EP0690709A1

EP0690709A1 - Pharmaceutical composition for the treatment of certain viruses and autoimmune diseases and the procedure of its preparation

Info

Publication number: EP0690709A1
Application number: EP94904353A
Authority: EP
Inventors: Constantin Romulus Dinu; Ileana Dana Dinu
Original assignee: Individual
Current assignee: Individual
Priority date: 1993-03-26
Filing date: 1993-12-29
Publication date: 1996-01-10
Also published as: CA2159116A1; WO1994022438A1

Abstract

A new pharmaceutical composition and the procedure of its preparation are disclosed, useful in the treatment of AIDS of certain viruses and autoimmune diseases and formulated as an aqueous injectable, or orally administered solution. Four examples are given of the pharmaceutical composition according to the invention. Example I: the pharmaceutical composition is composed of the calcium and sodium double salt of ethylenediaminetetraacetic acid, associated with calcium gluconate, reduced glutathione and sodium metabisulfite, wherein the weight ratio of the ingredients ranges from 5-10.5 g to 0.2-2 g to 0.01-0.1 g to 0.01-0.2 g respectively per 100 ml of water. Example II: the calcium disodium salt of ethylenediaminetetraacetic acid associated with calcium gluconate and L-histidine wherein the weight ratio for the active ingredients ranges from 5-10.5 g to 0.3-1 g to 0.05-0.2 g respectively per 100 ml of water. Example III: the calcium and sodium salt of diethylenetriaminepentaacetic acid (DTPA Na3Ca) associated with calcium gluconate, L-cysteine hydrochloride and sodium metabisulfite, the association weight ratio of the ingredients is 5-10 g to 0.4-1 g to 0.03-0.15 g to 0.05-0.15 g respectively per 100 ml water. Example IV: the calcium disodium salt of ethylenediaminetetraacetic acid associated with calcium gluconate, L-cysteine hydrochloride and sodium metabisulfite, wherein the weight ratio of the four active ingredients ranges from 9-11 g to 0.4-0.8 g to 0.1-0.3 g to 0.1-0.3 g respectively per 100 ml of water.

Description

PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF CERTAIN VIRUSES AND AUTOIMMUNE DISEASES

AND THE PROCEDURE OF ITS PREPARATION Description The present invention refers to a pharmaceutical composition, in the form of an aqueous injectable, or orally administered solution, for the treatment of AIDS, and for the treatment of one of the neuroviruses selected from herpes simplex recurrens, herpes Zoster, encephalomyelitis, polyradiculoneuritis, cranial nerve paralysis, or for the treatment of the autoimmune disease- multiple sclerosis. Background Art The manipulation of metal ions equilibria, so important in the health-disease correlation investigated in laboratory trials (D.D. Perrin and H. Stύnzi, Pharmac.Ther. vol. 12 p. 255-297,Pergamon Press Ltd. 1981) has suggested to us the possibility of applying "non-toxic chelatotherapy" as a new method of treatment in AIDS, in some neuroviruses and in autoimmune diseases. Metal ions are involved in viral infection. Some chelating agents exert an antiviral action by binding certain metal ions (United States Patent, number 4, 689,347, Aug.25, 1987, inventors Romulus C. Dinu, Ileana D. Dinu, Bucharest, Romania; Brevet Europeen no. 0101685, Priorite 23/02/82 RO 106709, inventeurs Romulus C. Dinu, Ileana D. Dinu, Romanian patent no.107092, 31.08.1993, titular Dinu Constantin

Romulus and Dinu Ileana Dana) . An initial viral infection may be followed by an autoimmune response, as in the case of recurrent herpes, multiple sclerosis and most likely in AIDS. Some chelating agents may prevent such autoimmune processes since they also act on the immunologically active cells, also through the metal ions. Some classes of chelating agents having proved to be active in the prophylaxis of AIDS, as D-penicillamine, Foscarnet and Imuthiol, are not indicated for humans owing to the fact that the therapeutic dose is very close to the toxic dose, with very severe side effects. Summary of the Invention

The best known multidentate ligand is EDTA (ethylenedia inetetraacetate) with six donor atoms and DTPA (diethylenetriaminepentaacetate) with eight donor atoms. A metal ion M and a ligand L may combine reversibly to form complexeS'ML_n where the equilibrium concentrations of the different species depend on the total concentrations of M and L and the stabilities of the complexes. In the presence of two or more different ligands mixed complexes ML'L' 'L¹ ' ' may be very important, increasing the stability of the complexes. The presence of many ligands in a biological environment also leads to an extensive formation of mixed ligand, ternary, complexes such as M(ligand A) (ligand B) . Often, especially where the ligands are of different types, the mixed complexes are more stable than expected. These reasons gave us the idea to combine several chelating agents, out of which a stronger one, like EDTA or DTPA, with mild chelating agents as cysteine, reduced gluthione, histidine and calcium gluconate. Disclosure of the invention

Four examples are given below on the making of the pharmaceutical composition according to the invention. Example I

The pharmaceutical composition of the present invention consisting essentially of: a) the calcium disodium salt of ethylenediaminetetraacetic acid b) calcium gluconate c) reduced glutathione d) sodium metabisulfite wherein the weight ratio for the active ingredients ranges from 5-10.5 g to 0.2-2.0 g to 0.01-0.1 g to 0.01-0.2 g, respectively per 100 ml of water.

The advantages of the preparation as per the invention are the following: The drug has a specific curative effect in AIDS, and some other viruses, as well as in autoimmune diseases.

Reduced glutathione (glutamil-cysteinyl-glycine) natural tripeptide, found in all animal tissues, is involved in the organism's defence mechanisms and considered to have the ability to bind through its thiols groups the metal ions being thus a mild chelating agent. It is also an antioxidant substance with a proeminent role in the immunologic and inflammatory processes. The association of reduced glutathione with calcium sodium edetate and calcium gluconate leads to an extensive formation of mixed-ligands, where the metal-complexes are more stable, thus increasing the antiviral action of the drug. Sodiummetabisulfite is a glutathione stabilizer, but at the same time the system glutathione-reduced- metabisulfite is a redox system intensified by the S₂0 -2

E anion, between the two substances appearing a synergism to the benefit of the inflammatory processes. Since it is not metabolized in the organism, the product is rapidly eliminated, the drug being toxicity- free in the dosage prescribed for the above mentioned diseases.

See below a concrete example: The composition of ingredients for 100 ml of solution as per the invention is the following:

Calcium and sodium salt of the ethylenediaminetetraacetic acid lOg Calcium gluconate 0.6g Glutathione reduced 0.07g

Sodium metabisulfite 0.lg

Distilled water ad 100 ml pH of the solution 6.4-6.7

The preparation of 1000 ml solution for injection or for orally administration as per the invention is the following:

All the water used for the manufacturing was water for injection purged with nitrogen gas. 78 g ethylenediaminetetraacetic acid (EDTA) p.a. was dissolved in 600-700 ml water in a borosilicate glass container at 110°C. 26.7g calcium carbonate p.a. was added little by little during l h by continuous stirring. Thereafter 106.5 ml 5 N sodium hydroxide (2l.3g) was gradually added into the solution that containes CaH₂EDTA. The solution has been purged with nitrogen gas for 30 minutes and it turned clear. 6g calcium gluconate were dissolved in 50 ml water at approximately 80°C by minimal stirring and thereafter it was added to the initial solution. Then the solution was kept for 30 minutes in the container at 110°C. The solution was then cooled down to approximately 20°C; charcoal activated powder p.a. lg was added and filtered through a Seitz filter under a low vacuum. The pH was adjusted to about 6.4 by adding l N sodium hydroxide. Thereafter 0.7 g reduced glutathione was added, the matter being dissolved in a little water (20-30 ml) , and lg sodium metabisulfite also priorly dissolved in a little water. Water was added to approximately 950 ml. The pH was redetermined and readjusted with sodium hidroxide to about 6.4-6.7. Water was added up to 1000 ml. The solution was filtered through a Millipore^R filter under a steam of nitrogen gas. The solution was divided in borosilicate colourless 10 ml ampoules under nitrogen gas and the vials were sterilized at 120°C for 30 minutes. Example II

The pharmaceutical composition of the present invention consisting essentially of: a) the calcium disodium salt of ethylenediaminetetraacetic acid b) calcium gluconate c) L-histidine wherein the weight ratio for the active ingredients ranges from 5-10.5 g to 0.3-1 g to 0.05-0.2 g, respectively per 100 ml of water. The advantages of the preparation as per the invention are the following: The drug has a specific curative effect in AIDS and other viruses and autoimmune diseases.

Histidine is included in the composition of the present invention because the mixed complexes with metal- histidinate are more stable than expected. For instance, for the complex copper-histidinate-phosphonoacetate the stability is enhanced by a factor of 16 (Perrin and Stύnzi Pharmac.Ther.Vol.12,p.262,PergamonPress 1981) . Histidine proved also to be the best stabilizer of the complex EDTA- Me^*2, hence bringing about an increased capacity of EDTA to bind metal ions like zinc, included in the structure of HIV (human immunodeficiency virus) .

The pharmaceutical composition is neither metabolized nor stored in the organism, the product is rapidly eliminated and it is toxicity-free in the dosage prescribed for the mentioned diseases. See below a concrete example.

The composition of ingredients for 100 ml of solution as per the invention is the following: Calcium and sodium salt of the ethylenediaminetetraacetic acid lOg Calcium gluconate 0.5g

L-histidine 0.lg

Distilled water ad lOOml pH of the solution 6.4-6.7

The manufacturing techniques were the same as in example I, except for glutathione, that was substituted with 1 g L-histidine and except for sodium metabisulfite that was not included. Example III

The composition as per the present invention, formulated as injectable solution or orally administered solution contains the calcium and sodium salt of diethylenetriaminepentaacetic acid (DTPA.Na₃Ca)asociated with calcium gluconate, as a source of calcium and mild chelating agent, with L-cysteine hydrocloride, and sodium metabisulfite; the association weight ratio of the 4 active ingredients is 5-10 g to 0.4-1 g to 0.03-0.15 g to 0.05-0.15 g respectively per 100 ml of water. The advantages of the preparation as per the invention are the following: The drug has a curative effect in AIDS and other viruses and autoimmune diseases.

DTPA.Na₃Ca is a multidentate chelating agent, easily capable to form complexes with M_e ^*2 or M ³ and in the presence of other different ligands these mixed-ligand complexes are more stable, thus exercising an antiviral and immunomodulatory action. It has the advantage of a very low toxicity (LD_so=6600mg/Kg of body weight) being neither metabolized nor stored in the organism and it is rapidly eliminated. L-cysteine and sodium metabisulfite were included in the composition for the same reasons as in example IV. See below a concrete example.

The composition of ingredients for 100 ml solution of the invention is the following: Calcium and sdium salt of the diethylenetriaminepentaacetic acid 7.5g

Calcium gluconate 0.6g

L-cysteine hydrochloride 0.lg

Sodium metabisulfite 0.lg Distiled water ad 100 ml pH of the solution 6.4-6.7

The preparation of 100 ml solution is the following:

All the water used was water for injection purged with nitrogen gas. 59.3 g diethylenetriaminepentaacetic acid (DTPA) p.a. was disolved in 600 ml water in a borosilicate glass container at 110°C. 18 g sodium hydroxide p.a. (solution 5N) was gradually added. Thereafter 15.1 g calcium carbonate p.a. was added little by little during 1 h by continouε stirring. The solution has been purged with nitrogen gas for 30 minutes and it turned clear. 6g calcium gluconate dissolved in 100 ml water was added. Then the solution was kept for 30 minutes in the container at 110°C. The solution was then cooled and 1 g charcoal activated powder p.a. was added and the solution was filtered through a Seitz filter. The pH was adjusted to about 6.4 with 1 N sodium hydroxide solution, and 1 g of L-cysteine hydrochloride and 1 g of sodium metabisulfite (both priorly dissolved in 20-30 ml water) were added. Water was added to approximately 950 ml, the pH readjusted, and the volume was completed to 1000 ml with water. The solution was filtered through a Millipore^R filter under a steam of nitrogen gas. The solution was divided in borosilicate colourless 10 ml ampoules under nitrogen gas and the vials were sterilized. Example IV The pharmaceutical composition of the present invention consisting essentialy of: a) the calcium disodium salt of ethylenediaminetetraacetic acid b) calcium gluconate c) L-cysteine hydrochloride d) sodium metabisulfite wherein the weight ratio for the four active ingredients ranges from 9-11 g to 0.4-0.8 g to 0.1-0.3 g to 0.1-0.3 g respectively per 100 ml. of water. The advantages of the preparation as per the invention are the following:

The drug has an antiviral, anti-inflammatory, immunomodulatory and neurotrophic effect in AIDS, herpes simplex, herpes Zoster, and some other neuroviruses as cranial nerves paralysis, encephalomyelitiε, polyradiculoneuritis as well as in multiple sclerosis.

L-cysteine was included as a mild metal chelating agent, that strongly increases the chelating capacity of EDTA. In the presence of two or more different ligands, mixed complexes are more stable. The administration of EDTA-cysteine-gluconate lower zinc level in serum leading thus to the use of such mixed ligands to control viral diseases . The importance of thiols groups in immunological and inflammatory processes is known very well already.

Sodium metabisulfite is a cysteine stabilizer but at the same time it is a redox system: 2S₂O_s ^-2 → 2S0₄ ^"2 + S0₂ + S

+0₂

-o₂

The cysteine-metabisulfite system is also a redox system potentated by the anion S₂O_s ^"2 with a benefic synergia in the inflammatory process.

The pharmaceutical composition is neither metabolized nor stored in the organism, being rapidly eliminated and toxicity-free in the dosage prescribed (10-15 mg/Kg of body weight) for the above mentioned diseases. See below a concrete example:

The composition of ingredients for 100 ml solution of the invention is the following:

Calcium and sodium salt of the ethylenediaminetetraacetic acid 10 g

Calcium gluconate 0.5 g

L-cysteine hydrochloride 0.1 g

Sodium metabisulfite 0.1 g

Distiled water ad 100 ml pH of the solution 6.4-6.7

All the water used for the manufacturing was water for injection purged with nitrogen gas. 78 g ethylenediaminetetraacetic acid (EDTA) p.a. was dissolved in 600-700 ml water in a borosilicate glass container at 110°C. 26.7 g calcium carbonate p.a. was added little by little during l h by continous stirring. Thereafter 106.5 ml 5N sodium hydroxide (21.3 g) was gradually added into the solution that contains CaH₂ EDTA. The solution has been purged with nitrogen gas for 30 minutes and it turned clear.

5 g calcium gluconate were dissolved in 50 ml water at approximately 80°C by minimal stirring and thereafter it was added to the initial solution. Then the solution was kept fo 30 minutes in the container at 110°C. The solution was then cooled down to aproximately 20°C; charcoal activated powder p.a. l g was added and filtered through a Seiz filter under a low vacuum. The pH was adjusted to about 6.4 by adding 1 N sodium hydroxide. Thereafter 1 g L-cysteine hydrochloride was added, the matter being dissolved in a little water (20-30 ml), and 1 g sodium metabisulfite also priorly dissolved in a little water. Water was added to approximately 950 ml. The pH was redetermined and readjusted with sodium hidroxide to about 6.4-6.7. Water was added up to 1000 ml. The solution was filtered through a Millipore^R filter under a steam of nitrogen gas. The solution was divided in borosilicate colourless 10 ml ampoules under nitrogen gas and the vials were sterilized at 120°C for 30 minutes. THE DOSAGE AND ADMINISTRATION OF THE DRUG AS PER INVENTION Genital or labial herpes: a series of 4-6 i.m. injections, or in orally administration (one ampoule/day) . Recurrent old herpes: one series of 6 ampoules (one/day) i.m. administered according to the first symptoms of a relapse (burns, pruritus, pain) . After the first series, with the occurence of the first symptoms of a relapse, 6 ampoules (one/day) orally administered.

Ocular herpes - keratitis - a series of 6-10 i.m. injections. Children: in herpes or varicella, 5-6 ampoules (one/day) orally administered.

Viral peripheral facial paralysis: series of 10-12 i.m. injections, with an interval of 4-5 days between the two series, if and when a follow-up of the treatment was required (one injection/day).

Multiple sclerosis: 6 i.m. injections (one/day) monthly, or at longer intervals, according to the clinical type of MS, the evolution of the symptoms and the medical advice. AIDS: After the diagnosis was set and the immunological determinations were performed through the tests of the total lymphocytes and the CD₄ and CD₈ subpopula ions, the therapy is commenced by one or two ampoules per day, according to the case, orally administered, over a 10-day period, monthly. After 2-3 months of treatment, the immunological survey must be performed. The treatment is continual with no time limit. In some cases, a slight decrease of CD₄ after 1-3 months of treatment can be seen, followed-up by a significant increase of CD₄ values. The CD_β (the cytotoxic subpopulation) proves an evolution specific to the treatment: it often grows exceeding the normal values, beginning with the first series of treatment.

The treatment is non toxic, very well tolerated by children and adults, with no notable side effects. The serum and urinary level of calcium is not modified under the treatment. During the period of treatment any medicinal calcium product and dairy product is forbidden. The pharmaceutical preparation as per this invention has "in vivo" antiviral, antiinflammatory, immunomodulatory and neurotrophic action. THE HYPOTHESIS on the antiviral mechanism of action is supported by the fact that the chelating agents exert an antiviral action by binding certain metal ions involved in the structure of the viruses and in their multiplication. Examples where chelating agents, as EDTA show "in vitro" antiviral effects include RNA-dependent DNA polymerases (reverse transcriptases) , zinc enzymes, by zinc complexation (D.D.Perrin and H.Stunzi, Pharmac. Ther. vol.12,p 267, 1981) .

The "in vivo" capacity of EDTA to bind zinc ions in stable complexes is much increased by the presence of other different types of ligands such as cysteine, histidine and reduced glutathione from the given examples as per this invention. Zinc is directly involved in HIV (human immunodeficiency virus) structure. HIV-l has got a viral nucleoid core - the capsid - that contains the most stable element of the virus the nucleocapsid. This nucleocapsid is constituted of many small molecules of NC_p7 protein which binds directly to the genomic RNA through two zinc fingers structural motif. These two zinc fingers are strongly structurated around the zinc atoms, providing the structural characteristic of the protein NC_p7, consequently to the nucleocapsid. This nucleocapsid contains also various preformed viral enzymes, including the reverse-transcriptase, a zinc enzyme (Medecine/sciences, 1993,9,p.952-8 Greene W.C., N.Engl. J.Med.1991, v.324, 5,p.308-17) . Our hyphotesiε is based on the "in vivo" removing of zinc from the nucleo capsid of HIV-l and from its zinc-enzymes, with the multi-ligand systems as presented in the invention. In this way the viral genome can be affected, involving an instability of the virus and its deterioration by the nucleases. Consequently, the attack target of zinc-chelating agents, presented in this invention, might be in AIDS, simply the zinc-protein of the nucleocapsid, whose plurifunctionality was demonstrated (Medecine/sciences, 1993,9,p.952-8) . The transport of calcium into tissues, by passive or various mechanisms of active transport, was specifically increased, and it exhibits tissue specificities, under the influence of EDTA associated with other different types of ligands, exerting in this way a control over the extent to wich the viral infection induces injuries. At the same time, the drug according to the present invention, acts on contractile protein systems, on cell membrane receptors, and signal transduction enzymes - protein kinase C - by a manipulation of the sulphide groups. Thus, the antiviral and immunomodulatory actions of the pharmaceutical composition from the present invention at least at a fundamental level may be better understood. The entry of calcium into cell may be considerered the first molecular event in the immunological modulation of lymphocytary subpopulation CD₄ and CD_S. The immunomodulatory action of metal-chelating agents is already known in the therapy of multiple sclerosis (United States Patent number 4,689,347, Aug.25, 1987 and Brevet Europeen no.010685, inventors Romulus Constantin Dinu and Ileana-Dana Dinu, Bucharest, Romania) and in AIDS (Patent nr.107092, 13.08.1993 titular Dinu Constantin Romulus and Dinu Ileana-Dana, Bucharest Romania) .

Thus the pharmaceutical composition consisting of the calcium disodium salt of ethylenediaminetetraacetic acid, calcium gluconate, L-cysteine and sodium metabisulfite in certain weight ratio is a very strong stimulator of lymphocytes involved inAIDS (CD₄, CD_e, total lymphocytes) . The drug stimulates succesively first the cytotoxic subpopulation CD₈ (suppresor) which starts,possibly, to eliminate the infected CD₄ cells. The lymphocytes CD_β called also CD₄ demographic control, proved "in vitro" their capacity to attack the HIV infected CD₄ cells. (Le journal du SIDA,Mars 1993,no.48 p.9). The level of CD₈ grows exceeding the normal values after 1-2 series of oral administration of 10 ampoules (one/day) monthly of the above mentioned pharmaceutical composition. This CD_Θ growth is in some cases associated with a small, temporary, diminution of the CD₄ subpopulation. But after 2-3 month of treatment the CD₄ begins to grow towards a normal level.

Therefore our hypothesis on the immunomodulatory action of the present invention is based on the fact that priorly it is about a stimulation of an active CD₈ lymphocytary subpopulation in some neuroviruses (herpes simplex, herpes Zoster, viral polyneuritis) and autoimmune diseases (multiple sclerosis) leading then to a recovery of a normal CD₄/CD₈ ratio, and further on to an increase of CD₄ in AIDS patients, due also to the cytotoxic activity of an active CD_e subpopulation. The problem the present invention resolves, consists in the "in vivo" antiviral action and in the stimulation of the lymphocytes involved in AIDS and, first of all, the cytotoxic subpopulation CD₈, that commences to carry on an activity on the infected CD₄ cells, this stimulation being continuous for the rise of the CD₄/CD₈ ratio up to a normal value. We think that subsequent to the viral attack, an autoimmune process is being launched, controled by the pharmaceutical composition from the present invention. The neurotrophic effect of the claimed pharmaceutical composition is based on the correcting of the effects of cellular Ca^2* deficiency, in the case of an injured nerve, by facilitating its in-flow into the nervous cell, re¬ establishing the nervous in-flow. The depolarizationphenomena increase by accumulating Ca^2* in the synapses and neural transmission also by facilitating the discharge of acetylcholine quanta.

The anti-inflammatory effect was determined by means of preclinical pharmacology and clinically manifested as a decrease of pain in herpes and herpes Zoster, as a diminishing of neuroganglia inflammation. Clinical trials

We have been following up on 10 AIDS adult patients, 12 children, and 7 HIV-l infected adults, the total lymphocytes and the T total, CD₄, CD₈ subpopulations developments, simultaneously with the clinical evolution of the patient having been treated with the drug according to the invention for 3-4 years long. The comparison was made versus a control group made up also of 10 AIDS patients. Thus it could be found in the treated groups that a significant growth in the total lymphocytes (TL) and in the T studied subpopulations counts did occur after 4-5 months period of i.m. and orally administered drug. An important increase in CD₄ parallelly with the favorable clinical evolution of the patients was evidenced as well. The patients clinically recorded on the Walter-Reed scale do get from an average stage of 4 up to stage 2. The control group showed a progressive decrease in TL and in all T subpopulations within 4-17 months of survey. The drug according to the invention was highly efficacious in the treatment of a group of 150 patients with recurrent herpes accompanied by neuralgia, under the dosage regime described. Pain rapidly diminishes after 24-48 hours, cutaneous lesions are arrested and rapidly evolve to epithelization (2.6 ± 0.4 days) (p<0.001; significant according to Student's test) . The drug increases the intervals between relapses and the remission speed increases after the administration of two or three series of 6 injections (or oral administration) , simultaneously with the first symptoms of a relapse. In the most cases, relapses occur, if at all, after 200% longer periods, with an eruption reduced "miscarried" .

The most important event in herpes Zoster is pain cessation (6.45 days) . Even if intense, pain was influenced within 24-48 hours after initiation of the therapy.

An intensely favorable and statistically highly significant evolution (p<0.00l) was noted as compared with the control group (chi squared test) . The eruption disappered in 6.19 days, a statistically significant difference appered between the test group of 127 patients and the control group (chi squared test) . In the herpetic Keratitis in 21 out of 23 cases the epithelial layer of cornea recovered within 3 to 6 days after 3 to 6 i.m. injections. By comparing the results with other antiherpetic drugs like Acyclovir it was found that this cures herpetic corneal ulcers in ten days on an average, but 10-30% of the cases show no improvement. Under this therapy, patients affected with frequent relapses were followed up over a period of one year, and no recurrences occurred. In an immunological survey 46 patients with herpes simplex, recurrent form, were investigated regarding the levels of their suppressor T cells (CD₈) and L cells bearing labile surface-bound igG. Abnormal values of CD₈ and L cells were detected in almost all patients. T lymphocyte subsets were identified by rosette formation and by monoclonal antibodies. The immunological abnormalities found in the active disease reached normal levels at 2 weeks after the therapy (p<0.00l) . Such positive immunological response was found to be correlated with clinical improvements, such as the increase of the intervals between relapses and the reduction of the relapse episodes. During the longitudinal study, the percentages of CD₈ and L subsets were serially monitored for 2-3 years. Whenever abnormal values were found the treatment was again resumed and the recurrences were either avoided or mildly expressed. Theraphy in peripheral facial paralysis. The test group(I) consisted of 27 patients, with idiopathic facial paralysis and one case with post-zosterian paralysis (Ramsey-Hunt syndrome) . These patients started the treatment up to 11 days since onset without having previously followed any other treatments. The control group (II) consisted of 17 patients. These patients started the treatment from an average of 42.0 days since onset, having previously been treated with corticoid, vitamins, physiotherapeutic procedures with hardly any or no effects at all. The drug was administered in daily i.m. injections in series of 6- 10 ampoules. The clinical examination produced a score calculated on the basis of remanent motility and blink reflex measurements. The electro-physiological examination produced the prompt reflex (PR) and delayed reflex (DR) . After an average period of 14 days treatment, full recovery in group I was achieved in 85% of the cases, whilst 15% still had discrete disabilities. Both the prompt reflex response and the delayed reflex response were present in 100% of cases in gr. I. Full recovery in group II was obtained in only 24% of the cases and the reflex response was absent in 33% of the cases. In the test group postparalytic spasms were not observed with a follow-up period of one year. It can also be remarked that the difference noted between the final mean scores is statistically significant (p<0.00l, ilcoxon test) in favor of group I. Therapy in multiple sclerosis. In a follow-up trial of 12 months in 40 cases with chronic progressive MS(CPD) and 88 patients with the clinically stable form (CSD) , the assessment was made in keeping with Kurtzke's disability status scale (DSS) andHauser's ambulation index (AI) . The best results were recorded in the group with initial quantitative DSS score between 1-3. The findings are significant (p<0.001) after one year as compared with the state of the patients before entering the treatment. Outstanding clinical signs not included in the scale levels and which were improved, include: nystagmus, oculomotor nerve paralysis, disorders of sensitiveness, trigeminal neuralgia, optical neuritis, sphincteral impairments. From the data it follows that in CPD out of the 40 patients treated during one year (every 2 month or even seldomer a series of 6 i.m. injections) 80% were improved, 17.5% were unchanged, and 2.5% grew worse. In CSD out of 88 patients 83% were improved, 15.9% were unchanged, and one patient (1.1%) worsened. T-lymphocyte subsets were measured before the admission to the therapy in 15 patients. It was found that the drug balances the Th/Ts ratio by increasing the CD₈ lymphocyte number and without decreasing the total leucocyte count inperipheral blood, and increases significantly the EAG population. These lymphocyte population followed up monthly, for 8 to 12 months showed a significant correspondence to the patient's clinical evolution. In another trial out of a number of 587 MS cases treated in the last years, a sample of 81 patients were selected, having started the treatment within the first three years since onset. They were followed-up in an out-patient treatment for 4 years. According to the MS clinical form, 30 patients were on acute attack, 18 chronic progressive form, 11 relapsing- remitting, and 22 clinically stable. The disability degree was indexed in keeping with the Kurtzke ten point Disability Status Scale. After four years of treatment, out of the total number of 81 patients, 63% were improved, 18.5% unchanged and 18.5% worsened. (SibleyW.A., Patients Management: Treatment of the Patients with Multiple Sclerosis, 1985) . By comparing our data with the group of W.Sibley of 170 patients treated with all the modern therapeutic methods, these patients presented a deterioration of 0.78 points whilst the group of 81 patients treated showed an improvement of 1.10 point on the Kurtzke DSS in four years. We consider that the improvements obtained, statistically significant, (the non parametric Mann-Whitney-Wilcoxon test for paired observation) are the results of its antiviral, immunomodulatory, anti-inflammatory and neurotrophic actions. From the group of 81 MS patients followed-up in an out-patient treatment for 4 years, 25 patients were investigated regarding the levels of their circulating suppressor T cells (CD₈) and L cells bearing labile surface-bound igG. Abnormal values of CD₈ and L cells associated with disease activity were observed in 21 out of 25 MS patients. After 3-4 weeks of the drug administration, improvements of these abnormal data were recorded. Results demonstrate that the difference betweeen the mean + SD percentage of CD₈ lymphocytes established on day 21-35 as compared to that found on day 0 was highly significant statistically (p<0.001 Student test). The abnormalities of CD₈ and L subsets found on MS patients were usually corrected after the therapy. The positive immunological response in 20 MS patients (out of 21) which responsed to therapy variedly from 33% (in one case) and 100% (in 13 cases) . The majority of the patients have entered the monitored study at intervals of 1 to 3 months over periods of time up to 2-3 years. New series of the drug were administered during the longitudinal study. Out of the large variety of neuroviruses that induce encephalities, the majority remains still unknown. The treatment with corticosteroids is very hazardous in the case of herpetic encephalitis, Zoster or varicellosus encephalitis. The great efficacy in neuroviruses of the drug according to the invention determined successful application of this treatment in more than 30 encephalitis cases which we^'re cured. After 10-12 i.m. injections (one/day) the patients were saved even from coma, but the treatment was continued, orally administered in series of 12 ampoules (one/day) , a break of 7 days and repeated again under the same conditions, according to EEG (electroencephalography) aspect. The same treatment is prescribed in immediate encephalitis sequelae, especially in children. In older sequelae with frequent epileptic seizures the administration of the drug is counter- indicated.

Claims

1. A pharmaceutical composition for the treatment of certain viruses and autoimmune diseases in the form of an aqueous injectable or orally administered solution used especially for the treatment of AIDS, for the treatment of viruses and autoimmune diseases, said composition consisting of: a) the calcium and sodium double salt of ethylenediaminetetraacetic acid b) calcium gluconate c) reduced glutathione; and d) sodium metabisulfite wherein che weighu ratio of the ingredients ranges from 5- 10.5 g to 0.2-2 g to 0.01-0.1 g to 0.01-0.2 g respectively per 100 ml of water.

2. A pharmaceutical composition in the form of an aqueous injectable, or orally administered solution for the treatment of AIDS, for the treatment of viruses and autoimmune diseases, said composition consisting of: a) the calcium and sodium double salt of ethylenediaminetetraacetic acid b) calcium gluconate; and c) L-histidine wherein the weight ratio of the ingredients ranges from 5- 10.5 g to 0.3-1 g to 0.05-0.2 g respectively per 100 ml of water.

3. A pharmaceutical composition in the form of an aqueous injectable, or orally administered solution for the treatment of AIDS, for the treatment of viruses and autoimmune diseases, said composition consisting of: a) the calcium and sodium salt of diethylenetriaminepentaacetic acid (DTPA Na₃Ca) b) calcium gluconate c) L-cysteine hydrochloride; and d) sodium metabisulfite wherein the weight ratio of the ingredients ranges from 5-10 g to 0.4-1 g to 0.03-0.15 g to 0.05-0.15 g respectively per 100 ml of water.

4. A pharmaceutical composition in the form of an aqueous injectable, or orally administered solution for the treatment of AIDS, for the treatment of a neurovirus selected from herpes simplex recurrens, herpes Zoster, encephalomyelitis, polyradiculoneuritis, cranial nerve paralysis, as well as multiple sclerosis, said composition consisting of: a) the calcium and sodium double salt of ethylenediaminetetraacetic acid b) calcium gluconate c) L-cysteine hydrochloride; and d) sodium metabisulfite wherein the weight ratio of the four active ingredients ranges from 9-11 g to 0.4-0.8 g to 0.1-0.3 g to 0.1-0.3 g respectively per 100 ml of water.

5. Procedure for the preparation of the pharmaceutical composition according to the claime 4. The preparation of 1000 ml. solution for injection or for orally administration as per the invention is the following: all the water used for the manufacturing was water for injection purged with nitrogen gas. 70...85.8 g ethylenediaminetetraacetic acid (EDTA) p.a was disolved in 600-700 ml water in a borosilicate glass container at 110°C. 24...29 g calcium carbonate p.a. was added little by little during 1 h by continuous stirring. Thereafter 5N sodium hydroxide solution (19.2...23.4 g) was gradually added into the solution that contains CaH₂EDTA. The solution has been purged with nitrogen gas for 30 minutes and it turned clear. 4.5...5.5 g calcium gluconate were dissolved in 50 ml water at approximately 80°C by minimal stirring and thereafter it was added to the initial solution. Then the solution was kept for 30 minutes in the container at 110°C. The solution was then cooled down to approximately 20°C; charcoal activated powder p.a. l g was added and filtered through a Seitz filter under a low vacuum. The pH was adjusted to about 6.4 adding l N sodium hydroxide. Thereafter 0.9...1.1 g L-cysteine hydrochloride was added, the matter being dissolved in a little water (20-30 ml), and 0.5...l.i g metabisulfite also priorly dissolved in a little water. Water was added to approximately 950 ml. The pH was redetermined and readjusted with sodium hidroxide to about 6.4-6.7. Water was added up to 1000 ml. The solution was filtered through a Millipore^R filter under a steam of nitrogen gas. The solution was divided in borosilicate colourless 10 ml ampoules under nitrogen gas and the vials were sterilized at 120°C for 30 minutes.