Treatment of a latent infection of herpes virus

A method for the treatment of latent infection of herpesviruses in mammals, including humans, which method comprises administering to the mammal in need of such treatment, an effective amount of a compound of formula (A): ##STR1## or a bioprecursor, or a pharmaceutically acceptable salt, phosphate ester and/or acyl derivative of either of the foregoing.

This invention relates to treatment of latent infection of herpesviruses. 
When used herein, `treatment` includes prophylaxis as appropriate. 
EP-A-141927 (Beecham Group p.l.c.) discloses penciclovir, the compound of 
formula (A): 
##STR2## 
and salts, phosphate esters and acyl derivatives thereof, as antiviral 
agents. The sodium salt hydrate of penciclovir is disclosed in EP-A-216459 
(Beecham Group p.l.c.). Penciclovir and its antiviral activity is also 
disclosed in Abstract P.V11-5 p.193 of `Abstracts of 14th Int. Congress of 
Microbiology`, Manchester, England 7-13 Sep.1986 (Boyd et. al.). 
Orally active bioprecursors of the compound of formula (A) are of formula 
(B): 
##STR3## 
and salts and derivatives thereof as defined under formula (A); wherein X 
is C.sub.1-6 alkoxy, NH.sub.2 or hydrogen. The compounds of formula (B) 
wherein X is C.sub.1-6 alkoxy or NH.sub.2 are disclosed in EP-A-141927 and 
the compounds of formula (B) wherein X is hydrogen, disclosed in 
EP-A-182024 (Beecham Group p.l.c.) are preferred prodrugs. A particularly 
preferred example of a compound of formula (B) is that wherein X is 
hydrogen and wherein the two OH groups are in the form of the acetyl 
derivative, described in Example 2 of EP-A-182024, hereinafter referred to 
as famciclovir. 
The compounds of formulae (A) and (B) and salts and derivatives thereof 
have been described as useful in the treatment of infections caused by 
herpesviruses, such as herpes simplex type 1 and herpes simplex type 2. 
Previous work has shown that if antiviral treatment is delayed beyond a few 
hours after infection then latency is established. Once a latent infection 
is established, the infection can recurr. 
It has now been shown in mice that famciclovir treatment can prevent the 
establishment of competent latency when treatment is commenced 18 h (first 
experiment) and up to 4 days (second experiment) after infection. It has 
also now been shown that latency can be prevented in an experiment in 
immunocompromised mice. The potential clinical advantage is that a 
patient, within 4 days of contact, may be treated with famciclovir to 
prevent not only the acute infection but also the development of latency 
and so avoid recurrences. Furthermore, it is thought that there may be a 
slow natural loss of latently infected cells and recurrent infections may 
be required in order to maintain the burden of latently infected cells by 
establishing latency in new cells. Therefore, suppressive treatment with 
famciclovir over a prolonged period (up to several years) may prevent new 
cells becoming latently infected. The result would then be curative 
treatment, the patient having no recurrences thereafter. 
Accordingly, the present invention provides a method of treatment of latent 
infection of herpesviruses in humans, which method comprises the 
administration to the human in need of such treatment, an effective amount 
of a compound of formula (A): 
##STR4## 
or a bioprecursor, or a pharmaceutically acceptable salt, phosphate ester 
and/or acyl derivative of either of the foregoing. 
The term `acyl derivative` is used herein to include any derivative of the 
compounds of formula (A) in which one or more acyl groups are present. 
Such derivatives are included as bioprecursors of the compounds of formula 
(A) in addition to those derivatives which are per se biologically active. 
The compound of formula (A) may be in one of the forms disclosed in 
EP-A-216459 (Beecham Group p.l.c.). 
Examples of bioprecursors, pharmaceutically acceptable salts and 
derivatives are as described in the aforementioned European Patent 
references, the subject matter of which are incorporated herein by 
reference. 
A particular compound of formula (B) of interest is 
9-(4-acetoxy-3-acetoxymethylbut-1-yl)-2-aminopurine, known as famciclovir 
(FCV), the well-absorbed oral form of penciclovir (PCV). 
The compound of formula (A), bioprecursors, salts and derivatives may be 
prepared as described in the aforementioned European Patent references. 
The compound, in particular, famciclovir, may be administered by the oral 
route to humans and may be compounded in the form of syrup, tablets or 
capsule. When in the form of a tablet, any pharmaceutical carrier suitable 
for formulating such solid compositions may be used, for example magnesium 
stearate, starch, lactose, glucose, rice, flour and chalk. The compound 
may also be in the form of an ingestible capsule, for example of gelatin, 
to contain the compound, or in the form of a syrup, a solution or a 
suspension. Suitable liquid pharmaceutical carriers include ethyl alcohol, 
glycerine, saline and water to which flavouring or colouring agents may be 
added to form syrups. Sustained release formulations, for example tablets 
containing an enteric coating, are also envisaged. 
For parenteral administration, fluid unit dose forms are prepared 
containing the compound and a sterile vehicle. The compound depending on 
the vehicle and the concentration, can be either suspended or dissolved. 
Parenteral solutions are normally prepared by dissolving the compound in a 
vehicle and filter sterilising before filling into a suitable vial or 
ampoule and sealing. Advantageously, adjuvants such as a local 
anaesthetic, preservatives and buffering agents are also dissolved in the 
vehicle. To enhance the stability, the composition can be frozen after 
filling into the vial and the water removed under vacuum. 
Parenteral suspensions are prepared in substantially the same manner except 
that the compound is suspended in the vehicle instead of being dissolved 
and sterilised by exposure to ethylene oxide before suspending in the 
sterile vehicle. Advantageously, a surfactant or wetting agent is included 
in the composition to facilitate uniform distribution of the compound of 
the invention. 
Preferred parenteral formulations include aqueous formulations using 
sterile water or normal saline, at a pH of around 7.4 or greater, in 
particular, containing penciclovir sodium salt hydrate. 
As is common practice, the compositions will usually be accompanied by 
written or printed directions for use in the medical treatment concerned. 
A suitable dosage unit might contain from 50 mg to 1 g of active 
ingredient, for example 100 to 500 mg. Such doses may be administered 1 to 
4 times a day or more usually 2 or 3 times a day. The effective dose of 
compound will, in general, be in the range of from 0.2 to 40 mg per 
kilogram of body weight per day or, more usually, 10 to 20 mg/kg per day. 
in the case of famciclovir, the dosage unit would be 125 mg, 250 mg, 500 
mg or 750 mg, preferably 125 mg or 250 mg. 
For prevention of establishment of competent latency, the treatment is 
preferably carried out as soon as possible after contact with the virus, 
preferably within 18 hours, although up to five days or possibly longer is 
acceptable. The treatment period is usually 3 to 14 days, more usually 5 
to 10 days, often 5 or 7 days. 
For treatment of established recurrent disease, the treatment period is up 
to 5 years, for example, up to 1, 2, 3, 4, and 5 years. 
The present invention also provides the use of a compound of formula (A) or 
a bioprecursor, or a pharmaceutically acceptable salt, phosphate ester 
and/or acyl derivative of either of the foregoing, in the preparation of a 
medicament for use in the treatment of latent infection of herpesviruses. 
Such treatment may be carried out in the manner as hereinbefore described. 
The present invention further provides a pharmaceutical composition for use 
in the treatment of latent infection of herpesviruses, which comprises an 
effective amount of a compound of formula (A) or a bioprecursor, or a 
pharmaceutically acceptable salt, phosphate ester and/or acyl derivative 
of either of the foregoing, and a pharmaceutically acceptable carrier. 
Such compositions may be prepared in the manner as hereinafter described. 
The compound of formula (A) and its prodrugs show a synergistic antiviral 
effect in conjunction with interferons; and treatment using combination 
products comprising these two components for sequential or concomitant 
administration, by the same or different routes, are therefore within the 
ambit of the present invention. Such products are described in EP-A-271270 
(Beecham Group p.l.c.). 
The following results from animal studies illustrate the invention.

EXPERIMENTS IN MICE INFECTED WITH HSV-1 VIRUS 
A cutaneous infection was established by inoculation of the ear pinnae of 
mice with HSV-1 (SC16) and the effects of oral famciclovir on the latent 
virus infection was investigated. 
BALB/c female mice (Bantin and Kingman, Kingston, Hull, UK) were purchased 
at 3 to 4 weeks old and inoculated one week later. Virus suspension (10 
ul) containing 5.times.10.sup.4 p.f.u. were inoculated into the skin of 
the left ear pinna. Skin thickness was measured daily in individual mice 
by means of an Engineers' micrometre screw gauge. (ref. Nash et al, 1980, 
J. Gen. Virol. 48, 351-357). These mice were kept for 3 (Experiment 1) or 
4 (Experiment 2) months and then killled. The trigeminal ganglia and 
cervical dorsal root ganglia were removed and co-cultivated. Those 
cultures showing virus replication were recorded as positive. 
Experiment 1 
In a first experiment, mice were treated within 18h and treatment ceased on 
day 10 post infection. 
Of the 24 untreated control mice, 12 showed latent infection in the 
trigeminal ganglia (TG) and 20 showed latent infection in the cervical 
dorsal route ganglia (DRG). All 24 control mice showed either TG or DRG 
latency. None of the FCV treated mice showed any latency. 
Experiment 2 
In a second experiment, antiviral treatment was initiated on days 1, 2, 3, 
4 or 5 post-infection (p.i.) and and ceased on day 10 p.i.. The compounds 
were administered ad libitum in the drinking water, at 1 mg/ml 
(approximately 100 mg/kg/day). 
The results are as shown in the following table: 
Note: The groups 1 and 2 received the same treatment regimens but the 
results ere assayed separately.) 
__________________________________________________________________________ 
Latency Total 
Acute Total 
Latency Latency % Mice with 
% Mice with 
(Group 1) (Group 2) virus + ve 
virus + ve 
Antiviral 
TG + DRG + 
TG + DRG + 
ganglia on day 
ganglia on day 
Therapy 
ve/8 ve/8 ve/8 ve/8 120 (n = 16) 
8 (n = 8) 
(days) 
Lt 
Rt Lt 
Rt Lt 
Rt Lt 
Rt L/RTG + DRG 
L/RTG + DRG 
__________________________________________________________________________ 
None 8 4 8 5 8 6 8 2 100 100 
5-10 4 0 4 0 2 0 2 0 38 100 
4-10 2 0 2 0 0 0 0 0 13 100 
3-10 0 0 0 0 0 0 0 0 0 0 
2-10 0 0 0 0 0 0 0 0 0 0 
1-10 0 0 0 0 0 0 0 0 0 0 
__________________________________________________________________________ 
Four months later, latent virus could be reactivated in ganglia explants 
(ipsilateral and contralateral trigeminal and dorsal root) from all of 16 
control mice. Latent virus was not reactivated from the ganglia of 
FCV-treated mice, except ipsilateral ganglia, and only when the start of 
therapy was delayed until days 4 p.i. (2/16) or 5 p.i (6/16). 
Similar results were obtained when compounds were administered twice daily 
by gavage at 50 mg/kg per dose. 
Experiment 3 
Mice were immunosupressed with Cyclosporin A (CyA) from day -2 to day =10 
(day 0 being the day of infection). Groups of mice were untreated 
(control), or treated with famciclovir orally at 50 mg/kg twice daily from 
22 h after infection to 5.5 or 10.5 days. The ganglia were examined for 
reactivation of infectious virus 1 or 4 months later and the results are 
shown in the Table below. 
______________________________________ 
LTG RTG LDR RDR 
(n = 6) 
(n = 6) (n = 6) (n = 6) 
______________________________________ 
Control 6 4 6 3 
FCV 0 0 0 0 
______________________________________ 
TG = trigeminal DRG = dorsal root ganglia L/R = left/right 
EXPERIMENTS IN MICE INFECTED WITH HSV-2 VIRUS 
A cutaneous infection was established by inoculation of the ear pinnae of 
mice with HSV-2 (Bry) and the effects of oral famciclovir on the latent 
virus infection was investigated. Treatment was 50 mg/kg twice daily for 5 
days starting 22 h post-infection. 
The table shows the number of mice/group with positive latent infection in 
the trigeminal or cervical dorsal root ganglia. 
______________________________________ 
No. of mice with +ve ganglia 
mice/ % 
number of mice tested yielding 
ganglion at least 
Group Left T/G Right T/G 
Left CDR 
Right CDR 
one +ve 
______________________________________ 
Control 10/10 10/10 10/10 6/10 100 
famciclovir 
0/10 0/10 0/10 0/10 0 
______________________________________ 
T/G = trigeminal CRG = Cervical dorsal root ganglia 
Evidence from a murine immunosuppression model that famciclovir is 
effective against an on-going chronic herpes simplex virus infection. 
Immunosuppression was induced in mice by means of cyclosporin administered 
on alternate days. When such mice were inoculated in the ear pinna with 
HSV-1, a chronic infection was established. Virus replication continued in 
both skin and central nervous system for 20 days, the duration of the 
experiment. During this time, clinical signs including inflammation of the 
ear, weight loss and cumulative mortality were monitored. Famciclovir or 
valaciclovir (50 mg/kg b.i.d). was commenced 5 days after virus 
inoculation for a period of 5 days. Famciclovir prevented mortality (which 
in untreated mice reached 50% by day 20). Furthermore, weight gain was 
restored to uninfected control levels within 3 days of starting treatment 
and inflammation (as judged by skin thickness) returned to normal levels 
within 4 days. Virus titres were measured daily throughout the experiment. 
Infectious virus was cleared from both skin and neural tissues 
(representing a reduction in approx. 4 log 10 p.f.u./organ) and, following 
cessation of famciclovir therapy, there was no recurrence of infectious 
virus despite the continuation of immunosuppression for a further 10 days. 
In, recurrence of virus replication on several days was observed in mice 
that had been treated with valaciclovir in both skin and brainstem. It was 
of interest that in a separate group of mice that were infected without 
immunosuppression, recurrence on cessation of valaciclovir still occurred, 
but only in brainstem and not in the ear pinna. Our results provide 
further evidence of the potent antiviral activity of famciclovir in a 
relevant animal model for chronic herpes simplex infection. 
Further investigation into the recurrence of infectious virus in the 
nervous system of mice infected with HSV-1 on cessation of valaciclovir 
therapy. 
Previously we have reported the contrasting effects of famciclovir and 
valaciclovir in murine infections models for HSV- 1 and HSV-2. A striking 
result was the reproducible recurrence of infectious virus in the nervous 
system of mice following cessation of valaciclovir therapy for 5 or 10 
days while no such recurrence was observed after famciclovir therapy (J. 
Inf. Dis. 73, 291-299 & Antimicrob. Ag. Chemother. 40(4) in press). We had 
noted a recurrence of viral replication with aciclovir (ip, once daily) 
(Field et al, 1979, Antimicrob. Ag. Chemother. 15:554-561). We therefore 
compared aciclovir with its prodrug, valaciclovir. Mice were infected with 
HSV- 1 in the skin of the ear. Valaciclovir was administered as before (50 
mg/Kg b.i.d. from day 1 to 5 post infection). Further groups of mice were 
treated with aciclovir (50 mg/Kg b.i.d, ip.) or aciclovir ad. lib. in the 
drinking water (approx. 160 mg/Kg/day). The clinical signs were noted and 
mortality, weight-gain/loss, inflammation in the ear were measured daily. 
Virus replication in the skin of the ear and in brain stem and trigeminal 
ganglion was measured daily from day 1 to 14 post infection. Aciclovir 
i.p. was the most, and valaciclovir the least, effective of the three 
regimens. When treatment was terminated on day 5; recurrence of infectious 
virus was observed in brain stem on day 8 in mice that had been treated 
with aciclovir in drinking water and on day 9 in mice that had been given 
valaciclovir. Recurrence in trigeminal ganglia was seen on day 8 but only 
in mice that had been given valaciclovir. No recurrences of infectious 
virus were recorded in the skin of the ear. These results confirm that, in 
contrast to famciclovir, neither oral aciclovir or valaciclovir therapy 
prevents virus recurrence, a factor which may limit their antiviral 
efficacy in man.