Antiviral Activities of 2-Fluorinated ArabinosylPyrimidine Nucleosides

FMAU, was highly effective in preventing rash and reducing viremia without apparent toxicity at doses of. 30, 10, or 3 mg/kg/day x 10 administered int...
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Chapter 10

Antiviral Activities of 2'-Fluorinated Arabinosyl—Pyrimidine Nucleosides Downloaded by KUNGLIGA TEKNISKA HOGSKOLAN on October 5, 2014 | http://pubs.acs.org Publication Date: August 2, 1988 | doi: 10.1021/bk-1988-0374.ch010

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J. J. Fox , K. A. Watanabe , T. C. Chou , R. F. Schinazi , K. F. Soike , I. Fourel , G. Hantz , and C. Trepo 4

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1Memorial Sloan-Kettering Cancer Center, New York, NY 10021 Emory University-Veterans Administration Medical Center, Atlanta, GA 30033 Delta Regional Primate Research Center, Covington, LA 70433 Hepatitis Virus Unit, Institut National de la Santéet de la Recherche Médicale, U 271, 69003 Lyon, France 2

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Comparative biochemical and antiviral studies are described for the 2'-fluoro-substituted arabinosyl-pyrimidine nucleosides 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)-5-methyluracil [FMAU] and 1-(2'deoxy-2'-fluoro-β-D-arabinofuranosyl)-5-ethyluracil [FEAU]. Biochemical studies indicated that FEAU should be a selective antiherpesvirus agent that is less toxic than FMAU. FEAU was evaluated against simian varicella virus infection in African green monkeys and, like FMAU, was highly effective in preventing rash and reducing viremia without apparent toxicity at doses of 30, 10, or 3 mg/kg/day x 10 administered intravenously. Oral administration of FEAU in those monkeys at 10, 3, and 1 mg/kg/day x 10 was equally effective. FEAU and FMAU were evaluated against woodchuck hepatitis virus (WHV) in chronically-infected woodchucks (an animal model of choice for evaluation of potential antihepatitis B virus agents in humans). FEAU inhibits WHV replication at 2.0 and 0.2 mg/kg/day x 10 in a l l animals tested. The inhibitory effect was immediate, nontoxic and long-lasting. Preliminary studies indicated that FEAU is also effective against WHV when given by the oral route. FMAU also produced immediate inhibitory effects against WHV replication at doses of 2.0 and 0.2 mg/kg/day x 5: however, unacceptable toxicities were observed with FMAU at these dosages. FEAU appears to be the most promising of the nucleoside analogs tested thus far against Hepadnaviridae and may be an effective agent clinically against Hepatitis B virus. 0097-6156/88/0374-0176$06.00/0 • 1988 American Chemical Society

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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10. FOX ET AL.

Arabinosyl—Pyrimidine Nucleosides

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Since our o r i g i n a l report (1.) on the synthesis and anti-herpes v i r u s a c t i v i t i e s of several 5-substituted pyrimidine nucleosides bearing the l-(2'-deoxy-2'-fluoro-0-D-arabinofuranosyl) moiety, structurea c t i v i t y studies have indicated that the 2'-fluoro substituent i n the "up" (arabino) configuration conferred more potent a n t i v i r a l a c t i v i t y than did a 2'-OH, hydrogen, or other halogens (2). Moreover, where studied (3.,4), the 2'-fluoro nucleosides were resistant to catabolic cleavage by nucleoside phosphorylases, presumably a r e s u l t of the increased metabolic s t a b i l i t y of the Ng l y c o s y l linkage imposed by this electronegative 2'-substituent. Of the several 2'-fluoro-5-substituted-arabinosyl pyrimidine nucleosides synthesized (1), l-(2'-deoxy-2'-fluoro-0-D-arabinofuranosy 1 )-5-iodocytosine [FIAC] has demonstrated c l i n i c a l e f f i c a c y against Herpesv i r u s infections i n Phase 1 (5.) and against V a r i c e l l a zoster v i r u s i n Phase 2 (6) c l i n i c a l t r i a l s i n immunocompromised cancer patients. The corresponding 5-methyluracil analog, l-(2'-deoxy-2'-fluoro-p-Darabinofuranosyl)-5-methyluracil [FMAU] was found to be more potent in mice infected with Herpes simplex v i r u s (HSV) types 1 and 2 without t o x i c i t y at e f f e c t i v e dose l e v e l s . FMAU was a l s o found to be active i n v i t r o and i n v i v o against P-815 and L-1210 leukemia c e l l l i n e s resistant to the antileukemic agent, 1-0-D-arabinofuranosylcytosine [Ara-C]. A Phase 1 t r i a l of FMAU i n patients with advanced cancer showed that drug-induced central nervous system (CNS) dysfunction was the dose-limiting t o x i c i t y (J).

FIAC

FMAU

With FMAU as a lead compound, the syntheses of other 5 - a l k y l substituted 2'-fluoro-ara-uracils were undertaken (8,£) including 1(2 '-deoxy-2'-f 1 uoro-0-D-arab inof urano sy 1 )-5-e thy 1 urac i 1 [FEAU]. As shown i n Table I, though FEAU was approximately one log order less potent than FMAU against HSV-1 and HSV-2 infected Vero c e l l s i n culture, the former exhibited f a r less host c e l l t o x i c i t y , r e s u l t i n g in an extremely favorable therapeutic index (9,10). A comparison of the a n t i v i r a l a c t i v i t y of FMAU, FEAU, and 5ethyl-2'-deoxyuridine (EDU) i n mice inoculated intracerebral l y with HSV-2 i s given i n Table II (10). FEAU showed a c t i v i t y at 50 mg/kg/day f o r 4 days and was h i g h l y e f f e c t i v e i n reducing m o r t a l i t y in these mice at doses of 100 - 200 mg/kg/day. At these dose l e v e l s , no t o x i c i t y was observed. In normal (uninfected) mice, FEAU

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Table I. Comparative Anti-HSV A c t i v i t y of FMAU and FEAU i n Plaque Reduction Assays i n Vero C e l l s

HSV-KF)*

HSV-2 (G)*

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^90 (uM)

ID*

E D

^50

90 (jiM)

(

»

# ft

l M )

Therapeutic Index I D

/ E D

50 90 HSV-1 HSV-2

FMAU

0.010

0.042

0.023

0.09

2.8

67

FEAU

0.024

0.26

0.24

0.91

>200

>769

31 >220

•Correlation c o e f f i c i e n t >. 0.86. #Cytotoxic effect measured i n r a p i d l y d i v i d i n g c e l l s . ID^Q = concentration required to i n h i b i t c e l l growth by 50%. **ED = E f f e c t i v e dose required to i n h i b i t HSV r e p l i c a t i o n by the percentage indicated.

Table I I .

Treatment

A n t i v i r a l Effects of FEAU, FMAU and EDU i n Mice Inoculated Intracerebrally with HSV-2 (Strain G) Dose* mg/kg/day

MDD**

-

% Survivors

%Wt Gain on Day 7 14 21 30

16 17

6.7

100 7

-3 -22

9 -14

13 8

10 50 100 200

9.4 11.8 11.0^ (14)

8 25 72 93

-7 3 2 0

-13 -10 7 1

+

31 16 13 9

36 25 13 13

FMAU

0.5

9.4

67

1

4

5

7

EDU

800 1000

7.6 8.6

13 33

-10 3

-6 13

Neg Control (no virus) PBSA (virus control)

FEAU

#

+

+

+

+

+

2 6 34 43

•Given 5 hr after intracerebral inoculation. Dose schedule, twice a day for 4 days. "Median day of death c a l c u l a t e d on day 21. #Numbers i n parentheses indicate death of a single animal. §As compared to day 1. Based on weight of a single surviving animal only. APhosphate buffer s a l i n e . +

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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10. FOX ET AL.

Arabinosyl—Pyrimidine Nucleosides

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was nontoxic at doses of 800 mg/kg/day given twice d a i l y for four days. I t i s a l s o noteworthy that EDU, which d i f f e r s s t r u c t u r a l l y from FEAU only by the absence of the 2'-fluoro substituent, i s much less e f f e c t i v e than FEAU i n this HSV-2 mouse encephalitis model. This finding i s consistent with our previous observation (1) attesting to the importance of the 2'-fluoro substituent f o r the anti-HSV a c t i v i t y exhibited by these arabinofuranosyl-pyrimidine nucleosides. It was concluded p r e v i o u s l y (11) that FMAU i s a most potent and s e l e c t i v e a n t i v i r a l compound f o r the treatment of mouse encephalitis caused by HSV-2 and therefore deserved consideration as a p o t e n t i a l agent i n human t r i a l s f o r the treatment of HSV encephalitis i n neonates and adults at low dose l e v e l s . The preliminary data described i n Tables I and II f o r FEAU suggested (10) that this compound may a l s o be worthy of s i m i l a r consideration. Based upon these e a r l i e r findings (10) and on our preliminary biochemical report (12), further comparative biochemical and a n t i v i r a l studies were undertaken with FMAU and FEAU, including their r e l a t i v e a c t i v i t i e s against Simian v a r i c e l l a v i r u s i n the African green monkey and against h e p a t i t i s v i r u s i n the woodchuck animal model. In these studies (13) an a l t e r n a t i v e synthesis of FEAU i s a l s o described. COMPARATIVE BIOCHEMICAL STUDIES Biochemical studies at Memorial Sloan-Kettering Cancer Center (13) on the r e l a t i v e effects of FEAU and FMAU on the growth of mammalian c e l l s i s shown i n Table III. Note that against the human

Table I I I . Comparison of E f f e c t s of FEAU and FMAU i n Mammalian C e l l s * ED (in |iM) f o r Inhibiting C e l l growth i n :

FEAU

FMAU

HL-60 C e l l s Vero C e l l s

2060 >200

15.4 2.8

ED (jiM) f o r i n h i b i t i n g thymidine incorporation into DNA

FEAU

FMAU

700 630 3,700

14.0 28.0 8.9

5Q

5Q

P-815 C e l l s L-1210 Cells Rat Bone Marrow C e l l s •See reference 13 f o r experimental

FEAU/ 'FMAU 133 >71

FEAU/ 'FMAU 50 22 415

procedures

promyelocytic leukemia c e l l l i n e (HL-60) as w e l l as against Vero c e l l s (derived from the African green monkey), FEAU i s f a r less growth-inhibitory than FMAU, which compares favorably to the i n v i t r o study given i n Table I. S i m i l a r l y , against rodent c e l l l i n e s (P-815, L-1210 and rat bone marrow c e l l s ) , FEAU i s s u b s t a n t i a l l y l e s s inhibitory of thymidine incorporation than FMAU (Table III).

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

C e l l u l a r k i n e t i c constants (K^) were a l s o determined (13) f o r the i n h i b i t i o n of natural precursor incorporation into the DNA of L1210 c e l l s by FEAU and FMAU. Using t r i t i u m - l a b e l led natural precursors as substrates (thymidine, 2'-deoxyuridine and 2'deoxycytidine), i t was found that the FEAU/FMAU K^ ratios were 61, 111, and 8.1, r e s p e c t i v e l y . These r e s u l t s a l s o indicated that FEAU is a weaker i n h i b i t o r of natural nucleoside anabolism than FMAU i n these mammalian c e l l s .

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Table IV. Incorporation of [2- C] FEAU or [2- C]FMAU Into DNA of Mammalian Cells Incorporation at 10 uM ( i n p mole/10 c e l l s / h r ) 6

C e l l Line

FEAU

FMAU

L-1210 Vero HSV-1 Infected Vero C e l l s

ND* ND* 0.48

0.69 1.3 3.4

•Not detectable.

Also not detectable at 100 jiM.

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Studies on the incorporation of 2 - C - l a b e l led FEAU and FMAU into the DNA of mammalian c e l l s showed very s i g n i f i c a n t differences (Table IV). There was no detectable incorporation of FEAU radioa c t i v i t y into the DNA of either L-1210 or Vero e e l 1 l i n e s , but substantial amounts of FMAU r a d i o a c t i v i t y were incorporated into the DNA of both c e l l l i n e s . When HSV-1 infected Vero c e l l s were exposed to C - l a b e l l e d FEAU and FMAU, both nucleosides were incorporated into the DNA of these v i r u s - i n f e c t e d c e l l s . Under these conditions, FMAU incorporation into HSV-1 infected Vero c e l l s was 7-fold greater than that observed for FEAU. This difference in incorporation may be due to the greater a f f i n i t y of FMAU for viral-encoded DNA polymerase and i s comparable to the magnitude of the difference of t h e i r a n t i he rpe t i c potencies i n v i t r o (13). It i s g e n e r a l l y accepted that the s e l e c t i v e antiherpes a c t i v i t y of 2'-fluoro-arabinosylpyrimidine nucleosides i s associated i n large measure with their a b i l i t y to be recognized as good substrates f o r HSV-specified thymidine kinase (TK), but not by the host TK (14*11). As shown in Table V, FMAU i s a good substrate ( r e l a t i v e to thymidine) for cytosol and mitochondrial TK's derived from the HL-60 human c e l l l i n e , as w e l l as f o r HSV-1 and HSV-2 derived TK's. By contrast, FEAU is a very poor substrate for the host HL-60 derived cytosol TK and an excellent substrate for HSV-1 and HSV-2 derived TK's (13). These data are consistent with the recent report by Mansuri et a l . , (16) who showed that FEAU has a very low a f f i n i t y for Vero c e l l TK (compared to thymidine) but a high a f f i n i t y toward HSV-1 and HSV-2 encoded TK's. Their data a l s o indicate that, in uninfected Vero c e l l s , FEAU would be phosphorylated at a very low rate i n the presence of thymidine. Their report (16) a l s o describes a modified chemical synthesis of FEAU.

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

10. FOX ET AL.

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Arabinosyl-Pyrimidine Nucleosides

Table V. Percentage Rates of Phosphorylation of FEAU and FMAU (Relative to Thymidine*) by Various Thymidine Kinases** Enzyme Source

% Rates of Phosphorylation of Various Thymidine Kinases

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FEAU

FMAU

HL-60 C e l l s Cytosol TK Mitochondrial TK

0 - 2.1 N/A#

81.7 219.0

HSV-1 (Strain KOS) TK HSV-2 (Strain 333) TK

82.7 203.2

42.0 146.6

•Thymidine at 400 jiM. **Measured by procedure of Cheng, et a l . , (14). #Not assayed.

These biochemical and a n t i v i r a l screening studies (10,12-16) suggest that FEAU should be more s e l e c t i v e i n i t s a n t i v i r a l a c t i v i t y and thus offer less host t o x i c i t y . In v i v o experiments i n mice (10,13) show that both FMAU and FEAU are r e l a t i v e l y nontoxic. However, FMAU i s very neurotoxic i n dogs ( l e t h a l dose 2.5 mg/kg/day, i.v. x 5), while preliminary studies on FEAU i n dogs at 50 mg/kg/day x 10 show no encephalopathy ( l e t h a l dose 100 mg/kg/day x 10). As mentioned previously, FMAU exhibited dosel i m i t i n g CNS t o x i c i t y i n patients with advanced cancer (1) at an intravenous dose of 0.8 mg/kg/day x 5. The t o x i c i t y of FEAU i n humans i s not known. COMPARATIVE STUDIES IN MONKEYS AGAINST SIMIAN VARICELLA VIRUS. Studies at the Delta Regional Primate Research Center compared the a c t i v i t i e s of FEAU and FMAU i n African green monkeys infected with simian v a r i c e l l a v i r u s (SVV). As shown i n Table VI, the three untreated controls exhibited marked viremia and died by day 11. Monkeys treated with FEAU at three dose l e v e l s (intravenous route) showed no apparent t o x i c i t y even at the higher dose of 30 mg/kg/day x 10. Hematology tests and serum chemistries f o r a l l treated monkeys were normal and viremia ( r e l a t i v e to the controls) was minimal even at the low dose of 3 mg/kg/day. While the control monkeys developed severe rash, none of the FEAU-treated monkeys developed rash at these drug l e v e l s . Further studies showed that a lower dose of 1 mg/kg/day prevented development of rash but did not reduce viremia i n two out of three monkeys. These data suggest that the minimal e f f e c t i v e dose i n this system for FEAU i s ~1 mg/kg/day. Concurrent studies with FMAU showed i t to be ~40-fold more potent against SVV with a minimal e f f e c t i v e dose of ~0.04 mg/kg/day x 10. FEAU was a l s o h i g h l y e f f e c t i v e i n the treatment of Simian v a r i c e l l a v i r u s by the o r a l route. Oral administration at dose l e v e l s of 10, 3, or 1 mg/kg/day x 10 prevented rash (Table VII) and reduced viremia s i g n i f i c a n t l y . Even at the 1 mg dose, rash was almost e n t i r e l y prevented (two v e s i c l e s appeared on day 9, then promptly disappeared). Doses at 10 mg/kg/day x 10 by the o r a l route

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Table VI. Evaluation of FEAU in Treatment of Simian V a r i c e l l a Virus Infection in the African Green Monkey: Effect on Viremia Viremia** - Mean PFU on Days p . i . Treatment Group*

Monkey Number

3

5

7

9

G029 GO 30 G031

1 3 1

140 163 99

>400 >400 >400

Dead >400 Dead

Dead

FEAU 30 mg/kg/day

G023 G024

1 2

0 0

0 0

0 0

0 0

FEAU 10 mg/kg/day

G025 G026

1 0

14 1

5 1

0 0

0 0

FEAU 3 mg/kg/day

G027 GO 2 8

1 0

8 1

0 1

0 0

0 0

Control H0

-

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•Treatment was administered by i.v. i n j e c t i o n twice d a i l y beginning 48 hours after virus inoculation and continuing for ten days ••Viremia was determined by culture of lymphocytes c o l l e c t e d from 3 mL of heparinized blood on indicated days post-infection (p.i.). The mean plaque-forming units expressed i s the average number of plaques present in two f l a s k s of Vero c e l l co-cultures inoculated with each lymphocyte suspension.

were without any observed t o x i c i t y . Thus, FEAU i s shown to be a highly e f f e c t i v e and s e l e c t i v e a n t i v i r a l in the treatment of SVV infection by both the intravenous and o r a l routes. In view of: a) the in v i t r o a c t i v i t y (15,18) of FIAC and FMAU against v a r i c e l l a zoster v i r u s (VZV, a member of the human herpesv i r u s group), b) the reported e f f i c a c y of FIAC for the treatment of v a r i c e l l a zoster v i r u s in Phase 2 t r i a l s in immunosuppressed patients (6), and c) the in v i v o a c t i v i t i e s of FMAU and FEAU against simian v a r i c e l l a v i r u s described herein and elsewhere ( 18), one may expect that FEAU w i l l also exhibit s i g n i f i c a n t s e l e c t i v e a c t i v i t y against VZV in humans. COMPARATIVE ANTI-HEPATITIS VIRUS STUDIES IN WOODCHUCKS. Hepatitis B v i r u s (HBV), a member of the Hepadna viruses, causes acute and chronic h e p a t i t i s in humans. It i s estimated that ~200 m i l l i o n people are c a r r i e r s of this v i r u s . HBV may be the primary causative agent of h e p a t o c e l l u l a r carcinoma (19). Hepadna viruses have also been discovered in other animals such as the woodchuck (Marmota Monax). The close s t r u c t u r a l and c l i n i c a l pathological s i m i l a r i t i e s , including nucleic acid homology (20), noted between woodchuck h e p a t i t i s v i r u s (WHV) and HBV suggest that the woodchuck may represent a promising model for studying persistent h e p a t i t i s v i r u s infections as w e l l as t h e i r r e l a t i o n s h i p to the development of l i v e r cancer (21). Like HBV, the woodchuck h e p a t i t i s v i r u s

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

10. FOX ET AL.

Arabinosyl-Pyrimidine Nucleosides

elaborates a very s i m i l a r DNA polymerase for i t s r e p l i c a t i o n and integration. Potential anti-HBV agents can be detected by their i n h i b i t i o n of endogenous WHV or HBV DNA polymerase obtained from sera prepared from chronic-carrier woodchucks and from an immunosuppressed patient p o s i t i v e for the hepatitis B surface antigen. Such studies were undertaken at the Hepatitis Virus Unit, INSERM, Lyon, France, to measure the inhibitory effects of certain nucleoside triphosphates on these endogenous v i r a l DNA polymerases. In a series of assays (23,24* « Trepo, personal communication), the r e l a t i v e s e n s i t i v i t i e s of HBV and WHV DNA polymerases to several nucleoside triphosphates were determined (Table VIII). Of the six nucleoside triphosphates examined, FMAU was the most e f f i c i e n t i n h i b i t o r of both HBV and WHV DNA polymerases, followed c l o s e l y by FIAC. Moreover, the potencies (ID Q» ) of each of these six triphosphates against HBV or WHV DNA polymerases, though not i d e n t i c a l , were rather close. More important, the orders of potency as i n h i b i t o r s of these v i r a l DNA polymerases were i d e n t i c a l . These r e s u l t s attest further to the s t r i k i n g s i m i l a r i t i e s between HBV and WHV and point to the v a l i d i t y of the woodchuck as an animal model of choice for the i n v i v o evaluation of p o t e n t i a l a n t i h e p a t i t i s B v i r u s agents (24,). Studies were then undertaken to evaluate FMAU and FEAU i n this animal model using woodchucks n a t u r a l l y c h r o n i c a l l y - i n f e c t e d with woodchuck h e p a t i t i s v i r u s (25). [It was assumed that these nucleosides would be anabolized to their nucleoside triphosphates within the animal.] WHV r e p l i c a t i o n was measured p e r i o d i c a l l y by the endogenous DNA polymerase a c t i v i t y and by the detection of WHV DNA by the dot-blot hybridization assay. The v i r a l DNA polymerase a c t i v i t i e s of untreated woodchucks are given i n Figure 1. Three d i f f e r e n t dosages of FMAU and FEAU were investigated. FMAU at 2.0 mg/kg/day x 5, (given i n t r a p e r i t o n e a l l y beginning at day 0), produced marked i n h i b i t i o n of WHV r e p l i c a t i o n as shown by the complete suppression of WHV DNA polymerase a c t i v i t y (Figure 2). However, at this dose, severe CNS t o x i c i t y was observed, which was e v e n t u a l l y l e t h a l . FEAU administered i.p. at 2.0 mg/kg/day x 10 a l s o produced almost immediate suppression of WHV r e p l i c a t i o n and did not exhibit any toxic effects. At 0.2 mg/kg/day, FMAU (given x 5) and FEAU (given x 10) were e q u a l l y e f f e c t i v e i n suppressing v i r a l r e p l i c a t i o n (Figure 3). At this dose, FMAU exhibited a less severe and delayed t o x i c i t y , whereas FEAU was again nontoxic. Even at doses of 0.04 mg/kg/day x 10, FEAU exerted a somewhat diminished anti-WHV effect. A preliminary study with one woodchuck given FEAU by o r a l administration at a dose of 0.2 mg/kg/day x 10 gave s i g n i f i c a n t suppression of WHV r e p l i c a t i o n , again without any observed t o x i c i t y (Figure 4). After cessation of drug administration, the i n h i b i t o r y a c t i v i t y of FEAU at 0.2 or 2.0 mg doses remained s i g n i f i c a n t over a six-week period while returning slowly to pretreatment l e v e l s . FEAU i n h i b i t i o n of WHV r e p l i c a t i o n was almost immediate and was markedly more sustained than i s the case with other a n t i v i r a l s such as 6deoxyacyclovir [9-(2-hydroxyethoxymethyl)-2-aminopurine], DHPG [9(l,3-dihydroxy-2-propoxymethyl)guanine], or Ara-AMP [9-(0-Da

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183

n

d c

5

s

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

184

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Table VII. Evaluation of Oral Dosage of FEAU i n the Treatment of Simian V a r i c e l l a Virus Infection i n the African Green Monkey: Effect on Rash*

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Rash _ jSeverity on Days p . i . Treatment Group*

Monkey Number

6

Control PBS**

F644 G668 G604



FEAU 10 mg/kg/day

G249 G267 G264

FEAU 3 mg/kg/day

G665

FEAU 1 mg/kg/day

7

8

9

10

11

12

14

+

3+

4+

-

-

+

±

3+

Dead 1+ 4+

±

+

4+ 2+ 4+

2+

+

+

-

-

-

-

-

G257 G268 G269 G270 G274

+

•Treatment was administered by stomach tube twice d a i l y beginning 48 hours after v i r u s inoculation and continuing f o r ten days. ••Phosphate buffer saline. #Severity of rash was graded on a scale of + to 4+. A + rash was scored when several v e s i c l e s were observed while a 4+ rash indicated the widespread d i s t r i b u t i o n of rash over the body surface. Table VIII. Comparative Inhibitory A c t i v i t i e s of Nucleoside Triphosphate Analogs on DNA Polymerases of Human Hepatitis Virus (HBV) and Woodchuck Hepatitis Virus (WHV) ID Inhibitors" FMAU-TP FIAC-TP BVDU-TP Ara T-TP ACV-TP Ara C-TP

5 0

HBV DNA Polymerase 0.025 0.05 0.25 0.30 0.90 1.10

+

(uM)* 1

WHV DNA Polymerase" " 0.05 0.10 0.30 0.40 0.70 1.20

'ID50 = Concentration of i n h i b i t o r giving 50% i n h i b i t i o n of DNA polymerase a c t i v i t y . ••These inhibitors are the nucleoside 5'triphosphate d e r i v a t i v e s of: FMAU, l-(2'-deoxy-2'-fluoro-0-Darabinofuranosy 1 )-5-methyluraci 1; FIAC, l-(2'-deoxy-2'-fluoro-p-Darabinofuranosyl)-5-iodocytosine; BVDU, E-5(2-bromovinyl)-2 deoxyuridine; Ara-T, 1-0-D-arabinofuranosylthymine; ACV, 9-(2hydroxyethoxymethy 1)guanine; Ara-A, 9-0-D-arabinofuranosy 1 adenine. DNA polymerase was assayed as described by Kaplan et a l . (22), as modified by Hantz et a l . (23) r

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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10.

FOXETAL.

Arabinosyl—Pyrimidine Nucleosides

185

Figure 1. WHV DNA Polymerase A c t i v i t y in Untreated C h r o n i c a l l y Infected Woodchucks (Controls). Each symbol (o—0, T—T, or •) represents i n d i v i d u a l woodchucks, with points on the l i n e indicating the day when blood was taken for polymerase assay. DNA polymerase a c t i v i t y was measured as described by Kaplan et a l . (22), as m o d i f i e d by Hantz et a l . (23.).

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FMAU

-10

0

10

20

DAYS

Figure 2. Comparison of Inhibition of WHV R e p l i c a t i o n in C h r o n i c a l l y infected Woodchucks by Treatment with FMAU and FEAU at Dosage and Schedule Indicated. The length of the bar gives the drug treatment time period in days indicated on the abscissa. Each of the symbols (A—A, • — o r A—A) refers to i n d i v i d u a l woodchucks, with points on the l i n e representing the days when bloods were drawn for polymerase assay. WHV DNA polymerase a c t i v i t y was measured according to Kaplan et a l . (22), as modified by Hantz et a l . (23.).

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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10. FOX ET AL.

Arabinosyl-PyrimidineNucleosides

F i g u r e 3. I n h i b i t i o n o f WHV R e p l i c a t i o n i n C h r o n i c a l l y I n f e c t e d Woodchucks by Treatment w i t h FMAU and FEAU at Lower Dosages. (See F i g u r e 2 f o r l e g e n d ) .

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Figure 4. I n h i b i t i o n of WHV Replication i n C h r o n i c a l l y Infected Woodchucks Treated with FEAU by the Oral Route. FEAU was administered by stomach tube. (See Figures 1 and 2 for legend.)

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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189

arabinofuranosyl)adenine 5'-phosphate. These l a t t e r drugs a l s o diminished WHV DNA polymerase l e v e l s , but they soon rebounded to pretreatment l e v e l s after cessation of drug administration (C, Trepo, personal communication). In contrast to previous r e s u l t s obtained with FMAU and FIAC (where l e t h a l t o x i c i t y was demonstrated), only an ~10% weight loss was observed following FEAU treatment (24). The observed e f f i c a c y of FEAU against woodchuck h e p a t i t i s v i r u s r e p l i c a t i o n at these low doses was rather surprising. On the basis of the i n v i t r o studies on Herpes simplex viruses (9,10), the i n v i v o studies on the herpes encephalitis model in mice (10), and the simian v a r i c e l l a v i r u s studies i n the African green monkey reported herein, one would have expected that FEAU would be much less potent than FMAU. The data suggest that FEAU may be an e f f e c t i v e agent c l i n i c a l l y against h e p a t i t i s B v i r u s . On the basis of i t s potency and s e l e c t i v i t y , i t appears to be the most promising of the nucleoside analogs tested thus far. Further evaluation of FEAU i s i n progress.

ACKNOWLEDGMENTS These studies were supported i n part by National Cancer Institute grants CA-08748, 18601 and 18856 (JJF, KAW, TCC), by CA-44094 and The Veterans Administration (RFS), by the National Institute of A l l e r g y and Infectious Diseases grant NO-A1-62521 (KFS), and by the I n s t i t u t National de l a Sante' et de Recherche Medicale (IF, OH, CT).

LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9.

Watanabe, K. A.; Reichman, U.; Hirota, K.; Lopez, C.; Fox, J. J. J. Med. Chem. 1979, 22 21-24. Fox, J. J.; Lopez, C.; Watanabe, K. A., in Medicinal Chemistry Advances; de las Heras, F. G.; Vega, S., Eds.; Pergamon Press: Oxford, 1981; pp 27-40. Chou, T. C.; Feinberg, A.; Grant, A. J.; Vidal, P.; Reichman, U.; Watanabe, K. A.; Fox, J. J. Cancer Res. 1981, 41, 3336-3342. Coderre, J. A.; Santi, D. V.; Matsuda, A.; Watanabe, K. A.; Fox, J. J. J. Med. Chem. 1983, 26, 1149-1152. Young, C. W.; Schneider, R.; Leyland-Jones, B.; Armstrong, D.; Tan, C.; Lopez, C.; Watanabe, K. A.; Fox, J. J.; Philips, F. S. Cancer Res. 1983, 43, 5006-5009. Leyland-Jones, B.; Donnelly, H.; Groshen, S.; Myskowski, P.; Donner, A. L.; Fanucchi, M.; Fox, J. J. J. Infectious Dis. 1986, 154, 430-436. Fanucchi, M. P.; Leyland-Jones, B.; Young, C. W.; Burchenal, J. H.; Watanabe, K. A.; Fox, J. J . Cancer Treatment Rep. 1985, 69, 55-59. Watanabe, K. A.; Su, T-L.; Reichman, U.; Greenberg, N.; Lopez, C.; Fox, J. J. J. Med. Chem. 1984, 27, 91-94. Perlman, M. E.; Watanabe, K. A.; Schinazi, R. F.; Fox, J. J. J. Med. Chem. 1985, 28, 741-748.

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10. Fox, J. J.; Watanabe, K. A.; Schinazi, R. F.; Lopez, C. In Herpes Viruses and Virus Chemotherapy; Kano, R.; Nakajima, A., Eds.; Excerpta Medica: Amsterdam, 1985; pp 53-56. Based upon proceedings of International Symposium on Pharmacological and Clinical Approaches to Herpes Viruses and Virus chemotherapy presented in Oiso, Japan, September, 1984. 11. Schinazi, R. F.; Peters, J.; Sokol, M. K.; Nahmias, A. J. Antimicrobial Agents Chemother. 1983, 24, 95-103. 12. Chou, T-C.; Kong, X-B.; Potter, V. P.; Schmid, F. A.; Fox, J . J . ; Watanabe, K. A.; Fanucchi, M. Proc. Amer. Assoc. Cancer Res., 1985, 26, 333. 13. Chou, T-C.; Kong, X-B.; Fanucchi, M. P.; Cheng, Y-C.; Takahashi, K.; Watanabe, K. A.; Fox, J. J. Antimicrobial Agents Chemother. 1987, 31, 1355-1358. 14. Cheng, Y-C.; Dutschman, G.; Fox, J. J.; Watanabe, K. A.; Machida, H. Antimicrobial Agents Chemother. 1981, 20, 420-423. 15. Lopez, C.; Watanabe, K. A.; Fox, J. J. Antimicrobial Agents Chemother. 1980, 17, 803-806. 16. Mansuri, M. M.; Ghazzouli, I.; Chen, M. S.; Howel, H. G.; Brodfuehrer, P. R.; Benigni, D. A.; Martin, J. C. J. Med. Chem. 1987, 30, 867-871. 17. Machida, H.; Kuninaka, A.; Yoshino, H. Antimicrobial Agents Chemother. 1982, 21, 358-361. 18. Soike, K. F.; Cantrell, C.; Gerone, P. J. Ibid., 1986, 29, 2025. 19. Blumberg, B. S.; London, W. T. In Clinical Management of Gastrointestinal Cancer: Decosse, J. J.; Sherlock, P., Eds.; Martinus Nijhoff Publishers: Boston, 1984; pp. 71-91. 20. Galibert, F.; Chen, T. V.; Mandart, E. Proc. Natl. Acad. Sci. U.S.A., 1981, 78, 5315-5319. 21. Blumberg, B. S. Hum. Pathol. 1981, 12, 1107-1113. 22. Kaplan, P. M.; Greenman, R. I.; Gerin, J. L.; Purcell, R. H.; Robinson, W. S. J . Virol. 1973, 12, 995-1005. 23. Hantz, O.; Ooka, T.; Vitvitski, L.; Pichoud, C.; Trepo, C. Antimicrobial Agents Chemother. 1984, 25, 242-246. 24. Hantz, O.; Allaudeen, H. S.; Ooka, T.; De Clercq, E.; Trepo, C. Antiviral Res. 1984, 4, 187-199. 25. Fourel, I.; Hantz, O.; Watanabe, K. A.; Fox, J. J.; Trepo, C. Hepatology 1987, 7, 1122. RECEIVED January 15, 1988

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