Method of treating humans and animals infected with viruses of the retrovirus group

Compositions useful in methods of treating humans and animals infected with viruses of the retrovirus group contain azoic compounds of the general formula ##STR1## wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are indentical or different and each represents an atom of hydrogen or an aliphatic or aromatic hydrocarbon radical comprising from 1 to 6 carbon atoms, R.sup.1 and R.sup.2 may be bonded together to form a heterocyclic ring with their adjacent nitrogen atom, and R.sup.3 and R.sup.4 may be bonded together to form a heterocyclic ring with their adjacent nitrogen atom; X.sup.1 and X.sup.2 are indentical or different and each represents an oxygen atom or an NR.sup.5 group, wherein R.sup.5 is a hydrogen atom, an aliphatic or aromatic hydrocarbon radical comprising from 1 to 6 carbon atoms, or a nitro group; and wherein when two NR.sup.5 groups are simultaneously present each R.sup.5 may be indentical to or different from the other.

FIELD OF THE INVENTION 
The present invention relates to azoic derivatives and to pharmaceutical 
and disinfectant compositions containing these derivatives. 
The present invention also relates to methods of treating humans and 
animals infected with viruses of the retroviruses group, by using the 
derivatives and compositions. 
BACKGROUND OF THE INVENTION 
The retroviruses are defined according to the invention as viruses wherein 
the genetic material carried on a chain of ribonucleic acid is transcribed 
inside a target cell of desoxyribonucleic acid by means of an enzyme 
called reverse transcriptase. 
These viruses are responsible for pathologies in the vegetal and animal 
worlds. A non-exhaustive list of said viruses is to be found in J. M. 
HURAUX et al., Virologie, Flammarion Medecine-Science, 1985, Paris. 
When the integration stage in the target cell chromosomes has been reached, 
the recovery likelihoods (return to the previous condition) are low. As a 
matter of fact, these viruses infect cell series of various types and no 
drug able to extract the viral genetic material from infected cells seems 
to be probable at the present time. 
Besides, these viruses have mutation properties and they are screened by 
animal pools which allow them to occur as new antigenic forms (by use of 
cellular fragments of the host cell, for example), which causes the 
vaccination to be complex. 
Today only one treatment is known which extends the survival of the 
patients, however not allowing a cure. This treatment comprises 
administration of 3'-azido-3'-desoxythymidine (see EP-A196185). This 
substance acts by reverse transcriptase inhibition. 
Other substances are known as inhibiting replication of viruses HIV through 
their action on the reverse transcriptase. Some didesoxynucleotides (see 
EP-A-307914) may be, for example, cited. 
It has also been found that some substances have as an effect to block the 
penetration of the viruses into the cells. As substances having this 
effect, it may be cited for example oligo-saccharides or polysaccharides 
(see EP-A-240098) or also castonospermine (B. D. WALKER, Inhibition of 
human Immunodeficiency virus suncytium formation and virus replication by 
castonospermine, Proc. Nail. Acad. Sci. USA, vol. 84, p. 8120-8124, Nov. 
1987). 
According to these treatments, it may be hoped that the virus having 
infected a patient will not follow its development and its propagation. 
However, the patient does not return to this condition before the 
affection because the provirus is not affected and it subsists inside the 
cell which has been previously attacked. The treatment is thus palliative 
and not curative. 
This kind of treatment has the important danger to allow resistance of 
viruses to compounds to appear. It seems already established that the 
virus becomes resistant to 3'-azido-3'-desoxythymidine after a more or 
less long term, in particular after about 12 to 18 months (SCHULHAFER E. 
et al., Acquired immuno-deficiency syndrome . . . , In Vivo 3(2):61-78 
(1989)). 
Finally, it has already been considered to use, as a drug against 
retroviruses, some benzidine derivatives which bear amongst others azoic 
groups (see FR-A-2612515). However, in this document there is only a 
simple affirmation concerning the activity of these compounds. 
The transmission way of the viruses have been determined in case of a 
direct blood contact and contact through wounds by infected material. The 
risk of a transmission from infected objects and particularly medical 
material is not to be neglected. 
On the other hand, the sexual transmission and the transmission to children 
via infected mother's milk are also established, which shows that the 
passage of infecting particles through sound mucosas is possible. (P. 
LEPAGE et al., Postnatal Transmission of HIV from Mother to Child, The 
Lancet, Aug. 15, 1987 p. 400; C. J. MILLER et al., Genital Mucosal 
Transmission of Simian Immunodeficiency Virus, Journal of Virology, Oct. 
1989, pp. 4277-4284). 
The existence of a virus, inoculation by way of a simple contact, i.e. 
through skin or mucosa, seems more and more likely. According to the 
opinion of some searchers, the inoculated viruses would seem to go through 
a replication phase during which they remain in the area of the mucosa or 
skin of the carrier. This phase could continue for several months. It 
would be in the second phase only that the viruses and/or the constituents 
thereof would spread from the mucosa. (R. ZITTOUN, Syndrome Immuno 
Deficitaire Acquis, Doins Editeurs, Paris, 1986, p. 183-184). 
Thus for the eradication of the disease, it is appeared as necessary to 
prepare molecules able to disinfect inanimated surfaces and objects, as 
well as materials and products which come into contact with mucosa and 
skin. It is appeared as essential to impede as far as possible the 
retrovirus transmission from a carrier to a healthy person. 
Compositions and a method for disinfecting, which use natural or synthetic 
oliosaccharides or polysaccharides having at least one S-oxoacid group, 
are already known (see EP-A-285357). However, from the Examples, it 
results clearly that, even if these compositions are active against the 
retroviruses, a part of the treated viruses always subsist, with the 
enormous risk to see after a term the generation of a still more dangerous 
resistant virus population. 
In an international patent application WO-A-90/01935, products are already 
provided, which are able to come locally into contact with skin, mucosas 
or body secretions, these products comprising an agent active against the 
viruses of the retrovirus group, for example sodium suramine, as well as 
some complex azoic derivatives such as pyridium, neotropine, Congo red, 
trypan blue, trypan red and trypan violet. 
The action of usual chemical disinfectants, such as ethanol, 
glutaraldehyde, sodium hypochlorite, formalin, .beta.-propiolactone, 
methylated spirit amongst others, has been examined against retroviruses. 
(V. B. SPIRE et al., Inactivation of Lymphadenopathy associated virus by 
chemical disinfectants, The Lancet, Oct. 20, 1984, pp. 899-901; L. RESNICK 
et al., Stability and inactivation of HTLV-III/LAV under clinical and 
laboratory environments, JAMA, Apr. 11, 1986, Volume 255, No. 14 ; L. S. 
MARTIN et al., Disinfection and inactivation of the human T lymphotropic 
Virus type III/lymphadenopathy-associated virus, The Journal of Infectious 
Diseases, Vol. 152, No. 2, Aug. 1985; P. J. V. HANSON et al., Chemical 
inactivation of HIV on surfaces, Br. Med. J., 1989, 198:862-4). 
It results from these assays that most of tile disinfectants used in 
hospitals are inefficacious or not very efficient against retroviruses 
HIV, and consequently potentially dangerous. Those which are the most 
efficient require rather long contact times, sometimes 10 minutes and 
more, which is concretely difficult to apply for cleaning grounds, tables, 
for example. Moreover, some of said disinfectants seem to lose their 
effectiveness in the presence of proteinaceous materials or are not of 
application if they must come into contact with the skin or the mucosa of 
a living body, due to their chemical agressivity or their cellular 
toxicity. 
SUMMARY OF THE INVENTION 
Consequently, the present invention has for its object to provide an active 
agent against the viruses, particularly of the retrovirus group, in 
particular the human immunodeficiency viruses HIV, this agent being able 
to be radically active on said viruses, preferably with a complete removal 
of the latter, while maintaining its activity at very low concentrations. 
Advantagelously, said agent will maintain its active power within a 
cellular medium, an aqueous medium as well as in the presence of organic, 
particularly proteinaceous materials. Its toxicity will preferably be low. 
According to a preferred embodiment, the virucidal action will be very 
rapid against the viruses HIV. 
The invention has also for its object to provide a pharmaceutical 
composition allowing the treatment or prophylaxis of viral diseases. 
The invention has also for its object to prepare a composition for 
disinfecting inanimate objects against viruses. 
It is obvious that, according to its applications, either as active 
substance in a pharmaceutical composition, or as disinfecting agent in a 
cleaning product, a cosmetical composition or other products of this kind, 
the active agent will have to meet different requirements concerning 
solubility, toxicity or stability. It will be the same if application of 
the pharmaceutical composition must be made orally, parenterally, 
intravenously, topically or following another administration way, or if 
the disinfection concerns inanimate objects such as instruments or 
grounds, or on the contrary organic wastes or liquids to be absorbed. 
Suprisingly, it has been found that some azoic compounds are able to 
particularly effectively and radically play the part of the searched 
active agent. 
DETAILED DESCRIPTION OF THE INVENTION 
To solve the raised problems, it has been provided, according to the 
invention, azoic derivatives having the general formula: 
##STR2## 
wherein R.sub.1 to R.sub.4 are identical or different and each represent 
an atom of hydrogen or halogen, or a substituted or not, aliphatic or 
aromatic hydrocarbon radical, comprising from 1 to 6 carbon atoms; X.sub.1 
and X.sub.2 are identical or different and each represent an oxygen atom 
or a NR.sub.5 group, in which R.sub.5 is a hydrogen or halogen atom, an 
aliphatic or aromatic hydrocarbon radical comprising from 1 to 6 carbon 
atoms or a nitro group; R.sub.5 when two NR.sub.5 groups are 
simultaneously present, may have an identical or different meaning in each 
of said groups, and R.sub.5 having a meaning other than an atom of 
chlorine simultaneously in both NR.sub.5 groups when R.sub.1 to R.sub.4 
represent hydrogen, as well as their salts, esters and isomers, as 
therapeutically active substances. According to one embodiment, R.sup.1 
and R.sup.2 are bonded together and form a heterocyclic ring with their 
adjacent nitrogen atom. According to one embodiment, R.sup.3 and R4 are 
bonded together and form a heterocyclic ring with their adjacent nitrogen 
atom. According to yet another embodiment, R.sup.1 and R.sup.2 are bonded 
together and form a heterocyclic ring with their adjacent nitrogen atom 
and R.sup.3 and R4 are bonded together and form a heterocyclic ring with 
their adjacent nitrogen atom. 
According to the invention, in these derivatives, R.sub.1 to R.sub.5 may 
advantageously represent independently a lower aliphatic hydrocarbon 
radical, in particular a methyl, ethyl, propyl or butyl group. Benzyl 
groups may also be provided. In the compounds according to formula 1, the 
halogen atoms are in particular those of chlorine, bromine, iodine and 
fluorine. 
As azoic derivatives according to the invention, one may in particular 
consider 1.1'-azobisdimethylformamide, 1.1'-azobisformamidine, 
1.1'-azobisdimethylformamide, 1.1'-azobisnitroformamidine. Particular 
active substances used in the compositions of the present invention 
include azobisformamide, 1,1'-azobisformamide, and 
1,1'-(azodicarbonyl)dipiperidine. 
Single doses of between 7 and 140 mg per kg body weight are tolerable and 
effective in increasing CD4 cell count and enabling weight gain without 
causing diarrhea. Single doses of between 7 and 140 mg per kg body weight 
1,1'-azobisformamide (ADA) have proven tolerable and effective. The total 
amount per single dose can vary and may fall within the range of 500 and 
12,000 mg ADA per single dose. 
Administered three times a day, individual doses may range from 20 mg per 
kg body weight or less, to 90 mg per kg body weight or more. The dosage 
and dosage per kg body weight will vary depending upon a number of factors 
such as the overall health of the patient, the stage of the disease, and 
the effective body weight of the patient or volunteer. Total daily dosages 
of up to 18,000 mg per day may be administered. 
Treatments according to the present invention are expected to be effective 
against HIV and other viruses of the retrovirus group. Treatments using 
ADA have proven particularly effective, as discussed below. 
It has to be understood that the invention is not limited to 
1.1'-derivatives and that those in 2.2'-position are also included in the 
invention, as well as all the isomers and their mixtures. 
Preparation of 1.1'-azobisformamidine and 1.1'-azobisformamide has already 
been carried at the end of the last century by J. THIELE (see The Merck 
Index, 10 ed., 919, Rahway, 1983; F. C. SCHMELKES et al., 
N,N'-Dichloroazodicarbonamidine (azochloramide), an N-chloro derivative of 
the oxidant in an oxidation-reduction system, Journal of American Chemical 
Society, 56, 1610, 1934). 1.1'-Azobisformamide has been known as an 
adjuvant in food flour. 1.1'-Azobisdimethylformamide has also been known 
for a long time due to its intracellular oxidising action on human blood 
cell glutathione (N. S. KOSOWER et al., Diamide, a new reagent for the 
intracellular oxidation of glutathione to the disulfide, Biochemical and 
Biophysical Research Communications, vol. 37, No. 4, 1969). 
1.1'-Azobisnitroformamidine has also been known from a long time. (W. D. 
KUMLER, The Dipole Moments, Ultraviolet spectra and structure of 
azo-bis-(chloroformamidine) and azo-bis-(nitroformamidine), Journal of 
American Chemical Society, 75, 3092, 1953). It is clearly apparent from 
the documents of this state of the art that it has been fully unexpected 
to obtain the searched effect from these relatively simple substances 
which are unexpensive to manufacture and known for a very long time. 
A still more unexpected effect has been seen. It is appeared that some 
derivatives have a selective toxicity against cells infected by HIV virus, 
while the Supt-1 cells and the lymphocyte cells of the human body are not 
or only a little altered by the tested compounds. These observations have 
allowed to consider the possibility of a chemiotherapy which selectively 
destroys the infected cells while maintaining the sound cells of the 
patient. One may thus consider a recovery to the previous condition of the 
treated patient, i.e. a cure of the latter. 
According to the invention, it is thus provided a pharmaceutical 
composition comprising, as active substance, at least one azoic derivative 
having the general formula of claim 1 or a salt, ester or isomer which is 
pharmaceutically acceptable of said derivatives, and at least one 
pharmaceutically compatible excipient, as well as if necessary one or more 
pharmaceutically current adjuvants. This composition may be administered 
as any form, orally or sublingually, rectally or vaginally, by injection 
or perfusion, topically, transcutaneously or transmucosally, or by any 
other current form in therapeutical or veterinary medecine. The excipient 
and possible current adjuvants are selected according to the selected 
administration way. Advantageously, preservation or solubilization agents, 
pH neutrality agents, isotonicity agents, buffer agents or other agents 
may be added to the composition. 
The selected excipient or vehicle can be solid or liquid. The composition 
will be at the option as a powder, ointment, tablets, capsules, aerosols, 
liquid to be injected and the like. 
Also some formulas which release the active substance with late effect can 
also be provided. 
According to an advantageous embodiment of the invention, the 
pharmaceutical composition according to the invention comprises a 
disinfectant as a supplement. As a matter of fact, in addition to the 
curative effect of the composition, it may be advantageous for the virus 
carrier that the composition defends him against external aggressions 
which, while stimulating his immunitary defence system, promote the 
proliferation of the HIV viruses that he carries. 
Also according to the invention, the same azoic derivatives having the 
above-mentioned formula are provided as active substances to combat, on 
and/or in inanimate objects, with the viruses, in particular of the 
retrovirus groups, more particularly the HIV human immunodeficiency 
viruses. 
According to the invention, it is also provided compositions to disinfect 
inanimate objects, containing at least one of these active azoic 
derivatives as well as a suitable vehicle. As vehicle, one may 
advantageously provide water or any other suitable solvent in which the 
active agent is in solution. Other current disinfectant or adjuvant agents 
can be if necessary provided in supplement. 
According to the invention, use of the active azoic derivatives or of 
above-mentioned disinfection compositions is made for the disinfection of 
inanimate object against the viruses, in particular the retrovirus group, 
more particularly HIV human immunodeficiency viruses. 
As inanimate objects to be disinfected, use of the present invention can be 
made, lot example with: 
plastic, rubber or textile sanitary materials: wadding, absorbent 
cotton-wool, gauze, bandages, toilet paper, packing films, and the like. 
medical, veterinary or dentist instruments and apparatuses: syringes, 
cannulas, sounds, clips, scissors, stomachal washing kits, surgical 
tables, basins, and the like. 
medical, veterinary or dentist clothes: gloves, dresses, towels, and the 
like. 
cosmetology instruments: material and equipment for hairdresser, 
manicurist, chiropodist, beautician, and the like. 
objects requiring a handling in the alimentary field: feeding-bottles, 
pans, bottles or cans, in particular for beverages, and the like. 
sanitary vehicules: ambulances, rolling tables, and the like. 
ground or wall surfaces: quarters or blocks, particularly in hospitals, and 
the like. 
sanitary and hygienic apparatuses: wash-hand basins, urinal vessels, 
dishes, bath-tubs, and the like. 
beverages: water necessary for beverages, milk, and the like. 
water for swimming pools. 
The disinfection of excrements, wastes of analysis laboratories and 
particularly of samples taken off from a human or animal body, for example 
lot an analysis, can be provided. 
According to the invention, some cosmetic compositions can advantageously 
be provided in order to include also at least an active azoic derivative 
of the invention. Obviously in this case, various usual carriers and 
additives in this field can be applied. 
A particular use of an active agent or of an active composition according 
to the invention can be provided in or on products which can come into 
contact with the skin or the mucosa of a human or animal body, which 
optionally is a virus carrier. In or on some of said products, such as 
physician or dentist gloves, pessaries, contraceptive sheaths, and the 
like, the active agent or composition can in addition to its disinfecting 
action form a barrier medium for the transmission of retroviruses from a 
carrier to a healthy person. For example, it is possible to lubricate a 
contraceptive rubber sheath with a petrolatum including an active agent 
according to the invention. For physician gloves, it is also possible to 
provide two rubber films between which is for example located an amylase 
amylopectin powder including an active agent according to the invention. 
In the last case, the disinfecting powder to be used as barrier is thus 
not in direct contact with the skin. 
The disinfection composition according to the invention may also optionally 
contain a disinfectant agent as a supplement, preferably with a wide 
spectrum of germicidal action. The so obtained composition has thus an 
appreciable defense against the presence of pathogenic or allogenic 
agents, other than retroviruses HIV. 
This last property is very important not only due to the important 
disinfection action such as obtained, but also because it can be useful 
for the virus carrier himself. As a matter of fact, the latter must avoid 
as much as possible any activation of his lymphocytar cells. Such an 
activation, for the HIV carrier, has as an effect the replication of the 
virus and the proliferation thereof in his cells. A HIV carrier must 
advantageously follow hygienic life habits in order to avoid at the 
maximum any risk of infected cell activation and consequently an 
immunitary reaction of his organism. 
By the use of agents and compositions according to the invention, the virus 
carrier has the possibility to disinfect himself, but also additionally to 
obtain a protection against immunitary reactions from another source.

The invention will now be illustrated in a more detailed manner by means of 
some non-limiting examples. 
EXAMPLE 1 
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Tablets 
______________________________________ 
1.1'-azobisdimethylformamide 
200-300 mgr 
Microcristalline cellulose (Avicel PH 101) 
60 mgr 
Povidone BP 15 mgr 
Sodium starch glycolate 20 mgr 
Magnesium stearate 5 mgr 
lactose, q.s. for 500 mgr 
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EXAMPLE 2 
______________________________________ 
Capsules 
______________________________________ 
1.1'-azobisformamide 400-600 mgr 
Microcrystalline cellulose (Avicel) for one capsule 
50 mgr 
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EXAMPLE 3 
______________________________________ 
Long-release composition (6 to 8 hours) 
______________________________________ 
1.1'azobisdimethylformamaide 
400-600 mgr 
Hydroxypropyl methyl cellulose 
111 mgr 
Lactose 53 mgr 
Povidone BP 28 mgr 
Magnesium stearate 7 mgr 
______________________________________ 
First, hydroxypropyl methyl cellulose is mixed with 
1.1'-azobisdimethylformamide, then the other components are added. 
EXAMPLE 4 
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Injectables 
______________________________________ 
1.1'-azobisformamide 
10-30 mgr 
Hydrochloric acid 
0.1 M 
Sodium hydroxide 0.1 M 
______________________________________ 
pH is adjusted to a value of 4 to 7 by means of either the acid or the 
base, the product is hot dissolved and filtration is made on sterile 
micropore. The liquid is then poured into brown sterile ampoules. 
EXAMPLE 5 
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Injectables 
______________________________________ 
1.1'-azobisformamidine 
50-150 mgr 
Benzyl alcohol 10 mgr 
Glycofurol 75 mgr 
Water for injection 
3 ml 
______________________________________ 
The product is prepared by dissolution of the active substance in 
glycofurol, then mixing the so obtained solution with filtered water and 
benzyl alcohol. The whole is poured into a sterile brown ampoule. 
EXAMPLE 6 
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Injectables by intravenous way 
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1.1'-azobisnitroformamidine 
50-150 mgr 
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Sterile water without pyrogen is added to this component, this water being 
buffered by a phosphate buffer at pH 7, in order to obtain 27 ml. 
EXAMPLE 7 
Suppository 
To 150-250 gr of micronized powder of 1.1'-azobisformamidine, 2 gr of 
glycerin are added in order to form a suppository. 
Experimental tests have been made by the laboratory of Institut Pasteur of 
Brabant, Belgium, in order to examine the efficiency of the azoic 
derivatives according to the invention., 
EXAMPLE 8 
Starting Materials of the Test 
1.1'-azobisformamidine of a purity level of 98%. The purity has been 
examined by a carbon-hydrogen test, 
a supernatent of cell cultures (Molt-3) which continuously produce HIV-1 
viruses, The used viral supernatent has a reverse transcriptase activity 
of 1.times.10.sup.6 cpm/ml, 
cells of a continue human T line of T.sub.4 phenotype (Supt-1) cultivated 
in a RPMI medium additionally supplied with 10% of foetal calf serum and 
1% of glutamine, 
The effectiveness of 1.1'-azobisformamidine has been investigated on two 
aspects: 
its toxicity against sound cells 
its action on the infectious power of HIV-1 viruses for the Supt-1 line. 
(a) Examination of the Toxicity against Sound Cells 
The cellular mortality has been determined by exclusion with trypan blue. 
To dilutions of 1/500, 1/1500 and 1/3000, 1.1'-azobisformamidine does not 
decrease the cellular viability of Supt-1 cells. 
(b) Examination of the Infectious Power 
Vital preparations of HIV-1 of high titer are incubated in the presence of 
dilutions of 1.1'-azobisformamidine of 1/500, 1/1500 and 1/3000 and for 
different incubation periods: 1 minute, 10 minutes and 30 minutes. For the 
incubation, 0.3 ml of virus concentrate and the active substance are 
brought together at a double concentration (once for the virus volume and 
once for the product volume). For 30 minutes at 37.degree. C., the Supt-1 
cells are previously treated with 10 .mu.gr/ml of polybren, then they are 
inoculated with the preparations of treated viruses. As controls, 
non-inoculated cells and cells inoculated with a non-treated virus 
preparation are provided. 
Then, the infectious power of said preparations is determined, for each 
cellular passage, the vital production (measure of the expressed p24 
antigen) being followed in the solubilized cellular lysates of the 
examined Supt-1 cells. 
The measures of the infectious power of HIV-1 virus after incubation with 
1.1'-azobisformamidine appear from the following Table 1, by comparison 
with a non-infected control and with a control infected by non-treated 
viruses. 
EXAMPLE 9 
The starting material for the test differs from that of Example 8 by using 
1.1'-azobisdimethylformamide instead of 1.1'-azobisformamidine. 
(a) Toxicity Examination 
At dilutions at 1/500, 1/1500 and 1/3000, the 1.1'-azobisdimethylformamide 
does not decrease the cellular viability of the Supt-1 cells. 
(b) Examination of the Infectious Power 
One proceeds in the same way as in Example 8b. The measures of the 
infectious power of the HIV-1 virus after incubation with 
1.1'-azobisdimethylformamide appear from the following Table I. 
TABLE 1 
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Measure of the infectious power of tthe HIV-1 on 
the 14th day after inoculation of Supt-1 cells. 
Duration of the 
treatment of the 
Active substance (dilution) 
viruses 
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1.1'-Azobisformamidine 
1/500 1 min 0.234 .+-. 0.008 
10 min 0.267 .+-. 0.021 
30 min 0.245 .+-. 0.020 
1/1500 1 min 0.469 .+-. 0.013 
10 min 0.439 .+-. 0.008 
30 min 0.406 .+-. 0.032 
1/3000 1 min 0.809 .+-. 0.049 
10 min 0.505 .+-. 0.023 
30 min 0.740 .+-. 0.013 
1.1'-Azobisdimethylformamide 
1/500 1 min 0.275 .+-. 0.049 
10 min 0.206 .+-. 0.006 
30 min 0.203 .+-. 0.008 
1.1500 1 min 0.278 .+-. 0.050 
10 min 0.217 .+-. 0.028 
30 min 0.193 .+-. 0.004 
1/3000 1 min 0.239 .+-. 0.016 
10 min 0.283 .+-. 0.042 
30 min 0.198-0.009 
Virus control 1.033 .+-. 0.081 
Cell control 0.206 .+-. 0.005 
(optical density 492 nm). 
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It is clearly apparent from this Table that at a dilution of 1/500, 
1.1'-azobisformamidine protects the Supt-1 cells after a treatment of the 
viruses for 1 minute only. 1.1'-azobisdimethylformamide has this effect 
even at dilutions of 1/3000. For the latter substance, the test has been 
extended up to the 25th day. The effectiveness of this active substance is 
maintained for all the dilutions, when the viruses have been treated for 
30 mintues. 
On the other hand, with a phase-contrast microscope, no cytopathogenic 
effect was observed for the cells inoculated with virus previously treated 
with 1.1'-azobisdimethylformamide and 1.1'-azobisformamidine. On the 
contrary, syncytia appear from the 14th day on after infection with the 
control viral preparation. 
(c) 1.1'-azobisdimethylformamide Has Moreover Been Examined Concerning Its 
Toxicity Against Cells Infected with Viruses 
To this end, the cellular mortality has been determined by exclusion with 
trypan blue. 
This examination has been made on Molt-3 cells infected by HTLVIII-B which 
are producing HIV-1 viruses. 
TABLE 2 
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% of 
Molt-3 cells Number of Number of dead 
(HIV-1) living cells 
dead cells 
cells 
______________________________________ 
Control 57 5 8 
Cells treated witth 
1.1'-azobisdimethylformamide 
Dilution Duration (min.) 
1/500 1 62 9 12.7 
10 67 11 14 
30 58 12 17.1 
1/1500 30 70 15 17.6 
1/3000 30 58 12 17.1 
______________________________________ 
It results very clearly from the Table 2 that the mortality of the infected 
cells is doubled in the presence of 1.1-azobisdimethylformamide after 30 
minutes of action, even at the dilution of 1/3000, while this molecule 
shows no toxicity for the human Supt-1 and lymphocyte cellular lines, even 
after 24 th hours. 
EXAMPLE 10 
The test material is different from that of Example 9 in that 
1.1'-azobisdimethylformamide is dosed at a dilution of 1/100 (10 mgr/ml). 
(a) Examination of the Cellular Toxicity Against Normal Cells, Namely 
Non-Infected Cells (Supt- 1 line) 
The cellular mortality has been determined by exclusion with trypan blue. 
TABLE 3 
______________________________________ 
% of 
Duration Number of Number of 
dead 
Supt-1 cells (hours) living cells 
dead cells 
cells 
______________________________________ 
Controls 0 61 19 24 
1 57 18 32 
5 55 20 36 
Cells treated with 
0 61 21 26 
1.1'-azobisdimethyl- 
1 63 20 24 
formamide (dilu- 
5 56 22 28 
tion 1/100) 
______________________________________ 
These results are given by ml of culture which was taken off. 
It is clearly apparent from the Table that, even at a relatively high 
concentration, the 1.1'-azobisdimethylformamide has no toxicity against 
sound Supt-1 cells. 
(b) Examination of the Infectious Power of the HIV-1 Virus 
One proceeds as in Example 8(b) by treating the viruses for 30 minutes with 
1.1'-azobisdimethylformamide at a dilution of 1/1000. After inoculation of 
Supt-1 cells with this viral preparation, the p24antigens are determined, 
being expressed by the optical density, after 14, 18 and 21 days. 
TABLE 4 
______________________________________ 
p24 Antigens expressed by optical density 
Day 14 Day 18 Day 21 
______________________________________ 
Control cells 0.125 0.126 0.168 
Control cells infected 
0.224 1.078 0.765 
by HIV 
Cells + HIV-1 treated 
0.144 0.160 0,130 
by the acive substance 
______________________________________ 
It is apparent from this experience that the HIV treated with 
1.1'-azobisdimethylformamide is not able to infect the sound Supt-1 cells. 
(c) Examination of the Integration of the Viral Genome into the Chromosomic 
DNA of Supt-1 Cells 
By means of a genetic amplification through a thermostable polymerase, the 
presence of genes "GAG", "LTR" and "ENV" of the HIV-1 provirus is 
evidenced. This method is called P.C.R. Polymerase chain Reaction) 
(Chin-Yih Ou et al., Sciences 239, 295-297, 1988). 
Said method is applied for detecting HIV-1 provirus in the genome of Supt-1 
cells inoculated with HIV-1 virus incubated with 
1.1'-azobisdimethylformamide (dilution 1/100), as described in Example 8 
Amplified oligonucleotides, UV visible by means of ethidium bromide appear 
only in the tubes corresponding to an inoculation with HIV-1 viruses which 
were not treated with the active substance. 
Moreover, after hybridaton with specific p.sup.32 marked moulds for to the 
three searched genes, it is possible to conclude that HIV-1 provirus is 
totally absent within Supt-1 cells which have received inoculates of HIV-1 
virion treated with a 1/100 dilution of to the active substance. 
It may thus be deducted that this active substance protects the cells 
against the infection by HIV-1. 
EXAMPLE 11 
The test material is different from that of Example 8 in that the used 
substance is 1.1'-azobisformamide dosed at a concentration of 35 mgr/l, 
i.e. 35 .mu.gr/ml, namely at an extremely low concentration (obtained by 
the supernatent of a suspension of 0.5 gr/l in RPMI). 
(a) Examination of the Cellular Toxicity Against Sound Cells 
No toxical effect has been noted for to the Supt-1 cells, even after 24 
hours of incubation. 
(b) Examination of the Infectious Power 
One proceeds as in Example 8(b) with incubation periods of the viruses of 
15 minutes 30 minutes, 60 minutes and 120 minutes. No cytopathogenic 
effect has been observed (no formation of syncytia). 
TABLE 5 
______________________________________ 
p24 Antigens expressed by optical densiy 
Day 14 Day 18 Day 21 
______________________________________ 
Control cells 0.140 0.142 0.136 
Infected conrol cells 
1.014 1.510 0.730 
Cells + HIV-1 treated 
0.134 0.150 0.140 
15 min. 
Cells HIV-1 treated 
0.152 0.170 0.146 
30 min. 
Cells + HIV-1 treated 
0.142 0.162 0.135 
60 min. 
Cells + HIV-1 treated 
0.145 0.168 0.154 
120 min. 
______________________________________ 
It can thus be concluded that the 1.1'-azobisformamide protects the 
cellular cultures against the infectious power of HIV-1. 
(c) Examination of the Integration of the Viral Genome into Chromosomic DNA 
of Supt-1 Cells 
One proceeds as in Example 11(c). The search of genes "GAG", "LTR" and 
"ENV" has been made in cellular cultures inoculated with a virus incubated 
for 15 minutes with the active substance. This search is appeared as being 
negative, and thus it can be concluded that 1.1'-azobisformamide at the 
dosage of 35 .mu.gr/ml protects the cells against infection by HIV-1. 
EXAMPLE 12 
The absence of toxicity of azobisformamide for the humans has already been 
described by B. L. OSER et al., Studies of the Safety of Azodicarbonamide 
as a Flour-Maturing agent, Toxicology and applied Pharmacology 7, 445-472 
(1965). 
Three sound volunteers were treated for 30 days with 1500 mgr a day of a 
azobisformamide in three portions of 500 mgr. 
No secondary effect was reported and the hematological parameters remained 
perfectly normal for the experimental treatment of 30 days. 
On the other handy three patients presenting different stages of AIDS were 
also treated with azobisformamide in the same dosages as he sound 
volunteers. 
The first patient in final phase presented at least 30 T.sub.4 
lymphocytes/mm.sup.3, abundant diarrheas and a complete space-time 
disorientation. 
The second patient in "ARC" phase presented a lymphocytar T.sub.4 
population of 190/ mm.sup.3, in constant decrease, as well as occasional 
diarrheas and anal herpes. 
The third patient was a sound seropositive, he had a population of 350 
T.sub.4 lymphocytes/mm.sup.3 and he had no sign of pathology. 
The first patient had on day 0 of the treatment 1850 white corpuscules 
including 17% of lymphocytes of which 5% presented the T.sub.4 receptor 
(namely 24 cells). On day 30 of the treatment, the white corpuscules were 
in an amount of 2600 including 20% of lymphocytes of which 9% presented 
the T.sub.4 receptor (namely 49 cells/mm.sup.3)., which represented an 
improvement of 100%. On the other hand, the diarrhea has ended, the 
patient had recovered his possibility of coherent talking with his near 
relations, as well as a limited walking autonomy. 
The second patient had on day 30 280 T.sub.4 lymphocytes/ mm.sup.3 , the 
diarrhea had completely disappeared as well as the anal herpes. 
The third patient has on day 30 440 T.sub.4 lymphocytes/ mm.sup.3. 
These results, even if they concern a restricted sampling, are remarkable 
and surprising and they could in no way be expected by the ones skilled in 
the art. 
EXAMPLE 13 
Disinfectant effevescent tablet for 100 cm.sup.3 of water. 
______________________________________ 
1.1'-Azobisdimethylformamide 
33 mgr 
Citric acid 15 mgr 
Tartaric acid 17,5 mgr 
NaH CO.sub.3 37,5 mgr 
Avicel PH 1C.sub.2 27 mgr 
Lactose EFK 45 mgr 
For one tablet: 175 mgr 
______________________________________ 
EXAMPLE 14 
Disinfectant effervescent tablet, for 1000 cm.sup.3 of water. 
______________________________________ 
2.2-Azobismethylformamidine 
330 mgr 
Citric acid 150 mgr 
Tartaric acid 175 mgr 
NaHCO.sub.3 375 mgr 
Avicel PH IC.sub.2 180 mgr 
Lactose EFK 522.2 mgr 
Sodium lauryl sulfate 
64 mgr 
Aerosil 200 3.8 mgr 
For one tablet: 1800 mgr 
______________________________________ 
EXAMPLE 15 
Tooth-paste 
To 100 gr of a tooth-paste comprising 4% of ricinilate, 30 mgr of 
1.1'-azobisfluoroformamidine (for 30 days) were added. 
EXAMPLE 16 
______________________________________ 
Mouth-wash 
______________________________________ 
Sodium perfluorate 8 gr 
Borax 32 gr 
Sodium chloride 20 gr 
Sodium bicarbonate 40 gr 
1.1'-azobisfluoroformamidine 
30 gr 
Mint oil 3 drops 
1 coffee spoonful in a lukewarm water cup. 
______________________________________ 
EXAMPLE 17 
______________________________________ 
Cream 
______________________________________ 
1.1'-Azobisformamide 1 gr 
Triethanolamine 1 gr 
Glycerol 2.5 gr 
White wax 2.5 gr 
Stearic acid 6 gr 
Almond oil 7.5 gr 
Lavender oil 2 drops 
Aqua conservans, ad 50 gr FMS 
for 50 gr of cream. 
______________________________________ 
EXAMPLE 18 
______________________________________ 
Talc 
______________________________________ 
1.1'-Azobisformamide 2 gr 
Lavender oil 5 drops 
Talc ad 100 gr 
for 100 gr of talc. 
______________________________________ 
EXAMPLE 19 
______________________________________ 
Liquid soap 
______________________________________ 
1-Monochloro-azobisformamidine 
3 gr 
Potassium soap 60 gr 
Lavender oil 10 drops 
Antiseptic solution ad 100 gr 
for 100 gr of soap. 
______________________________________ 
EXAMPLE 20 
Toilet paper, hygienic bands and tampons, wadding and the like. 
A powder to be sprayed was first prepared, the composition of which is for 
example as follows: 
______________________________________ 
1.1'-Azobisdimethylformamide 
20 mgr 
Bismuth sub-gallate 50 gr 
Zinc peroxide 100 gr 
Talc 840 gr 
for 1 kg of powder to be sprayed. 
______________________________________ 
This powder which adheres to the fibers of the treated products was then 
sprayed in a usual way. 
EXAMPLE 21 
______________________________________ 
Oil for preservative sheath 
______________________________________ 
Silicone oil 100 gr 
1.1'-Azobisformamide 
1 gr 
______________________________________ 
The preservative sheaths are coated with the preparation as well as on the 
internal face as on the external one. 
It has to be understood that the present invention is in no way limited to 
the hereinabove described embodiments and that many variants may be 
brought therein without departing from the scope of this invention. 
Many other disinfectant of pharmaceutical compositions may be provided in 
addition to those given as Examples, by simply using the formulations such 
as used in general in the concerned fields, such as cleaning products, 
cosmetic compositions, pharmaceutical products and the like. 
Discussion 
I. First Type 
The assays according to Examples 8b, 9b, 10b, 10c, 11b, and 11c of the 
present specification. 
All these experiments comprise isolating free virus, exposing the virus to 
the active substance, and finally infecting healthy cells with the exposed 
virus. 
The infectious power of HIV-1 is measured according to the well-known ELISA 
test technique. These tests are quantitative tests which consist of 
binding to the p24 protein of the HIV-1 a marker which absorbs light in a 
predetermined spectrum. The more virus that is present in the cellular 
lysates, the more the marker is present and the higher the optical 
density. 
Moreover, an examination of the inoculated cells was carried out with a 
phase-contrast microscope. If the substance to examine is without action 
on the virus, the infected cells are binding to other cells and form giant 
multinuclear cells, called syncytia. If the substance is active against 
the virus, there is no syncytia. 
Examples 10c and 11c disclose a more accurate method than the ELISA test 
for examining whether the inoculated cells contain HIV-1 at the end of the 
assay, i.e., the PCR-Method. 
All these assays are conducted in order to demonstrate whether the virus 
shows an infectious power or not after a treatment with an active 
substance, i.e., whether the virus is able to multiply or not in the 
infected cells after the treatment. It is impossible to deduce therefrom 
if the virus has been destroyed or not, but it is possible to determine if 
the virus continues to replicate or not, in the cells. 
II. Second Type 
The assay according to Example 9c of the present application. 
Example 9c discloses an assay conducted on previously infected cells in 
order to examine them after treatment with an active substance. 
In example 9c, Molt-3 cells infected by HTLVIII-B are examined. The 
cellular mortality is measured by the usual method of exclusion by means 
of trypan blue. 
It results from this assay that the mortality of the infected cells is 
doubled, with respect to the mortality of controls. 
It is possible to deduce from this assay that the active substance has a 
selective toxicity against cells infected by HIV. 
III. Third Type 
EXAMPLE 22 
A new assay was conducted by the AIDS laboratory of the REGA Institute for 
Medical Research of the Katholieke Universiteit of Leuven. This assay was 
performed in order to examine whether the active substance inhibits the 
replication of the retroviruses within infected cells. 
1,1'-azobisformamide was purchased from Riedel de Haen and stored in a 
refrigerator (4.degree.-8.degree. C.) under light-protected conditions. 
Dilutions were prepared in order to obtain concentrations of 0.4 .mu.g/ml, 
2 .mu.g/ml, 10 .mu.g/ml, 50 .mu.g/ml and 250 .mu.g/ml. 
Peripheral Blood Lymphocytes (PBLs) were freshly collected from a single 
donor. PBLs were activated by phytohemagglutinin at 2 .mu.g/ml during 3 
days, and then cultured in the presence of IL-2 for 1 week. 
HIV-1 strain III.sub.B /LAI was provided by R. C. Gallo (Popovic M. et al, 
Science 1984, 224:497-500). Stocks were obtained from the culture 
supernatants of HIV-1 infected cell lines (HUT 78/III.sub.B /LAI). 
The inhibitory effects of 1,1'-azobisformamide on the replication of strain 
III.sub.B /LAI in PBLs were monitored by the detection of HIV-1 p24 core 
antigens. PBLs were incubated with an excess of strain III.sub.B /LAI at 
37.degree. C. for 60 min. All cells were washed once with RPMI 1640 
medium. Thereafter cells were plated at 1.times.10.sup.6 cells per ml in 
the presence of various concentrations of the test compound. Seven days 
after plating PBLs, p24 antigens were detected by enzyme-linked 
immunosorbent assay (ELISA) (HIV-1 p24 Core Profile ELISA from Du Pont). 
This method comprises measuring the concentration of the viral antigen p24 
in the supernatant, i.e., the concentration of free viruses produced by 
replication of the viruses present in the infected cells. This measure is 
expressed as a measure of protection (see Table I below), where 
##EQU1## 
TABLE I 
______________________________________ 
Inhibition of HIV-1 replication 
in acutely infected PBLs by 1,1'-azobisformamide 
Cell line: PBLs acutely infected with III.sub.B /LAI 
Concentration 
Protection 
Compound .mu.g/ml (%) 
______________________________________ 
1,1'azobisformamide 
0 0 
0.4 15.2 
2 45.8 
10 77.6 
50 Toxic 
250 Toxic 
2.47 IC50 
______________________________________ 
"Toxic" means that the cells are destroyed, which renders impossible the 
ELISA test. However, this toxicity is maybe the result of the selective 
toxicity of the active substance against the infected cells. This result 
could be a supplementary advantage of the active substance. In any case, 
from concentration of 10 .mu.g/ml, the substance inhibits advantageously 
the replication of the viruses. 
Discussion 
From the three types of assays discussed immediately above, the main 
effects of the active substances according to the invention may be 
summarized as follows: 
(1) First type of assay: inactivation of free viruses. 
(2) Second type of assay: selective toxicity of the active substance 
against cells infected by the viruses. 
(3) Third type of assay: inhibition of the replication of the viruses 
within infected cells. 
EXAMPLE 23 
An additional assay was conducted in order to verify whether the active 
substances are also active against other viruses than the retroviruses. 
The assay consists of investigating the ability of 1,1'-azobisformamide 
(ADA) to inactivate Vesicular Stomatitis Virus (VSV) in plain RMI tissue 
culture medium. 
The active substance was obtained from Aldrich Chemical Co., as an orange 
powder. The material was dissolved in DMSO to obtain a final concentration 
of 20 mg/ml. 
One ml of VSV was thawed at room temperature and a sample of 0.06 ml of VSV 
is added to 29.94 ml of RPMI 1640 to form a 1/500 dilution. This dilution 
was shaken during 10 minutes at approximately 70 rpm. 
After agitation, an aliquot of 1 ml of the viral suspension was removed and 
kept at room temperature as the non-treated process control sample. 
Aliquots from the VSV suspension were distributed to four 15 ml sterile 
tubes as shown in Table II, to which were added aliquots of the ADA/DMSO 
solution as shown in the table. The final concentrations of ADA in the 
viral medium are shown in Table II. 
TABLE II 
______________________________________ 
Final concentrations of ADA in the VSV media 
Volume of Concentration 
Volume of VSV 
ADA/DMSO of ADA in 
suspension solution VSV medium 
Tube # (in ml) (in ml) (in .mu.g/ml) 
______________________________________ 
1 4.938 0.062 250 
2 4.963 0.037 150 
3 4.975 0.025 100 
4 4.987 0.012 50 
______________________________________ 
At 10, 60 and 1200 minutes after addition of the ADA solution to the viral 
suspension, 1 ml was sampled from each tube, 0.055 ml of which was added 
to the first well of a limiting dilution assay series, whereas the 
remainder was frozen immediately and stored. 
The dilution assay consists in tenfold dilutions of the VSV medium placed 
into wells containing monolayers of VERO cells. After incubation, cells 
are evaluated for cytopathic effects by light microscopy. The assay 
consists of determining the dilution of VSV medium at which 50% of the 
wells to which the dilution has been added show surviving cells. This 
dilution, which inhibits cell death in half the wells, is called the TCID 
50, and it is expressed as the logarithm of the dilution factor. Hence a 
TCID 50 corresponding to a dilution of the viral medium of 1/10,000 will 
be expressed as log.sub.10 10,000 (dilution factor=10,000), i.e. 4.0. To 
compare the inhibitory effects of a protective compound on cell death with 
a non-treated control VSV medium, the difference between the TCID 50 of 
the viral medium with a given concentration of the compound and the TCID 
50 of the viral medium without the compound (control) is used to express 
to the potency of the protective effect of the compound. This difference 
between the two TCID 50 values expressed in logarithms is called the "log 
reduction". 
TCID 50 determinations were made on the process control sample and on the 
four tubes as described in table II, each at three different times after 
addition of ADA to the viral suspension (i.e. 10, 60, and 1200 min.). Log 
reductions are therefore available for each of the four ADA concentrations 
tested at each of the three time points. 
Results 
No toxicity against VERO cells was observed, even at the highest ADA/DMSO 
concentrations tested and after exposure of the cells during 20 hours. 
The TCID 50 values and corresponding log reductions are presented in Table 
III. 
TABLE III 
______________________________________ 
TCID 50 and Log reduction (log red) results 
TIME 
POINT END- ADA CONCENTRATION (.mu.g/ml) 
(min) POINT 0 50 100 150 250 
______________________________________ 
10 TCID 50 7.95 6.90 6.73 6.73 6.55 
Log Red -- 1.05 1.22 1.22 1.40 
60 TCID 50 7.95 6.55 6.73 6.55 6.55 
Log Red -- 1.40 1.22 1.40 1.40 
1200 TCID 50 6.03 4.28 3.75 3.58 3.93 
Log Red -- 1.75 2.28 2.45 2.10 
______________________________________ 
TCID 50 of control--TCID 50 of ADA treated sample=log reduction, i.e., for 
example 7.95-6.90=1.05. 
The strongest effect with a log reduction of 2.28, 2.45 and 2.10 is 
observed after exposure at an ADA concentration of 150 .mu.g/ml. 
It can therefore be concluded that ADA dissolved in DMSO is effective 
against VSV in the foregoing type of VERO cell assay, with log reductions 
in excess of 2.0 at ADA concentrations of .gtoreq.100 .mu.g/ml after a 
duration of exposure of between 1 and 20 hours. 
EXAMPLE 24 
Liquid media was provided wherein azobisformamide (ADA, also referred to as 
azodicarbonamide) was present in the liquid at a concentration of from 0.4 
.mu.gr/ml to 250.0 .mu.gr/ml. The media was found to inhibit the 
replication of HIV-1 (strain IIIB(LAI)) and wild HIV-1 strains including 
strains phenotypically resistant to AZT (zidovudine), in Peripheral Blood 
Lymphocytes (PBLs) derived from HIV-1-negative human donors, and inhibits 
the replication of HIV-1 and HIV-2 (ROD) in MT4 cells. 
Target cells were infected with HIV stock solution and cultured in the 
liquid media. After collection from donors, cells were separated using the 
Ficoll/Hypaque cell separation technique, and PBL's were washed twice in 
phosphate buffered saline. Target cells were cultured in the presence of 
phytohemagglutinin for three days at 37.degree. C. in a CO.sub.2 
-controlled incubator. This was followed by a 90 minute incubation in the 
presence of the viral solution. Non-absorbed virus was removed during 5 
washing steps with fresh culture medium, and HIV-infected and 
mock-infected PBL's and MT4 cells were resuspended at 2.times.10.sup.5 and 
5.times.10.sup.5 cells per ml, respectively, in complete medium (RPMI 1640 
DM medium supplemented with 10% fetal calf serum and 20 .mu.gr/ml 
gentamycin) supplemented with 5% recombinant interleukin 2, to which the 
liquid media was added in order to reach the desired final concentrations. 
Fresh medium without ADA was added at day 4 after infection of the target 
cells. HIV p24 antigen in the cell culture media was quantified 7 to 8 
days after infection by an antigen-capture assay using a sandwich ELISA 
technique. The median IC50 (concentration in .mu.gr/ml inhibiting viral 
replication by 50%) was found to be 8 .mu.gr/ml calculated from an IC50 
range of from 1.6 .mu.gr/ml to 14.8 .mu.gr/ml as shown in the Table below. 
Complete inhibition (.gtoreq.90% inhibition, also referred to as IC90) was 
achieved with wild strains from patients, at a median concentration of 18 
.mu.gr/ml, calculated from the range of 17.4 .mu.gr/ml to 21.2 .mu.gr/ml 
as shown in Table IV below. Individual test results are also summarized in 
the Table below. From Table IV, it can be seen that the lowest observed 
IC50 value is 1.6 .mu.gr/ml in MT4 cells infected with HIV-1 strain 
LAI/IIIB. 
TABLE IV 
______________________________________ 
PBL's MT4 CELLS 
TYPE STRAIN Exp. # IC50 IC90 Exp. # 
IC50 IC90 
______________________________________ 
HIV-1 LAI/ 1 2.5 &gt;10.0 13 4.5 &gt;25.5 
IIIB 2 7.0 47.0 14 9.9 &gt;15.7 
3 13.8 39.7 15 9.7 12.7 
4 3.2 35.0 16 14.8 &gt;31.1 
5 3.9 &gt;10.0 17 1.6 8.0 
18 5.1 8.0 
Wild A* 6 8.9 17.4 
Wild B* 7 11.6 18.6 
Wild C* 9 11.1 18.0 
Wild D 9 10.0 18.1 
Wild E 10 8.3 18.4 
Wild F* 11 6.4 19.0 
Wild G* 12 11.2 21.2 
HIV-2 ROD 19 4.1 7.6 
20 6.0 &gt;29.0 
21 14.3 &gt;17.5 
22 6.7 8.3 
______________________________________ 
*Phenotypically resistant to AZT; wild strains F and G are also syncytium 
inducers. 
EXAMPLE 25 
Pharmaceutical grade ADA in gelatin capsules or enteric-coated tablets, 
each containing 500 mg, was administered as single oral doses to healthy 
volunteers in order to estimate the maximum tolerated dose (MTD) in humans 
and to detect urinary excretion of ADA. Enteric-coated tablets are 
generally considered as having a delayed distribution in the 
gastro-enteric tractus. Doses of 35 mg/kg (mgADA per kg body weight), 70 
mg/kg, and 140 mg/kg were administered. No adverse effects such as changes 
in hemodynamic parameters or subjective well-being were observed with the 
lower doses, whereas a flu-like syndrome was observed from 6 hours after 
intake of the highest dose. The flu-like symptoms lasted several hours. 
The MTD for a single dose was therefore estimated to be 140 mg/kg. In the 
urine collected 2 to 3 hours after intake of a single dose of 3 g (6 
gelatin capsules.apprxeq.35 mg/kg), ADA was detected in the urine of one 
volunteer at a concentration of 25-30 .mu.gr/ml, showing that the drug can 
be absorbed and even excreted in its active form. 
EXAMPLE 26 
Pharmaceutical grade ADA in gelatin capsules, each containing 500 mg, was 
administered as single oral doses to healthy volunteers to determine its 
tolerability and safety. A dose-related increase in methemoglobin was 
observed in one volunteer, with Cmax at 1-1.5 hours after intake. 
Methemoglobin returned to normal within half an hour. Glutathione and 
methemoglobin reduces in peripheral blood were slightly depressed in the 
volunteers from doses of 40 mg/kg. These changes were reversible within 
4-6 hours from intake. No adverse effects were observed after either 40 
mg/kg or 60 mg/kg doses on a broad range of hematological and chemical 
parameters in the blood and urine of the volunteers. No adverse effects 
were observed at any of the tested dosages on blood pressure, pulse, or 
respiratory rate. It is therefore concluded that (1) ADA is absorbed via 
the gastro-intestinal route, (2) ADA distributes readily to intracellular 
compartments, with an intracellular peak 1-1.5 hours after intake, and (3) 
ADA is well tolerated at single oral dosages of up to 60 mg per kg body 
weight. 
EXAMPLE 27 
Pharmaceutical grade ADA in gelatin capsules, containing 250 mg or 500 mg, 
were administered to one patient with stage IV AIDS. The patient started 
ADA treatment with a 500 mg dose three times a day corresponding to a 
total of about 25 mg/kg body weight taken three times a day. The total 
daily dose was therefore 1.5 gram. Absolute CD4 lymphocyte counts 
increased from 77 cells/mm.sup.3 to 144 cells/mm.sup.3 over a 26-week 
period. Concomitant medication with DDI (didanosine, from Bristol Myers 
Squibb), and subsequently with a combination of DDI and DDC (zalcitabine, 
from Hoffman-LaRoche) was stopped because of resistance, and another 
experimental reverse-transcriptase (RT) inhibitor (d4T from Bristol-Meyers 
Squibb) was administered instead. As CD4 lymphocyte counts continued to 
improve, ADA was voluntarily withdrawn in order to test the effect on CD4 
lymphocyte counts. Under RT medication, but without ADA, the CD4 
lymphocyte count returned to baseline within two months, the patient lost 
weight and developed intractable diarrhea requiring rehydration. ADA was 
restarted at a dose of 1 gram three times a day, twice the initial dose, 
whereupon the CD4 lymphocyte count returned to 144 cells/mm.sup.3 within 
two weeks. The patient gained considerable weight and was free of diarrhea 
12 weeks after restarting ADA. Fifteen weeks after restarting ADA, the CD4 
lymphocyte count increased further to 190 cells/mm.sup.3. No adverse 
effects that could be drug-related were reported, despite occasional 
dosage increases of up to 2 grams three times a day. Gene sequencing based 
on viral RNA-extraction from the patient's blood shows that this strain 
has mutations in codons 67, 70, 184, 215 and 219, indicating that it is 
genetically resistant to AZT, DDI, DDC, and 3TC (lamivudine available from 
BioChem Pharma/Glaxo). 
EXAMPLE 28 
ADA in a liquid medium was tested in an acellular in-vitro system for any 
inhibitory effect on HIV-1 reverse transcriptase (RT) using the 
polyrC-oligodG template-primer-complex. ADA is not an RT-inhibitor at 
concentrations up to the IC90. From concentrations of 60 .mu.gr/ml upward, 
there is a 50% inhibition of RT. ADA is not a Tat-antagonist. ADA does not 
inhibit pepsin, an enzyme with an active site almost identical to that of 
HIV-1 protease. Therefore, ADA inhibits HIV replication with an as yet 
unknown but original mode of action. In-vitro, ADA showed additive effects 
to those of didanosine at concentrations at and below the IC50. 
EXAMPLE 29 
Four patients with advanced AIDS have been treated with ADA. One patient 
has been treated during two periods, i.e., treated was restarted after a 
return to baseline subsequent to the withdrawal of ADA. The patients 
received capsules containing 500 mg. The dosage regimen was 500 mg three 
times a day except during the second treatment period in the patient who 
is also the patient of Example 27 where the dosage regimen was increased 
to 1 gram three times a day. The graph represents the median value of all 
CD4 lymphocyte counts measured in the patients. All dosage regimens are 
combined. There were five periods of ADA treatment. Due to the small 
number of values, medians were calculated by carrying forward the last 
values of the patients with no data point in the corresponding treatment 
week. Values of patients who dropped out of the study were carried 
forward. The linear regression is represented by the equation Y=77.2 +4.8 
X, where Y is the absolute CD4 count/ml and X is the number of weeks after 
start of ADA treatment. The correlation coefficient is 0.79. 
Although the present invention has been described in connection with 
preferred embodiments, it will be appreciated by those skilled in the art 
that additions, modifications, substitutions and deletions not 
specifically described may be made without departing from the spirit and 
scope of the invention defined in the appended claims.