WO1996034612A1

WO1996034612A1 - Compositions for the treatment of infections caused by hiv, aids and lymphomas using hydrogen peroxide

Info

Publication number: WO1996034612A1
Application number: PCT/IB1996/000466
Authority: WO
Inventors: Francisco Javier Castanedo Vera
Original assignee: Promex Investments Limited
Priority date: 1995-05-05
Filing date: 1996-05-03
Publication date: 1996-11-07
Also published as: AU5511896A; PT101698A

Abstract

The present invention relates to a method for the treatment of infections caused by HIV, AIDS and lymphomas using hydrogen peroxide, to the use of hydrogen peroxide in such treatment, to the use of hydrogen peroxide in the preparation of a medicament used in such treatment, to pharmaceutical preparations containing hydrogen peroxide used in such treatment, to the process for the preparation of pharmaceutical compositions containing hydrogen peroxide used in such treatment and to a method for enabling hydrogen peroxide to be absorbed by the human body without toxic effects. Normally the hydrogen peroxide is administered by infusion over a long period of time in the form of a diluted solution.

Description

- 1 -

DESCRIPTION

COMPOSITIONS FOR THE TREATMENT OF INFECTIONS CAUSED BY HIV, AIDS AND LYMPHOMAS USING HYDROGEN PEROXIDE

Field of the Invention

The present invention relates to the use of hydrogen peroxide in the treatment of infections caused by HIV, AIDS and lymphomas.

Background to the Invention

AIDS is an extremely serious disease and thousands of people have been mobilised and vast sums of money spent in the search for a cure. According to the relatively conservative estimates of the WHO, it is thought that by the year 2000 approximately 200 million people will be affected all over the world. It is thus a scourge which could destroy humanity if it is not stopped.

It was ascertained in the very early days of infection by HIV that the depletion of the CD4+ cells (T lymphocyte helpers) was the direct cause of opportunist infections and consequently responsible for AIDS. As well as the change in the number of these cells, innumerous studies showed that these cells were also altered functionally, thus affecting other dependent immune T functions of both the specific system (humoral response) and the non-specific system (NK or ADCC activity). Thus, the development of immunodeficiency has been attributed to this situation, it being recognised that the virus is also directly responsible for this depressive effect on the CD4 cells. More recently, it was understood that the virus, after infection of the organism, has a high capacity to multiply and is able to destroy the cells of the immune system. It was also understood that the immune system actively responds to infection by HIV, which altered the approach adopted in view of this pathological situation.

Today it is recognised that the immune system is able from the very outset to reply to viral destruction and can control the growth and multiplication of this agent. Proof of this concept lies in the fact that many patients show no symptoms for many years.

This immune response, which affects the cytotoxic cells, is thought to be responsible for the destruction of the CD4 cells which are affected and display viral proteins, which, together with the cytopathic effect of the virus, would explain the decrease in the number of T lymphocyte helpers. In spite of this, in view of the vigorous immune response which begins at the very outset, it would be expected that it would be possible to remove the agent, which is not the case. The HIV virus thus has a high capacity to mutate, which allows it to constantly escape the response of the immune system. This great genie variability is itself affected by the action of reverse transcriptase and it is estimated that every time the enzyme copies the RNA onto DNA, the latter differs in an average of one place to the previous copy. This data, together with the calculation that a billion new viral particles are produced every day in an infected patient, gives us an idea of the capacity for viral mutatation and the effort thus required on the part of the immune system in order to recognise and respond to these new antigenic forms. Together with this data, we should also recognise that some of these mutations may lead to the appearence of less antigenic epitotes, which would allow these strains to multiply at a higher rate until they were recognised and the immune system were to give an efficient response, and in this case the possible role of the antigenes of histocompatability in the processing and presentation of these new epitotes should also be taken into account.

Thus, as regards immunity, bearing in mind that the organism is able to control the disease for relatively long periods of time despite all the resistance put up by the HIV virus, it would be expected that by adopting a different approach and possibly by means of positive immunomodulation of the cytotoxic functions, it would somehow be possible to alter the natural development of this pathology.

The immune response produces various bioactive molecules, such as cytokines and oxidising molecules, an example of the latter being hydrogen peroxide. Oxidising molecules in themselves are able to potentialise the production of IL1, IL8 and TNF, which are powerful immunomodulating agents. TNF and IL1 are inflammation inducers, the former of these molecules being a powerful T cell activator. Thus, TNF can directly or indirectly affect the stimulus necessary for increasing specific and non-specific cytotoxic activity.

The treatments using antiretroviruses which are currently used all over the world are able to increase the number of T lymphocytes, but patients improve only for a short period of time and also build up a resistance after approximately 6 months of use. Side effects are also caused, which on many occasions make it necessary to suspend treatment or change to other antiretroviruses. For example, 3'-azido-3'-deoxytimidine (AZT) which is the ant etrovirus most commonly used in the treatment of AIDS, either in isolation or in combination with other antiretroviruses, acts in this way. This strategy has proved counterproductive since the antiretroviruses used do not increase the number of T₈ lymphocytes or natural killer lymphocytes or B cells. They only increase the number of T₄ cells, which are the cells infected by the virus. In other words, the greater the number of T cells produced, the greater the number of T cells infected, which means that we are "feeding" the virus.

It is also known that the HIV viruses are extremely sensitive to oxygen and are extremely unstable in the presence thereof and die in only a few moments. It is known that the human body produces oxygen naturally in the cells and that it is even more active than atmospheric oxygen (i.e. molecular oxygen, 0₂). The oxygen produced in the cells takes the form of a free radical or nascent oxygen (atomic oxygen O). If the level of oxygen can be increased, the HIV virus can be more efficiently combatted. However, all the therapies based on the introduction of oxygen into the bloodstream have come up against the problem of embolism, which is caused by this method.

The present invention is based precisely on the possibility of increasing the level of oxygen in the blood and cells without causing the problems mentioned above.

In accordance with the invention, the new method of treatment for AIDS and lymphomas is based on the use of hydrogen peroxide, which is a powerful oxidizing agent, as a supplier of oxygen to the body. Administration is intravenous, in particular by infusion. This use is based on the fact that it has been discovered that this product not only destroys the HIV virus but also that it simultaneously increases the number of natural T₈ lymphocytes and natural killer CD₅₆ lymphocytes. These subpopulations of lymphocytes are the ones that destroy the cells infected by the virus and the tumorous cells. As well as this, it increases the total number of lymphocytes and the number of B lymphocytes, which produce antibodies. Hydrogen peroxide has never been used intravenously in the treatment of disease and is normally applied topically.

When the HIV virus attacks the T₄ lymphocytes, it eventually destroys them and these lymphocytes are the ones that activate the T₈ lymphocytes, the natural killer lymohocytes and the B lymphocytes. If there are no T lymphocytes, there is a decrease in the number of the other lymphocytes. The main characteristic of hydrogen peroxide is that, owing to the fact that it increases the number of T₈ lymphocytes, natural killer lymohocytes and B lymphocytes, it destroys the cells infected by the virus. It also increases the number of monocytes which are then converted into macrophages, which also have a certain level of antiviral activity.

Normally, this treatment is accompanied by the administration of antioxidants, preferably vitamin C or alopurinol, in order to eliminate the free radicals which may remain in the body.

The results obtained with this treatment have so far been quite enlightening and encouraging. Cultures of HIV which before treatment gave positive results, gave negative results after treatment. Also, the polymerase chain reaction test carried out in one case did not detect the presence of the HIV virus after treatment. This case is the only one of its kind in the world since the patient has been clinically cured.

It is also very significant that seven of the first patients to be treated have not received any further treatment for three years and lead a totally normal life completely free of symptoms. Hydrogen peroxide is an oxidising agent used as an antiseptic and disinfectant. It has low antibacterial activity and is also effective against viruses, including HIV. Its antiseptic action is attributed to the fact that oxygen is rapidly released when it is applied to tissues. It is generally used in aqueous solutions for topical application.

There are numerous references in literature to the endovenous application of this compound. Its capacity for transferring and capturing free oxygen radicals led to the recommendation that it be used in oral or endovenous administration in a variety of pathologies. Recent investigations have shown that this compound plays a vital role in the intracellular mechanisms of bacterial death (Clifford & Repine 1982; Baggiolini 1984), which has caused an increase in the interest and credibility of the aforementioned possibility. Various studies have been published in literature on this point.

Hollcroft & Lorenz, in their study of irradiation in experimental leukaemia, ascertained that IV administration of this compound during the minute follwing the start of irradiation led to tumorous regression of a level significantly greater than in the case of a tumour irradiated normally.

Holman ascertained that mice implanted with 256 walker adenocarcinoma and treated, per os, with water containing the diluted oxidising agent were cured at a rate of about 50 to 60%, 72 mice having been cured. The total disappearance of the tumours took between 15 and 60 days. Of four mice which underwent similar treatment, two showed a marked clinical improvement with a decrease in the size of the liver (metastatic) and the progressive decrease of the serous polysaccharide, which Keyser showed to be an indication of therapeutic efficacy in neoplastic situations. In various studies, Nathan et al. showed that activated macrophages and granulocytes could be pharmacologically stimulated, in vitro, for the lysis of a variety of target cells, and that hydrogen peroxide mediated this extracellular lysis.

Nathan & Cohn (1981), having ascertained that mononuclear phagocytes and granulocytes segregate hydrigen peroxide for cytolysis, which destroys tumorous cells by lysis, studied the parenteral administration of glucose oxydase, which produces hydrogen peroxide. The glucose oxydase was covalently joined to polystyrene microspheres. They concluded that an oxygen- generating system confined to the tumorous mass had a clear antitumorous effect with only a small level of toxicity for the host.

It was also demonstrated that the compound in concentrations as low as 10^"5 M was effective in the death of non-lethal yoelli plasmodium and lethal berghei plasmodium (Dockrell & Playfair 1983). Its efficacy in vivo in parasites in mice, after IV administration, was also demonstrated.

In single IV administration acute toxicity studies carried out on 20 wistar mice (10 of each sex) aged between 8 and 10 weeks, it was ascertained that:

- all the animals survived until they were put down;

- no significant alterations in behaviour or external habit were observed during the test;

- the average increase in weight in the females was 33.38 g;

- the necroscopic examination did not reveal any morphological, macroscopic or histopathological alterations of the internal organs (kidney, liver, spleen and lungs). In the study carried out no alterations were observed which gave evidence of toxicity of the product tested.

Summary of the Invention

The present invention has as its subject-matter a method for the treatment of infections caused by HIV, AIDS and lymphomas using hydrogen peroxide, the use of hydrogen peroxide in the treatment of infections caused by HIV, AIDS and lymphomas, the use of hydrogen peroxide in the preparation of a medicament used in the treatment of infections caused by HIV, AIDS and lymphomas, pharmaceutical preparations containing hydrogen peroxide used in the treatment of infections caused by HIV, AIDS and lymphomas, a process for the preparation of pharmaceutical compositions containing hydrogen peroxide used in the treatment of infections caused by HIV, AIDS and lymphomas and a method for enabling hydrogen peroxide to be absorbed by the human body without toxic effects.

Detailed Description of the Invention

Hydrogen peroxide (H₂0₂) is a substance that is cheaply and readily available. It is sold in the form of an aqueous solution, normally between 30 and 80%, although aqueous solutions of 3% are commercially available, for example. Hydrogen peroxide can be obtained, for example, using electrolytic methods (from ammonium persulphate) or chemical methods (from barium peroxide). The first process is more commonly used as it is more economic. H₂0₂ is a powerful oxidant, which gives off nascent oxygen (O) and can be used as a germicide and bacteriocide. It reacts quickly with catalase, the enzyme present in nearly all organic tissues, and it is not toxic.

The main subject-matter of the invention is a method for the treatment of infections caused by HIV, AIDS and lymphomas using hydrogen peroxide.

Hydrogen peroxide is administered in the form of a solution, preferably an aqueous solution in sterile bidistilled water or a glucosade solution. The concentration of the solution to be administered is situated between 0.0001% and 1%, preferably between 0.0001875% and 0.5%. The quantity administered per session of treatment is normally between 200 and 1000 cm³, preferably 500 cm

Administration is carried out intravenously. The method of administration is preferably by means of infusion. In accordance with this method, the length of time for which the solution is administered is between 1 and 10 hours, preferably around 3 hours. This administration is carried out once every fortnight, 5 times a week, for 3 to 30 weeks. Administration should preferably be carried out 3 times a week for 5 weeks or once a week for 15 weeks.

It is advisable for the patient to be prepared for treatment in order to minimise the harmful effects of this treatment. Thus, it is advisable to administer to the patient one or two antioxidants before, during and after treatment. The preferred antioxidants are vitamin C and alopurinol. This administration is carried out for about 5 to 10 days before treatment, preferably a week, with 1 to 10 g per day of vitamin C, preferably about 3 g per day, and - 10 -

about 100 to 2000 mg per day of alopurinol, preferably about 300 to 900 mg per day. These doses of vitamin C and alopurinol should be continued during treatment and it is recommended that the administration of vitamin C and alopurinol should be continued indefinitely.

In some patients, suitable doses of interferon alpha can be administered before treatment, due to its antiviral effects and its capacity for activating the immune response, as well as for helping to avoid infection of normal healthy cells. Interferon alpha attaches itself to the receptors of other interferon cells and produces an antiviral state, which causes the infection to be limited. Interferon alpha stimulates the prenatural killer cells, activating them and differentiating them from natural killer cells. This constitues a rapid natural defence and the spreading of the virus is limited. The suitable dose varies between 1 and 15 million units, 1 to 5 times a week. The recommended dose is preferably between 2 and 10 million units 3 times a week.

These doses refer to normal cases. However, the doctor prescribing treatment may, in special cases, administer lower or higher doses than those given above, depending on the state of the patient, the seriousness of the disease, etc. For similar reasons, the length of treatment can be lower or higher than the limits given above.

Normally, this treatment is applied successfully during the fist three of the four stages of AIDS that are normally considered. When applied during the fourth stage of the disease (terminal stage), the results are not as efficient.

The second subject-matter of the invention is the use of hydrogen peroxide for the treatment of infections caused by HIV, AIDS and lymphomas. As mentioned above, this is the first time that hydrogen peroxide has been used. The concentrations and the doses used are as mentioned above.

The third subject-matter of the invention is the use of hydrogen peroxide in the preparation of a medicament for the treatment of infections caused by HIV, AIDS and lymphomas The medicament obtained takes the form of a solution in the concentrations mentioned above.

The fourth subject-matter of the invention is a pharmaceutical composition containing hydrogen peroxide for the treatment of infections caused by HIV, AIDS and lymphomas The solution takes the form of an aqueous solution in sterile bidistilled water or a glucosade solution. The concentration of the solution to be administered is situated between 0.0001% and 1%, preferably between 0.0001875% and 0.5%. The composition is packed in suitable dosage forms or it can be prepared immediately before administration. In the first case, the composition is packed, for example, in bottles normally containing between 200 and 1000 cm , preferably 500 cm , ready for administration. In the second case, more concentrated hydrogen peroxide is contained, for example, in phials to be dissolved, which contain the quantity of hydrogen peroxide suitable for one session of treatment. It is advisable in both cases to add a stabiliser, in order to avoid decomposition of the hydrogen peroxide and prolong the life of the medicament.

The fifth subject-matter of the invention is a process for the preparation of pharmaceutical composition containing hydrogen peroxide for the treatment of infections caused by HIV, AIDS and lymphomas. The process consists of associating the hydrogen peroxide to at least one pharmaceutically acceptable vehicle and, optionally, to a stabiliser. As mentioned above, the preferred vehicles are solvents, namely sterile bidistilled water or an aqueous glucose solution. Finally, the sixth subject-matter of the invention is a method for enabling hydrogen peroxide to be absorbed by the human body without toxic effects. This method consists of preparing a hydrogen peroxide solution, as mentioned above, and administering this solution intravenously, preferably by infusion, over a long period of time of not less than an hour. The toxic effects can also be minimised even further by simultaneously administering antioxidants, as mentioned above.

The present invention is illustrated in the non-restrictive examples given hereunder.

EXAMPLE I

Preparation of a composition containing hydrogen peroxide and sterile bidistilled water

40% hydrogen peroxide solution 1.25 ml

Sterile bisdistilled water q.s. to 500 ml

The composition is prepared using a method of succesive dilutions starting with 12.5 ml of 40% hydrogen peroxide solution used for medicinal purposes and sterile bidistilled water of a quantity sufficient for making up 500 ml. A solution of 0.1% hydrogen peroxide is thus obtained. This composition is then introduced into a bottle suitable for administration by infusion. If the solution is intended to be stored it is advisable to add a stabiliser. EXAMPLE II

Preparation of a composition containing hydrogen peroxide and a aqueous glucose solution

80% hydrogen peroxide solution 0.234 ml

5% aqueous glucose solution q.s. to 500 ml

The composition is prepared as above, using a 5% aqueous glucose solution instead of sterile bidistilled water. A solution of 0.0375% hydrogen peroxide is thus obtained, which is processed in the same way as in Example I.

EXAMPLE III

Result of the Blood Test in Patients Treated in Accordance with the Method of the Invention

Blood tests were carried out on two patients A and B infected with the HTV virus to determine the total number of leucocytes and absolute number of lymphocytes. An analysis of the subpopulations of lymphocytes was also carried out based on 5000 lymphoid cells. The method used was flow cytofluorometry. The cytofluorographic study of the subpopulations of lymphocytes, using monoclone antibodies, gave the results shown in Table 1, below. The patients were treated in accordance with the method of the invention for 6 weeks (1 week's preparation and 5 weeks' administration of hydrogen peroxide). About 5 months later in the case of patient A and 9 months later in the case of patient B the tests were repeated under the same conditions, using the same procedure as described above. The results are also shown in Table 1. TABLE 1

A B

Before After Before After

LEU¬ COCY¬ Total 7.3 6.8 3.6 7.8 TES

L

Absolute Total 1321 1842 1166 1895 Y

Mature Cells (%) 57.1 72.1 61.0 67.2

M T Cells¹ Cells/μl 755 1328 711 1274

S Total Cells (%) 81.9 84.3 91.5 87.7 P U T Cells² Cells/μl 1083 1553 1067 1661 B Cooperating Cells (%) 22.9 21.4 21.9 27.9 H P T Cells³ Cells/μl 303 394 256 528 O Supression- Cells (%) 8.5 7.6 16.7 13.5 p Inducing T Cells⁴ Cells/μl 112 140 194 256 u Supressing/Cyto- Cells (%) 33.9 48.0 28.5 42.8

C L toxic T Cells⁵ Cells/μl 448 884 333 811 A Y T CD^CDg Ratio 0.7 0.4 0.8 0.7 I T o Total Cells (%) 6.7 5.6 1.7 5.1

N B Celkls⁶ Cells/μl 103 89 20 96

^{E S} "NK" Cells (%) 5.4 17.5 15.9 18.2 Cells⁷ Cells/μl 72 322 186 346 s

CD₃ Cells

2

CD₂ Cells

3

CD₄ Cells 4 CD₄/CD45 or CD.45 Cells 5

CDg/CDa or CD₈ Cells 6

CD20 Cells

7

CDg_β Cells 15

From an analysis of the results it can be concluded that in the case of both patient A and patient B there was an increase in the absolute total number of lymphocytes. The percentage of CD₈ and CD₅₆ cells also rose. In the case of patient A there was a slight drop in the percentage of B cells (CD₂o), but in the case of patient B there was a large increase in the percentage of these cells. The percentage of CD₄ cells increased in one case (patient B) and decreased in the other (patient A), but these variations are not very significant. As regards the CD-j/CD₈ relationship, it decreased in both cases, which shows that the treatment increased the number of CD₈ cells in respect of the CD cells.

EXAMPLE IV

Detection of the HIV Virus in Blood Samples

Blood samples were analysed after treatment in accordance with the method of the invention carried out on 7 patients (A, B, C, D, E, F and G). The length of treatment was 6 weeks (1 week's preparation and 5 weeks' administration of hydrogen peroxide). Various techniques were used, namely detection in cultures of lymphocytes, "western" tests, ELISA tests, etc. The interval between the two analyses varied from patient to patient. The results are shown in Table 2.

TABLE 2

A B C D E F G

Result of the Test (Before) + + + + + + +

Result of the Test (Ater) - - - - - - - - 16 -

As shown by the analysis of the results, all the patients gave negative results in the detection of the HIV virus after partial or complete treatment, whereas at the beginning they were all infected with HIV. After treatment, patient G underwent a further test using the polymerase chain reaction technique (PCR). In this test the result was also negative and no viral DNA was detected, proving that this patient has undoubtedly been clinically cured.

EXAMPLE V

Progress of the Patients after the Final Treatment

Seven patients suffering from AIDS underwent treatment in accordance with the method of the invention, over a period fo 6 weeks, after which time it was considered that the disease was under control. After three years without treatment, the patients were examined again and in all cases they showed no symptoms of the disease and were leading a perfectly normal life.

Various alterations to the methods of carrying out this invention are possible, which are obvious to persons skilled in the art, provided that these alterations do not fall outside the scope and spirit of the invention, as defined in the claims set forth hereunder.

Claims

1. Method for the treatment of a human being who is carrying the HTV virus, is suffering from AIDS or has a lymphoma, characterised by the intravenous administration to such patient of an efficient quantity of hydrogen peroxide solution.

2. Method in accordance with claim 1, characterised in that the hydrogen peroxide is administered by infusion in the form of an aqueous or glucose solution over a long period of time.

3. Method in accordance with claims 1 or 2, characterised in that the concentration of hydrogen peroxide in the solution is situated between 0.0001% and 1% and the quantity administered per session of treatment is 200 to 1000 cm³.

4. Method in accordance with any of claims 1 to 3, characterised in that each session of administration lasts for 1 to 10 hours and is carried out 5 times a week every fortnight, for 3 to 30 weeks.

5. Method in accordance with any of claims 1 to 4, characterised in that before, during and after treatment one or more antioxidants are also admimstered to the patient, preferably vitamin C and alopurinol, in order to reduce the harmful effects of the treatment.

6. Method in accordance with any of claims 1 to 5, characterised in that interferon alpha is also administered to the patient, in order to activate the immune response, as well as to help avoid infection of normal healthy cells.

7. Hydrogen peroxide, characterised in that it is used in the treatment of an infection caused by HIV.

8. Hydrogen peroxide, characterised in that it is used in the treatment of AIDS.

9. Hydrogen peroxide, characterised in that it is used in the treatment of lymphomas.

10. Hydrogen peroxide in accordance with any of claims 7 to 9, characterised in that it is used in conjunction with an antioxidising agent, preferably vitamin C or alopurinol, and/or with interferon alpha.

11. Use of hydrogen peroxide, characterised in that such product is used in the preparation of a medicament for the treatment of an infection caused by HIV.

12. Use of hydrogen peroxide, characterised in that such product is used in the preparation of a medicament for the treatment of AIDS.

13. Use of hydrogen peroxide, characterised in that such product is used in the preparation of a medicament for the treatment of lymphomas.

14. Pharmaceutical composition for the treatment of infections caused by HIV, AIDS and lymphomas in humans, characterised in that it contains as an active ingredient hydrogen peroxide and at least one pharmaceutically acceptable vehicle.

15. Pharmaceutical composition in accordance with claim 14, characterised in that the vehicle is water or an aqueous glucose solution.

16. Pharmaceutical composition in accordance with claim 14 or 15, characterised in that it may also contain a stabiliser.

17. Pharmaceutical composition in accordance with any of claims 14 to 16, characterised in that it contains hydrogen peroxide in a concentration of 0.0001% to 1%.

18. Pharmaceutical composition in accordance with claim 17, characterised in that it contains hydrogen peroxide in a concentration of 0.0001875% to 0.5%.

19. Pharmaceutical composition in accordance with any of claims 14 to 18, characterised in that it is adapted to intravenous administration, preferably by infusion.

20. Pharmaceutical composition in accordance with any of claims 14 to 19, characterised in that it is presented in the form of a unit dosage, being a bottle containing between 200 and 1000 cm³ of such composition, ready to be administered.

21. Pharmaceutical composition in accordance with any of claims 14 to 19, characterised in that it is prepared at the place of admuiistration, using phials to be dissolved, which contain more concentrated hydrogen peroxide of a quantity suitable for one session of treatment.

22. Process for the preparation of a pharmaceutical composition in accordance with any of claims 14 to 21, characterised in that it comprises the association of hydrogen peroxide to at least one pharmaceutically acceptable vehicle and, optionally, to a stabiliser.

23. Method for enabling hydrogen peroxide to be absorbed by the human body without toxic effects, characterised in that a diluted hydrogen peroxide composition is administered intravenously, in accordance with claims 14 to 21, preferably by infusion, over a long period of time of not less than an hour.