GB2310138A - Immunomodulating plant polyprenols - Google Patents

Description

IMMUNOMODULATING ACTIVE SUBSTANCE This invention relates to medicine and in particular to the manufacture of medicinal immunomodulating active preparations.

The artificial correction of immune homeostasis, especially in the cases of acquired or genetically predetermined immune deficient states (e.g. sepsis, AIDS, etc.,) is a very actual scientific and practical task. There is therefore a special need for substances which combine modulating properties (i.e. stimulatordepressor).

Inosiplex (isoprinosin) provides such a combined effect and is now widely used as an antiviral and immunostimulating preparation, being used in the treatment of acquired immunodeficiency syndrome (AIDS) (1,2).

However, the production of Inosiplex is a difficult synthetic process which requires the importation of both component parts and reagents. Thus the search for new substances with immunomodulating properties and being derived from raw materials available by wasteless technology is of great importance.

The purpose of this invention is the elaboration of an immunomodulating active substance.

This is achieved by using as immunomodulating active substances Ropren (Rp)-plant polyprenols with a carbon chain of fifty or more carbon atoms having the chemical formula (I):

where: n = 8 - 18 Hereinafter, the term "ropren" or Rp is used to refer to compounds of formula (I) having a carbon chain of fifty or more carbon atoms.

Thus, in one aspect the present invention provides use of a compound of formula (I)

where: n = 8 - 18 or an isomer (eg. stereoisomer) thereof, in the preparation of a medicament for use as an immunomodulating agent.

Compounds of formula (I), or Rp as referred to herein, as mentioned above are known plant polyprenols and have been described for example in Chojnacki et al., Chemica Scripta, 1987, 27, 209-214 and Rip et al., Prog.

Lipid Res., 1975, 24, 269-309.

The compounds may be prepared by isolation from natural plant materials for example arboreal foliage.

Exemplary sources include the foliage (needles and shoots) of the European spruce Pica abies (L.) Karst and the ordinary pines Pinus silvestris.

Ropren may be prepared by grinding the plant foliage and extracting with a solvent such as light petroleum, eg.

for 6 hours in a Socselets' apparatus, portions of 200-220 grams being sequentially loaded into the apparatus. The solvent eg. light petroleum is distilled off, to leave a residue. This may be chromatographed on a column with silica gel. The fraction eluted after carotene is made up of polyesters and contains acetates of polyprenols. In order to obtain free polyprenols, the polyester fraction, after distilling off the solvent, is exposed to hydrolysis, eg. in a boiling solution containing 0.5N potassium hydroxide and ethanol for 30 minutes. Then the solution may be cooled and placed into a dropping funnel containing a 2% aqueous solution of sodium hydroxide. The polyprenols (ropren) obtained as a result of the hydrolysis of acetate polyprenols are extracted with solvent eg. light petroleum. For example, the elution may be carried out using light petroleum with 15% diethyl ether. The chromatography process as well as the purity of the eluted polyprenols, may be monitored by layer chromatography with a standard "marking" of polyprenols.

The fractions containing polyprenols may be amalgamated, and the solvent distilled off. The yield of polyprenols per unit of dehydrated foliage usually ranges from 0.9 1.1%. The characteristics of ropren obtained by such a process are usually as follows: State oily Colour yellow-amber Odour specific Content of main product not less than 95% Density, 20"C + 2 no more than 0.902-0.905 g/cm3 Carotenoids per unit of weight 20 mg per 100g of dry product Triterpenoids per unit of weight no more than 5% Rp compounds are related to the dolichols found in mammals and have previously been proposed for the treatment of certain forms of diabetes, certain diseases of the central nervous system, and disturbances in spermatogenosis.

Inosiplex (In) and Rp have the following properties in common: 1. Stimulation of the production of antibody-forming cells in the spleen.

2. Stimulation of the humoral immune response of weak responding animals and strong-responding animals immunised with a sub-optimum dose of antigen.

3. Suppression of the humoral immune response of strong-responding animals during immunisation by an optimal dose of antigen.

4. Stimulation of phagocytosis and digesting ability of macrophages.

However, Rp and In may be distinguished by the following characteristics: ROPREN 1. Chemical formula and structu.

2. Derived from raw plant materials.

3. Does not possess toxicity; LD 50 is not reached at a dosage in excess of 10000 mg/kg.

4. During 10 days internal administration at a dosage of 100 mg/kg, the lowering of the humoral immune response and the inhibition of macrophage functions caused by emotional stress, is prevented.

INOSIPLEX 1. Chemical formula and structure.

2. Derived by chemical synthesis.

3. Toxicity: LD 50 at 7500-10000 mg/kg.

As a result of investigations it was ascertained that Rp has an immunomodulating effect on the development of both cell and humoral immunity. The LD 50 for Rp was determined on white mongrel mice during oral administration and was not revealed for a dosage in excess of 10000 mg/kg. Rp is thus extremely safe to use.

As will be described in more detail below, Rp has been shown to have both immunosuppressive and immunostimulatory properties depending on the circumstances of use. For example, as mentioned above the humoral immune response of animals immunised with a sub-optimal dose of antigen may be stimulated or enhanced. On the other hand, in animals immunised with an optimum antigen dose, the immune response may in certain situations (eg. in a strongly responding animal) be suppressed.

In particular, Rp has been shown to stimulate the development of the humoral immune response, and also to modulate the degree of the humoral immune response. The adjuvant properties of Rp have also been demonstrated.

These various immunomodulating effects may be put to a number of uses in combatting various disorders of the immune system and diseases associated therewith, including autoimmune diseases as well as microbial infections. The term "immune disorder" as used herewith encompasses all imbalances of the immune system. As used herein, the term "combatting" includes both prophylaxis and therapy.

Thus for example, the effect of Rp in stimulating the humoral immune response in subjects immunised with sub-optimum doses of antigen, may be employed in the treatment or prophylaxis of microbial infections eg.

bacterial or viral infections eg. bacterial sepsis. As such Rp may be used in conjunction with other antimicrobial agents eg. antibiotics.

The stimulation of humoral immune response development eg. enhancement of antibody-producing cell production, indicates a role for Rp as an adjuvant.

The immunosuppressive effect of Rp may usefully be employed in correcting a pathologically enhanced immune response as occurs for example in various auto immune or allergic diseases eg. bronchial asthma, non-specific ulcerative colitis, psoriasis, systemic lupus erythematosus, rheumatic arthritis, chronic glomerulonephritis and the like.

A vital task of extreme practical importance for modern immunology is a search for biologically active substances which would be able, by affecting the immune system directly or indirectly (via the central nervous system), to protect an organism against the oppressive immunosuppressive effects of chronic emotional or other stress, which is known frequently to result in various diseases (including oncological ones), due to the failure of fine specific protection mechanisms. With proper consideration of the above, Rp was investigated for antistressor activity in mice. When administered per os at a dose of 100.0 mg/kg for 10 days, Rp precluded a 1.25-2.2 fold decrease in the humoral immune response, and macrophage functional disturbance, as induced by chronic emotional stress.

This effect of Rp can be utilized for the prophylaxis and treatment of many diseases (including cancers) where a significant trigger link may include decreased immunological resistance due to stress eg.

chronic emotional stress, as well as for prophylaxis of possible recurrences. Antistressor and adaptogenic effects of Rp can be used in cases of long term psychoemotional or physical overloads, particularly when under confined room conditions, e.g. in ships, polar stations or during long-term space flights, etc.

When administered intraperitoneally, Rp does not affect the development of cell-type immune reactions, eg. delayed skin reaction (DSR) or graft versus host reaction (GVHR). In has similar properties.

For example, when given per os to mice at a dose of 100.0 mg/kg for 5 days, Rp causes a 1.2 fold suppression of DSR development. Thus, cell-based immunity is affected by Rp only under certain circumstances eg. mode of administration, dosage, immune status of the patient or animal, etc. and this can also be used to achieve a desired clinical effect.

The fact that Rp exerts no effect on cellular immunity whilst stimulating humoral immune response, can be of much practical importance in the treatment of, for example, secondary immune deficient states in transplantation cases and related immunosuppressive therapy necessitating prophylaxis of bacterial sepsis.

Different effects of Rp on phagocytic activity have also been observed. For example, in mice when intraperitonealy administered at doses of 10.0 and 100.0 mg/kg, Rp induces a 1.3-1.85 fold stimulation of phagocytosis completion; when given per os at a dose of 100.0 mg/kg, it leads to a 1.3 fold increase of phagocytic cell count in peritoneal exudate.

For comparison, when administered intraperitoneally at doses of 5.0 and 50.0 mg/kg, In also stimulates phagocytosis completion, but to a lesser degree than Rp (1.4-1.7 times).

The stimulating effect of Rp on phagocytosis in combination with its stimulating effect on humoral immune response can be used for the treatment of septic states and bacterial or viral infections as well as for conservative action on metastases or primary tumors following radical oncological operations.

Thus, it has been shown that Rp is a new immunomodulator which selectively affects the humoral arm of the immune response and the non-specific phagocytic activity of macrophages; therefore, it can be used in the treatment of various diseases whose principal pathogenic mechanism is the imbalance of the immune system which is responsible for the genetic integrity and uniformity of an organism and for the protection of the latter against foreign living bodies or substances bearing genetically heterologous signs. In addition, Rp is an efficient antistressor and adaptogenic drug capable of protecting the immune system against the suppressive effects of chronic emotional or other stress and ensuring prophylaxis of various diseases associated with any degree of immune disbalance.

The biological investigation of Rp has shown it to be completely innocuous, to have no effect on reproductive function, to induce no undesirable mutations, and to have neither embryotoxic nor teratogenic effects, making it extremely safe for use as a pharmaceutical.

The compounds useful in practising the invention are preferably formulated prior to administration. The present invention therefore also provides a pharmaceutical composition comprising a compound of formula (I) or isomer thereof as defined above, and one or more further pharmacologically or biologically active agents. Such agents may include for example antimicrobial agents such as antibacterials eg.

antibiotics as anti-viral agents, or antigens in vaccine compositions, together with at least one physiologically acceptable carrier, diluent or excipient.

The compound of formula (I) or isomer thereof need not, however, necessarily be co-formulated with the further active agent and could for example be coadministered in a separate formulation sequentially or simultaneously.

Thus, the present invention also provides a product containing a compound of formula (I) or isomer thereof as defined above, and one or more further pharmacologically or biologically active agents as a combined preparation for simultaneous, separate or sequential use in combatting microbial diseases or disorders of the immune system and diseases associated therein.

The compound of the invention in such compositions may comprise from about 0.1t to about 99 by weight of the formulation. By "pharmaceutically acceptable" is meant that the ingredient must be compatible with other ingredients of the compositions as well as physiologically acceptable to the recipient.

Pharmaceutical compositions for use according to the present invention may be formulated in conventional manner using readily available ingredients, for example as widely described in the pharmaceutical literature.

Thus, the active ingredient may be incorporated, optionally together with other active substances, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions sterile packaged powders, and the like.

Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, aglinates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol, water/ glycol, water/polyethylene, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof. The compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.

The compositions are preferably formulated in a unit dosage form, eg. with each dosage containing from about 0.1 to about 500 mg of the active ingredient.

The precise dosage of the active compound to be administered and the length of the cause of treatment will, of course, depend on a number of factors including for example, the age and weight of the patient, the specific condition requiring treatment and its severity, and the route of administration. Generally however, an effective dose may lie in the range of from about 0.1 mg/kg to about 500 mg/kg per day, eg from about 0.5 to about 100 mg/kg per day, administered one or more times per day.

The administration may be by any suitable method known in the medicinal arts, including for example oral, parenteral (eg. intramuscular, subcutaneous, intraperitoneal or intravenous) rectal or topical administration or administration by inhalation.

The invention will now be described in more detail by way of the following non-limiting Example: Example The immunomodulating activity of Rp was investigated experimentally in animal lines having a genetically predetermined immune disbalance/mice corresponding to lines C57B1/6 CBA, and F1 (CBA x C57B1/6) (1).

An emulsion of Rp was prepared in 0.025% twin-80 solution (available from Merck). Experimental animals were intraperitoneally administered 0.1 ml doses of 10.0 and 100.0 mg/kg in parallel to antigen administration; similar doses were given per os for 5 days before antigen administration. 0.025k twin-80 solution was used as an intraperitoneal control exerting no direct effect on the antibody-forming cell count in mouse spleen, delayed skin reaction (DSR), graft-versus-host reaction (GVHR) or phagocytosis, whilst causing a slight stimulation of antibody production (activity index 1.2); persic oil was used as a per os administration control leading to no alterations in the immune system (2,3).

Experimental emotional stress was created by overcrowding (40 mice in a 17 x 25 x 8 cm cage) for 10 days. During the experiment, control animals were daily given per os doses of twin-80 solution, and experimental animals were intraperitoneally administered RSL-1/88-1 "BF" at a dose of 100.0 mg/kg (4,5) dissolved in physiological solution and administered intraperitonerally as single doses together with antigen (sheep erythrocytes). Physiological solution was used as a control. It was administered at doses of 0.5 to 250.0 mg/kg.

The following investigative techniques were used to determine the effects produced by the substances on the animals immune system: 1. Determination of the antibody-forming cell count in mouse spleen on the 5th day following immunisation using optimum (lox108 cells) and sub-optimum (2x107 cells) doses of sheep erythrocytes, i.e. local hemolysis reaction (6).

2. Determination of hemagglutinin titres for sheep erythrocytes on the 7th and 14th days following immunisation with the optimum and sub-optimum doses, using 96-hole round-bottom plates. Results were accounted for macroscopically and expressed in log2 of a titre. Administration patterns used were similar to those practised when determining antibody-forming cell counts (7).

3. DSR determination using intraveneous immunisation of mice with sheep erythrocytes administered at a dose of 2x105 cells. A resolution dose (lox108 cells in 0.04 ml of physiological solution) was injected into a hind footpad on the 5th day following immunisation. Local inflammatory reaction was accounted for 24 hours later by the difference in mass between experimental (Re) and control (Rc) footpads. A reaction index (RI) was calculated for each mouse using the following formula: RI Re -Rc x 100% (8) Rc 4. GVHR determination. GVHR was induced by subcutaneous administration of 2x106 lymph node cells of host genotype (CBA) into right hind footpads of line F1 (CBA x C57B1/6) mice.

Syngeneic lymphocyles were administered into left footpads. The cell count was determined in 5 ml of homogenate of left (control) and right (experiment) lymph nodes.

A reaction index (RI) was calculated using the following formula: RI control (9) experiment Administrations were performed via the intraperitoneal route simultaneously with GVHR induction.

5. Estimation of phagocytic activity of peritoneal macrophages in mice was performed, using IgG opsonized sheep erythrocytes as the phagocytosis target (10). Macrophages were attracted by 4% hydrolyzed starch solution. On the 3rd or 4th day, 0.5 ml of 5% sheep erythrocyte suspension was administered intraperitoneally. After 60 minutes the exudate was removed. Non-phagocytic sheep erythrocytes were subjected to lysis. Cell suspension was washed out. The phagocytic cell percentage of the preparation stained by Romanovsky-Gimze was determined. To determine the phagocytic index (PI1), the number of sheep erythrocytes captured was calcaulated spectrophotometrically (11) by hemoglobin concentration using a calibration curve. Phagocytic index (PI2) was determined again following a 3 hour incubation period at 37"C. The phagocytosis completion index (PCI) was calculated using the following formula: Pli -P12 x 100% PI2 Phagocytic activity determination in vivo in mice was performed by intravenous injection of 0.5 ml of 25% ink solution. Then, for 15 minutes, blood was taken from the orbital sinus (0.020 ml) every 3 minutes and introduced into 1 ml of 3k acetic acid.

After the blood taking was completed, the animals were killed and their body, liver and spleen weights were determined. The optical density of the hemolyzed blood was determined spectrophometrically at a wavelength of 610 nm, and the f(D)t curve was plotted. Points at log (0) (zero time) and log (10) (on the curve) were located. The phagocytosis constant (K) was calculated using the following formula: log0-log10 @@@@ logic and then the true phagocytosis index (a) was calculated using the following formula: a body weight 3 liver weight +spleen weight 7. Complement activity determination using the titration technique employed rabbit erythrocytes treated with the hyperimmune antiserum of guinea pigs (12) for the direct activation route and employed rabbit erythrocytes treated with potassium iodide solution (13) for the alternative activation route. Activity was expressed in units of 50k lysis per ml of serum (CH50).

8. The statistical processing of data thus obtained was performed on a digital computer using an application program package. Both the Student's and Whitney-Mann-Wilcoxon techniques were applied.

Logarithmic indices were used for the data processing of antibody-forming cell counts and hemagglutinin titres.

Results 1. Effects exerted by Rp and In on humoral immune response development Investigative data concerning the effect of Rp on antibody-forming cell formation in mouse spleen and on hemagglutinin titres in the serum of mice of various lines immunised with the optimum and sub-optimum doses of sheep erythrocytes are summarised in Tables 1 and 3.

As can be seen from these tables, when administered intraperitoneally Rp leads to an approximately 2-fold increase in the production of antibody-forming cells in the spleen and to a two-times higher hemagglutinin level in the serum of line C57B1/6 mice that are lowresponsive to sheep erythrocytes, when immunised with optimum or sub-optimum antigen doses. More intensive production of antibody-forming cells in high-responsive mice Fl in the sub-optimum immunisation mode with Rp administered at doses of 10.0 and 100.0 mg/kg, and higher hemagglutinin titres with Rp administered at a dose of 100.0 mg/kg, indicate the presence of adjuvant properties.

With RP administered at a dose of 100.0 mg/kg for 5 days, antibody-forming cell counts in the spleen of line C57Bl/6 mice increased both in the optimum and suboptimum immunisation modes, whereas hemagglutinin titres increased only in the optimum immunisation mode.

It is of interest to note the very fact of dosedependent suppression of antibody-forming cell production in the spleen of line Fl mice with sheep erythrocytes administered at the optimum dose (Table 1), and the decrease of hemagglutinin titres in serum with Rp administered at a dose of 100.0 mg/kg via intraperitoneral or per os route. This phenomenon might be related to simultaneous administration of considerable amounts of sheep erythrocytes and substance Rp activating a respiratory enzyme chain (14) as well as monooxygenases, e.g. cytochrome P-450 (15). The latter circumstance, related to a functional connection between the immune system and metabolism, leads to the accelerated degradation of erythrocytes in phagocytic cells, weaker antigenic properties of sheep erythrocytes and thus, less pronounced humoral immune response in intact animals with high-quality physiological parameters. According to literature data, similar immunosuppressive properties are exhibited by alkyl- and aryl-substituted 5- oxypyrimidines (16), 3-oxypyridine derivatives, and aqueous extracts of pea tissues (17).

As can be seen from Table 3, the decrease in hemagglutinin titres is of a phasewise character; no valid difference can be detected in the experimental and control group indices by the 14th day following immunisation.

Thus, a conclusion can be made that Rp of vegetable origin stimulates development of the humoral immune response, possesses adjuvant properties, and modulates the degree of the humoral immune response in highlyresponsive mice.

As a comprison, a series of experiments was set up to investigate the effect of In on the production of antibody-forming cells in the spleen of mice of lines that are opposite responsive to T-dependant antigen, i.e. sheep erythrocytes (see Table 1). Three dosage values (0.5, 5.0 and 50.0 mg/kg) and two immunisation modes (optimum and sub-optimum) were used. As can be seen from Table 2, when given at a dose of 0.5 mg/kg in the optimum immunisation mode only, inosiplex enhances the formation of antibody-producing cells by 90% (p 0.05) in low-response line C57Bl/6 mice.

When given at a dose of 5.0 mg/kg in the suboptimum immunisation mode, In induces a poorly expressed and statistically invalid effect, increasing the antibody-forming cell count by 38%. In the optimum immunisation mode, it increases the antibody-forming cell count by 82% (p 0.05).

When given to low responsive line mice at a dose of 50.0 mg/kg in the sub-optimum and optimum immunisation modes, the stimulating effect of inosiplex amounts to 32% and 139%, respectively.

A series of experiments was set up to investigate the effect of inosiplex on the antibody formation level (hemagglutinin titre for sheep erythrocytes) in the suboptimum T-dependent antigen immunisation of mice, with inosiplex administered at doses of 0.5, 5.0, 25.0, 50.0, and 250.0 mg/kg.

When In was given at doses of 0.5, 5.0 and 25.0 mg/kg to line CBA mice, no stimulating effect (p 0.05) was observed both on the 7th and 14th day following immunisation (see Table 4).

When given at a dose of 250.0 mg/kg (Table 4), inosiplex stimulated hemagglutinin production by as much as two times (lxlog2 of hemagglulinin titre).

2. Effects exerted by Rp and by In on cell immune response development Data concerning the effect of Rp on DSR and GVHR are summarised in Table 5. As can be seen from this table, this substance induces no change in the cell-type reaction rates when administered at a single intraperitoneal dose. When given per os at a dose of 100 mg/kg for 5 days to line C57B1/6 mice whose DSR is 2.0 to 2.5 times more vigorous than that in line CBA mice, Rp suppresses the intensity of this reaction. Per os administrations of Rp exerted no effect on GVHR development.

Investigation of the effect of In on DSR development was performed in mice of two lines, with the drug administered at doses of 0.5, 5.0 and 50.0 mg/kg.

The drug induced no changes in the reaction intensity (see Table 6).

Investigation of the effect of In on GVHR was also made using doses of 0.5, 5.0 and 50.0 mg/kg. A slight inhibitory effect was noted when given at a dose of 50.0 mg/kg (p 0.05) (see Table 6).

3. Effects exerted by Rp and by In on phagocytic activity of macrophages Investigative data concerning the effect of Rp on phagocytic activity of peritoneal macrophages and on ink clearance are summarised in Tables 7 and 9.

As can be seen from Table 7, when administered at single intraperitoneal doses, Rp induces neither changes in phagocytic cell count in peritoneal exudate nor alterations in the ability of phagocytes to capture opsonized sheep erythrocytes, while causing a 1.5-2.0 fold increase in the digestive capacity of macrophages.

When administered per os at a dose of 100.0 mg/kg, Rp increases the phagocytic cell count in exudute while inducing no changes in the phagocytic index or in the phagocytosis completion index. When administered intraperitoneally, Rp is able to stimulate phagocytosis completion; this can be related to the local effect of the substance.

When administered intraperitoneally or per os, Rp induces no changes in the capturing intensity of inert ink particles and exerts no influence on the blood clearance rate (Table 9).

The effect of In at doses of 0.5, 5.0 and 50.0 mg/kg on the phagocytosis index was studied in hybrid line F1 (CBA x C57B1/6) mice, with the drug being administered intraperitoneally 30 minutes before injection of the phagocytosis target, i.e. sheep erythrocytes.

At a dose of 0.5 mg/kg, no stimulation effect was noted.

At a dose of 5.0 or 50.0 mg/kg, the phagocytosis completion index increased by 39% (p 0.02) and 76% (p 0.01), respectively (see Table 8).

The effect of In on the ink clearance rate (phagocytosis in vivo) was studied with the drug administered intraperitoneally at doses of 0.5, 5.0 and 50.0 mg/kg 30 minutes before intravenous ink injection.

As can be seen from Table 10, when administered days results in the suppress ion of the humoral immune response (antibody forming cell count and hemagglutinin count) and in the suppression of the phagocytosis completion and inert ink particle clearance rates (approximately by 1.25 to 2.2 times). All these disturbances (except for the latter) are precluded by per os administration of Rp at a dose of 100.0 mg/kg for 10 days.

Thus, per os administration of Rp protects the immune system against the depressive effects of emotional stress.

The present experimental study has resulted in the following findings. Rp causes a 1.9-2.3 fold stimulation of humoral immune response development in low responsive line C57B1/6 mice when given at doses of 10.0 and 100.0 mg/kg, and in high responsive line F1 (CBA x C57B1/6) mice immunised with the sub-optimum dose of antigen (sheep erythrocytes). This Rp effect can be utilised in various bacterial-origin infectious diseases, and particularly in septic cases when humoral immunity to pathologic micro-organisms is considerably reduced. Combined usage of an immunostimulator and antibiotics should yield a higher therapeutical effect.

When given at doses of 5.0 and 50.0 mg/kg, In causes a 1.3-1.8 fold stimulation of humoral immune response in animals that are low responsive to antigen.

When administered at doses of 10.0 and 100.0 mg/kg intraperitoneally and at a dose of 100.0 mg/kg per os to high responsive line F1 (CBA x C57B1/6) mice immunised with the sub-optimum antigen dose, Rp leads to a 4.711.8 fold suppression of humoral immune response development. This suppressive effect of Rp can be utilised for correction of a pathologically enhanced humoral immune response, for example in various allergic and autoimmune cases, such as bronchial asthma, nonspecific ulcerative colitis, psoriasis, systemic lupus erythematosus, rheumatic arthritis, and the like.

When given at doses of 5.0 and 50.0 mg/kg, In also suppresses the humoral immune response in line CBA mice that are high-responsive to antigen in the optimum immunisation mode, but to a lower extent than Rp (1.74 to 2.3 times).

Table 1 ROPREN EFFECT ON PRODUCTION OF ANTIBODY FORMING CELLS (AFC) IN MOUSE SPLEEN

N Administration route Mouse Sheep Ropren dose AFC count per p < AFC count per entire p < line, sex, weight1 erythrocyte dose (Mg/kg) 106 nucleated spleen organ (cells) cells 1. Single C57B1/6 2x107 Control 5.4(6.0'4.8) 0.01 1072(1202'955) intraperitoncal males 10.0 11.5(13.5'9.8) 0.01 1122(1202'1047) dose 22-25g 100.0 10.2(11.7'8.9) 0.01 1288(1479'1122) 2. ditto C57B1/6 108 Control 5.5(6.3'4.8) 0.001 794(891'708) 0.001 females 10.0 12.6(12.9'12.3) 0.001 1585(1820'1380) 0.001 21-22g 100.0 1585(1820'1380) 3. ditto F.(CBAxC57B1) males 2x107 Control 6.9(7.6'6.3) 0.001 764(851'741) 0.001 21-23g 10.0 12.6(13.2'12.0) 0.001 1259(1318'1202) 0.001 100.0 10.7(11.0'10.5) 1259(1288'1000) 4. ditto F1(CBAxC57B1) females 108 Control 23.7(25.1'21.9) 0.001 3981(4541'3467) 0.001 21-23g 10.0 5.0(5.4'4.7) 0.001 1000(1072'933) 0.001 100.0 2.0(2.4'1.7) 398(473'363) 5. ditto ditto ditto Control 44.7(49.0'41.0) 0.05 9772(11220'8511) 0.02 10.0 25.1(31.0'20.4) 0.001 5012(5754'4365) 0.001 100.0 10.0(12.0'8.3) 3981(4467'3548) 6. Per os, for 5 days C57B1/6 2x107 Control 6.3(7.9'5.0) 0.05 794(1120'562) 0.01 males 10.0 7.9(10.2'6.2) 1000(1230'813) 18-20g 100.0 15.8(18.2'13.8) 3162(3548'2818) 7. Per os, for 5 C57B1/6 108 Control 5.4(6.2'4.7) 0.05 631(794'501) females 10.0 7.1(8.1'6.2) 1000(1259'794) 20-22g 100.0 7.8(8.5'7.1) 955(1023'891) 8. ditto F1(CBAxC57B1) 2x107 Control 16.0(17.0'15.0) 1995(2344-1698) males 10.0 20(23.0'17.0) 3162(3802'2630) 21-23g 100.0 16(19.0'13.012) 1995(2239'1778) 9. ditto F1(CBAxC57B1) ditto Control 10.0(11.7'8.5) 1000(1072'933) 0.02 females 10.0 12.6(14.5'11.0) 1585(1820'1380) 23-25g 100.0 7.9(8.9'7.1) 1175(1288'1072)

10 ditto F1(CBAxC57B1) 108 Control 17.0(18.2'15.8) 0.01 3152(3467'2884) 0.001 females 10.0 11.0(13.5'8.9) 1996(2455'1622) 21-23g 100.0 10.7(12.0'9.5) 1585(1955'1259) 1) 8 animals in a group Table 2 INOSIPLEX EFFECT ON ANTIBODY FORMING CELL (AFC) POOL PRODUCTION IN MOUSE SPLEEN

Mouse Line, Sheep Drug weight, erythrocyte AFC COUNT PER 106 NUCLEATED SPLEEN CELLS number in a dose (cells) Control 0.5 gm/kg stimulation 5.0 mg/kg stimulation 50.0 mg/kg stimulation group degree, % degree, % degree, % C57B1/6, 2x107 inosiplex 85(98-74) 52(63'44) 117(149'98) 3882 112(141'89) 32139 males, 108 inosiplex 83(102-68) 158(186'134) 151(169'134) 199(245'162) 20-22 g n=8 p < 0.05 90 p < 0.05 p < 0.01) Table 3 ROPREN EFFECT ON HEMAGGLUTININ (GA) TITRE FOR SHEEP ERYTHROCYTES IN MOUSE SERUM1

N Administration Mouse line, sex, Sheep Ropren HA TITRES FOLLOWING IMMUNISATION route weight erythrocyte dose dose(cells) (mg/kg) 7th day P < 14th day P < 1. Single C57B1/6, males 2x107 Control 6.75)0.20 0.02 4.92)0.26 0.01 intraperitoneal 21-23g 10.0 7.50)0.15 0.02 6.17)0.31 0.05 dose 100.0 7.50)0.17 5.70)0.15 2. ditto C57B1/6, males, 108 Control 5.58)0.21 0.01 7.42)0.28 20-22g 6.50)0.17 0.01 8.08)0.20 6.50)0.12 8.00)0.17 3. ditto ditto ditto 10.0 8.50)0.179 0.01 7.92)0.19 0.01 100.0 9.42)0.23 0.02 8.67)0.17 0.001 9.67)0.39 9.17)0.21 4. ditto F1(CBAxC57B1) 2x107 ditto 7.3)0.3 0.02 8.4)0.3 0.01 males. 22-24g 7.2)0.4 7.7)0.4 8.3)0.2 9.6)0.2 5. ditto ditto ditto ditto 6.92)0.17 0.001 8.5)0.41 8.00)0.12 9.1)0.25 7.25)0.21 9.5)0.41 6. ditto CBA, females 108 ditto 8.5).0.45 0.05 8.5)0.31 0.01 18-22g 7.6)0.25 7.3)0.22 7.3)0.13 8.0)0.31 7. ditto F1(CBAxC57B1) ditto ditto 6.9)0.25 0.001 10.2)0.4 0.01 males. 18-20g 6.4)0.14 10.9)0.6 5.8)0.17 10.5)0.4 8. Per os, for 5 days C57B1/6, 2x107 ditto 8.10)0.42 6.80)0.25 males, 7.60)0.65 7.60)0.42 18-20g 7.70)0.58 7.20)0.47

9 ditto C57B1/6, 108 ditto 5.80)0.12 6.10)0.09 0.01 females, 6.70)0.25 0.01 7.30)0.30 21-23g 6.80)0.20 0.01 6.30)0.20 10. ditto F1(CBAxC57B1) 2x107 ditto 9.00)0.25 7.10)0.30 males, 20-22g 10.70)0.69 7.30)0.30 6.70)0.37 0.001 7.30)0.30 11. ditto ditto 108 ditto 7.90)0.22 0.001 8.20)0.20 7.90)0.30 8.25)0.27 6.90)0.14 8.30)0.38 1) 6 animals in a group Table 4 INOIPLEX EFFECT ON HEMAGGLUTININ (HA) TITRE IN SERUM OF LINE CBA MICE IMMUNISED WITH SUBOTIMUM SHEEP ERYTHROCYTE DOSE

Day after Mouse sex, weight Dose (median) immunisation number in a group (mg/kg) HA TITRE ---------- Males, 20-22 g, n = 10 M # m Control Inosiplex P < 7th day Males, 20-22 g, n = 10 0.5 8.0 8.0 8.10 # 0.48 8.20 # 0.35 Males, 20-22 g, n = 10 5.0 9.0 9.0 8.80 # 0.49 9.17 # 0.24 Males, 20-22 g, n = 12 25.0 7.5 7.0 7.67 # 0.31 7.30 # 0.21 14th day Males, 20-22 g, n = 8 250.0 7.5 8.5 0.02 Table 5 ROPREN ON CELLULAR IMMUNITY REACTION DEVELOPMENT N MICE1

Reaction type Administration Mouse line, sex, REACTION INDEX (M)m) route Co 10 mg/kg P < 100 mg/kg P < ntr ol DSR Single C57B1/6, males, 28.5)3.1 23.2)2.1 24.6)2.9 intraperitoneal 20-22 g dose ditto ditto CBA, males, 17.2)1.5 21.3)2.9 22.2)2.4 19-21 ditto Per os, for 5 C57B1/6, 45.7)3.7 46.8)4.7 35.3)3.1 0.05 days males, 20-22 g ditto ditto ditto 36.2)4.3 31.2)3.6 25.8)3.3 0.05 ditto ditto CBA, males, 19.1)2.0 19.0)1.8 17.5)1.5 20-22 g GVHR Single F1 (CBAxc57B1) 1.8)0.2 2.2)0.2 2.0)0.1 intraperitoneal males, 18-20 g dose ditto ditto ditto 2.6)0.2 2.5)0.2 2.6)0.3 ditto Per os, for 5 ditto 3.9)0.4 3.7)0.4 3.7)0.4 days 1) 8 animals in a group Table 6 INOSIPLEX EFFECT ON CELLULAR IMMUNITY REACTION DEVELOPMENT IN MICE1

Reaction Mouse line, sex, weight Dose, mg/kg REACTION INDEX, % (M)m) type Control Inosiplex P < DSR C57B1/6, males, 0.5 20.6)2.70 13.9)2.41 18-20 g n = 6 n = 4 5.0 26.8)2.33 27.2)2.38 n = 8 n = 8 19.4)1.34 22.58)2.44 50.0 n = 7 n = 5 0.05 ditto CBA, males, 18-20 g 0.5 12.1)1.4 11.9)0.8 n = 8 n = 9 5.0 9.02)1.09 8.9)0.89 n = 10 n = 9 50.0 23.7)1.25 25.7)2.57 n = 7 n = 6 0.05 GVHR F1 (CBAxC57B1/6) 0.5 2.12)0.21 2.35)0.18 n = 9 n = 8 5.0 2.04)0.19 2.51)0.15 n = 10 n = 10 50.0 3.66)0.31 2.66)0.31 n = 10 n = 10 0.05 1) 6 animals in a group Table 7 ROPREN EFFECT ON PHAGOCYTIC ACTIVITY OF PERITONEAL MACROPHAGES'

N Administration route Dose, Phagocyting cells%, P < Phagocytosis index P < Phagocytosts index P < mg/kg (M)m) (M)m) (M)m) 1. Single intraperitoneal dose Control 64.1) 6.4 12.4)1.0 37.1)4.1 10.0 71.7)3.4 14.8)2.8 68.7)3.4 0.001 100.0 73.2)0.6 12.0)3.7 49.0)1.8 0.05 2. ditto Control 49.2)5.1 24.4)4.1 29.0)5.7 10.0 48.2)7.1 10.5)0.44 0.02 71.3)1.4 0.001 100.0 48.8)5.0 17.9)2.7 65.6)1.6 0.01 3. Per os, for 5 days Control 56.6)6.2 7.7)0.6 30.9)2.4 10.0 63.0)3.7 8.7)1.4 28.9)2.5 100.00 72.5)3.6 0.05 8.0)1.1 17.8)2.6 4. ditto Control 56.0)5.7 7.0)0.9 13.5)5.1 10.0 61.0)3.9 8.3)0.7 24.5)5.5 100.0 68.7)1.3 0.05 0.3)1.1 31.4)12.8 0.02 5. ditto Control 60.5)3.62 7.7)1.0 23.3)5.1 10.0 75.3)4.0 0.05 6.2)0.8 19.4)0.9 100.00 80.8)1.6 0.01 5.0)0.6 35.8)1.8 1) 6 animals in a group Table 8 INOSIPLEX EFFECT ON PHAGOCYTIC ACTIVITY OF PERITONEAL MACROPHAGES IN LINE (CBA x C57/6) MICE (Males, weight 20-22g)

Phagocytosis index, %(M)m) Dose Number of animals in groups mg/kg Control Inosiplex Stimulation degree, % 0.5 7 37.1)2.8 29.7)7.8 5.0 6 37.7)3.2 52.4)3.9 39 P < 0.02 50.0 6 36.0)8.2 63.5)4.5 76 P < 0.05 P - validity of deviation from the control Table 9 ROPREN EFFECT ON INK CLEARANCE IN LINE CBA MICE

N Administration route Dose, mg/kg Phagocytosis index (M)m) P < 1. Single intraperitoneal dose Control 4.85)0.19 10.0 4.63)0.24 100.0 4.71)0.09 0.05 2. ditto Control 3.96)0.1 10.0 4.04)0.1 100.0 4.38)0.28 0.05 3. Per os, for 5 days Control 4.22)0.13 10.0 4.01)0.18 100.0 3.70)0.20 0.05 4. ditto Control 4.52)0.14 10.0 4.46)0.15 100.0 4.58)0.27 0.5 Table 10 INOSIPLEX EFFECT ON INK CLEARANCE'

Dose, mg/kg Compound True phagocytosis index P < 0.5 Control 5.20)0.20 Inosiplex 5.00)0.16 0.05 5.0 Control 5.56)0.26 Inosiplex 5.59)0.18 0.05 50.0 Control 5.44)0.30 Inosiplex 5.70)0.18 0.05 1) Investigation made on line F1 (CBA x C57 B1), 7 animals in a group Table 11 ROPREN EFFECT ON BLOOD SERUM COMPLEMENT ACTIVITY IN LINE F1(CBA x C57B1/6) MICE'

Complement activity, CH50/ml N Administration route Dose, mg/kg Direct acti Alternative activation pathway 1 Intraperitoneal Control 20.0 25.0 10.0 25.0 25.0 100.0 25.0 33.3 2 Per os, for 5 days Control 16.7 20.0 10.0 14.3 20.0 100.0 16.7 14.3 1) Serum pool of 5 mice was used for analysis in each group Table 12 ROPREN EFFECT ON IMMUNE SYSTEM OF STRESSED MICE

Immunoreactivity index, Date of experiment Intact animals STRESSED ANIMALS unit of measurement Control P1 < Experiment P2 < (0.025% twin) (prenols, 100 mg/kg) AFC per 106 cells per spleen 11.05-26.05.87 4.8(5.4+4.3) 2.4(2.8+2.0) 0.01 6.5(6.6+6.3) 0.001 (M)m) 1000(1122+891) 398(490+324) 0.001 0.01 HA titre on the 14th day 11.05-4.06.87 5.4)0.2 4.3)0.1 0.001 5.0)0.4 (M)m) DSR (M)m of reaction index) 11.05-27.05.87 20.4)1.9 24.5)3.33 25.3)2.8 GVHR (M)m of reaction 11.05-28.05.87 2.06)0.12 1.96)0.14 2.08)0.11 index) Phagocytosis Phagocytic index (M)m) 15.05-29l.05.87 13.8)3.6 16.4)3.2 16.0)2.0 Phagocytosis completion index, % 15.05-29.05.87 37.3)7.4 (M)m) 16.8)3.5 0.05 34.6)5.1 0.01 Ink clearance index 11.05-27.05.87 4.32)0.1 3.02)0.23 0.01 3.60)0.01 (M)m) P1 < 0.01 P1 - validity of deviations from indices of intact animals P2 - validity of deviations from indices of stressed animals REFERENCES 1. Blandova Z.K., Doushkin V.A., Malshenko A.M.., Smidt E.F., Laboratory animals lines for medico biological studies, Moscow, Nauka, 1983,190 pages 2. Methodological materials for experimental (pharmacological) and clinical testing of pharmaceuticals for immunomodulating activity.

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Claims (9)

1. Use of a compound of formula (I)

where: n = 8 - 18 or an isomer thereof, in the preparation of a medicament for use as an immunomodulating agent.

2. Use as claimed in claim 1, for combatting microbial diseases, or disorders of the immune system and diseases associated therewith.

3. Use as claimed in claim 2, for combatting immunodeficiency or a pathologically enhanced immune response.

4. Use as claimed in claim 1, for combatting stress.

5. Use as claimed in claim 1, wherein said compound of formula I or isomer thereof is used as an adjuvant.

6. A pharmaceutical composition comprising a compound of formula I as defined in claim 1 or an isomer thereof, and one or more further pharmacologically or biologically active agents together with at least one physiologically acceptable carrier, diluent or excipient.

7. A product containing a compound of formula (I) or isomer thereof as defined in claim 1, and one or more further pharmacologically or biologically active agents as a combined preparation for simultaneous, separate or sequential use in combatting microbial diseases or disorders of the immune system and diseases associated therein.

8. A composition as claimed in claim 6 or a product as claimed in claim 7, wherein said further active agent is an antimicrobial agent or a vaccine antigen.

9. Each and every novel compound, composition, method and use herein disclosed.