Anti-retroviral activity of 2',3'-dideoxy-3'-fluoronucleosides

Use of a compound of the formula ##STR1## wherein Base is thymine, Cytosine, adenine or guanine, or a physiologically acceptable salt thereof, for therapeutic treatment of hepatitis B virus infections and to a method for such treatment.

FIELD OF THE INVENTION 
The present invention relates to the use of known chemical compounds and 
physiologically acceptable salts thereof for the therapeutic treatment of 
the Acquired Immuno Deficiency Syndrome (AIDS), infections by Human 
Immunodeficiency Virus, hepatitis B virus infections and retrovirus 
infections for such control and treatment in animals and man. 
BACKGROUND OF THE INVENTION 
In the late seventies a new disease was reported, which subsequently was 
referred to as Acquired Immuno Deficiency Syndrome (AIDS). It is now 
generally accepted that a retrovirus referred to as HIV (Human Immuno 
Deficiency Virus, formerly known as Human T-cell Lymphotropic Virus 
(HTLV-III) or Lymphadenopathy Associated Virus (LAV) plays an essential 
role in the etiology of AIDS. 
AIDS is characterized by a profound immunodeficiency due to low numbers of 
lymphocyte-T-helper cells, which are the targets for HIV (also called 
HTLV-III/LAV) infection. The profound immunodeficiency in AIDS patients 
makes these patients highly susceptible to a variety of opportunistic 
infections of bacterial, fungal, protozoal or viral etiology. The 
etiological agents among viral opportunistic infections are often found in 
the herpes virus group, i.e., Herpes simplex virus (HSV), Varicella Zoster 
virus (VZV), Epstein-Barr virus (EBV) and, especially, cytomegalovirus 
(CMV). Other retroviruses affecting humans are HTLV-I and II and examples 
of retroviruses affecting animals are feline leukemia virus and equine 
infectious anaemia virus. 
Hepatitis B virus infections cause severe disease such as acute hepatitis, 
chronic hepatitis, fulminant hepatitis in a considerable number of 
persons. It is estimated that there are 200 million patients with chronic 
hepatitis B infection in the world. A considerable number of the chronic 
cases progress to liver cirrosis and liver tumours. In some cases the 
hepatitis infections also take a rapid and severe course as in fulminant B 
hepatitis with about 90% mortality. At present there is no known effective 
treatment against hepatitis B infections. 
PRIOR ART 
The compound 3'-deoxy-3'-fluoro-thymidine is described in Journal f. prakt. 
Chemie, Vol. 315, 895-900 (1973) as having cytostatic and virustatic 
activity as selective inhibitor of DNA synthesis. In the same article the 
synthesis of the compound 2',3'-dideoxy-3'-fluorocytidine is described. 
The compounds 2',3'-dideoxy-3'-fluoroadenosine and 
2',3'-dideoxy-3'-fluoroguanosine are described in the East-German patents 
DD 158903 and DD 209197, respectively, as virostatic agents. 
DISCLOSURE OF THE INVENTION 
It has been found according to the present invention that the compounds of 
the formula 
##STR2## 
wherein Base is thenine, cytosine, adenine or guanine, or a 
physiologically acceptable salt thereof, present a new possibility to 
block the multiplication of hepatitis B virus, by use of a nucleoside 
analogue of said formula. Accordingly, the nucleoside analogue of said 
formula and physiologically acceptable salts thereof, have beneficial 
properties as therapeutic agents in the treatment of hepatitis B virus 
infections, respectively. 
Hepatitis B virus (HBV) is a DNA virus with a unique circular 
double-stranded DNA genome which is partly single-stranded. It contains a 
specific DNA polymerase required for vital replication. This DNA 
polymerase also acts as a reverse transcriptase during the replication of 
HBV DNA via an RNA intermediate. 
The compounds of the invention are transformed by cells/or enzymes to 
triphosphates which inhibit the activity of DNA polymerase of hepatitis B 
virus. 
The following known compounds constitute part of the invention as 
therapeutic agents in treatment of hepatitis B virus infections: 
##STR3## 
3'-Deoxy-3'-fluorothymidine is especially preferred as an agent for 
treatment of hepatitis B virus infections in animal and man. 
In clinical practice the nucleosides of the invention will normally be 
administered orally, by injection or by infusion in the form of a 
pharmaceutical preparation comprising the active ingredient in the form of 
the original compound or optionally in the form of a pharmaceutically 
acceptable salt thereof, in association with a pharmaceutically acceptable 
carrier which may be a solid, semi-solid or liquid diluent or an 
ingestible capsule. The compound may also be used without carrier 
material. As examples of pharmaceutical preparations may be mentioned 
tablets, dragees, capsules, granulates, suspensions, elixirs, syrups, 
solutions etc. Usually the active substance will comprise between 0.05 and 
20% for preparations intended for injection and between 10 and 90% for 
preparations intended for oral administration. 
In the treatment of patients suffering from hepatitis B virus infections, 
it will be preferred to administer the compounds by any suitable route 
including the oral, parenteral, rectal, nasal, topical and vaginal route. 
The parenteral route includes subcutaneous, intramuscular, intravenous and 
sublingual administration. The topical route includes buccal and 
sublingual administration. The dosage at which the active ingredients ape 
administered may vary within a wide range and will depend on various 
factors such as the severity of the infection, the age of patient etc., 
and may have to be individually adjusted. As a possible range for the 
amount of the compounds of the invention or a physiologically acceptable 
salt thereof be administered per day may be mentioned from about 10 mg to 
about 10 000 mg, preferentially 100-500 mg for intravenous administration 
and preferentially 100-1000 mg for oral administration. 
SALTS 
The physiologically acceptable salts of the nucleosides of the invention 
are suitable acid addition salts, derived from non-toxic acids. Such acid 
addition salts include, for example, those derived from inorganic acids 
such as hydrochloric acid, hydroiodic acid, sulphuric acid, phosphoric 
acid and sulfamic acid, organic sulphonic acids such as p-toluenesulphonic 
acid, methanesulphonic acid, p-chlorobenzonesulphonic acid, ethanesulfonic 
acid, and benzensulfonic acid and organic carboxylic acids such as maleic 
acid, malic acid, lactic acid, citric acid, tartaric acid, succinic acid, 
oxalic acid, acetic acid, isethionic acid, gluconic acid, pantothenic acid 
and lactobionic acid.

EXPERIMENTAL TESTS 
Test I. Effect of 3'-deoxy-3'-fluorothymidine as a triphosphate on the DNA 
polymerase of hepatitis B virus (HBV) in cell free assay 
Since hepatitis B virus cannot be grown in cell cultures, a cell-free assay 
system of the hepatitis B virus DNA polymerase has been used to investigate 
the effect of 3'-deoxy-3'-fluorothymidine. 3'-Deoxy-3'-fluorothymidine in 
cells is transformed to 3'-deoxy- 3'-fluorothymidine- 5'-triphosphate. 
The HBV associated DNA polymerase activity can be measured in vitro (Kaplan 
et al., J. Virol., 12,995-1005, 1973). A slight modification of this method 
has been used to test the substance for inhibition of this DNA polymerase 
activity. (Nordenfelt E., (Oberg, B., Helgstrand E. and Miller E. Acta 
path., Microbiol. Scand. Sect B, 88:169-175, 1980). With this assay the 
3'-deoxy-3'-fluorothymidine-5'-triphosphate has been tested, instead of 
the prodrug 3'-deoxy- 3'-fluorothymidine, to evaluate its potential 
against hepatitis B virus DNA polymerase. 
3'-deoxy-3'-fluorothymidine-5'-triphosphate was added to the final 
concentrations of 0.01 .mu.M, 0.05 .mu.M, 0.1 .mu.M, 0.5 .mu.M and 1.0 
.mu.M in the reaction mixture. The inhibition is calculated after 3 hours 
incubation at 37.degree. C. and based on cpm compared to control with 
added water. The test result is shown in Table I. 
TABLE I 
______________________________________ 
Inhibition of hepatitis B virus (HBV) DNA polymerase activity 
by 3'-deoxy-3'-fluorothymidine-5'-triphosphate 
Concentration of 
3'-deoxy-3'-fluorothymidine- 
5'-triphosphate (.mu.M) 
% inhibition 
______________________________________ 
0.01 17 
0.05 17 
0.1 39 
0.5 79 
1 88 
______________________________________ 
From the results shown in Table I the apparent ID.sub.50 -value of 
3'-deoxy-3'-fluorothymidine was found to be 0.16 .mu.M. 
Test II. Effect of 3'-deoxy-3'-fluorothymidine as a triphosphate on the 
reverse transcriptase of HIV (HTLV-III/LAV) in cell free assay. 
A cell-free assay system has been used to investigate the inhibition of 
3'-deoxy-3'-fluorothymidine on reverse transcriptase of HIV 
(HTLV-III/LAV). The assay was performed as described by Vrang et Oberg, 
Antimicrob. Agents Chemother. 29,867-872 (1986). With this assay the 
3'-deoxy-3'-fluoro-thymidine-5'-triphosphate has been tested, instead of 
the prodrug 3'-deoxy-3'-fluorothymidine to evaluate its potential against 
reverse transcriptase from HIV (HTLV-III/LAV). The test result is shown in 
Table II. 
TABLE II 
______________________________________ 
Inhibition of HIV (HTLV-III/LAV) reverse transcriptase by 
3'-deoxy-3'-fluorothymidine-5'-triphosphate 
Concentration of 5'-triphosphate 
3'-deoxy-3'-fluorothymidine (.mu.M) 
% inhibition 
______________________________________ 
0.01 12 
0.05 27 
0.1 38 
______________________________________ 
From the results in Table II, the ID.sub.50 -value of 
3'-deoxy-3'-fluoro-thymidine with regard to the activity of HIV reverse 
transcriptase was found to be 0.2 .mu.M by extrapolation. 
Test III. Effect of 3'-deoxy-3'-fluorothymidine on HIV (HTLV-III/LAV) in H9 
cells 
MATERIAL AND METHODS; HIV INFECTION OF H9 CELLS 
H9-cells (2.times.1O.sup.6) were preincubated overnight with 
3'-deoxy-3'-fluorothymidine at various concentrations. The cells were then 
pelleted and dispersed in 2.5 ml phosphate buffered saline (PBS) including 
2 .mu.g/ml Polybrene. After incubation for 30 min the cells were pelleted 
and infected with HIV. After an adsorption period of 1 hour the cells were 
pelleted and washed once with 2.5 ml PBS. To each culture 7 ml media 
including 3'-deoxy-3'-fluorothymidine at studied concentrations was added. 
Samples for reverse transcriptase activity tests were taken as indicated. 
ASSAY 
1.3 ml samples from the supernatant of each culture were centrifuged at 18 
000 rpm in a 55-34 rotor for 1.5 hours and the virus pellet resuspended in 
100 .mu.l buffer containing 50 mM Tris-HC1, pH 7.5; 35 mM KC1; 4 mM DTT; 1 
mM EDTA; 1.3 % Triton X-100.50 .mu.l samples were taken to the reverse 
transcriptase activity tests and analyzed in a 100 .mu.l reaction mixture 
containing 75 mM Tris-HC1, pH 8.0; 60 mM KC1; 6.2 mM MgC1.sub.2 ; 6 mM 
DTT; 0.5 mM EDTA; 0.65 % Triton X-100; 100 .mu.g/ml BSA; 25 .mu.Ci/ml 
.sup.3 H-dTTP (spec activity 80 Ci/mmol); 2.5 .mu.g/ml (dT).sub.12-18 ; 
and 2.0 .mu.g/ml (rA).sub.n. Incubation was for 1 hour at 37.degree. C. 
and the TCA-insoluble product precipitated onto Whatman GF/A filter 
papers, washed and dried, and counted in a liquid scintillation counter. 
The test result is shown in Table III. 
The amounts of reverse transcriptase molecules and their total activity 
expressed in HIV-infected cell cultures is correlated to the amount of HIV 
particles present. The addition of an effective antiviral agent which 
inhibits the production of new HIV particles, also decreases the amount of 
reverse transcriptase molecules and is expressed as a decreased total 
activity. 
TABLE III 
______________________________________ 
Effect of 3'- 
deoxy-3'-fluorothymidine on the expressed reverse transcriptase 
activity in HIV (HTLV-III/LAV)-infected H9 cells. 
Reverse transcriptase activity (cpm .times. 10.sup.-3) in the 
presence of indicated amounts (.mu.M) of 3'-deoxy-3'- 
Days 
fluorothymidine 
post-infection 
0 0.01 0.05 0.1 0.5 1.0 
______________________________________ 
4 0.9 0.5 0.3 0.2 0.3 0.3 
8 19 1.3 0.3 0.2 0.3 0.2 
11 17 1.9 0.4 0.5 0.5 0.5 
15 11 2.7 0.6 0.4 0.3 0.4 
18 27 28 0.5 0.4 0.6 0.5 
22 46 52 1.6 0.2 0.1 0.1 
29 16 21 3.1 0.2 0.2 0.2 
______________________________________ 
In Table III is shown the effects of different concentrations of 
3'-deoxy-3'-fluorothymidine on the reverse transcriptase activity of HIV 
(HTLV-III/LAV) during an incubation period of several weeks. The presence 
of 3'-deoxy-3'-fluorothymidine at 0.1 .mu.M and higher concentrations 
completely prevents the enzyme activity during at least 29 days. At 0.05 
.mu.M of 3'-deoxy-3'-fluorothymidine no significant enzyme activity was 
detected up to 22 days and at 0.01 .mu.M concentration, enzyme activity 
was not detected up to 11 days.