Antiviral effects of phosphonoformate (PFA, foscarnet sodium)

Pharmac. Ther. Vol. 19. pp. 387 to 415, 1983

0163-7258/83/030387-29514.50/0 Copyright © 1983 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

Specialist Subject Editor: D. SHUGAR

ANTIVIRAL

EFFECTS OF PHOSPHONOFORMATE (PFA, FOSCARNET SODIUM) Bo

OB ER G

Department of Antiviral Chemotherapy, Research and Development Laboratories, Astra Liikemedel AB, S-151 85 S6dertiilje, Sweden

1. INTRODUCTION The number of new antiviral compounds has increased sharply in recent years. This has been due to both increased efforts to synthesize new inhibitors and to a more systematic design of compounds likely to interfere with viral enzymes (Helgstrand and (3berg, 1980). The main progress has been within the area of herpesvirus inhibitors where the viral thymidine kinase and DNA polymerase have been the primary targets. The reasons for the concentration on antiherpes drugs have been the high frequency of herpesvirus infections and the doubtful outlook for vaccination against labial, genital or ocular herpes infections. One of the new compounds showing promising effects against these infections is trisodium phosphonoformate (International Nonproprietory Name: foscarnet sodium). About ten years ago we observed that pyrophosphate and oxalic acid inhibited influenza virus RNA polymerase activity. This served as a starting point for structural variations around these compounds and led to the synthesis of trisodium phosphonoformate (Fig. 1). It was found that trisodium phosphonoformate inhibits influenza virus RNA polymerase (Helgstrand et al., 1978; Stridh et al., 1979). Mao et al. (1975) reported that phosphonoacetic acid inhibits herpesvirus DNA polymerase, and this explained the earlier observed antiherpes activity in cell-culture and in animals (Shipkowitz et al., 1973). Trisodium phosphonoformate, through independent experiments by Helgstrand et al. (1978) and Reno et al. (1978), was also found to inhibit herpesvirus DNA polymerase and herpesvirus replication in a manner similar to that of phosphonoacetic acid. However, several differences in the activities of phosphonoformate and phosphonoacetate have been detected, both concerning the range of enzymes and viruses inhibited and the toxicity. In the literature, trisodium phosphonoformate, phosphonoformate, phosphonoformic acid, PFA and foscarnet sodium have been used to denote the same compound. The free phosphonoformic acid is not stable, but the name has been used in analogy to the stable phosphonoacetic acid (PAA). In most published reports PFA denotes trisodium phosphonoformate and PAA phosphonoacetic acid. In both cases the actual charge of the acid groups will depend on the pH of the solution. The name foscarnet will be used in this paper. A few reviews have appeared describing PAA (Overby et al., 1977; Hay et al., 1977; Boezi, 1979) and foscarnet (Helgstrand et al., 1980b). This paper will discuss mainly the antiviral properties of foscarnet. For comparison, PAA and some other compounds will also be included. Some toxicological and pharmacokinetic properties of foscarnet will be presented. 2. CHEMISTRY The structure of foscarnet sodium is shown in Fig. 1. The compound was first synthesized by NylOn (1924) by alkaline hydrolysis of triethyl phosphonoformate. Naqui et al. 387

388

B. O~ER¢~

-0 --I~--C\o

-

3Na

O-

FiG. I. Structure of foscarnet sodium (trisodium phosphonoformate, PFA).

(1971) determined the crystal structure of foscarnet sodium and found an unusually long P - - C bond. At low pH values foscarnet decomposes to form carbon dioxide and phosphorous acid (Warren and Williams, 1971). The pK, values of foscarnet are 7.27, 3.41 and 0.49 (Warren and Williams, 1971), which are lower than the corresponding values for PAA, 8.60, 5.40 and 2.30 (Boezi, 1979). The solubility in water at pH 7 is about 5 ~ (w/w), and it has a low solubility in dimethylsulfoxide. The possibility of forming metal complexes of foscarnet and PAA has been discussed by Perrin and Sttinzi (1981). Phosphonoacetate forms a &ring chelate with metals such as Mg 2+, Ca 2+, Cu 2+ and Zn 2+. Similar chelate 5-rings would be expected for foscarnet. On the other hand, phosphonopropionate, which has no antiviral activity, should form a 7-membered ring, which is likely to be less stable.

3. E F F E C T S ON ENZYMES The effect of foscarnet on a wide range of polymerases and two nucleases has been determined, and the results are summarized in Table 1.

3.1. RNA POLYMERASES The only RNA polymerase which is inhibited by low concentrations of foscarnet is influenza virus RNA polymerase. The degree of inhibition depends on the ionic conditions and the presence of primers in the assay (Stridh et al., 1979). If Mn 2+ is used instead of Mg 2 + the concentration of foscarnet required for a 50% inhibition of the RNA polymerase decreases from 30-60/tM to 0.1-0.4/~M for different influenza strains. The inhibition of vesicular stomatitis virus RNA polymerase by foscarnet has been used to confirm the sequential transcription of the viral genes N NS M G (Chanda and Banerjee, 1980). A 90% inhibition of the polymerase activity is obtained at a concentration of 5 m~a foscarnet, and the inhibition is not dependent on the Mg 2 + concentration in the assay. 3.2. REVERSE TRANSCRIPTASES As shown in Table 1 the inhibition of reverse transcriptase by foscarnet differs between reverse transcriptases from different retroviruses. Reverse transcriptase activities from Rauscher murine leukemia virus (RMuLV), simian sarcoma virus (SSV), baboon endogenous virus (BaEV), bovine leukemia virus (BLV), avian myeloblastosis virus (AMV) and and visna virus (VV) are inhibited by 90~, or more at 100 llM foscarnet when (rA),, .(dT)10 is used as template (Sundqvist and Oberg, 1979). Reverse transcriptase from AMV is inhibited by 50~,; at 7 10/,M foscarnet (Sundqvist and Oberg, 1979; Reno et ell., 1980; Modak et al., 1980; Eriksson et ell., 1982b) and from Moloney murine leukemia virus at 10/tM foscarnet (Margalith et el/., 1982) when poly(rA)-oligo(dT) is used as template. Using the same template, Varnier et al. (1982) found that 100/*m foscarnet inhibits reverse transcriptase from Friend murine leukemia virus by 61~,] and from AKR murine leukemia virus by 30%. Using activated DNA as template,

Antiviral effects of phosphonoformate

389

TABLE t. Inhibition o f E n z y n w Activities by Foscarnet

Enzyme RNA polymerases Influenza A Victoria Influenza B HK Vesicular stomatitis virus Reovirus Calf thymus I Calf thymus II Escherichia coil

Reverse transcriptases Avian myeloblastosis virus

Avian myeloblastosis virus Rauscher murine leukemia virus Rauscher murine leukemia virus Moloney murine leukemia virus DNA polymerases HSV-1, several strains HSV-I KOS strain HSV-2 strain 91075 HSV-2 333 strain Human cytomegalo virus Ad169 Epstein-Barr virus Herpesvirus of turkeys Hepatitis B virus Hepatitis B virus Woodchuck hepatitis B virus Calf thymus ~ Hela cell ~ Mouse thymus c~ Hela cell fl Hela celt ~, Calf thymus 7 Mouse thymus fl Micrococcus luteus Escherichia coil

RNase AMV, RNase H DNase HSV-I 3'-5'-exonuclease

Concentration (#M) of foscarnet giving 50% inhibition 29 61 500 > 500 > 500 > 500 > 500 5-8

1(~55 0.7 2

10 0.4-3.5 0.(~18 0.5 0.(~22 0.3 2 3 2.5 10~20 100 < t00 50 32 55 not inhibited not inhibited > 500 > 500 > 500 > 500 > 500 2.4

Reference Stridh et al., 1979 Stridh et al., 1979 Chanda and Banerjee, 1980 Helgstrand et al., 1980b Helgstrand et al., 1978 Helgstrand et al., 1978 Helgstrand et al., 1978 Sundquist and Oberg, 1979 Reno et al., 1980 Eriksson and Oberg, 1982b Modak et al., 1980 Sundquist and Oberg, 1979 Modak et al., 1980 Margalith et al., 1982 Helgstrand et al., 1978 Eriksson and C)berg, 1979 Ostrander and Cheng, 1980 B. Eriksson (per. comm.) Ostrander and Cheng, 1980 Eriksson et al., 1982a Datta and Hood, 1981 Reno et al., 1978 Nordenfelt et al., 1980 Hess et al., 1980 Nordenfelt and Werner, 1980 Reno et al., 1978 Reno et al., 1978 Modak et al., 1980 Reno et al., 1978 Reno et al., 1978 B. Eriksson (per. comm.) Modak et al., 1980 Helgstrand et al., 1978 Helgstrand et al., 1978 Modak et al., 1980 Derse and Cheng, 1981

the 50~o inhibition of AMV reverse transcriptase has been reported to occur at 55/AM (Modak et al., 1980) and 5 #M foscarnet (Eriksson et al., 1982b) and at 80 #M foscarnet when Moloney murine leukemia virus reverse transcriptase is used (Margalith et al., 1982). The reason for this difference is not clear. Reverse transcriptase from RMuLV is more sensitive to foscarnet, showing a 50~o inhibition at 0.7/tM (Sundqvist and Oberg, 1979) and 2 #M (Modak et al., 1980) in two different studies using (rA),'(dT)lo as template. The influence of the assay conditions on the effect of foscarnet is illustrated in Table 2, in which different templates are used (Sundqvist and Oberg, 1979). Both the RNA and the DNA dependent reactions are inhibited, but the pattern of inhibition depends on the reverse transcriptases. In Table 2 is also given results from Modak et al. (1980). It is not clear why the results for AMV reverse transcriptase differ when (rC),.(dG)12 18 is used as template, and why the results for RMuLV reverse transcriptase disagree when (rC),'(dG)12_xs and (dC),'(dG)12_18 are used as templates. Margalith et al. (1982) also found that a high concentration (250 pM) of foscarnet is required to inhibit Moloney murine leukemia virus reverse transcriptase when (rC),,'(dG)12 18 is used as template. The endogenous reaction (without added template) is less sensitive to foscarnet

B. (~)BERG

390

TABLE 2. Inhibition o! Reverse Transcriptase Activity by lOOpM Foscarm, t Percent inhibition

Template/Primer

Avian myeloblastosis virus

Rauscher murine leukemia virus

Bovine leukemia virus

(rA),,. (dT h o (rC),. (dG)l 2_ 18 (dC),,' (dG)~ 2-18

96 (90) 2 (50) 44 (50)

96 (98) 97 ( < 50) 84 ( < 50)

88 82 14

The figures are taken from Sundquist and Oberg (1979). Figures from Modak et al. (1980) are given in parenthesis.

than the reaction primed by poly(rA), oligo (dT) (Sundquist and Oberg, 1979; Margalith et al., 1982). 3.3. DNA POLYMERASES All tested herpesvirus DNA polymerase are inhibited by foscarnet. As shown in Table, 1, the sensitivity of herpes simplex virus (HSV) type 1 and 2 DNA polymerase depends on the virus strain. The HSV-1 strain C42 DNA polymerase is inhibited by 500L at 0.4 p~ and HSV-1 strain 124 at 3.5 M foscarnet (Helgstrand et al., 1978; Helgstrand and Oberg, 1978; Eriksson et al., 1980). In cell-culture the C42 strain has an average sensitivity to foscarnet (B. Oberg, unpublished observation). Ostrander and Cheng (1980) found that the degree of purification of HSV-1 and 2 DNA polymerase and the ionic strength in the assay strongly influence the sensitivity to foscarnet. At an ionic strength of 0.21, crude, phosphocellulose and DNA cellulose-purified HSV-2 DNA polymerase are inhibited by 50~,, at 0.6, 1.7 and 3 pM foscarnet, respectively. At an ionic strength of 0.31, the 50~, inhibition is seen at 16 pM, and at an ionic strength of 0.38 at 24 pM foscarnet (Ostrander and Cheng, 1980) when the DNA cellulose-purified enzyme is used. The human cytomegalovirus (HCMV) strain Ad 169 DNA polymerase is inhibited by 50% at 0.3 pN foscarnet (Eriksson et al., 1982a), herpesvirus of turkeys DNA polymerase at 2.5/~M foscarnet (Reno et al., 1978) and Epstein-Barr virus DNA polymerase at 2-3 l~M foscarnet (Datta and Hood, 1981). It is likely that other herpesvirus DNA polymerases are also inhibited by foscarnet, considering the sensitivity of herpesviruses to foscarnet in cell-culture (see Table 4), and in analogy to the pattern of inhibition shown by PAA (Boezi, 1979). Both human hepatitis B virus and woodchuck hepatitis virus DNA polymerases are inhibited by foscarnet (Nordenfelt et al., 1979, 1980; Nordenfelt and Werner, 1980). It is probable that the degree of inhibition depends on the assay conditions, and Hess et al. (1980) have reported that 100gM foscarnet is required to obtain a 50?/,, inhibition of human hepatitis B virus DNA polymerase. A strain variation in sensitivity is also possible, as indicated by the inhibition of DNA polymerase from different hepatitis B patients (Nordenfelt et at., 1979, 1980). Eucaryotic DNA polymerase c~ is inhibited by 50~£ at 32-55 pg foscarnet (Helgstrand et al., 1978; Reno et al., 1978; Modak et al., 1980}. The influence of the purity of the enzyme~ the ionic strength, or other assay conditions on the sensitivity to foscarnet have not been described. No other cellular DNA polymerase has shown any sensitivity to foscarnet. 3.4. RNASE The RNase H activity associated with AMV reverse transcriptase is not atlected by foscarnet (Modak et at., 1980; Reno et al., 1980; Margalith et al., 1982).

Antiviral effectsof phosphonoformate

391

3.5. DNASE A 3'-5'-exonuclease activity associated with HSV-l DNA polymerase is inhibited by foscarnet (Derse and Cheng, 1981). The inhibition of the exonuclease by foscarnet seems to be analogous to that reported by Knopf (1979) for PAA. It has been found that foscarnet, as well as PAA and zinc ions, prevents the chromosomal abnormalities induced by HSV in cell-cultures (Nachtigal and Nachtigal, 1980). This could be explained by an inhibition of the 3'-5'-exonuclease. 4. STRUCTURE-ACTIVITY RELATIONS Several studies on the structure-activity relations for the inhibition of polymerases by pyrophosphate analogues have been published (Shipkowitz et al., 1973; Leinbach et al., 1976; Lee et al., 1976; Herrin et al., 1977; Boezi, 1979; Stridh et al., 1979; Ostrander and Cheng, 1980; Nordenfelt et al., 1980; Helgstrand et al., 1980a,b, 1981a; Eriksson et al., 1980, 1982a,b). Some results have been summarized in Table 3. These results should be comparable since the same assay conditions were used for all compounds tested with each enzyme. Furthermore, the assay conditions for HSV-I, HSV-2 and HCMV DNA polymerases were the same. However, it should be pointed out that the strain dependence has not been systematically evaluated, and it is not unlikely that this will influence the spectrum of sensitivity. It is clear from Table 3 that active inhibitors are confined within a narrow range of structures. The inhibition of herpesvirus DNA polymerases by foscarnet and PAA is similar. Other viral polymerases like those of hepatitis B virus, AMV and influenza virus are much more sensitive to foscarnet than PAA. Hepatitis B virus DNA polymerase shows an interesting inhibition profile, being sensitive to hypophosphonate and foscarnet but not to oxalic acid or PAA. The AMV reverse transcriptase responds in a similar manner. A considerable number of other pyrophosphate analogues have been tested and found to be inactive. An ester group at either the C or P end of foscarnet eliminates the inhibitory effect as shown in Table 4. Depending on the type of ester, herpesvirus multiplication can be inhibited in cell-cultures and in animal experiments (Helgstrand et al., 1980a; Nor6n et al., 1982). An ester of foscarnet can be inactive in the HSV-1 DNA polymerase assay and without effect on HSV-1 multiplication in cell-culture but still inhibit the viral infection in guinea pig skin. This suggests that an ester like p-methyl foscarnet is hydrolyzed to foscarnet in guinea-pig skin but not in a cell-culture.

5. EFFECTS ON VIRUS MULTIPLICATION Several DNA and RNA viruses have been assayed for sensitivity to foscarnet as shown in Table 5. Viruses using cellular DNA polymerases, such as polyoma and adeno, are not inhibited by 100 pM foscarnet. The multiplication of vaccinia virus is not inhibited by foscarnet, in contrast to the inhibition seen with PAA (Overby et al., 1977). All herpesviruses tested are inhibited by 100 pM foscarnet. Different primary isolates of HSV (Svennerholm et al., 1979) and different strains of bovine rhinotracheitis virus (Schwers et al., 1980a) and pigeon herpesvirus (Schwers et al., 1981) differ in their sensitivity to foscarnet. This is also the case for different strains of human cytomegalovirus (Wahren and Oberg, 1980). It should be pointed out that, due to its lack of thymidine kinase, human cytomegalovirus is not inhibited by most pyrimidine analogues. The inhibition of herpesviruses by foscarnet is similar to that by PAA (Boezi, 1979). The inhibition by foscarnet is dependent on the multiplicity of infection as shown by Svennerholm et al. (1979) for HSV-1 and by Arsenakis and May (1981) for HSV-2. An increased multiplicity of infection decreases the inhibition of human cytomegalovirus foci formation (Wahren and Oberg, 1980). It has also been reported for several other selective

/

\

O

\

/

rl

O

II

(PFA)

0

i

OH

r

OH

HO-- P--C--P--O H

II il ql

0

OH

HO

0

I

I

HO--P--CH:--P--OH

O

Ir

OH

O

OH

I

I

OH

O

\

O

OH

O

O

OH

I

//

OHOH

I I

O

C--C

HO---P--C

HO

O

Compound

10

> 500

> 500

0.3

150

115

HSV-1" DNA pol

27

--

500

0.5

12

400

HSV-2 h D N A pol

6

> 500

500

0.3

150

130

HCMV c DNA pol

- 500

> 500

~ 500

20

97°~i at 500

> 500

Hepatitis B a DNA pol

200

> 500

500

8

25

> 500

AMV" DNA pol

Concentration (IzM) giving 50°,i inhibition

TABLE 3. Inhibition qf Polymerase Activities hy Pyrophosphate Analogues

280

> 500

450

30

> 500

> 500

Influenza j RNA pol

100

> 500

> 500

40

250

> 500

Cellular" DNA pol

g: ©:

OH

O

OH

O

O

OH

[

H

O

O

OH

O

H

0

\

H

O

\

OH

I

I

H

C

I

II

C

OH

O

HOoP

C9H19

OH

I

\

//

\

.o_~ ~ J

C3H 7

OH

I

.oA ~ J

CH 3

OH

I

OH

O

OH

O

OH

0

OH

.o_; '~__J

H

OH

\

H

O

I

CH 3 OH

I

OH

I

.o_; ~ Lo.

H

OH

I

.o_~__~ Lo.

O

10

200

15

0.5

>500

120

160

550

0.7

260

1.5

30

18

0.4

> 500

105

>500

> 500

> 500

> 500

> 500

>500

> 500

> 500

> 500

> 500

> 500

> 500

> 500

> 500

> 500

300

>500

> 500

100

160

> 500

> 500

35

> 500

> 500

o

I

o

I

I

C

\

OH

0

\

CH2CH2--C ~

CH 3

I

C

OH

OH

O > 500

> 500

HSV-1 a D N A pol

"Eriksson et al. (1980) and B. Eriksson (pers. comm.) bB. Eriksson (pers. comm.) CEriksson et al. (1982a) aNordenfelt et al. (1980) ~Eriksson et al. (1982b) /Stridh et al. (1979) and S. Stridh (pers. comm.) ~Eriksson et al. (1980) and B. Eriksson (pers. comm.)

OH

HO--~

O

OH

HO--P

0

Compound

--

HSV_2 ~ D N A pol

500

HCMV c D N A pol

> 500

> 500

Hepatitis B d D N A pol

> 500

> 500

AMV e D N A pol

Concentration (/~M)giving 50'!J;i inhibition

TABLE 3. (Cored)

> 500

> 500

Influenza I RNA pol

> 500

> 500

Cellular s D N A pol :~

M

©:

4~

Antiviral effects of phosphonoformate

395

TABLE 4. Inhibition of HSV-I D N A polymerase, HSV-I Multiplication and HSV-1 Cutaneous h f e c t i o n by Esters of Fosearnet* O

O

II Ir

Conc. (/aM) giving 50~o inhibition of HSV-I DNA polymerase

R 10---P--C I ~ R20 OR3

Rl

R2

R3

Na CH3CH 2

Na CH3CH 2 CH3CH 2 Na

Na(PFA) CH 3 CH3CH 2 CH3CH2 Na

Na Na Na Na Na Na Na

Na H3C ~

~

Conc. (/am) giving 50~o inhibition of HSV-I (C-42) plaque formation

Effects on cutaneous HSV-1 infection in guinea-pigs

45 500 500 500 500 15 60 15 3

active not active not active not active active active active active active

0.35 35 35 35 35 35 10 10 > 35

> > > > >

"~ Na Na

> > > >

*The results are from Helgstrand et al. (1980a) and Nor6n et al. (1982).

inhibitors that an increased multiplicity of infection decreases the inhibition (Harmenberg et al., 1980; Arsenakis and May, 1981). One irido virus, African swine fever virus, is also inhibited by foscarnet (E. Vinuela, cited in Helgstrand et al., 1980b) in analogy to its inhibition by PAA (Moreno et al., 1978). Visna virus multiplication is inhibited at 100~m foscarnet (Sundquist and Larner, 1979). The relative sensitivity of reverse transcriptase from Friend murine leukemia virus TABLE 5. Inhibition of Virus Multiplication #1 Cell-Culture by Foscarnet Virus DNA viruses Polyoma Adeno type 2 HSV-I, several strains HSV-2, several strains HCMV Ad169 Epstein-Barr Simian varicella Pseudorabies Infectious rhinotracheitis Herpes virus of turkeys Marek's disease herpes virus Pigeon herpes virus Herpesvirus platyrrhine Herpesvirus saimiri Herpesvirus ateles Herpesvirus sylvilagus Murine cytomegalovirus African swine fever virus Vaccina virus RNA viruses Polio type 1 Semliki Forest Influenza, WSN Influenza Victoria Mumps Measles Vesicular stomatitis Rabies Lymphocytic choriomeningitis Visna Friend murine leukemia

~o inhibition at 100/aM PFA

Reference

not inhibited not inhibited 92-95 91 96 >90 inhibited inhibited 90 >90 70 > 90 90 ~90 >90 > 90 > 90 < 50 inhibited not inhibited

G. Magnusson, cited in Helgstrand et al., 1980b Helgstrand et al., 1978 Helgstrand et al., 1978 Helgstrand et al., 1978 Wahren and Oberg, 1980 Dana and Hood, 1981 A. D. Felsenfeld, cited in Helgstrand et al., 1980b Helgstrand et al., 1978 Helgstrand et al., 1978 Reno et al., 1978 Reno et al., 1978 Schwers et al., 1980b Daniel et al., 1980 Daniel et al., 1980 Daniel et al., 1980 Goodrich et al., 1980 Kern et al., 1978 E. Vinuela, cited in Helgstrand et al., 1980b Helgstrand et al., 1978

not not not not not not not not not

Helgstrand et al., 1978 L. K~i~iri~iinen, cited in Helgstrand et al., 1980b Helgstrand et al., 1978 L. Vrang, cited in Helgstrand et al., 1980b C. Orwell, cited in Helgstrand et al., 1980b C. Orwell, cited in Helgstrand et al., 1980b A. Banerjee, cited in Helgstrand et al., 1980b J. C. Chermann, cited in Helgstrand et al., 1980b I. Rode Pedersen, cited in Helgstrand et al., 1980b Sundquist and Larner, 1979 Varnier et al., 1982

inhibited inhibited inhibited inhibited inhibited inhibited inhibited inhibited inhibited > 90 34

396

B. OBI:R~;

and AKR murine leukemia virus corresponds to their inhibition by foscarnet in cellculture (Varnier et al., 1982). At 500 #M, foscarnet reduces the replication of AKR, Friend and Moloney murine leukemia virus in SC-I cells with a factor of 61, 457 and 1000, respectively (Varnier et al., 1982). Influenza A virus (WSN) is inhibited at 400/~M foscarnet (Stridh et al., 1979). A strain-dependent sensitivity to foscarnet has been noted for different influenza virus strains (J. Ortin, personal communication). Retroviruses and influenza virus are not inhibited by PAA in accordance with its lack of effect on reverse transcriptases and influenza RNA polymerase (Sundquist and Oberg, 1979; Stridh et al., 1979). The reversibility of inhibition by foscarnet has been studied in cell-cultures infected with herpesviruses. The multiplication of HSV-I in Vero cells determined as plaque formation is irreversibly inhibited by 100/2M foscarnet present for 24hr or more (Schn~irer and Oberg, 1981). Svennerholm et al. (1979) observed that the removal of foscarnet after 2 or 7 days of incubation from HSV-I and HSV-2 infected green monkey kidney cells only partially reverses the inhibition and reduces the final titers. A partial reversibility for HSV-1 and an irreversible inhibition for HSV-2 was found by Cheng et al. (1981) by determination of the virus yield. The replication of human cytomegalovirus in human lung fibroblasts is reversibly inhibited by foscarnet even if the time for inhibition is extended to 35 days. No foscarnet resistant virus emerges during this period (Wahren and Oberg, 1980). A similar reversibility of inhibition by foscarnet and also by PAA, acycloguanosine and interferon has been reported for two oncogenic herpesviruses, herpesvirus saimiri and herpesvirus ateles (Daniel et al., 1980). The inhibition of visna virus replication in sheep choroid plexus cells by foscarnet is also reversible after 150 hr, and the results indicate that virus replication is blocked at an early event in the lytic cycle (Sundquist and Lamer, 1979). Although different methods to determine reversibility have been used, it seems probable that there are differences between different viruses in the intracellular survival of their genomes in the presence of foscarnet. It is presently not clear if these results in cell-cultures have any implications for the in vivo situation in which host defence mechanisms are also involved.

6. M E C H A N I S M O F A C T I O N 6.1. CELL-FREE SYSTEMS Several studies on the mechanism of action of foscarnet have been published, and the results seem to agree with those reported for the action of PAA on DNA polymerases. The inhibition by foscarnet extends however to enzymes not inhibited by PAA, and some results are summarized in Table 6. The two RNA polymerases inhibited by foscarnet show different patterns of inhibition. Influenza virus RNA polymerase is inhibited in a noncompetitive manner with respect to nucleoside triphosphates (Stridh et al., 1979; S. Stridh, personal communication). The inhibition of vesicular stomatitis virus RNA polymerase is competitive with respect to ATP (Chanda and Banerjee, 1980). In this case the inhibition appears to be at the level of RNA chain initiation. The inhibition of reverse transcriptase by foscarnet is noncompetitive with respect to both substrate and template (Sundquist and Oberg, 1979; Eriksson et al., 1982b; Margalith et al., 1982). Both the RNA- and the DNA-dependent polymerase activities show the same pattern of inhibition. The lack of effect on RNase H (Modak el al., 1980; Margalith el al., 1982) is in agreement with the noncompetitive inhibition with respect to template. A rapid shutoff of the reverse transcriptase activity indicates that the elongation of the polynucleotide is affected (Sundquist and Oberg, 1979; Margalith el al., 1982). Reno el al. (1980) have reported that foscarnet is a competitive inhibitor of the PP exchange activity of AMV reverse transcriptase.

Antiviral effects of phosphonoformate

397

TABLE6. Mechanism of Inhibition by Foscarnet and Inhibition Constants

Enzyme HSV-I (C42) DNA polymerase Variable dNTPs Variable DNA HSV-I (KOS) DNA polymerase Variable dTTP Variable DNA HSV-2 (333) DNA polymerase Variable dTTP Variable DNA Human cytomegalovirus (Ad169) DNA polymerase Variable dNTPs Herpesvirus of turkeys DNA polymerase Variable dNTPs Variable DNA Epstein-Barr virus DNA polymerase Variable dNTPs Variable DNA Hepatitis B DNA polymerase Variable dNTPs Cellular DNA polymerase Variable dNTPs Variable DNA Avian myeloblastosis virus reverse transcriptase Variable dTTP Variable (rA), •(dT) l 0 Variable dTTP Variable dGTP template: (rC).. (dG) 12-18 template: (dC)..(dG)l 2_1a Variable dNTP template DNA Influenza (A Victoria) RNA polymerase Variable ATP Vesicular stomatitis virus RNA polymerase Variable ATP

Type of inhibition NC UC

Reference

Ki

Kil

Kis

0.4 0.4

0.4 0.4

0.8 25

Eriksson et al. (1980)

17 15

16

Ostrander and Cheng (1980)

16 14

18

Ostrander and Cheng (1980)

NC UC NC UC

--

NC

0.4

0.28

NC NC

---

NC UC

0.82

Eriksson et al. (1982a) B. Eriksson (pers. comm.)

0.9 2.3

Reno et al. (1978)

2.6

---

2.4 1.9

2.3 15

Datta and Hood (1981)

NC

7.2

--

NC NC

---

24 32

NC NC NC

16 9 8

-----

----

NC NC

100 0.25

---

---

Eriksson et al. (1982b)

NC

4.0

--

--

Eriksson et al. (1982b)

--

--

S. Stridh (pers. comm.)

--

--

Chanda and Banerjee (1980)

NC C

33

1.1

--

59 176

Nordenfelt et al. (1980) Sabourin et al. (1978)

Sundquist and E)berg (1979) Eriksson et al. (1982b)

Noncompetitive: NC. Uncompetitive: UC. Competitive: C. T h e i n h i b i t i o n of H S V - 1 D N A p o l y m e r a s e by foscarnet is n o n c o m p e t i t i v e with respect to the d N T P s as varied substrates and u n c o m p e t i t i v e with respect to v a r i a t i o n in the c o n c e n t r a t i o n of a c t i v a t e d D N A (with the N T P s at sat u r at ed level) as s h o w n in Figs 2 and 3 (Eriksson et al., 1980; O s t r a n d e r and Cheng, 1980). T h e same p at t er n is also s h o w n by H S V - 2 D N A p o l y m e r a s e ( O s t r a n d e r and Cheng, 1980) an d E p s t e i n - B a r r virus D N A p o l y m e r a s e ( D a t t a a n d H o o d , 1981). T h e inhibition of herpesvirus of turkeys D N A p o l y m e r a s e by foscarnet has a n o n c o m p e t i t i v e p at t er n with respect to b o t h d N T P an d D N A (Reno et al., 1978). T h e inhibition with varied t e m p l a t e seems to be n o n c o m p e t i t i v e w h e n the substrates are held at Km level an d u n c o m p e t i t i v e at s a t u r a t i n g level. T h e i n h i b i t i o n of h u m a n c y t o m e g a l o v i r u s D N A p o l y m e r a s e by foscarnet shows the same kinetic properties as for HSV-1 D N A p o l y m e r a s e (Eriksson et al., 1982a). H e r p e s v i r u s D N A p o l y m e r a s e s induced by m u t a n t s resistant to foscarnet an d P A A have been used to d e t e r m i n e the m e c h a n i s m of inhibition. It was f o u n d that D N A p o l y m e r a s e i n d u c e d by herpesvirus of turkeys resistant to P A A in a cell-free assay has a decreased sensitivity to foscarnet, indicating that these c o m p o u n d s interact at the same site on the e n z y m e ( R e n o et al., 1978). Eriksson and (3berg (1979) f o u n d that HSV-1

B. ~)BER(i

398

0.12~

,°t

0.10

,_I! 0.75-

0.06

0.04.

0.25"

0.02 0.025

1

0.

dNTP (jIM)

0.075

1 DNA (jU~l/ml)

0. 0

FIG. 3. Inhibition of HSV-I (C42) DNA polymerase by foscarnet under varied DNA concentrations. Activated DNA was used as the varied substrate and the four dNTPs were used at saturating levels.The concentrations of foscarnet were 0 (@), 0.1 ]/M (O), 0.25pM (A}, 0.50/2M (~) and 1.0 ttM (ll). The data are from Eriksson et al. (1980) with permission.

FIG. 2. Inhibition of HSV-] (C42) DNA po]y-

merase by foscarnet under varied nucleotide concentration. The four dNTPs were used as the varied substrates and activated DNA was at a saturating level. The concentrations of foscarnet were 0 (e), 0.25/~M (O), 0.50pM (A), l pM (A). The data are from Eriksson et al. (1980) with permission.

D N A polymerases from strains resistant to foscarnet a n d P A A have a cross-wise resistance to these c o m p o u n d s . These foscarnet a n d PAA resistant polymerases also have an increased resistance to i n h i b i t i o n by p y r o p h o s p h a t e but n o t to v i d a r a b i n e t r i p h o s p h a t e ( a r a A T P ) when c o m p a r e d to p o l y m e r a s e from wild-type virus. This is s h o w n in T a b l e 7 for the foscarnet resistant p o l y m e r a s e a n d indicates that foscarnet as well as PAA interacts at a p y r o p h o s p h a t e b i n d i n g site o n the p o l y m e r a s e (Eriksson a n d Oberg, 1979). This was previously p r o p o s e d by M a o a n d R o b i s h a w (1975) a n d L e i n b a c h e t al. (1976) as the m e c h a n i s m of i n h i b i t i o n for PAA. It has also been found that the presence of pyrophosphate reduces the i n h i b i t o r y action of foscarnet on E p s t e i n - B a r r virus D N A polymerase (Datta a n d H o o d , 1981). T h e i n h i b i t i o n of H e L a cell D N A polymerase ~ by foscarnet a n d PAA shows that these c o m p o u n d s are m u t u a l l y exclusive, i.e. act at the same site ( S a b o u r i n et al., 1978). The m e c h a n i s m is n o n c o m p e t i t i v e with respect to d N T P s a n d D N A ( S a b o u r i n et al., 1978). T h e D N A p o l y m e r a s e of hepatitis B virus ( D a n e particles) is inhibited in the same m a n n e r as H S V - I D N A polymerase, e.g. the reaction is n o n c o m p e t i t i v e with respect to d N T P (Nordenfelt e t al., 1980). Hess e t al. (1980) also found that this i n h i b i t i o n is not competitive to d N T P ' s . T h e p o l y m e r a s e reaction is rapidly stopped by the a d d i t i o n of foscarnet (Nordenfelt e t al., 1980; Hess e t al., 1980).

TABLE 7. Inhibition of HSV-I DNA Polymerases from Wild-Type and Foscarnet Resistant Virus*

Per cent inhibition HSV-I DNA Foscarnet polymerase 1 #M C42 C42, PFA r

70 10

Pyrophosphate 500 ,UM 1000pM 41 19

74 46

Vidarabine triphosphate 100 #~a 500 pM 57 56

77 73

*The results are taken from Eriksson and Oberg (1979). C42 = wild type; C42, PFA r = foscarnet resistant mutant of C42.

Antiviral effects of phosphonoformate

399

The inhibition by foscarnet of the 3'-5'-exonuclease associated to HSV-1 DNA polymerase is uncompetitive with respect to activated DNA, and the K~ is 2.4 #M at an ionic strength of 0.25. The same salt dependence for the 3'-5'-exonuclease as for HSV-1 DNA polymerase was also found (Derse and Cheng, 1981) indicating that these enzyme activities may have the same active site. 6.2. CELLCULTURES The time dependence for inhibition of HSV-I replication shows that foscarnet has to be added before eight hours post infection to inhibit virus production (Schn~irer and C)berg, 1981), and to obtain maximal inhibition it should be added before three hours post infection (Cheng et al., 1981). As mentioned above there are differences between different viruses in their ability to survive intracellularly in the presence of foscarnet. No virucidal effect of foscarnet has been observed. This is in accordance with an inhibition of the HSV DNA synthesis, and similar results have been observed with PAA (Overby et al., 1974). A selective inhibition of HSV- 1 DNA synthesis by foscarnet is seen when the synthesis of viral and cellular DNA is determined by isotopic labeling of the DNA and density gradient separation of viral and cellular DNA (Larsson and Oberg, 1981, 1982). The selectivity of this inhibition is shown in Fig. 4. This result is similar to that obtained with cpm x 10 .3

20* i~io

10

1.710

1.710

10

20

30

40

Fraction number FIG. 4. Selective inhibition of HSV-I DNA synthesis by foscarnet. HSV-I (C42) infected green monkey kidney cells were labeled with ortho-1-32p]phosphate at different concentrations of foscarnet. The DNA was prepared and viral and cellular DNA were separated on CsCI gradients. The striped area shows viral, DNA and the density of the peaks are given at the arrows. (a) No foscarnet added. (b) 100 I~M foscarnet. (c) 500 pM foscarnet. The figure is from Larsson and Oberg (1981) with permission.

400

B. Ot~l~G

PAA by Overby et al. (1974) and by Drach and Shipman (1977). The synthesis of Epstein-Barr virus DNA in Raji cells is also selectively inhibited by foscarnet (Datta and Hood, 1981). The presence of foscarnet will not inhibit the formation of early cytomegalovirus antigens, but late antigens are inhibited (Wahren and Oberg, 1979). The formation of Epstein-Barr virus early (EA) and nuclear antigen (EBNA) are not affected by foscarnet, while the capsid antigens are inhibited (Margalith et al., 1980; Datta and Hood, 1981). This is in accordance with the results obtained with PAA showing an inhibition of late viral capsid antigens but not of EBNA or early antigens which are not dependent on viral DNA synthesis (Summers and Klein, 1976). It has also been shown that foscarnet, as well as acycloguanosine and some other inhibitors, does not inhibit the formation of HSV-2 early antigen (Arsenakis and May, 1981). The mutiplication of HSV-I strains selected for resistance to either foscarnet or PAA shows a cross-wise resistance to these compounds (Eriksson and Oberg, 1979; Cheng et al., 1981). A PAA resistant herpesvirus of turkeys is resistant to foscarnet (Reno et al., 1978). The foscarnet resistant HSV-1 has an increased resistance to vidarabine (araA) (Eriksson and Oberg, 1979), indicating that this mutation could affect a binding-site on the DNA polymerase common to foscarnet and araA. However, the wild-type and foscarnet resistant DNA polymerases show the same sensitivity to vidarabine triphosphate (Table 7). Field et al. (1981) have described an ACG resistant HSV-1 strain [C1(101)P2C5] having an intact thymidine kinase but probably mutated in the DNA polymerase. This strain has an increased sensitivity to foscarnet and araA, indicating that a change in the binding-site for ACG triphosphate can affect the interaction of foscarnet and vidarabine triphosphate to the polymerase. The assumption is further strengthened by the observations of Schnipper and Crumpacker (1980), Coen and Schaffer (1980) and Knopf et al. (1981) that PAA resistant mutants can acquire coresistance to ACG. The replication of HSV-1 mutants with a defective thymidine kinase or lacking thymidine kinase is sensitive to foscarnet (De Clercq et al., 1980) as also shown in Table 8, and addition of nucleosides does not decrease the effect of foscarnet (Schntirer and Oberg, 1981). This clearly shows that the viral thymidine kinase is not involved in the mechanism of action of foscarnet, and it has also been observed that the HSV-1 thymidine kinase in a cell-free assay is not inhibited by foscarnet (A. karsson, personal communication). It is furthermore obvious that the lack of crosswise resistance shown in Table 8 offers the possibility of combination therapy. The effects of different combinations of antiviral drugs have been studied, and R. Schinazi and A. Nahmias (personal communication) found that foscarnet and BVDU can give a synergistic effect, and foscarnet and ACG an additive effect. The pattern of inhibition seems to depend on the virus strain used. 7. C E L L U L A R E F F E C T S The cellular toxicity of several pyrophosphate analogues has been determined and corresponds to their animal toxicity (Stenberg, 1981). Foscarnet has a low cellular toxicity (Stenberg and Larsson, 1978), and the inhibition of cell proliferation at high concentrations is reversible. The DNA synthesis and/or cell proliferation are reversibly inhibited by 50~o after incubation at 500-1000/~M PFA for 24-48 hr using HeLa cells and primary human lung fibroblasts (Stenberg and Larsson, 1978), sheep choroid plexus cells (Sundquist and Larner, 1979), African green monkey cells and W1-38 cells (Stenberg, 1981). Penetration of foscarnet into cells seems to be a very slow process. The penetration coefficient for transcellular penetration in African green monkey kidney cells was 2.5 × 10-gcm/s, which can be compared to 9.1 × 10 4cm/s for water. This might lead to a lower intracellular than extracellular concentration (K. Stenberg, personal communication). The effect of foscarnet on the cell cycle progression of Madin-Darby canine kidney cells has been investigated (Stenberg et al., 1982). After an incubation with 5 mM of foscarnet the cell cycle is delayed in the S phase, and at 10 mM foscarnet progression is

401

Antiviral effects of phosphonoformate TABLE 8. Inhibition of Herpes Simplex Virus Mutants by Antiviral Compounds HSV-I strain

100/aM foscarnet

C42 C42 PFA r C42 ACG" C915 T K -

> 90 33 75 >90

1 /aM IDU > 90 94 32 71

50/aM araA

0.1 /aM ACG

0.05/aM FIAC

> 90 87 94 >90

> 90 65 0 0

> 90 80 0 14

2/aM TFT 90 > 90 > 90 >90

5/aM araT

0.02/aM BVDU

> 90 84 0 8

79 90 18 47

The results were obtained by a determination of the plaque inhibition on Vero cells using methods described earlier (Schniirer and ()berg, 1981). C42: wild-type (Eriksson and (3berg, 1979); C42 PFAr: foscarnet resistant mutant; C42 ACGr: acycloguanosine resistant mutant; C915 T K - : thymidine kinase negative strain. The following abbreviations are used; IDU: 5-iodo-2'-deoxyuridine; araA: 9-fl-D-arabinofuranosyladenine; ACG: 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine); FIAC: l-(2-fluoro-2-deoxy-fl-D-arabinofuranosyl)-5iodocytosine; TFT: 5-trifluoromethyl-2'-deoxyuridine; araT: l-fl-D-arabinofuranosylthymine; BVDU: E-5-(2bromovinyl)-2'-deoxyuridine.

blocked near the G1/S' border. This accumulation is not seen until 15 hr of incubation. Inhibited cells rapidly initiate DNA synthesis when foscarnet is removed although the cell-cycle is delayed. After treatment of quiescent human embryonic cells (large portion in G s phase) with up to 10 mM of foscarnet for 3 days and then subcultivating in drug-free medium, the treated cells passed through the cell-cycle in phase with control cells (Fig. 5). The mechanism by which foscarnet affects the cell cycle progression is probably through a reversible inhibition of cellular DNA polymerase(s), which is in agreement with its effect on cell-free DNA polymerase ~. The long term effect of foscarnet has been studied by serial passage of human embryonic lung fibroblasts in 100 #M foscarnet (A. Macieira-Coelho, personal communication). As shown in Fig. 6 the total number of passages possible is not affected by the continuous presence of foscarnet, indicating that the cells are not accumulating damages to the genetic material and are not immortalized. The growth of mycoplasma is not affected by foscarnet, nor is bacterial growth affected (S. Bengtsson and L. Magni, cited in Helgstrand et al., 1980b). 8. TREATMENT OF VIRUS INFECTIONS IN ANIMALS Most human herpesvirus infections are cutaneous, mucocutaneous, or involve corneal cells. In all these instances a topical medication should have the advantage of rapid drug 10.

(a)

t(b) SO

pC

5.

:1

o

2'0

40

6C) 8=0 Time(h)

100 165

2b

40

~0 810 Time(h)

FIG. 5. Cell cycle progression of human diploid fibroblasts after 72 hr treatment with foscarnet. Quiescent fibroblast cultures were treated with foscarnet for 72 hr. Each culture was washed twice, trypsinized and subcultivated on petri dishes. At indicated times cells were trypsinized and the cell number and volume were determined (Stenberg eta/. 1982). (a) Cell number (b) Cell volume. (11) Control; (A)1 mM foscarnet; (0) 10 mM foscarnet.

10=0 165

402

B. OB~RG Human lung flbroblasts o-o e--e

-

~ "'0" p " •

Control 100 j4M foscarnet

O.

b.

~

m -~

O105

o,

"

~o t

~O-Q

g

18 ~o

do

4'o

go

~o"

Number of subcultures

FIG. 6. Passage of human embryonic fibroblasts in the presence of foscarnet. Human embryonic lung fibroblasts were grown on glass coverslips and split one to two after outgrowth. One set of cells was grown without foscarnet and one with 100Fa~ foscarnet present. (A. Macieira-Coelho, personal communication).

delivery only to the site necessary. Several animal models have been used to mimic the human infections, and the effects of topically applied foscarnet have been investigated. The results are summarized in Table 9, 8.1. CUTANEOUS HERPES INFECTIONS

Alenius et al. (1978) determined the therapeutic effect of foscarnet on cutaneous HSV-1 infections in guinea-pigs. A therapeutic effect on the lesions is seen down to 5 mM foscarnet in water solution applied twice daily and starting 48 hr after virus inoculation. A total of four topical applications of 2~o (w/w) (60 m~a) foscarnet starting 48 hr after inoculation is sufficient to give a therapeutic effect on the lesions. Treatment can be delayed to three days after inoculation and still result in a therapeutic effect. The appearance of foscarnet and placebo treated infections is shown in Fig. 7. Foscarnet also reduces the virus titers in the skin after topical treatment (Alenius, 1980; Alenius et al., 1982). When starting treatment 48 hr after inoculation, two daily treatments for three days using a 2% (w/v) foscarnet water solution reduce the virus titre in the skin by 99~0 or more. An intraperitoneal treatment with 100mg/kg per day also has a therapeutic effect on the lesions (Alenius et al., 1978). Several different HSV strains have been used for the cutaneous infection of guinea pigs, and both HSV-I and 2 strains cause infections sensitive to topical treatment with foscarnet (Alenius et al., 1978, 1982). When a foscarnet resistant HSV-I mutant is used, the topical treatment with foscarnet has little effect on the virus titre in the skin (Alenius, 1980), suggesting that the mechanism of action of foscarnet in this model is an inhibition of the viral DNA polymerase. This is analogous to the lack of effect of PAA on an infection in mice by a PAA resistant strain of HSV-I (Klein and Friedman-Kien, 1975). The cutaneous HSV-I infection in guinea-pigs has been used as a model to compare the therapeutic effects of topically applied antiherpes compounds (Alenius and Oberg, 1978; Alenius, 1980; Alenius et al., 1978, 1982; Helgstrand et al., 1979; C)berg et al., 1982). Foscarnet and PAA have been superior to all other tested compounds applied topically in this model, according tc the lesion scores. This is surprising since foscarnet and PAA are considerably less active than several nucleoside analogues in cell-culture tests (De CIercq et al., 1980), which is shown for foscarnet in Table 8. The effects of topical treatment with foscarnet and ACG are shown in Figs 8a and 8b. Acyclovir, in both polyethylene glycol and dimethylsulfoxide, is considerably less active than foscarnet in this model (Alenius et al., 1982). The same picture emerges when the virus titre in the skin

MCMV Simian varicella HSV- 1 PH V- 1 SHV-1 Woodchuck hepatitis B

Mice

Patas monkey

Mice

Pigeon

Rabbit

Woodchuck

Latency

Pigeon herpesvirus infection Pseudorabies infection Hepatitis B

HSV- 1

Mice

Cytomegalovirus infection Simian varicella

HSV-2

Mice

Systemic herpes virus infection Herpes encephalitis

HSV-1 HSV-2 HSV-I

Mice

Rabbits

HSV-2

Mice

HSV-2

Guinea-pig

Herpes keratitis

Genital infection

HSV-2

HSV-I

Nude mice

Guinea-pig

HSV-I

Hairless mice

Genital herpes infection

HSV-I H SV-2

Guinea-pig

Cutaneous herpes infection

Virus

Animal

Disease

10~o foscarnet suspension, topical 10~o foscarnet suspension, topical 8~o foscarnet suspension, topical 3~o foscarnet solution 5~o foscarnet ointment 250 mg/kg of foscarnet twice daily, i.p. 400 mg/kg of foscarnet twice daily, i.p. 250 mg/kg of foscarnet twice daily, i.p. 100 mg/kg of foscarnet twice daily, i.p. 500 mg/kg/day of foscarnet, i.p. during 4 weeks 100 mg/kg/day of foscarnet, i.m. 100 mg/kg/day of foscarnet, i.m.

3~o foscarnet, topical

l~o foscarnet, topical

3~o foscarnet, topical

5 100 mr,I foscarnet (0.15-3~o) topical

Treatment

Kern et al. (1978) Kern et al. (1978) Kern et al. (1978) K.F. Soike (pers. comm.)

Increased mean day of death Decreased mortality Increased mean day of death Decreased mortality Decreased rash and viremia

Vindevogel et al. (1981) Vindevogel et al. (1981) Nordenfelt et al. (1982)

No effect No effect

Svennerholm et al. ( 1981) No effect

No effect on latency

Alenius et aL (1980)

Decreased keratitis

Kern et al. (1978)

Kern et al. (1978)

Alenius and Nordlinder (1979)

Descamps et al. (1979)

Kern et al. (1981)

Helgstrand e t a l . (1978) Alenius et al. 0978) Helgstrand et al. (1979) Alenius, 1980 Oberg et al. (1982) Klein et al. (1979)

References

Decreased lesions Decreased mortality Decreased mortality Decreased lesion formation Decreased lesions Decreased virus shedding Normal weight gain Prevention of paralysis Decreased lesions Decreased virus titers Decreased virus shedding Decreased mortality Reduced virus shedding

Decreased lesions Decreased time to healing Decreased virus titers in skin

Effects

TABLE 9. Efficacy o f Foscarnet on Herpesvirus Infections in Animals

,--1

0

0

=r O

t'~

404

B. Q)BFRG

FIG. 7. Effect of topically applied foscarnet on cutaneous HSV-1 infection m guinea-pigs. A guinea-pig was inoculated with HSV-I (strain C42) on four separate sites on the back. Treatment started 48 hr after inoculation and consisted of two daily applications of 30 ~1 2°~i (w/w) foscarnet in water containing 0.1'~i Tweeen and I00,, glycerol. The foscarnet treatment was given to the upper left and lower right areas. The upper right and lower left areas were used as controls receiving water containing 0.11~, Tween and 1()I~,, glycerol. The result of 3 days of treatment is shown. (S. Alenius, personal communication).

is determined as illustrated for foscarnet and ACG in Fig. 9 (Alenius et al., 1982). It is also clear that the effect of treatment can be strain dependent (Fig. 8). A cutaneous HSV- I infection in hairless mice has been used to evaluate the effect of a topically applied 100 mM (3')~) water solution of foscarnet (Klein et al., 1979). The lesion score and mortality rate decrease when treatment with foscarnet is started 3 hr after inoculation of virus in the lumbosacral area. The mortality rate is significantly reduced even when treatment is instituted 24 hr after inoculation. When an orofacial inoculation is used, topical treatment can be delayed until 24 hr after inoculation and still cause a significant reduction in lesion score. The effects of several antiherpes compounds were compared by Descamps et al. (1979) on a cutaneous HSV-I infection in athymic nude mice. In this model foscarnet, PAA, ACG and BVDU emerge as the most active agents when applied topically as 1~,, cream preparations twice daily starting immediately after virus inoculation. The establishment of latency in the hairless mouse model after topical treatment with foscarnet, initiated 3 hr after virus inoculation, was compared to the results of some other compounds (Klein et al., 1979). The frequency of latent infections is lower when A C G or PAA is used than when foscarnet is used. However, the comparison could have been influenced by the vehicles used. The clinical relevance of this difference is not clear since few patients with a primary genital infection are likely to apply a medication within a few hours after being infected. It has been found (R. J. Klein, personal communication) that

Antiviral effects of phosphonoformate

405 III

I 1

o o

I,S

g

_o .9

a

I ® Z

O,5

i

13 1/, DAYS POST INOCULATION

TREATMENTS

"re

I

2.5

®

2

lI

u

/\

® 1.5 u

o

z

%

'll

O.S

t

2 3 t. 5 lift tttt tttt TREATMENTS

6

7

8

9

"It

i

i

!

10

It

12

I

13

1/*

DAYS POST INOCULATION

FIG. 8. Effect of foscarnet and acyclovir treatment on the development of lesions in guinea-pigs inoculated with different strains of HSV-I. (a) Sixteen guinea-pigs were inoculated on four sites on their backs with HSV-1 strain C42. Topical treatment started 48 hr after inoculation and consisted of four daily applications of 30 kd cream, ointment or solution during three days. Each point represents the average score from 16 skin areas (four guinea-pigs). (,0) Untreated areas; (O O) 5~o Acylovir polyethylene glycol ointment; (111 II) 3~o Acyclovir in dimethylsulfoxide; (I-1 IS]) 3~o Foscarnet cream. (b) Fifteen guinea-pigs were inoculated on their backs with HSV-I strain 79 and treated as in A. Each point represents the average of 20 skin areas (five guinea-pigs). ( H ) Untreated areas; (O ©) 5~o Acyclovir polyethylene glycol ointment; ( D - - D ) 3~o Foscarnet cream. The figures are from Alenius et al. (1982) with permission.

incubation in vitro of HSV infected mouse ganglia in the presence of foscarnet, or other antiviral compounds, does not influence the possibility of reactivation once the drug is removed. Svennerholm et al. (1981) found that mice latently infected in their trigeminal ganglia and subsequently treated with foscarnet, vidarabine, acyclovir, idoxuridine or PAA retained their latent infections. To date no convincing evidence has been presented showing an effect of any antiherpes drug on an established latent infection. 8.2. GENITAL HERPES INFECTIONS Genital infections with HSV-2 in female guinea-pigs and mice have been used to evaluate the effect of foscarnet treatment (Alenius and Nordlinder 1979, Kern et al., 1978). In guinea-pigs, topical application of a 3~o (w/w) foscarnet gel or ointment has a therapeutic effect preventing any signs of infection when the treatment is started one or

406

B. OILERS; 7 ¸

6

E s

3u..

um

Z•

-:z Doql,

|

o

o 4 o

2

. . . . . . . . . . . . .

";"

I

°-I-

I

-- ~1h ~~°°~I- - - L I M I T I

I

OF VIRUS A S S A Y

PLACEBO 0 . 0 5 % 0,5% 3% PLACEBO 5% UNTREA- )MSO 3% ACYCLOVIR CREAM FOSCAR- FOSCAR-FOSCAR- OINT- ACYCLO- TED IN DMSO NET NET NET MENT VIR CREAM CREAM CREAM OINTMENT

FIG. 9. Effect of treatment with foscarnet and acyclovir on HSV-I strain C42 titer in guinea-pig skin. Thirtysix guinea-pigs were inoculated on four sites on their backs with HSV-I strain C42 and the virus titer in the skin at day five after inoculation was determined. The mean virus titer for 16 skin areas (four guinea-pigs) is shown for each treatment group. Each point represents the virus content in one skin area. The topical treatment started 48 hr after inoculation and consisted of four daily applications of 30 /d cream, ointment or solution during three days. The results are from Alenius et al. (1982) with permission. four hours after virus inoculation (Alenius and Nordlinder, 1979). W h e n treatment is delayed until 24 hr after inoculation, the development of lesions is not prevented. Kern et al. (1978) also found an early topical treatment of HSV-2 infected guinea-pigs more effective than a late. W h e n the intravaginal treatment using 10~,, foscarrlet in 0.4~, agarose is started 6 hr after inoculation, viral replication is prevented and no lesions develop. If the treatment is initiated 24 hr after inoculation, a 4-5 log decrease in virus titers is seen on days 3, 5 and 7, and a slight delay occurs in the development of lesions. In a genital HSV-2 infection in female mice, Kern et al. (1978) found a decrease in vaginal virus titers after topical treatment with 10~o foscarnet P F A in 0.4Yo agarose when treatment is initiated both at 3 and 24 hr after inoculation. A 5% P A A cream has a similar therapeutic effect. A c o m p a r i s o n of the effects of a 8~o foscarnet suspension in 0.4~o agarose and a 5~o P A A cream (equimolar concentrations) showed that both topical treatments are active on genital infections in mice (Kern et al., 1981). These infections were caused by several HSV-I and HSV-2 strains and the treatments reduce virus shedding and mortality. The P A A cream was m o r e effective but this might have been due to the difference in vehicle. It is presently unclear why effective treatment can be instituted later in the cutaneous than in the genital infection models.

8.3. HERPES KERATITIS A c o m p a r i s o n of the effects of topically applied foscarnet and idoxuridine on herpes keratitis in rabbits revealed that foscarnet has a therapeutic effect but is not as effective as idoxuridine in rapidly reducing the corneal lesions (Alenius et al., 1980). The same result was obtained using herpesvirus-immunized and n o n i m m u n i z e d animals. An effect of foscarnet treatment is only seen when frequent applications are given. It is possible that this depends on the slow penetration of foscarnet into cells, (K. Stenberg, personal c o m m u n i c a t i o n ) and that foscarnet in contrast to nucleoside analogues is not trapped intracellularly by p h o s p h o r y l a t i o n but is washed away by the tear fluid. A treatment with foscarnet, however, could be of importance for patients with thymidine kinase negative virus infections resistant to nucleoside analogues requiring this enzyme for activation. The mechanism of action of foscarnet on herpes keratitis seems to be an inhibition of the

Antiviral effects of phosphonoformate

407

viral DNA polymerase, since a HSV-I mutant with a PFA resistant polymerase gives an infection with increased resistance to treatment (Alenius et al., 1980). 8.4. SYSTEMIC AND INTRACEREBRAL INFECTIONS Herpes encephalitis in mice, induced by intracerebral inoculation with HSV-I, has been treated twice daily by intraperitoneal injections of foscarnet (400 mg/kg) for 5 days. This has only a slight effect on the mean day of death (Kern et al., 1978). A similar slight effect was observed when mice were inoculated intraperitoneally with HSV-2 and then treated intraperitoneally with foscarnet (250 mg/kg) twice daily for 5 days. These results are probably explained by a short half-life of foscarnet in serum and a limited penetration through the blood-brain barrier (Helgstrand et al., 1980b). K. F. Soike (personal communication) found a decrease in viremia and rash after intramuscular treatment of paras monkeys infected with simian varicella virus. These monkeys were given 100 mg/kg intramuscularly twice daily. A murine cytomegalovirus infection was treated intraperitoneally with foscarnet (250mg/kg) twice daily for 5 days, and a slightly decreased mortality or increased number of days till death was observed (Kern et al., 1978). The murine cytomegalovirus, however, is less sensitive to foscarnet than the human cytomegalovirus (Kern et al., 1978, Wahren and Oberg, 1979, 1980). Vindevogel et al. (1982) found that intramuscular administration of foscarnet four times daily (100mg/kg per day) does not affect the fatal outcome of herpes virus of pigeons infection in pigeons or pseudorabies infection in rabbits. The explanations for these results could be a low sensitivity to foscarnet for herpes virus of pigeons and a neural spread of pseudorabies-virus not affected by foscarnet (Schwers et al., 1980a,b, 1981). Woodchucks chronically infected with woodchuck hepatitis virus have been used to evaluate the effect of subcutaneous administration of foscarnet (Nordenfelt et al., 1982). The results show that even at serum levels sufficiently high to inhibit the enzyme by more than 50~, no effect on the level of DNA polymerase activity or other viral parameters in the blood is seen after two weeks of treatment. The interpretation of this result could be that the virion DNA polymerase is not necessary for the continued virus production in a chronic infection. This possibility has been suggested recently by Tiollais et al. (1981).

9. PHARMACOKINETICS Helgstrand et al. (1980b) have described some pharmacokinetic properties of foscarnet. After intravenous administration to young mice, foscarnet is distributed as shown in Fig. 10. No metabolic conversion of foscarnet takes place, and the major part of foscarnet is rapidly eliminated from soft tissues and excreted in the urine. A very limited penetration of the blood-brain barrier occurs. The blood half-life of foscarnet, administered as a single dose to different animal species, is shown in Table 10. In 17 g mice, about 30~o of the systemically available foscarnet is deposited in bone and cartilage. In adult mice, this figure decreases to less than 10~o (J. Lundstr6m, personal communication). The uptake is not concentration dependent, and foscarnet is only retained by bone tissue and not by bone marrow. The elimination of foscarnet from bone is shown in Fig. 11 in which mice have been followed for several months (J. LundstriSm, personal communication). In this case 17 g mice were used during the administration of foscarnet. Similar results were obtained with adult mice. Using a 3~o (w/w) foscarnet cream containing 14C-labeled foscarnet, the percutaneous adsorption has been studied in guinea-pigs (J. Lundstr6m, personal communication). The adsorption of foscarnet through intact skin is 2-4~o and through striped skin 60-75~o. Some foscarnet (7-10~o) remains in the skin 48 hr after administration and 24 hr after washing.

408

B. O~ERG

'4C.PFA nmollg I

,

/~ ~'~ 100

;

'

~

~

BONE

"

r;' i

lO

1.o-

0.1HEART

FIG. 10. Tissue levels of [~4C] foscarnet following i.v. administration in the mouse. A single dose of 80/~mol/kg of [14C] foscarnet was given to mice (20 g). At each time point three animals were used to determine the tissue levels (mean values + S.D.). The results are from Helgstrand et al. (1980b) with permission.

10. T O X I C O L O G Y Toxicity studies have been reported for foscarnet by Helgstrand et al., (1980b). The acute toxicity (LDso) in mice is 510 mg/kg when given intravenously and > 4 g/kg when given orally. In rats the corresponding figures are > 800 mg/kg and > 2 g/kg. In a general toxicity study, rats were given 4.5-195 mg/kg per day and dogs 1.5-122 mg/kg per day as a 2~o foscarnet solution administered subcutaneously each day during one month. The parameters followed in this study are indicated in Table 1 I. No difference was seen between the control group and any of the dose groups in the rat or the dog study. The possible effects on cartilage and bone were specifically followed in this study as shown in Table 12. No adverse effects were seen. In addition to these general toxicity studies, it was found that foscarnet does not affect vitamin D metabolism in rats, nor does it affect in vitro sulfate incorporation into cartilage tissue (E. Larsson and H. Brostr6m, cited in Helgstrand et al., 1980b). A fertility study in rats and teratogenicity studies in rats and rabbits were carried out according to FDA guide-lines. Rats were given 12-150 mg/kg per day and rabbits 12-75 mg/kg per day subcutaneously. Fertility and general reproductive performance are not adversely affected under these experimental conditions. No adverse effects of foscarnet on TABLE 10. Blood Elimination Half-life of Foscarnet after Single Dose Administration*

Dose (mg/kg)

Route of administration

Blood half-life time (hr)

Mouse (NMRI, 20 g) Monkey (cynomolgus, 4.2 kg)

24 36

Dog (beagle, 19.6 kg)

50

Pig (78 kg)

30

i.v. i.v. s.c. i.v. s.c. i.v. s.c.

0.7t 1.2 1.2 2.0 2.3 3.6 3.3

Animal

*The figures are taken from Helgstrand et al. (1980b). tEstimated from the second declination phase after fitting blood leveltime data to a three c o m p a r t m e n t model. The terminal half-life was 6.8 hr.

Antiviral effects of phosphonoformate

409

Retention of "C-foscamet in the femurs of mice following a single s.c. dose (24 mg/kg) foscarnet pg/g of femur 300 200 -

100-

!

50

o

'

lbo

'

26o

'

3~o

Days after s,c. dose

FIG. 11. Elimination of foscarnet from bone. Mice (17 g) were given [14C]foscarnet as an s.c. injection and the release of foscarnet from bone was followed (J. Lundstr6m, pers. comm.)

the dams were seen in any group in the teratogenicity studies. Furthermore, foscarnet does not adversely affect litter size, fetal loss, litter and mean pup masses or frequency of malformations. The dermal toxicity of topically applied foscarnet has been studied in pigs (Helgstrand et al., 1980b), and a one hour daily application of 3~o (w/w) foscarnet cream for 20 days does not result in any erythema, edema or microscopic changes. Foscarnet has a considerably lower dermal toxicity than PAA in guinea-pigs (Alenius et al., 1978) and cynomolgus monkeys (B. Oberg, unpublished observation). In a tolerance study in healthy male volunteers, no skin irritation was observed after patch tests with 3~o (w/w) foscarnet cream on the upper part of their backs. 11. CLINICAL STUDIES A clinical study on cutaneous herpes using a 3~o (w/w) foscarnet cream has been carried out in Sweden, and similar studies are presently in progress in several countries. TABLE l l. Toxicity after Repeated Administration of Foscarnet to Rats

and Dogs Daily dose ltM/kg (mg/kg)

Dose groups

Rat n = 6 per sex and group

Dog n = 1 per sex and group

1 2 3 4 5

15 (4.5) 40 (12) 100 (30) 250 (75) 650 (195)

5 (1.5) 15 (4.5) 45 (14) 135 (41) 405 (122)

Dosing: Route of administration: Test c o m p o u n d :

Records:

Result:

J.p.T. 19/3 H

Once a day, seven days a week for one month. Subcutaneous injection Phosphonoformic acid as 2~o (w/w) solution (pH 7) of trisodium salt hexahydrate. Clinical observation. Clinical chemistry. Pathology. No toxic effects. Modified after Helgstrand et al. (1980b).

B. Q)BERG

410

TABLE I2. Studies ()['Possible Ef.:fects on Cartilage and Bone q/ter Repeated Administration of Foscarnet to Rats and Dogs Clinical chemistry

Calcium Phosphate

Pathology Histochemical study of glucosaminoglycans

Tracheal cartilage Cartilage joint surface of knee-joint (rat) Cartilage joint surface of interphalangeal joint (dog) Epiphyseal growth plate of femure bone (rat) Cellular appearance and matrix of bone structure (dog)

Morphology

Dosing and administration of foscarnet as in Table I 1. Result: No adverse effects. (C. Lundberg, pers. comm.)

The design and results of the first Swedish study have been presented in preliminary reports (Wallin et al., 1980, 1982a,b; Helgstrand et al., 1981b). The study population consisted of patients having a history of recurrent cutaneous (labial) herpes. The study was carried out double-blind placebo controlled and after the first episode studied patients were put on a cross-over study for another two episodes, During the cross-over study medication was kept at home to ensure early treatment institution. The results from this study show that an early topical treatment with foscarnet cream shortens the vesicular period (Fig. 12). Furthermore, as compared to placebo treated, fewer foscarnet treated patients tend to develop new vesicles during treatment. A subjective preference for the foscarnet cream as compared to the placebo cream was also noted (p < 0.05). The topical treatment with 3% (w/w) foscarnet cream in labial and cutaneous herpes is well tolerated. 12. D I S C U S S I O N AND C O N C L U S I O N S Structure-activity studies on the effect of pyrophosphate analogues on RNA and DNA polymerases show a narrow spectrum of active structures, foscarnet and PAA being the most active on herpesvirus DNA polymerase (Tables 1, 3 and 4). Although similar in structure, foscarnet and PAA differ in their inhibition of influenza virus RNA polymerase, reverse transcriptases and DNA polymerase from hepatitis B viruses. The mechanism of action on polymerases is the same, however, and is noncompetitive with respect to

CumulaUve percentage of patients who have passed the vesicular stage. First treab'nent study- early treatment. CUM%

100•

Fo:

•

PIi

p
1

2

3

4

5

Days from start of symptoms

FIG. 12. Therapeutic effect of foscarnet on recurrent herpes labialis. Patients with recurrent herpes labialis were treated topically with 3% (w/w) foscarnet cream and the time to pass the vesicular stage was recorded. (Wallin et al., 1982b, with permission).

Antiviral effectsof phosphonoformate

411

NTP's and uncompetitive with respect to template when the substrate is at saturating level (Table 6). It is likely that both foscarnet and PAA interact at a pyrophosphate binding site on the polymerases (Lee et al., 1976; Boezi 1979; Eriksson and Oberg, 1979). This is also suggested by the increased resistance to (i) inhibition by pyrophosphate, observed when DNA polymerase from foscarnet resistant HSV-I was compared to that from wild-type virus (Table 7), and (ii) the reduced inhibitory action of foscarnet in the presence of pyrophosphate (Datta and Hood, 1981). The inhibition of viral replication by foscarnet in cell-cultures (Table 5) corresponds well to its effect on isolated viral enzymes. The relative inhibition of HSV-t and cellular DNA polymerase ~ in the cell-free assay by foscarnet is reflected in a selective inhibition of herpesvirus DNA synthesis in the cell (Fig. 4). Higher concentrations of foscarnet are required for inhibition of virus multiplication (Table 5) than for inhibition of viral polymerases (Table 1). This could partly be due to a slow penetration into cells and also to real differences in sensitivity of the polymerases, depending on the conditions in the cell and in the cell-free assay. This is indicated by the influence of purification of the enzyme on sensitivity to foscarnet (Ostrander and Cheng, 1980). Mutants of HSV-I selected for resistance to foscarnet induce a DNA polymerase resistant to foscarnet (Eriksson and Oberg, 1979). Animal infections with this foscarnet resistant virus have an increased resistance to foscarnet treatment, indicating that the mechanism of action in the infection animal is an inhibition of HSV-1 DNA polymerase (Alenius, 1980; Alenius et al., 1980). The selection of the vehicle and the use of a virus strain with a known and appropriate sensitivity to the drugs tested are obvious although sometimes neglected considerations for a meaningful evaluation of therapeutic effects in animal models. It is surprising that topical application of foscarnet has a better therapeutic effect on cutaneous HSV-1 infections in guinea-pigs than nucleoside analogues with considerably lower IDso values in cellculture experiments (Alenius and Oberg, 1978; Alenius et al., 1978, 1982). This might be due to a better skin-penetration of foscarnet than of nucleoside analogues and could also be due to a high thymidine concentration in the skin (J. Harmenberg, personal communication). Thymidine competes with several nucleoside analogues in the phosphorylation by viral thymidine kinase, whereas the inhibition by foscarnet is not affected by thymidine (SchniJrer and Oberg, 1981). Subcutaneous administration of foscarnet to woodchucks with chronic hepatitis did not result in any therapeutic effects (Nordenfelt et al., 1982), suggesting that treatment of chronic hepatitis B in man with foscarnet will not be feasible. However, a systemic administration of foscarnet to patients with severe cytomegalovirus infections seems motivated, especially since HCMV is sensitive to foscarnet but not to most nucleoside analogues. A short half-life in blood and soft tissues has been found when foscarnet is administered subcutaneously or intravenously, but a longer retention occurs in bone and cartilage tissue (Helgstrand et al., 1980b). The similarity to phosphate and pyrophosphate and a tendency to form calcium complexes are probably the reasons why foscarnet binds to the inorganic matrix of bone. In the one month general toxicity study (Table 11), however, no adverse effects were observed in rats and dogs at doses of up to 195 and 122 mg/kg per day subcutaneously, and this also includes careful histopathological studies on bone and cartilage (Table 12). It should be pointed out that the amount of foscarnet systemically available after topical application of 100pl 3% cream to the lips of a 70kg man can be estimated to 0.002 mg/kg of which most is likely to be rapidly eliminated. The wide safety margin and lack of skin irritation together with a good therapeutic effect on cutaneous infections in animals made clinical trials desirable. It is evident from the results of the first clinical trial (Wallin et al., 1980, 1982c) using a 3% (w/w) foscarnet cream that therapeutic effects have been obtained on labial herpes (Fig. 12). A therapeutic effect on recurrent labial herpes has probably not been observed earlier in any double-blind placebo controlled study (Overall, 1979; Spruance et al., 1980).

412

B, OBI!R¢;

The effect of foscarnet on genital herpes in animal models and the lack of any clinically effective treatment of genital herpes have prompted clinical studies to be started and these are now in progress. The possibility of resistance development has been studied by determination of the sensitivity to foscarnet of HSV isolates from patients treated during a few episodes of recurrent labial herpes. So far no resistance development has been observed and further studies are ongoing. The lack of cross-resistance for most mutants selected in cell-culture for resistance to foscarnet and nucleoside analogues gives the option of combination therapy or change of drug if resistance has developed to one drug. A combination therapy might also have the advantage of synergistic effects. In the clinical study on labial herpes using 33;, (w/'w) foscarnet cream, the effect on the recurrency rate is presently followed. The results with infected animals treated with antivirial drugs have not shown any effect on the latent infection. It is possible, however, that treatment of several episodes of recurrent herpes might lead to a decreased rate of recurrency. Note: Recent clinical results show that topical administration of 0.3'~, foscarnet cream has a significant therapeutic effect on recurrent genital herpes (J. Wallin, pets. comm.). Acknowledgements For kindly providing unpublished data and wlluable suggestions, 1 thank Drs. S. Alenius, B. Eriksson, J. Harmenberg, T. Hovi. N. G. Johansson, R. J. Klein. A. Larsson, J.-O. Lernestedt, C. Lundberg, J. Lundstr6n, E. Lycke, M. Margatith, A. Macieira-Coelho, J.-O. Noren, E. Nordenfelt, J. Ortin, P.-P. Pastoret, R. Schinazi, K. F. Soike, K. Stenberg, S. Stridh, J. Wallin, and O. E. Varnier. I also thank Mrs. G. Br~nnstrCJm for performing virus titrations, Mrs M. Kropp and Mrs. G. Norlin for typing the manuscript and Mrs. J. de Paulis for linguistic corrections.

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ERIKSSON, B., Q)BERG,B. and WAHREN,B. (1982a) Pyrophosphate analogues as inhibitors of DNA polymerases of cytomegalovirus, herpes simplex virus and cellular origin. Biochim. biophys. Acta. 696: 115-123. ERIKSSON, B., STENING, G. and OBERG, B. (1982b) Inhibition of reverse transcriptase activity of avian myeloblastosis virus by pyrophosphate analogues. Antiviral Res. 2:81-95. FIELD, H., MCMILLAN, A. and DARBY, G. (1981) The sensitivity of acyclovir-resistant mutants of herpes simplex virus to other antiviral drugs. J. infect. Dis. 143: 281-285. GOODRICH, J. M., LEE, K. W. and HINZE, H. C. (1980) In vitro inhibition of herpesvirus sylvilagus by phosphonoacetic acid and phosphonoformate. Arehs Virol. 66:261 264. HARMENBERG,J., WAHREN, B. and OBER6, B. (1980) Influence of cells and virus multiplicity on the inhibition of herpes viruses with acycloguanosine, lntervirology 14: 239-244. HAY, J., BROWN, S. M., JAMIESON,A. T., RIXON, F. J., MOSS, H., DARGAN, D. A. and SUBAK-SHARPE,J. H. 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