e a t c o n c e n t r a t i o n s >luM. In comparison t o c o n t r o l s , r e v e r s e t r a n s c r i p t a s e p r o d u c t i o n was p a r t i a l l y inhibited when these compounds were tested at levels >0.1uM. The detection of HIV from culture supernatants (infectious v i r a l yield) correlated with detectable levels of reverse transcriptase and HIV-induced syncytia formation. The n u c l e o t i d e dimers exhibited a higher degree of i n h i b i t i o n f o r the detection of i n f e c t i o u s v i r a l y i e l d when c o m p a r e d t o t h e m o n o m e r s .

3) Inhibition effect.

o f HIV a n t i g e n

expression

and c v t o p a t h i c

A f t e r fourteen days o f i n f e c t i o n , 84% o f M T - 2 c e l l s expressed HIV p24 a n t i g e n as detected by indirect immunofluorescence. At a f i n a l concentration of luM of AZT-P-ddA, AZT-P(CyE)-ddA, o r t h e combination o f AZT + ddA, a >70% i n h i b i t i o n of viral a n t i g e n e x p r e s s i o n was


161

Nucleotide Dimers

H A H N ET AL.

ο

R0 J

II

Ρ

OR

2

OR


3

Figure 1.

0 HO-

Structure of Dimers.

IB ] 1

II

IB]

IB]

AISOJX

-p—

I

°

OR Figure 2.

v

-

r

°

OR

Coupling Scheme for Dimer Synthesis.

120

ddA ·•- AZT -o AZT • ddA AZT-P(CyE)ddA • AZT-P-ddA

log c o n c e n t r a t i o n

(uM)

Figure 3. Syncytium Inhibition. The number of syncytia was determined 96 hours after exposure of HIV infected MT-2 cells to drug concentrations (lOOuM, lOuM, luM, 0.5uM, O.luM, 0.05uM, O.OluM and 0.005uM). Each value represents the arithmetic mean of triplicate cultures from two sets of experiments. The mean syncytium number in infected cultures without drug was 421 ± 27


162

N U C L E O T I D E ANALOGUES

achieved. AZT (luM) or ddA (luM) alone exhibited no inhibition. S i m i l a r r e s u l t s were o b t a i n e d when a s s e s s i n g the i n h i b i t i o n of cytopathic e f f e c t by t h e s e compounds (Fig. 5).


4)

Cvtotherapeutic

evaluation.

The c o m p a r a t i v e HIV i n h i b i t o r y e f f e c t s o f n u c l e o s i d e s and n u c l e o t i d e d i m e r s a r e shown on T a b l e 1 . Using a 14day a s s a y , m o n i t o r i n g c e l l v i a b i l i t y and the expression of HIV a n t i g e n by c e l l u l a r fluorescence, studies were performed t o determine the potency and t o x i c i t y of the compounds. L i n e a r r e g r e s s i o n a n a l y s i s was p e r f o r m e d to d e t e r m i n e t h e ID50 a n d ED50 f o r e a c h c o m p o u n d . The growth i n h i b i t o r y e f f e c t s o f t h e compounds on MT2 cells which were not exposed to the virus, were compared. According to their ID50, t h e compounds c o u l d be c l a s s i f i e d i n t o t h r e e m a j o r g r o u p s . The compounds w i t h the highest toxicity were AZT-P-AZT, AZT + ddA, AZTP(CyE), and AZT. Compounds w i t h m o d e r a t e t o x i c i t y were A Z T - P - d d A , A Z T - P - d d l , and A Z T - P ( C y E ) - d d A . The compounds w i t h t h e l e a s t t o x i c i t y were d d l , ddA, and ddA-P(CyE) . When t h e c y t o t o x i c e f f e c t s o f t h e compounds were tested a g a i n s t t h e h u m a n c e l l l i n e s , H9 a n d U 9 3 7 , s i m i l a r t o x i c p r o f i l e s were s e e n . The a n t i - H I V a c t i v i t y o f t h e compounds a c c o r d i n g to t h e i r ED50 r e v e a l e d two m a j o r p r o f i l e s . The most p o t e n t compounds were AZT + ddA, A Z T - P - d d A , A Z T - P ( C y E ) - d d A , AZTP - d d l , and A Z T - P - A Z T . AZT, ddA, d d l , ddA-P(CyE) , and AZTP(CyE) e x h i b i t e d weaker activities. However, the cytotherapeutic indices of AZT-P-ddA, A Z T - P - d d l , and A Z T - P ( C y E ) - d d A were t h e highest. Based on t h e data obtained from these studies we f o c u s e d on f u r t h e r e v a l u a t i o n o f A Z T - P - d d A and A Z T - P ( C y E ) ddA. F i g u r e 6 shows t h e r e s u l t s o f i n c u b a t i o n o f A Z T - P ddA i n human p l a s m a a t v a r i o u s t e m p e r a t u r e s . The compound is m e t a b o l i z e d a t a r a t e o f a p p r o x i m a t e l y 10% p e r hour. T h i s v a l u e appears t o be s p e c i e s dependent as i s seen i n Fig. 7. A n a l y s i s o f t h e m e t a b o l i t e s i n human p l a s m a ( F i g . 8) s h o w s t h a t t h e p r i n c i p a l c o m p o u n d s f o r m e d a r e A Z T a n d ddl. A s i m i l a r study of the s t a b i l i t y of AZT-P(CyE)-ddA (Fig. 9) at 37° shows that it is metabolized more e x t e n s i v e l y o v e r 3 h o u r a s s a y p e r i o d when c o m p a r e d t o A Z T P-ddA. F i g u r e 10 s h o w s t h a t t h e n a t u r e o f t h e products formed i s also species related. In human p l a s m a , the major metabolite is AZT-P-ddA which is a result of hydrolysis of the triester.


11. H A H N E T A L

163

Nucleotide Dimers


80000

Mi no drug B ddA • AZT 0 AZT • ddA • AZT-P(CyE)ddA 1 AZT-P-ddA

0.1

0.5

concentration

1 (uM)

F i g u r e 4 . I n h i b i t i o n o f Reverse T r a n s c r i p t a s e A c t i v i t y . I n h i b i t i o n o f r e v e r s e t r a n s c r i p t a s e a c t i v i t y from c u l t u r e s u p e r n a t a n t s was determined on day 8 . Each v a l u e r e p r e s e n t s the a r i t h m e t i c mean o f t r i p l i c a t e c u l t u r e s from two s e t s o f experiments. Detectable l e v e l s i n reverse t r a n s c r i p t a s e a c t i v i t y o c c u r when CPM/ml a r e g r e a t e r than 5,000 ( L i n e s , S D ) .

c

EN?

a

b

c

d

e

F i g u r e 5. Assessment o f HIV I n h i b i t i o n by F l u o r e s c e n t and V i a b l e C e l l Count. The i n h i b i t o r y e f f e c t o f luM o f drug on HIV e x p r e s s i o n was a s s e s s e d on day 14 by i n d i r e c t immunofluorescence ( s o l i d columns) and v i a b l e c e l l numbers (open c o l u m n s ) : (a) ddA, (b) AZT, (c) AZT + ddA, (d) AZT-P(CyE)-ddA, and (e) AZT-P-ddA. The r e s u l t s a r e the a r i t h m e t i c mean o f t r i p l i c a t e c u l t u r e s from two s e t s o f experiments ( L i n e s , S D ) .


164

NUCLE(XnDE ANALOGUES

Table 1. Comparative Inhibitory Effects o f C o m p o u n d s


25 57 60

4.0 7.0 7.5

100 400 450

AZT ddA ddl AZT + ddA AZT-P-AZT AZT-P(CyE)-ddA AZT-P-ddA AZT-P(CyE) AZT-P-ddl ddA-P(CyE)

CTI

ED50

ID50

Drug

133 40 300 250 30 240 80

0.6 1.5 0.7 0.8 3 1 5

80 60 210 200 90 240 400

ID50: of

uninfected

MT-2

cells

by

50% o n

day

14.

ED50:

D r u g c o n c e n t r a t i o n a c h i e v i n g 50% i n h i b i t i o n of HIV e x p r e s s i o n a s s e s s e d by i m m u n o f l u o r e s c e n c e o n day 14.

CTI:

Cytotherapeutic

index:

ID50/ED50.

The results are expressed as the arithmetic mean of t r i p l i c a t e c u l t u r e s f r o m two s e t s o f e x p e r i m e n t s . Linear r e g r e s s i o n a n a l y s i s was u s e d t o d e t e r m i n e t h e ID50 and ED50.

100 60 Percent

60

40 20

2

3 Hours

F i g u r e 6. S t a b i l i t y o f AZT-P-ddA i n Human Plasma ( i n v i t r o ) a t V a r i o u s Temperatures (4 u g / m l ) .



H A H N ET AL.

Nucleotide Dimers

Time (hours)

Figure 7. Stability of AZT-P-ddA in Plasma of Various Species at 37 C (4 ug/ml).

l

3

2

4

Hours

Figure 8 . Disposition of AZT-P-ddA in Human Plasma at 37° (4 ug/ml).



166

NUCLEOTIDE

ANALOGUES

Time (hours)

Figure 9 . Stability of AZT-P(CyE)ddA at 37°C.

2

3

Time (hours)

1

2

3

Figure 10. Stability of AZT-P(CyE)ddA in Human and Rat Plasma.


11.

HAHNETAL.

167

Nucleotide Dimers

T a b l e 2. Radioactivity i n Rats Following I.V. Administration o f A Z T (5 mg/kg) A Z T - P ( C y E ) d d A (11.5 mg/kg)

Total

Radioactivity (% o f d o s e )

(H ) 3


a

Time

AZT-P

AZT

(CyE)ddA

Plasma

Brain

Plasma

Brain

10

min

7.1

0.069

6.6

0.073

20

min

4.1

0.056

4.6

0.063

40

min

2.9

0.047

2.5

0.032

1 hr 0.028

1. 4

2

1. 1

hr

0 . 030

1.4

0 . 028

0.7

0 . 024

0.6

0.022 3 hr 0.026

a.

0. 5

Percent of radioactivity radioactivity

dose was calculated by present in plasma or brain administered

dividing by total

To assess in vivo disposition of AZT-P-ddA we synthesized radiolabelled substrate containing H in a b i o l o g i c a l l y s t a b l e p o s i t i o n i n t h e AZT m o l e c u l e . This compound was d i l u t e d w i t h u n l a b e l l e d d r u g a n d a d m i n i s t e r e d intravenously (6mg/kg) into rats. B l o o d samples were o b t a i n e d a t 1, 2 and 3 h o u r s a f t e r i n j e c t i o n and a n a l y z e d for radioactive content. The r e s u l t s showed a b o u t 5.5% o f t h e d o s e was p r e s e n t i n s e r u m a f t e r 1 h o u r . This value remained constant over the 3 hour sampling p e r i o d . A s i m i l a r s t u d y was c a r r i e d o u t u s i n g r a d i o l a b e l l e d AZTP ( C y E ) - d d A a n d t h e r e s u l t s w e r e c o m p a r e d t o A Z T ( T a b l e 2) . The d a t a shows t h a t at each time period examined the concentration of both drugs present i n p l a s m a was not significantly different. 3


168

NUCLEOTIDE ANALOGUES

Additional studies are ongoing to determine whether i n v i v o a d v a n t a g e s e x c e e d i n g t h o s e o b s e r v e d i n v i t r o may be a s s o c i a t e d w i t h a d m i n i s t r a t i o n of the dimers. This possibility arises because first they do not have to contend with the fate of the individual components in terms of in vivo metabolism, and second, as intact nucleosides both components will reach the cell simultaneously.


Acknowledgment: The a u t h o r s would l i k e thank Dr. C.C. L i n for h i s collaboration. LITERATURE

to

CITED

1.

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16.

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1879

Nucleotide Dimers as Anti Human Immunodeficiency Virus Agents

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