FIELD AND BACKGROUND OF THE INVENTION The present invention relates to the treatment of AIDS and immuno- allergical diseases. More particularly, the present invention relates to a therapeutic substance for use in the treatment of AIDS, HIV+ related infections and immuno- allergical diseases, and to pharmaceutical compositions for the treatment of same.

The search for effective means of treating the acquired immuno deficiency syndrome (AIDS), HIV+ related infections and immuno-allergical diseases is intense due to the potentially devastating effect that such diseases can have on humanity. Epidemiological statistics show an ever increasing prevalence of such diseases, with global and regional health organisations predicting catastrophic consequences on a mass scale, unless effective and easily applicable means are provided and implemented, for the control of such diseases.

The present invention involves the use of the total culture, extracts, filtrates, dialyzates, filtration or dialysis residues, either individually or in combination, in liquid form or as the dried solids, of an initially protein-free culture medium comprising bacillus subtilis var. indolasus, in the treatment, prevention and control of AIDS, HIV+ related diseases and immuno-allergical diseases, and in the preparation of improved pharmaceutical compositions for the treatment, prevention and control of AIDS, HIV+ related diseases and immuno-allergical diseases.

Consequently, it is an object of certain aspects of the present invention to provide substances for treating AIDS, HIVt related diseases and immuno- allergical diseases in human and in veterinary applications.

It is also an object of certain aspects of the present invention to provide substances for the preparation of pharmaceutical compositions for treating AIDS, HIV+ related diseases and immuno-allergical diseases in humans and in veterinary applications (where applicable).

It is a further object of certain aspects of the present invention to provide substances and pharmaceutical compositions for treating AIDS, HIV+ related diseases and immuno-allergical diseases in humans and in veterinary applications (where applicable).

It is yet a further object of certain aspects of the present invention to provide highly effective substances and pharmaceutical compositions for treating AIDS, HIV+ related diseases and a wide variety of immuno-allergical diseases in humans and in veterinary applications (where applicable), from readily available raw materials and by relatively simple processes.

It is yet a further object of certain aspects of the present invention to provide highly effective substances and pharmaceutical compositions for treating AIDS, HIV+ related diseases and a wide variety of immuno-allergical diseases in humans and in veterinary applications (where applicable), with minimal, negligible or no negative side effects.

Various other objects and benefits of the present invention will be obvious to anyone familiar with the relevant medical and pharmaceutical art, from the data and information describing certain typical, representative but not limiting embodiments of the invention and how it may be implemented, contained herein.

SUMMARY OF THE INVENTION According to the present invention there is provided a novel treatment for AIDS, HIV+ related diseases and immuno-allergical diseases.

According to further features in prefen-ed embodiments of the invention described below, the new treatment includes the use of one or more components or fractions of an initially protein-free culture medium comprising bacillus var. indolasus, including the total culture, extracts, filtrates, dialyzates, filtration or dialysis residues, either individually or in combination, in liquid form or as the dried solids, in the preparation of pharmaceutical compositions for the treatment, prevention or control of AIDS, HIV+ related diseases and immuno-allergical diseases in humans as well as veterinary applications (where applicable), said compositions comprising at least one or more components of the initially protein- free culture medium comprising bncillzrs s. .2thIiiis l ar. indolasles.

According to still further features in the described preferred embodiments provided are one or more components or fractions of the initial protein-free culture medium comprising bacillus szlbtilis i'ar indolasus, for treatment, control or prevention of AIDS, HIV+ related diseases and immuno-allergical diseases, such as Systemic Lupus Erythematosus, Bronchial Asthma, Allergic Rhinitis, Autoimmune Thyroiditis, Psoriasis and Crohn's Disease.

According to still further features in the described preferred embodiments the pharmaceutical composition further comprising at least one pharmaceutically acceptable inactive component selected ffoin the group consisting of carriers, coatings, diluents and adjuvants.

According to still further features in the described preferred embodiments the pharmaceutical composition is used for the treatments prevention or control of AIDS.

According to still further features in the described preferred embodiments the pharmaceutical composition is used for the treatments prevention or control of HIV infection.

According to still further features in the described preferred embodiments the pharmaceutical composition is used for the treatments prevention or control of Systemic Lupus Erythematosus.

According to still further features in the described preferred embodiments the pharmaceutical composition is used for the treatments prevention or control of Bronchial Asthma.

According to still further features in the described preferred embodiments the pharmaceutical composition is used for the treatments prevention or control of Allergic Rhinitis.

According to still further features in the described preferred embodiments the pharmaceutical composition is used for the treatments prevention or control of Autoimmune Thyroiditis.

According to still further features in the described preferred embodiments the pharmaceutical composition is used for the treatments prevention or control of Psoriasis.

According to still further features in the described preferred embodiments the pharmaceutical composition is used for the treatments prevention or control of Crohn's Disease.

According to still further features in the described preferred embodiments the component, components and fractions have been dried and milled prior to preparation of the pharmaceutical composition.

According to still further features in the described preferred embodiments said pharmaceutically acceptable inactive component includes at least one member of the group consisting of silica gel, starch cellulose, silicon dioxide, talc and fibers.

According to still further features in the described preferred embodiments said starch is selected from the group consisting of corn starch and potato starch.

According to still further features in the described preferred embodiments the pharmaceutical composition is formulated for oral or parenteral administration.

According to still further features in the described prefened embodiments the pharmaceutical composition is forlllulated as uncoated tablets, coated tablets or capsules.

According to still further features in the described preferred embodiments the pharmaceutical composition is formulated in liquid form to be administered parenterally.

According to still further features in the described preferred embodiments the liquid form includes one or more purified components or fractions of an initially protein-free culture medium comprising bacillus subtilis var. indolasus, in solution suitable for therapeutic administration.

According to still further features in the described preferred embodiments the pharmaceutical composition is formulated for rectal administration.

According to still further features in the described preferred embodiments the pharmaceutical composition is formulated as a suppository.

According to still further features in the described preferred embodiments the pharmaceutical composition is formulated for trans-delmal administration.

According to still further features in the described preferred embodiments the pharmaceutical composition is fabricated as a transdermal device.

According to still further features in the described preferred embodiments the pharmaceutical composition is fonnulated in liquid furth for administration as eye or nasal drops.

According to still further features in the described preferred embodiments the pharmaceutical composition is formulated in ointment, cream of gel form for external administration.

According to still further features in the described preferred embodiments the one or more components or fractions consist mainly or exclusively of the insoluble protein fraction of the culture.

According to still further features in the described preferred embodiments the one or more components or fractions consist mainly or exclusively of the insoluble protein fraction of the culture.

According to still further features in the described preferred embodiments the one or more components or fractions conslst mainly or exclusively of the soluble protein fraction of the cultule.

BRIEF DESCRIPTION OF THE DRAWINGS The invention herein described, by way of example only, with reference to the accompanying drawings, wherein: FIG. 1 shows plots of HIV-I p24 antigen levels (in O.D. units) in culture media of T-cell line cultures which were ctltured In the indicated concentrations of Antiviran and thereafter subjected to infection with the HlV-l virus; and FIG. 2 shows plots of HIV-1 p24 antigen levels (in O.D. units) in culture media of T-cell line cultures which were sllbjected to infection with the HIV-1 virus and were treated with the indicated substances at the indicated times postinfection.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of a therapeutic substance for use in the treatment of AIDS, HIV+ related infections and immuno-allergical diseases, and to pharmaceutical compositions for tlie treatment of same, which can be used to cure or relieve such conditions. Specifically, the present invention can be used for treating AIDS, HIV+ related diseases and a variety of immuno-allergical diseases in human and in veterinary applications (where applicable) with minimal, negligible or no negative side effects.

The principles of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and telminology employed herein is for the purpose of description and should not be regarded as limiting.

In accordance with the present invention, one or more components or products of an initial protein-free culture medium comprising bacillus subtilis var. indolasus, including the total culture, extracts filtrates, dialyzates, filtration or dialysis residues, either individually or in combination, in liquid form or as the dried solids, are used in the treatment, prevention and control of AIDS, HIV+ related diseases and a variety of immuno-allergical diseases, as well as in the preparation of pharmaceutical compositions for the treatment, prevention and control of AIDS, HIV+ related diseases and a variety of immuno-allergical diseases. The compositions comprising at least one or more components of the initially protein-free culture medium comprising bacilllls btilis var. indolasus, and optionally in addition, one or more pharmaceutically acceptable inactive components selected from, carries, coatings diluents and adjutants.

The substance used in the present invention is interchangeably referred to hereinbelow as "Imumodulan" (IM) with respect to immuno-allergical diseases or as Antiviran' (AV) with respect to AIDS, I IIV+ related diseases.

The substances of the present invelltioll caii be used alone or in combination with other therapeutic substances and methocls known for the treatment of AIDS, HIV+ related diseases, or immuno-allergicai diseases.

The substance referred to herein as Antiviran or Imumodulan, has been known previously as Subtilnyptasin. Its preparation and certain of its properties

are described in - German Patent No. 873,732, whose teachings are included herein by reference.

It has now been found that Antiviran (AV) is effective against HIV-1 type viruses. Its efficacy is manifested both therapeutically, i.e., allowing infected T- cells to recover, as well as preventively, i.e., preventing the infection of T-cells by the virus. While the mechanism of action of AV is not fully clarified, it appears to be related to a dual function of intelcellulal immunostimulation and intracellular regulation.

It has now been further found that Imumodulan (IM) is effective against a very wide variety of immuno-allergical diseases and conditions. Imumodulan has been tested for effectiveness in the treatment of the following immuno-allergical diseases: Systemic Lupus Erythematosus, Bronchial Asthma, Allergic Rhinitis, Autoimmune Thyroiditis, Psoriasis and Crohn's Disease. In every case dramatic recovery was observed, thus clearly demonstl-ating that it is effective against all such diseases.

Its efficacy is manifested both as therapeutic, i.e., facilitating recovery in immuno-allergical diseases, as well as preventive, i.e., preventing the immuno- allergical condition. While the mechanism of action of Imumodulan is not fully clarified, it appears to be related to one or more of the following functions, namely, intercellular immunostimul ation, immunomodulation and intracellular regulation.

Imumodulan or Antiviran can be e administered in various forms, such as but not limited to, solid or liquid, powders, tables. capsules suppositories, solutions, suspensions, ointments, creams, gels. etc. and in various ways of administration, such as, but not limited to, oral, parenteral. rectal dennal, transdermal, nasally, in the eyes(s), etc.

Pharmaceutically acceptable inactive components for pharmaceutical compositions comprising linumodulan or Antiviran can include, but are not limited to, silica gel, starch, cellulose, silicon dioxide. talc, fibers, and other substantially inert, acceptable pharmaceutical components. Typical examples of starches for such purposes include, corn starch. potato stanch or other edible starches.

Tablets may be uncoated or coated with gelatin or other suitable coating materials.

The form and the method of administration will obviously be selected or adapted in each case to suit the particulai needs and specific conditions involved.

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

EXAMPLES The following examples will serve to show the wide scope of effectiveness of the substances and compositions of the present invention, in dealing with AIDS.

EXAMPLE 1 Effect of Antiviran on HIV-1 Infected T-cell line The efficacy of Antiviran to inhibit the replication of HIV-1 in a T-cell line was evaluated by the measurement of the p24 antigen in the growth media. The p24 antigen is a marker of HIV-1 expression, which reflects the presence of the virus in the cells. An increase of tile virus load directly con-elates with an increase in the p24 antigen level, as measured in optical density (O.D.) units.

Table 1 below summarizes the results. Table 1 shows the HIV-1 p24 antigen O.D. units in days 0, 1, 2 and 7, as measured in the growth media by the DuPont HIV-1 Core Profile enzyme-linked immunosorbent assay, in duplicate experiments, in five assays wherein 50, 5, 0.5, 0.05, 0.005 or 0 (= negative control) pg/ml Antiviran were added to 0.5 x 106 C81-66-45/HIV-1 T-cells per assay.

The OD values of the negative control decreased from day 0 to day 7, as expected, reflecting the death of cells.

The cultures subjected to 0.5, 0.05 and 0.005 µg/ml Antiviran behaved like the control.

However, at 50 µg/ml Antiviran an apparent paradoxical effect was observed. On day 7 the level of the p24 antigen had the maximal value ( .D.

1.313).

Table 1 Antiviran effect on p24 antigen expression in HIV-1 preinfected T-cells p24 antigen - - O.D. units Time 0 µg/ml 50 µg/ml 5 µg/ml 0.5 µg/ml 0.05 µg/ml 0.005 µg/m AV AV AV AV AV Day 0: Value 1 1.134 1.154 ().85,S ().815 1.113 1.078 Avlue 2 1.189 0.875 1.442 0.838 1.158 0.749 Day 1: Value 1 0.744 1.035 1.05 1.087 0.479 0.722 Value 2 0.822 0.804 0.959 0.866 0.748 0.71 Day 2: Value 1 1.076 0.841 0.777 1.148 1.115 0.88 Value 2 0. 954 0.649 0.848 0.88 1.089 0.493 Day 7: Value 1 0.818 1.192 1.084 0.797 0.87 0.807 Value 2 0.811 1.313 l.()75 0.684 0.571 0.772 One conclusion could have been that the virus had been stimulated.

However, the explanation lies in Table 2, below, summarizing viability estimations for the above cultures (% living cells). With a starting value of viability for all assays of 85 % viable cells, it can be clearly seen that at the highest concentration of Antiviran (50 pJml) the viability on day 7 is 93 %, which means an increase in the number of living cells (in contrast to the control, which had decreased to 77 %).

Table 2 viability control Cell Viability (Evaluation / %) Time 0 µg/ml 50 µg/ml 5 µg/ml 0.5 lig/ml 0.05 ug/ml 0.005 pg/ml AV AV AV AV AV AV Day 0 +++ +++ +++ +++ +++ +++ Day 1 Day 2 Day 7 ++=77% +++=93% ++=82% ++=83% ++=83% ++=80% The meaning of this phenomenon is not entirely clear. It might be speculated that despite the presence of the virus in the cells, the cells do not die and continue to replicate and perhaps even being stimulated to do so.

Table 3 below denotes the results of a microscopic inspection for a cytopathic effect (CPE), which confinns the sprout of the virus and the validity of the experiment.

Table 3 Cytopathic effect Cytopathic Effect (Evaluation) Time 0 pg/ml 50 µg/ml 5 µg/ml 0.5 µg/ml 0.05 pLg/ml 0.005 pg/ml Day 0 +++ +++ +++ +++ +++ +++ Day I +++ +++ +++ +++ +++ +++ Day 2* Day 6** +++ N +++ N +++ N +++ N +++ N +++ N * Very advanced forming of syncytium in ill w cells. No differences between the assays.

** The cells are partially necrotic in all the cells (N)

This experiment shows that the addition of Antiviran to a culture medium with infected HIV-1 T-cells, has no influence on the replication of HIV-1.

However, with high concentrations Antiviran has a protective or recovery effect on the T-cells, since their number increased as compared to the control.

Thus Antiviran has a presumable protecting effect on a T-cell that is already infected with HIV-1, since, at higher concentrations a cultured T-cell line did recover after the addition of Antiviran to the culture, as compared to the control.

EXAMPLE 2 Inhibition of HIV-I infection of a T-cell llne kv Antiviran The efficacy of Antiviran to inhibit HIV- 1 infection was evaluated by measurements for HIV-1 p24 antigen in the medium of a T-cell line infected with HIV-1 after culturing in a medium containing the substance.

To this end, the media of cultures of the C8 l-66-45 T-cell line (not infected) were replaced by fresh media containing 50, 5, 0.5, 0.05, 0.005 or 0 (= negative control) g/ml Antiviran. The replaced media were collected for p24 antigen determinations as described for Example 1 above. Cultures were divided into two equal parts. One part served for viability control (or Antiviran cytotoxicity) and the other part was infected with a predefined HIV-1 quantity (10- 4), leaving the virus for two hours in the culture, and then washing it away. Media samples were drawn for p24 antigen determinations at various days postinfection.

Microscopic evaluations were made at various days for the detection of cytopathic effect (CPE) or syncytium formation.

Table 4 and Figure 1 present the p24 antigen values O.D. units following the indicated number of days postinfection. as was detected using the DuPont HIV-1 Core Profile enzyme-linked immunosorbent assay.

In the control culture (0 pg/ml Antiviran), the p24 antigen appeared in increasing concentrations, as expected. However, in the 3 lowest concentrations (0.5 ug/ml ; 0.05 ug/ml and 0.005 pLg/ml) there was substantially no expression of the p24 antigen, and only a slight increase over the lower cut-off value of p24 antigen in the two highest concentrations (5 and 50 µg/ml) was detected.

Considering the present result anti the results described under Example 1 above, it seems that apparently, HIV-I viz sews could not penetrate T-cells when preincubated with Antiviran.

The presence of the virus in the medium was confirmed by the increase of the p24 antigen in the negative control (> 3.0). The Cytopathic effect is shown in Table 5, below.

Table 4 Antiviran protection against HI V-i infection of a T-cell line as determined by p24 antigen expression p24 antigen (O.D. units) Time 0 µg/ml 50 µg/ml 5 µg/ml 0.5 µg/ml 0.05 µg/ml 0.005 µg/ml AV AV AV AV AV AV Day 0.028 0.028 0.028 0.026 0.028 0.027 Day 1 0.023 0.033 0.015 0.025 0.026 0.026 Day 4 0.03 0.064 0.037 0.031 0.019 0.021 Day 8 1.1 0.051 0. 129 0.025 0.026 0.031 Day 11 1.54 0.024 0.037 0.024 0.026 0.026 Day 15 >3.000 0.032 0.054 0.026 0.026 0.022 Table 5 Cytopathic effect Cytopathic Effect (Evaluation) Time 0 µg/ml 50 µg/ml 5 µg/ml 0.5 µg/ml 0.05 µg/ml 0.005 µg/ml AV AV AV AV AV AV Day 1 - - - - - Day 4 Day6 + + + + + + Day 8 + + ++ ++ ++ ++ Day 11 + +++ ++ +++ +++ Day 15 +++ ++ ++ +++ +++ The viability of the cells grown in presence of Antiviran was higher as compared to the control (Table 6).

Table 6 viability control Viability Control (%) Time 0 lig/ml 50 µg/ml 5 ptg/ml 0.5 ptg/ml 0.05 µg/ml 0.005 µg/ml AV AV AV AV AV AV Day 1 92% 92% 88% 89% 88% 85% Day 4 49% 37% 34% 67% 48% 40% Day 8 48% 52% 30% 54% 54% 60% Day 11 66% 66% 71% 59% 58% 54% Day 15 34% 72% 71% 54% 59% 48% Table 7 shows that there is no difference in the viability between the control cells and all the cells grown in Antiviran containing media. That means that there is no cytotoxic effect imposed by Antiviran on normal cells.

Table 7 Cytotoxic effect of Antiviran Viability of Uninfected cells (%) Time 0 µg/ml 50 µg/ml 5 µg/ml 0.5 µg/ml 0.05 µg/ml 0.005 µg/ml AV AV AV AV AV AV Day -2 89% 88% 83% 86% 87% 87% Day -1 90% 89% 87% 90% 86% 89% Day 0 87% 86% 89% 90% 91% 86% Day 1 88% 88% 95% 87% 75% 86% Day 4 40% 35% 30% 25% 46% 35% Day 8 535 34% 42% 42% $7% 50% Day 11 66% 71% 71% 73% 60% 58% Day 15 69% 65% 64% 65% 69% 69% Thus, addition of Antiviran to tlie culture before the infection with HIV-1, did confer protection to the cell by either preventing the infection or by rendering it negligible.

EXAMPLE 3 Antiviran selectivity index Preliminary anti-HIV-1 results obtained with Antiviran show activity against HIV-1 (IIIB strain) with an EC50 of 24.4 pg/ml of pure substance while it was toxic for the host cells at a concentration of 172.4 ug/rnl, resulting in a selectivity index of 7, as was determined by an MTT-assay.

The following experimental procedures were employed in Examples 3-6.

MT-4 cells were grown in RPMI 1640 medium (Life Technologies, Merelbeke, Belgium), supplemented with 10 % (v/v) heat-inactivated fetal calf scrum (FCS), 2 mM L-glutamine, 0.1 % sodium bicarbonate and 20 ug/ml gentamicin (Life Technologies, Merelbeke, Belgium). The cells were maintained at 37 "C in a humidified atmosphere of 5 % CO2 in air. Every 3-4 days, cells were seeded at 3 x 105 cells/ml.

Stocks of HIV-1 IIIB strain were obtained from the culture supematant of 4 x 105 MT-4 cells/ml infected with HIV at 400 CCID50 immediately after complete cytopathic effect (CPE) has appeared. The virus titer of the supernatant was determined in MT-4 cells using the Reed and Muench end-point dilution method. The virus stocks were aliquoted and stored at -70 "C until used.

Flat-bottom, 96-well plastic microtiter flays (Nunc, Roskilde, Denmark) were filled with 100 ml of complete medium using a Titertek Multidrop dispenser (ICN Biomedicals - Flow Laboratories). Stock solutions (10 x final test concentration) of various compounds tested including Antiviran (see also Examples 5 and 6 below) were added in 25 ttl volumes to two series of triplicate wells so as to allow simultaneous evaluations of their effects on HIV- and mock- infected cells. Serial five-fold dilutions were made directly in the microtiter trays using a Biomek 2000 robot (Beckman, Fulleflon. CA). Untreated control HIV-and mock-infected cell samples were included. 50 pl of HIV at 100 CCIDso or medium was added to either HIV infected or mock-infected part of a microtiter tray. Exponentially growing MT-4 cells were centrifuged for 5 min at 140 x g and the supernatants were discarded. The MT-4 cells were resuspended at 6 x cells/ml in a flask which was complected witli an autoclavable dispensing cassette of a Titertek Multidrop dispenser. Under slight magnetic stirring 50 pl volumes were then transferred to the microtiter tray wells. The outer row wells were filled with 200 pl of medium. The cell cultures wee incubated at 37 "C in a humidified atmosphere of 5 % CO2 in air. The cells remained in contact with the test compounds during the whole incubation period. Five days after infection the viability of mock-and HIV-infected cells was examined spectrophotometrically by the MTT method as described hereinbelow.

The MTT assay is based on the reduction of the yellow coloured 3-(4,5- dintethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (Sigma Chemical Co., St. Louis, MO) by mitochondrial dehydrogenase of metabolically active cells to a blue formazan which can be measured spectrophotometrically.

To each well of the microtiter trays, 20 p1 of a solution of MTT (7.5 mg/ml) in phosphate-buffered saline was added using the Titertek Multidrop. The trays were further incubated at 37 °C in a 5 % C02 incubator for 1 h. A fixed volume of medium (150 p1) was then removed from each cup using the Biomek 2000 robot without disturbing the MT-4 cell clusters containing the formazan crystals.

Solubilization of the formazan crystals was achieved by adding 100 pl of 10 % (v/v) Triton X-100 in acidified isopropanol (2 ml concentrated HCl per 500 ml solvent) using the Biomek 2000 robot. Complete dissolution of the formazan crystals could be obtained after the trays had been placed on a plate shaker for 10 min (ICN Biomedicals Flow Laboratories). Finally, the absorbance were read in an eight-channel computercontrolled Titertek Microplate reader and stacker (Multiskan MCC, ICN Biomedicals - Flow Laboratories) at two wavelengths (540 and 690 nm). The absorbance measured at 690 nm was automatically subtracted from the absorbance at 540 nm, so as to eliminate the effects of non-specific absorption. Blanking was carried out directly on the microtiter trays with the first column wells which contained all reagents except MT-4 cells, virus and compounds. All data represent the average values for a minimum of three wells.

The 50 % cytotoxic concentration (CC50) was defined as the concentration of compound that reduced the absorbance (OD540) of the mock-infected control sample by 50 %. The percent protection achieved by the compounds in HIV- infected cells was calculated by the following formula: (ODT)HIV-(ODC)HIV expressed in % (ODC)mock-(ODC)HIV whereby (ODT)HIV is the optical density measured with a given concentration of the test compound in HIV-infected cells; (ODC)HIV is the optical density measured for the control untreated HlV-infected cells; (ODC)mock is the optical density measured for the control untreated mock-infected cells; all OD values were determined at 540 nm. The concentration achieving 50% protection according to the above formula was defined as the 50 % effective concentration (EC50).

The results shoed that Antiviran has activity against HIV-1(IIIB) with an EC50 of 24.4 pg/ml of pure substance while it was toxic for the host cells at a

CC50 of 172.4 pg/ml, resulting in a selectivity index (SI) of 7. Thus, it can be concluded that Antiviran is an active substance against HIV-1.

EXAMPLE 4 Antiviran - time of action In a time-of-addition experiment, the question how long the addition of Antiviran could be delayed after HIV-1 infection and still be active against the replication of HIV, has been determined. Antiviran has been compared to the binding inhibitor dextran sulfate, to the reverse transcriptase inhibitor AZT and the protease inhibitor Saquinavir (Ro3 1-8959) as controls.

The results confirmed that Antiviran, like dextran sulfate, need to be present at the time of infection, in order to be active against the replication of HIV. It is believed that Antiviran interacts with the binding of HIV to the target cells. A slight inhibitory effect on the binding of gp120 to MT-4 cells has been noticed.

However, the exact mechanism has not yet been elucidated and experiments are still currently in progress.

MT-4 cells were infected with HIV- 1 (IIIB) at a multiplicity of infection (moi) of >1 and Antiviran, the binding inhibitor dextran sulfate (DS), the reverse transcriptase inhibitor AZT and the protease inhibitor Ro3 1-8959 (Saquinavir), were added at different times (0, 1, 2, 3 . 22, 23, or 24 h) after infection (the last 3 compounds served as controls). Viral p24 antigen production was determined as described hereinabove 29 h postinfection and is expressed in O.D. units. The compounds were added at a standardized concentration - that is, 100 times their EC50 required to reduce by 50 % the cytopathicity of HIV-1 (IIIB) (moi, 0.01) in MT-4 cells.

Table 8 p24 antigen (O.D. units) Time (hours) Dextran sulfate AZT Ro3 1-8959 Antiviran Control 0 0.045 0.022 0.104 0.083 0.829 1 0.316 0.062 0.068 1.199 1.365 2 0.372 0.02 ().()5 1.302 1.117 3 0.754 0.071 0.0001 1.513 1.07 4 1.057 0.137 (.009 1.643 0.991 5 1.732 0.?75 0.019 1 1.943 1.291 6 0.529 0.026 1.328 7 0.62 0.013 1.219 8 0.589 0.006 1.147 9 1.337 24 ().473 1.229

Figure 2 and liable 8 above clearly show that Antiviran, like dextran sulfate, needs to be present at the time of infection to be active against the replication of HIV (=time 0).

EXAMPLE 5 Andviran - mechanisnz of action Through Flow Cytometry experiments the interaction of Antiviran with the binding of recombinant gp120 with the CD4+ MT-4 cells has been studied.

Dextran sulfate has been included as a positive control. A slight inhibitory effect on the binding of gp 120 to MT-4 cells has been noticed. Since both, Antiviran and dextrin sulfate do not interfere with the binding of the OKT4a mouse antibody to its epitope on CD4, responsible for the binding to gp 120, it is believed that if there is an interaction with HIV binding to cells, its target will be gp120. However, the complete mechanism of action is yet not entirely clear and more experiments need to be done.

EXAMPLE 6 Fractionation of Antiviran Antiviran has been separated into 3 distinct components, namely (i) fraction 8a which contains polysaccharides and purines, (ii) fraction 3b which contains insoluble proteins and (iii) fraction 2b which contains soluble proteins. Each of these fractions has been tested against HIV, as in Example 3 above, and has been found to be active against HIV-1 infection. The same MT-4/MTT assay (as in Example 3) has been applied in duplicates at the present test.

Table 9 below shows the activity of the 3 components against HIV-1 (IIIB) infection. Fraction 2b shows activity as EC50 of 3 and 1.5 mg/ml; EC90 of 5.1 and 2.3 mg/ml; CC50 of > 10 and 15.5 mg/ml with a selectivity index (SI) of > 3 and 10, respectively. Fraction 3b shows activity as EC50 of 2.7 and 9.5 mg/ml; EC90 of4.l and > 25 mg/ml; CC50 of > 10 and > 25 mg/ml with a SI of > 4 and > 3, respectively. Whereas fraction 8a shows activity as EC50 of 2.4 and 2.7 mg/ml; EC90 of 3.6 and 4.1 mg/ml ; CCso of > 10 and > 10 mg/ml, with a SI of > 4 and > 4, respectively.

Thus, all the three components of Antiviran are active substances against HIV-1.

Table 9 Antiviran (mg/ml) Code EC50 EC90 CC50 SI Max Prot 2b 3 5.1 > 10 >3 141 2b 1.5 2.3 15.5 10 164 3b 2.7 4.1 >10 >4 176 3b 9.5 > 25 > 25 >3 71 8a 2.4 3.6 > 1 () > 1 188 8a 2.7 4.1 >1() >4 172 EXAMPLE 7 Lack of antibiotic activity against bacteria Antiviran was found to have no antibiotic action against pathogenic gram- positive or gram-negative organisms.

The following examples will serve to show the wide scope of effectiveness of the substances and compositions of the present invention, in dealing with immuno-allergical disorders.

EXAMPLE 8 Effect Imumodulan on the formation of cytokines in cell lines Cytokines consist of a broad class of glycoproteins which regulate intercellular communication. They play an important role in hematopoiesis, immune reactions and inflammation, both in physiological and pathological conditions. When stimulated, the cytokines are secreted by a variety of cells ubiquitously distributed in the body, such as lymphocytes, macrophages, fibroblasts and endothelial cells.

In order to investigate the cytokine-inducing capacity of Imumodulan and determine its cytotoxicity the following experimental procedures were employed.

1 x 106 human peripheral blood mononuclear cells (PBMCs) were incubated for 24 h in absence or presence of various concentrations of Imumodulan (10 mg/ml; 1 mg/ml ; 0.5 mg/ml; 0.1 mg/ml 0.01 mg/ml; and 0.001 mg/ml). Release of Interleukin 6 (IL-6). lnterleukin 10 (IL-10), Interleukin 12 (IL- 12) and Tumour Necrosis Factor-ot (TNFa) into the supernatant was determined, using commercial enzyme-linked immunosorbent assay (ELISA) test kits. The

sensitivity of these assays enables detection of cytokine levels as low as 18 pg/ml for IL-6, 5 pg/ml for IL-10, 10 pg/ml for IL-12 and 20 pg/ml for TNFa.

Endotoxin contamination in Imumodulan preparations was measured with a quantitative kinetic LAL assay [Ditter B, Becker KB, Urbaschek R, Urbaschek B: Quantitativer Endotoxin Nachweis. Automatisierter, kinetischer Limulus - Ambbozyten-Lysat-Microtiter-Test mit Messung probenabhagiger Interferenzen.

Arzeim Forsch 1983; 5: 681-687]. In an Imumodulan solution of 8.5 mg/rnl the endotoxin level (+SD) was 880 (+28. 2) pg/ml, i.e., a very low endotoxin contamination.

However, to rule out the effect of endotoxin on cytokine release, each concentration of Imumodulan was also incubated in the presence of 5 pJml of Polymyxin B (Polymyxin B has been shown to block in vitro effects of endotoxin).

In cultures of PBMC, Imumodulan induced a dose dependent and considerable enhancement of IL-6 secretion. In addition, elevated secretion of IL- 10 was also observed. TNFa was secreted only in relatively higher concentrations of Imumodulan (as compared to the concentrations which induced the release of IL-6 and IL-10). The release of IL-12 could not have been manifested. No cytotoxicity was observed. Table 10. below. summarizes the results for IL-6 and TNFa.

Table 10 Effect of Imumodulan on IL-6 and TNFα secretion from human peripheral blood snonosluclear cells without Polymyxin B in presence of Polymyxin B IM Concentration IL-6 TNFα IL-6 TNFot neg. control 0.4 ng/ml 0 1 ng/ml 0 10 mg/ml 75 ng/ml 300 pg/ml 67.5 ng/ml 310 pg/ml 1 mg/ml 7.5ng/ml 0 13.1 ng/ml 0 0.5 mg/ml 2.5 ng/ml 0 8 ng/ml 0 0. 1 mg/ml 0.5 ng/ml 0 1.4 ng/ml 0 0.01mg/ml 0.56ng/ml 0 l.4ng/ml 0 0.001 mg/ml 0.94 ng/ml 0 1.4 ng/ml 0 Thus, Imumodulan is a strong IL-6 induce and the coactivation of IL-10 may have beneficial effects on the imbalance between humoral and cellular

immunity observed at allergic and autoimmune diseases, mainly those diseases which belong to the group of "Delayed-Type Hypersensitivity" [=Thl], like Diabetes Mellitus type 1, Autoimmune Thyroid Diseases, Crohn's Disease, Acute Allograft Rejection, Acute Graft-Vs-Host Disease, HIV infection, Rheumatoid arthritis, Multiple Sclerosis etc.

EXAMPLE 9 Immumodulan cytotoxicity The cytotoxic effects of Imumodulan were examined in two cell systems.

A calorimetric assay with the reagent WST-1 in two tumor cell lines (K-562 cells and YAC-1 lymphoma cells). And a penneability assay with propidium iodide using flow cytometry in cultured PBMC.

The effect Imumodulan has on human K-562 leukemia and murine YAC-1 lymphoma cells after 72 h in culture was measured with a calorimetric assay, based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. Cells were cultured in microtiter plates in a final volume of 100 ul/well culture medium (serumfree low protein hybridoma medium purchased from Life Technologies, Basel) in a humidified atmosphere (37 °C, 5 % CO2) for 72 h, at a cell concentration of 2 x 104/well. After 72 h incubation of the cells in the presence of various concentrations of Imumodulan, as indicated in Table 11 below, 10 pl/well WST-1 reagent (purchased from Boehringer Mannheim) was added to each well. After 4 h incubation (37 °C, 5 % CO2) the absorbance of the samples against the background was measured at 450 nm. Table 11 summarizes the results.

Table 11 Immunodulan lack of cytoxicity Viability % Concentration of IM K-562 cells YAC-I cells 5 mg/ml 73 % 100 % 0.5mg/ml 100 % 100% 50pg/ml 100% 100% 5 µg/ml 100 % 100 % 500 ng/ml 100 % 100 % 50ng/ml 100% 100% <BR> <BR> 5ng/ml 100% <BR> <BR> <BR> <BR> 500pg/ml 100% 100%

Only at 5 mg/ml concentration Imumodulan was able to reduce the viability of K562 cells.

The change in the permeability of PBMC cell membrane during culture was used to differentiate living from dead cells by flow cytometty. Cells which have an intact plasma membrane exclude Propidium Iodide (PI), while dead cells with damaged plasma membranes do not. Cells (106) were treated with various concentrations of Imumodulan for 24 h. Then they were collected by centrifugation, resuspended in 0.5 ml phosphate buffered saline (PBS) containing 5 Rg/ml propidium iodide, and incubated for 15 min at room temperature. Results were obtained as dot plots of forward scatter versus fluorescence intensity obtained with a FACscan (Becton Dickinson) Lysis II program. The percentage of propidium iodide containing (dead) cells was 17.07 % at negative control. This value was practically the same observed at various concentrations of Imumodulan indicating that up to 10 mg/ml concentration did not affect viability of PBMC cultured cells (10 mg/ml - 19.92 % dead cells; 1mg/ml - 18.02 % dead cells; 0.5 mg/ml 19.88 % dead cells; 0.1 mg/ml 19.42 % dead cells; 0.01 mg/ml-1 8.42 % dead cells).

EXAMPLE 10 Imumodulan used for relieviiig Systemic l upus Erythematosus symptoms Observations on humans based on compassionate use treatment of patients suffering from Systemic Lupus Erythematosus (SLE) have been clearly shown definite activity of Imumodulan which was manifested by the control/cure of the underlying immuno-allergical disease or condition.

A female patient age 45. diagnosed as suffering from Systemic Lupus Erythematosus, was initially treated with Meticorten. Slight improvement was observed, but the Meticorten had to be discontinued because of severe side effects.

Recurrence of high fever, discomfoit, rush and joint aches ensued.

Imumodulan treatment was initiated. (No other drug was used in addition).

Subsequently, the skin cleared, the rush was eliminated, as well as the fever and chills. The patient experienced only mild joint aches.

Imumodulan treatment was continued (every 2-3 months) for several years.

There was no recurrence of acute symptoms such as fever or rush. Only occasional mild joint aches were experienced during changes of weather. The patient was able to resume her regttlar wollc routine and normal life style.

EXAMPLE 11 Imumodulan used for relieving Bronchial Asthma symptoms Observations on humans based on compassionate use treatment of patients suffering from Bronchial Asthma, have clearly shown a definite activity of Imumodulan which was manifested by the control/cure of the underlying immuno- allergical disease or condition.

A female patient, 8 years old, was diagnosed as suffering from Bronchial Asthma and Sinusitis since the age of two. The patient experienced frequent severe attacks of shortness of breath. She had been treated in hospital emergency rooms with injections. She received Elixophyllin and desensitization injections, as well as corticosteroids, without any improvement. She has also received Ventolin and antibiotics. There was Bronchial Asthma in the family. Change of climate was tried, without improvement.

Positive physical findings were as follows: Pale, nose congested, prolonged expiration, wheezes over both lungs, cervical glands tender, Spirometer FEV = 0.5 liters, after Ventolin inhalation, 0.9 liters.

June 15th - Received first dose of Imumodulan.

July 25th - Received second dose of Imumodulan. A slight improvement was observed. Only two attacks of Bronchial Asthma. FEV = 1.0 liter.

September 1st - Received third dose of Imumodulan. No attacks. Only dry cough. FEV = 1.2 liters.

October 15th - Received forth dose of Imumodulan. Cough stopped, lungs clear, physical appearance improved. FEV = 1.5 liters.

January 10th - Received fifth dose of Imumodulan. No attacks. FEV = 1.8 liters.

Treatment was terminated.

A follow up examination, some extended time later, showed that the patient had no attacks subsequent to the termination of treatment and was doing well.

EXAMPLE 12 Imumodulan usedfor relieving Allergic Rh in itis symptoms Observations on humans based on compassionate use treatment of patients suffering from Allergic Rhinitis have already shown a definite activity of Imumodulan which was manifested by the contlol/cul-e of the underlying immuno- allergical disease or condition.

A male 50 years of age had a history of 29 years of seasonal Allergic Rhinitis. No improvement was observed after desensitization. Antihistamines were occasionally administered.

March 8 - Treatment was initiated with Imumodulan, first dose. No change in the disease was noted.

April 19 - The second dose of Imumodulan was administered. There was immediate improvement and disappearance of all symptoms, i.e., complete remission. No relapse occurred. No side effects of any sort were experienced.

EXAMPLE 13 Imumodulan used for relieving Autoimmune Thyoiditis symptoms Observations on humans on compassionate use treatment of patients suffering from Autoimmune Thyoiditis have clearly shown a definite activity of Imumodulan which was manifested by the control/cure of the underlying immuno- allergical disease or condition.

A female patient, age 46 years, had been suffering many years from Insulin Dependent Diabetes Mellitus (IDDM). In late autumn of 1996, a diagnosis of an Autoimmune Thyroiditis with Hypothyroidism was made (low levels of T3 and T4, high levels of TSH). A replacement treatment with Eltroxin was ordered. On march 28th, 1997, the Eltroxin was stopped and the first dose of Imumodulan was administered. Since then (i.e., up to August, 1997), additional 4 doses have been given with intervals of 4-5 weeks between the doses. In different and regular laboratory controls, the hormonal levels (T3, T4 and TSH) remained within normal values (i.e., complete remission). Tables 12 and 13 summarize these observations.

Table 12 Before Imumodulan administration Test Units Normal Year: Year. Dates: * Values 1993 1995 11.11 96 17.11.96 # 12.12.96 12.02.97 Free T4 pmoI/L 10.5 - 28.1 7 7.7 16.5 18.4 Total T3 nmol/L 1.11-2.51 2.03 2. 14 TSH mlU/L 0.171-5.5 22.3 25.8 9.26 1.36 Anti Thyroid antibodies: -cytoplasm. 0 l:lU 1:2) l:8() - 1;20 - -microsomal 0 - - 1;40 - -anti TPO <75 -anti TG < 150 * Starting Eltroxin 50 µg/day.

Table 13 After Imumodulan administration Test Units Normal Dates: Values 13.04.97 30.04.97 21.05.97 04.06.97 04.09.97 31.10.97 Free T4 pmol/L 10.5-28.1 - 19.4 14.5 - 156. 22.5 Total T3 nmol/L 1.11-2.51 - 1.78 - - 1.7 1.86 TSH mlU/L 0.171-5.5 5.12 1.86 2.64 - 1.76 2.17 Anti Thyroid antibodies: -cytoplasm. 0 l:4() -microsomal 0 -anti TPO <75 - - - 284 148 106 -antiTG < 150 ~ ~ ~ 48 92 37 EXAMPLE 14 Imumodulan used for relieving Psoriasis symptoms Observations on humans based in compassionate use treatment of patients suffering from Psoriasis have clearly shown a definite activity of Imumodulan which was manifested by the control/cure of the underlying immuno-allergical disease or condition.

A female patient, 45 years of age, had a history of about 20 years of Psoriasis, localized mainly on both legs. Different cycles, with corticosteroids and non-corticosteroids containing ointments and creams, were administered.

Tentative treatment at the dead sea was also experienced. No improvement was observed. In August first dose of Imumodulan was administered. Three weeks later, there was a complete disappearance of all the typical lesions characteristic for the disease (i.e., complete remission). The patient continued treatment with 4 additional doses of Imumodulan with 4-6 week intervals and then stopped the treatment. No side effects were experienced. The complete remission lasted for one year, when a relapse was noted.

EXAMPLE 15 Imumodulan used for relieving Crohn's Disease symptoms Observations on humans on compassionate use treatment of patients suffering from Crohn's Disease. have already shown a definite activity of Imumodulan which was manifested by the contol/cure of the underlying immuno- allergical disease or condition.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many altel-natives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.