Pharmaceutical composition

Combinations of 3'-azido-3'deoxythymidine and phsphonoformate have been found to produce a synergistic inhibitory effect against human immunodeficiency virus (HIV).

The present invention relates to a new pharmaceutical composition 
comprising a combination of a first component which is 
3'-azido-3'-deoxythymidine (AZT) or its 5'-triphosphate (AZT-TP), and a 
second component which is phosphonoformic acid or a therapeutically 
acceptable salt thereof (PFA). This new combination has been found to give 
synergistic antiviral effect against human immunodeficiency virus (HIV), 
especially HIV type 1 (HIV-1). 
In a further aspect, the invention relates to a method for the treatment of 
HIV virus infection, especially HIV type 1 virus infections, and to a 
method for the treatment of AIDS by concomitant administration of AZT, or 
AZT/TP, and PFA. 
The novel pharmaceutical combinations of AZT or AZT-TP and PFA will 
comprise the respective components at a ratio of from 1:200 to 1:8000, 
suitably from 1:1000 to 1:4000, given as ration AZT or AZT-TP to PFA on 
molar basis. 
PFA is preferably used in the form of its tri-sodium salt. It was used in 
that form in the test described below. 
In the tests described below, the HIV type 1 virus has been used. 
Combinations of 3'-azido-3'-deoxythymidine and phosphonoformate have been 
found to produce a moderate synergistic inhibitory effect against human 
immunodeficiency virus type 1 in vitro at concentrations that are easily 
achieved in humans. The synergistic effect was more pronounced with 
increasing concentrations and was not secondary to toxic effects of the 
drugs. 3'-Azido-3'-deoxythymidine neither inhibited the replication of 
human cytomegalovirus in human embryonic lung fibroblasts nor interfered 
with the anti-cytomegalovirus effect of phosphonoformate. Using partially 
purified reverse transcriptase of human immunodeficiency virus type 1 and 
human cytomegalovirus DNA polymerase, various combinations of 
3'-azido-3'-deoxythymidine-5'-triphosphate and phosphonoformate produced 
strong indications of additive interactions. The synergistic interactions 
in infected cells and the additive effects observed at the reverse 
transcriptase level indicated that mechanisms other than the reverse 
transcriptase may be of importance for the inhibition of human 
immunodeficiency virus replication by these two compounds. A concomitant 
treatment of cytomegalovirus infections, such as cytomegalovirus 
retinitis, with phosphonoformate in AIDS patients receiving 
3'-azido-3'-deoxythymidine may be appropriate and this combination may 
also be useful in controlling the infection with the human 
immunodeficiency virus. 
Several reports have indicated that a number of agents can affect human 
immunodeficiency virus type 1 (HIV-1) replication in cell culture (4, 17, 
23, 34, 45) 3'-Azido-3'-deoxythymidine (zidovudine, AZT), a modified 
nucleoside, has received considerable attention because it was the first 
agent shown in a multicenter, double-blind, placebo-controlled study to 
prolong life and provide clinical improvement in certain patients with 
AIDS and advanced AIDS-related complex who had been treated during a 
period up to six months (10). However, AZT is associated with pronounced 
toxicities which have limited its use in individuals with established AIDS 
(27, 40). Opportunistic infections of human cytomegalovirus (HCMV) is one 
of the major problems in individuals with AIDS. Cytomegalovirus retinitis 
has been described in approximately one third of this population (26). In 
the absence of therapy this infection inevitably leads to irreversible 
retinal necrosis and can progress to permanent blindness. Only two drugs, 
9-(1,3-dihydroxy-2-propoxymethyl)guanine (ganciclovir, DHPG, 2'-NDG, or 
BIOLF-62) and phosphonoformate (foscavir, foscarnet, or PFA) have been 
investigated with some success as possible treatments against 
HCMV-infections (8, 20, 21, 25, 28, 36, 42). PFA, an analog of 
pyrophosphate, has the unique property of being effective against both 
HIV-1 and HCMV in vitro (5, 24, 30, 31, 41, 43). Recent observations by 
Jacobson et al. (19) have also demonstrated a significant reduction of 
HIV-1 p24 antigen concentrations in patients after a 14-day treatment with 
PFA. Since a concomitant treatment of PFA against CMV retinitis is 
currently being considered in HIV-infected individuals undergoing AZT 
therapy, it was important to determine the type of interaction produced by 
these drugs against HIV-1 and HCMV. 
The use of combinations of compounds with different modes of action is an 
attractive and logical extension of any therapeutic approach to enhance 
drug efficacy. Lower doses of the drugs might be used, which also may 
reduce the potential toxicity caused by either drug alone and the 
appearance of drug-resistant virus. In fact, almost all of the currently 
available antiretroviral agents have been evaluated in combinations with 
AZT (4, 13, 34). For example, using a rigorous definition of synergy for 
drug interactions, Hartshorn et al. (15) demonstrated synergistic effects 
against HIV-1 in cell culture by combinations of PFA and recombinant 
alpha-A interferon. In a similar in vitro system, combinations of AZT and 
alpha-A interferon gl granulocyte-macrophage colony-stimulating factor 
(GM-CSF) have been reported to produce synergy (14, 16). 
In this report, the effects of different combinations of AZT and PFA were 
examined on the replication of HIV-1 in human peripheral blood mononuclear 
(PBM) cells and HCMV Ad169 in human embryonic lung (HEL) fibroblasts. 
Since the major mode of action of PFA and the triphosphate derivative of 
AZT (AZT-TP) is an inhibition of the HIV-1 reverse transcriptase (RT) 
activity by different mechanisms (6, 7, 12, 43, 44), the effects of 
several combinations of the two compounds were also examined on the 
partially purified HIV-1 RT. For comparison, the effects of combinations 
of PFA and AZT-TP on the partially purified HCMV DNA polymerase were 
studied. (Parts of this work were presented at the Second International 
Conference on Antiviral Research in Williamsburg, Va., Apr. 10-14, 1988, 
and at the IV International Conference on AIDS in Stockholm, Sweden, Jun. 
12-16, 1988). 
MATERIALS AND METHODS 
Compounds. PFA was provided by Astra ALab, Sodertalje, Sweden. AZT and 
AZT-TP were synthesized and purified in our laboratory according to 
published methods (22, 44, 46). The purity of AZT and AZT-TP was 
established by reversed-phase and anion exchange HPLC methods and 
spectrophotometric analysis. 
Cells, virus strains, and cell culture assays. PBM cells from healthy HIV-1 
and hepatitis B virus seronegative donors were isolated and propagated as 
described previously (35). HIV-1 (strain LAV-1) was obtained from the 
Centers for Disease Control, Atlanta, and propagated in PHA-stimulated 
human PBM cells as described previously (35). The details of the methods 
used for infection and assaying the anti-HIV-1 effect in infected human 
PBM cells have been reported (35). 
HCMV strain Ad169 was a gift from Dr. Fred Rapp, Hersey, Pa. Human 
embryonic lung (HEL) cells, obtained from the American Type Culture 
Collection, Rockville, Md., were cultured in Dulbecco modified Eagle 
medium as decribed previously (39). Viable cells were conducted 
microscopically using a hemacytometer and the trypan blue exclusion 
method. Plaque reduction assays were performed in confluent monolayers of 
HEL cells in 6-well plates (Costar, Cambridge, Mass.) with 100 to 200 pfu 
of HCMV Ad169. After an adsorption period of 1 hour, unadsorbed virus was 
removed and the monolayers were overlaid with Dulbecco modified Eagle 
medium containing 0.75% SeaPlaque agarose (FMC BioProducts, Rockland, 
Me.), 2% heat inactivated fetal calf serum, and the appropriate 
concentration of drug. The plates were incubated at 37.degree. C. in a 
humidified atmosphere containing 5% CO.sub.2 for 10 to 12 days. The 
monolayers were then fixed with 10% buffered Formalin (Fisher Scientific 
Co, Fair Lawn, N.J.), stained with crystal violet, and the number of 
plaques were counted. 
Cell proliferation. The drugs alone and in combination were evaluated for 
their potential toxic effects on uninfected PHA-stimulated human PBM 
cells. Flasks were seeded so that the final cell concentration was 
3.times.10.sup.5 cells ml. The cells were cultured with and without drug 
for 6 days at which time aliquots were counted for cell viability by using 
the trypan blue exclusion method. 
RT activity assay with disrupted virions. Six ml supernatant from each 
culture was clarified from cells at 300.times. g for 10 minutes. Virus 
particles were then pelleted from 5 ml samples at 40,000 rpm for 30 
minutes using a Beckman 70.1 Ti rotor and suspended in 200 .mu.l of virus 
disrupting buffer 50 mM Tris-HCl pH 7.8, 800 mM NaCl, 20% glycerol, 0.5 
mM phenylmethyl sulfonyl fluoride (PMSF), and 0.5% Triton X-100!. 
The RT assays were performed at 37.degree. C. in 96-well microtiter plates 
using methods described previously (35, 38). The results were expressed as 
dpm/ml of the originally clarified supernatant. 
RT activity assay with partially purified enzyme. HIV-1 reverse 
transcriptase was isolated from detergent disrupted virions obtained from 
the cell-free supernantant of infected PHA-stimulated PBM cells. The 
enzyme was purified by passing the extract through ion-exchange 
chromatography columns as described previously (12). The enzyme was 
characterized as HIV-1 RT based on its specific requirements according to 
previous description (1, 18). The standard reaction mixture (100 .mu.l) 
contained 100 mM Tris-HCl (pH 8.0), 50 mM KCl, 2 mM MgCl.sub.2. 5 mM 
dithiothreitol 400 .mu.g/ml bovine serum albumin, 0.05 U/ml (3.1 .mu.g/ml) 
of (rA).sub.n (dT).sub.12-18, 1 .mu.M .sup.3 H! dTTP (spec. act. 18,000 
cpm/pmol) and 10 .mu.l of HIV-1 Rt. The reactions were incubated and 
processed as previously described (7). 
CMV DNA Polymerase Assay. The partially purified HCMV-specific DNA 
polymerase was a generous gift from Dr. B. Wahren, National 
Bacteriological Laboratory, Stockholm. The procedure for the purification 
of HCMV DNA polymerase by sequential chromatographic steps on 
DEAE-cellulose (Whatman DE-52) and phosphocellulose (Whatman P-11) and the 
measurement of enzyme activity have been described previously (5). 
Calculation of synergy. To determine whether synergistic, additive or 
antagonistic antiviral effects were achieved in virus-infected cell 
cultures treated with combinations of AZT and PFA, or in enzyme assays 
with combinations of AZT-TP and PFA, the multiple drug effect analysis 
developed by Chou and Talalay (2, 3) was used. Briefly, the method 
involves plotting of dose-effect curves for each compound alone and in 
combinations. It is essential to use a fixed ratio of the agents in 
multiply diluted combinations in order to use the median-effect equation: 
f.sub.a /f.sub.u =(C/C.sub.m).sup.m. In this equation, C is the 
concentration, f.sub.a and f.sub.u are the fractions of the system which 
are affected and unaffected, respectively, by concentration C. C.sub.m is 
the concentration required for a 50% effect (analogous to IC.sub.50 to 
EC.sub.50 values), and m is a Hill-type coefficient signifying the 
sigmoidicity of the dose-effect curve. The slopes (m) of the dose-effect 
plots, obtained for the studied compounds, provided information as to 
whether the compounds are mutually exclusive (i.e. similar modes of 
action) or mutually non-exclusive (i.e. different modes of action). When 
the plots of both compounds and their combination were all parallel, the 
effects of the two compounds were mutually exclusive. The interaction 
between two compounds was determined by calculating the combination index 
(C.I.) with assumptions, where appropriate, of either mutually exclusive 
or non-exclusive interactions. When uncertainty existed as to whether the 
drugs acted in similar or independent manners, the determination of 
C.I.-values were calculated under each assumption and compared. Values of 
C.I.&lt;1 indicated synergy, C.I.=1 indicated additive effects, and C.I.&gt;1 
indicated antagonism. A computer program obtained from Elsevier-Biosoft, 
Cambridge, U.K. was used for automatic analysis of all dose-effect data. 
Additional details on using this method have been reported previously 
(33). 
RESULTS 
Effects in HIV-1 infected and uninfected PBM cells. The effects of AZT and 
PFA, alone and in combinations, on the HIV-1 replication in human PBM 
cells (measured as RT activity of disrupted virions) are shown in Table 1. 
The supernatant of untreated virus-infected human PBM cells contained a 
mean RT activity of 772 kdpm/ml (equivalent to 11.8 pmoles of dTMP 
incorporated into acid-insoluble product). A concentration-dependent 
decrease in RT activity was observed in cultures treated with each 
compound alone and their combinations. A 50% reduction of HIV-1 
replication median effective concentration (EC.sub.50)!, was observed at 
0.006 .mu.M AZT and 22 .mu.M PFA when the drugs were tested alone and at 
3.5 .mu.M and 8.5 .mu.M when combinations of AZT and PFA were tested at 
ratios of 1:1,000 and 1:4,000, respectively. The ratios of AZT to PFA were 
selected according to the approximate ratio of their EC.sub.50 -values. 
The upper part of Table 2 summarizes the slopes, median effect values, and 
correlation coefficients obtained from the median-effect plots of AZT, 
PFA, and their combinations, respectively. The observed slope values (m) 
were greater than one for both compounds and their combinations in HIV-1 
infected cells, which indicated a sigmoidal nature of the dose-effect 
curves rather than hyperbolic (m=1). This is in accordance to what is 
normally observed in more organized biological systems where the 
dose-effect relationships of inhibitors are frequently sigmoidal rather 
than hyperbolic (2). Since the median-effect plots of AZT, PFA, and their 
combinations were not parallel to each other in HIV-1 infected cells (data 
not shown), the exclusively of the combined effects could not be 
established. Therefore, the C.I.-values were calculated under both 
mutually exclusive and mutually non-exclusive assumptions. As shown in 
Table 2, the C.I.-values of both combinations giving a 50%, 70%, or 90% 
reduction of the HIV-1 replication were all less than one, which suggested 
synergistic effects. The computer-generated C.I.-values for f.sub.a 
-values ranging from 0.05 to 0.95 for the studied combinations of AZT and 
PFA are shown in FIG. 1. Both combinations produced similar synergistic 
effect patterns which were concentration-dependent and increased with 
increasing concentrations. 
At a ratio of 1:4,000, the combination of AZT and PFA produced no toxicity 
greater than the agents alone to uninfected PHA-stimulated PBM cells; the 
highest combined concentration tested was 128.032 .mu.M. PFA was not toxic 
to PBM cells when tested up to 640 .mu.M (data not shown). 
Effects on HIV-1 reverse transcriptase. The synergistic effect observed in 
HIV-1 infected PBM cells by combinations of AZT and PFA could be a 
consequence of the different mechanisms involved in inhibiting the RT 
activity. A combination of AZT-TP and PFA may, therefore, produce 
synergistic effects at the RT level. To study whether this hypothesis 
would hold true, the effects of several different combinations of AZT-TP 
and PFA were investigated using partially purified HIV-1 RT. FIG. 2 shows 
the median-effect plot for the inhibition of HIV-1 RT by AZT-TP, PFA, and 
a representative combinations of the two components at a molar ratio of 
1:20. Both inhibitors followed first-order kinetics (i.e. m-values were 
close to 1) and from the parallel lines it was apparent hat AZT-TP and PFA 
were mutually exclusive inhibitors. The slopes, median-effect 
concentrations, and C.I.-values calculated for AZT-TP and PFA alone and in 
the four different combinations studied (1:20, 1:50, 1:100, and 1:200) 
against the HIV-1 RT are presented in the lower part of Table 2. In 
contrast to what was observed in HIV-1 infected cells, slope values closer 
to one were observed. The C.I.-values were found to be close to one for 
f.sub.a -values at 0.50, 0.70, and 0.90, which strongly suggested that all 
studied combinations produced additive effects. The computer-generated 
calculation of C.I.-values for combinations of AZT-TP and PFA at ratios of 
1:20 and 1:200 were found to be close to one over the entire range of 
f.sub.a -values (FIG. 3). 
Effect on HCMV plaque formation. To study whether AZT would interfer with 
the anti-HCMV effect of PFA, the effects of four appropriate combinations 
(1:1, 1:2, 1:5, and 1:10) of the two compounds on the multiplication of 
HCMV Ad169 in HEL fibroblasts were studied. Whereas AZT was found to be 
virtually without inhibitory effect even at concentrations up to 200 .mu.M 
(data not shown), a dose-dependent inhibition was obtained for PFA with an 
EC.sub.50 -value of 34 .mu.M (Table 3). Although AZT did not significantly 
inhibit HCMV multiplication, an EC.sub.50 -value of 383 .mu.M was 
extrapolated (data not shown) in order to calculate the C.I.-values for 
the studied combinations. As shown in Table 3, the obtained C.I.-values 
indicated an additive effect for all combinations of AZT and PFA examined. 
Furthermore, the EC.sub.50 -values obtained indicated that the 
contribution of PFA in each combination caused the antiviral effect. 
Effects on the HCMV DNA polymerase. Since AZT-TP and PFA are structurally 
different and presumably inhibit the HCMV DNA polymerase activity by 
different mechanisms, the interaction of the two drugs was investigated 
for four different combinations, at ratios 5:1, 10:1, 25:1, and 50:1. As 
summarized in the lower part of Table 3, AZT-TP was found to inhibit the 
HCMV DNA polymerase activity with an observed EC.sub.50 -value of 25 
.mu.M. For PFA the corresponding EC.sub.50 -value of 0.78 .mu.M was more 
than 30 times lower. All combinations of AZT-TP and PFA produced 
C.I.-values close to one when the HCMV DNA polymerase activity was 
inhibited between 50% and 90%. This indicated that all combinations of the 
two compounds produced an additive effect also at the HCMV DNA polymerase 
level. 
Table 1. Effects of AZT and PFA alone and in combinations at ratios 1:1,000 
and 1:4,000 on HIV-1 replication in human PBM cells. 
TABLE 1 
______________________________________ 
Effects of AZT and PFA alone and in combination at ratios 
1:1,000 and 1:4,000 on HIV-1 replication in human PBM cells. 
Treatment Concn. RT activity 
Inhibition 
(drug ratio) 
(.mu.M) (kdpm/ml).sup.a 
(%) 
______________________________________ 
AZT 0.002 750 3.0 
0.004 584 24.5 
0.008 343 55.7 
0.016 35.9 95.5 
PFA 8 703 9.1 
16 455 41.1 
32 190 75.5 
64 93.1 88.1 
128 35.9 95.5 
AZT/PFA 0.001/1 797 -3.1 
(1:1,000) 0.002/2 668 13.6 
0.004/4 247 68.1 
0.008/8 68.4 91.3 
AZT/PFA 0.001/4 635 17.8 
(1:4,000) 0.002/8 551 28.8 
0.004/16 238 69.3 
0.008/32 4.6 99.5 
______________________________________ 
.sup.a The activity of uninfected PBM cells was 5,100 dpm/ml. All values 
are corrected for the mean value of the blanks (995 dpm). The mean value 
of RT activity .+-. S.D. of triplicate untreated HIVinfected cells was 77 
.+-. 50.8 kdpm/ml. 
TABLE 2 
__________________________________________________________________________ 
Median effective concentration and combinations index (C.I.) values for 
AZT 
(AZT-TP) and PFA alone and at different drug ratios 
against HIV-1 replication in human PBM cells and purified HIV-1 reverse 
transcriptase. 
Parameter.sup.a 
C.I. at F.sub.a.sup.b of 
Treatment (drug ratio) 
m .+-. SE 
EC.sub.50 (.mu.M) 
r 0.50 0.70 0.90 
__________________________________________________________________________ 
HIV-1 infected cells 
AZT 3.02 .+-. 0.27 
0.006 
0.99 
PFA 1.89 .+-. 0.17 
22.0 0.99 
AZT/PFA (1:1,000) 
3.03 .+-. 0.10 
3.5 0.99 
0.71 (0.80) 
0.68 (0.76) 
0.65 (0.71) 
AZT/PFA (1:4,000) 
3.20 .+-. 0.90 
8.5 0.93 
0.72 (0.85) 
0.65 (0.76) 
0.56 (0.64) 
HIV-1 
reverse transcriptase 
AZT-TP 0.87 .+-. 0.03 
0.003 
1.00 
PFA 0.91 .+-. 0.03 
0.14 1.00 
AZT-TP/PFA (1:20) 
0.89 .+-. 0.04 
0.043 
0.99 
0.98 0.97 0.96 
AZT-TP/PFA (1:50) 
0.92 .+-. 0.02 
0.074 
1.00 
1.02 0.99 0.93 
AZT-TP/PFA (1:100) 
0.87 .+-. 0.02 
0.084 
1.00 
0.89 0.91 0.95 
AZT-TP/PFA (1:200) 
0.92 .+-. 0.02 
0.11 1.00 
0.95 0.93 0.90 
__________________________________________________________________________ 
.sup.a m is the slope (SEvalues are given when four or more concentration 
have been used in the median effect plot), EC.sub.50 is the median 
effective concentration, and r is the correlation coefficient, as 
determined from the median effect plot. 
.sup.b C.I. &lt; 1 indicates synergy, C.I. = 1 indicates additivity, and C.I 
&gt; 1 indicates antagonism (See Materials and Methods). F.sub.a is a 
component of the median effect equation referring to the fraction of the 
system affected (e.g., 0.50 means the C.I. at a 50% reduction of 
activity). C.I. values for HIV1 infected cells were determined under both 
mutually exclusive and mutually nonexclusive (numbers in paranthesis) 
assumptions. 
TABLE 3 
__________________________________________________________________________ 
Median effective concentration and combinations index (C.I.) values for 
AZT 
(AZT-TP) and PFA alone and at different drug ratios against CMV plaque 
formation in 
human fibroblasts and purified CMV DNA polymerase. 
Parameter.sup.a 
C.I. at F.sub.a.sup.b of 
Treatment (drug ratio) 
m .+-. SE 
EC.sub.50 (.mu.M) 
r 0.50 0.70 0.90 
__________________________________________________________________________ 
CMV infected cells 
AZT 5.95 383 0.97 
PFA 1.88 .+-. 0.10 
34 1.00 
AZT/PFA (1:1) 
2.16 .+-. 0.39 
61 0.97 
0.97 (1.04) 
0.94 (1.03) 
0.91 (1.03) 
AZT/PFA (1:2) 
1.71 .+-. 0.32 
41 0.95 
0.82 (0.85) 
0.87 (0.91) 
0.97 (1.05) 
AZT/PFA (1:5) 
2.65 .+-. 0.22 
52 0.99 
1.29 (1.32) 
1.14 (1.17) 
0.94 (0.97) 
AZT/PFA (1:10) 
2.30 33 0.92 
0.89 (0.90) 
0.82 (0.83) 
0.72 (0.74) 
CMV 
DNA Polymerase 
AZT-TP 0.88 .+-. 0.09 
25 0.99 
PFA 0.79 .+-. 0.02 
0.78 1.00 
AZT-TP/PFA (5:1) 
0.74 .+-. 0.05 
3.4 0.99 
0.84 0.92 1.07 
AZT-TP/PFA (10:1) 
0.70 .+-. 0.02 
6.1 1.00 
0.93 1.10 1.45 
AZT-TP/PFA (25:1) 
0.78 .+-. 0.03 
11 1.00 
0.93 1.00 1.13 
AZT-TP/PFA (50:1) 
0.87 .+-. 0.08 
16 0.99 
1.01 0.98 0.94 
__________________________________________________________________________ 
.sup.a m is the slope (SEvalues are given when four or more concentration 
have been used in the median effect plot), EC.sub.50 is the median 
effective concentration, and r is the correlation coefficient, as 
determined from the median effect plot. 
.sup.b C.I. &lt; 1 indicates synergy, C.I. = 1 indicates additivity, and C.I 
&gt; 1 indicates antagonism (See Materials and Methods). F.sub.a is a 
component of the median effect equation referring to the fraction of the 
system affected (e.g., 0.50 means the C.I. at a 50% reduction of 
activity). C.I. values for CMVinfected cells were determined under both 
mutually exclusive and mutually nonexclusive (numbers in paranthesis) 
assumptions.