GB2042558A - Interferon inducers, methods for their preparation, pharmaceutical compositions containing them and their use as medicaments - Google Patents

Abstract

An interieron inducer isolated from plant tissues, having an elemental analysis of H: 6.54 +/- 0.3 %, C: 41.9 +/- 0.3%, N: 2.39 +/- 0.3%, P: 0.28 +/- 0.03% and a molecular weight of 100,000 to 3,000,000 (preferably 500,000 to 1,000,000), which is believed to be a complex of protein and sugar containing phosphoric acid, which may be produced by extracting a substance having interferon inducing activity from the tissue of a plant belonging to the genus Carthamus of the family Compositae or a variant thereof containing said active substance and recovering the said active substance from the extract thereby obtained. Preferably, the extraction may be effected with water and the recovery may be effected by ultra filtration.

Description

SPECIFICATION Interferon inducers, methods for their preparation, pharmaceutical compositions containing them and their use as medicaments The present invention relates to interferon inducers, methods for their preparation, pharmaceutical compositions containing them and their use as medicaments.

Interferon, hereinafter also referred to as IF or virus inhibiting factor is a substance capable of acting upon animal cells to inhibit the growth of a virus and is a type of protein liberated from the cell in response to viral infection. The antiviral activity of IF is specific with respect to an animal species and non-specific with respect to a viral species, and may vary, with differing conditions used for its induction.

It is also known that the growth of certain animal tumour type viruses may significantly be inhibited by IF under certain conditions.

A substance capable of acting upon animal cells to induce IF is designated as an IF inducer. Thus and IF inducer is of potential interest in the prevention and treatment of various human and animal diseases caused by viral infection. However, various known IF inducers have never been used in practice for such a purpose due to certain serious defects. Thus, for example, US Patent3,583,893 (1971) discloses a double-stranded ribonucleic acid as an IF inducer, produced by using a microorganism and describes in its prior art statement that many substances including bacteria, viruses, polysaccharaides, mitogenic agents, endotoxin and the like stimulate interferon formation but none is of interest for routine use because ofinter alia toxicity, antigenicity and infectiousness.Other known IF inducers are described, for example, in US Patents 3,773,924 and 3,884,845 and Japanese Patent Application as laid open to public inspection as Kokai Koho 121919/78.

However, these IF inducers are not isolated from plants, and it is also known that IF inducers originating from microorganisms are in general disadvan tageousfortherapeutic purpose because of their high toxicity.

Examples of known mitogenic agents include phytohemagglutinin [Wheelock, Science, 149:310 (1965)], pokeweed mitogen[Friedman petal, Proc.

Soc. Exp. Biol. Med., 125:901 (1967)j and concanava lin A[Willen et al, Cell. Immunol., 6:110 (1973)], respectively isolated from the tissues of kidney bean, pokeweed and horse bean. Because of their extremely low IF inducing activity, however, no successful attempt has been made to use these mitogenic agents for preventing and curing various human and animal diseases caused by viral infection.

Other IF inducers isolated from the tissues of higher plants are also known. For instance, Kumazawa, Kojima et al (Japanese Patent Application as laid open to public inspection as Kokai Koho 32107/78) disclosed that an IF inducer, which is believed to be a kind of heteropolymeric saccharide containing as main constituents hexose (48%), protein (5%) and uronic acid (40%) and having amolecular weight of more than 100,000 is isolated from the root of Angel7ica acutiloba Kitagawa (known in Japan as Toki) by extraction with hot water to give an extracted solution, subjecting the same to dialysis to give a residue, adding acetone to the residue to give a precipitate and freeze-drying the same.If desired, the extracted solution may be made up to a suitable quantity by concentration under reduced pressure or by using a Diaflo membrane, followed by dialysis. Subsequently, Kojima and Tamamura (Japanese Patent Application as laid open to public inspection as Kokai Koho 99313/78) disclosed an IF inducer having a molecular weight of more than 20,000 (mainly more than 100,000) and containing as main constituents a 1-3 bonded glucose (hexose: more than 90%), which is produced by extracting the peeling of the rootofa mulberry such asMorusalba Linne (known in Japan as Maguwa) orMorus bombycis Koizdumi (known in Japan as Yamaguwa) with hot water, adding an organic solvent to the extracted solution to give a precipitate, adding to the precipitate a small amount of water, subjecting the solution to dialysis to give a residue and freeze-drying the same. If desired, the solutions after extraction andlor before dialysis may be made up to a suitable quantity by concentration under reduced pressure or by using a Diaflo membrane.

These two IF inducers have low toxicity and may easily be produced. However, the cheap and abundant supply of the plant tissues, from which the IF inducer is obtained, may cease as a result of the continued use of these plant tissues over many years as the source of traditional Sino-Japanese drugs.

The present invention is based upon the discovery that a substance which we have isolated from the tissues of various plants belonging to the genus Carthamus of the family Compositae and variants thereof show good IF inducing activity and low toxicity. Moreover, the active substance may readily and cheaply be isolated from the plant tissues.

According to one feature of the present invention, there is provided a substance having IF inducing activity, which is stable in the form of an amorphous whitish powder and which in substantially pure form possesses the following physico-chemical characteristics: (1) Elemental analysis: H:6.54t0.3%,C:41.9j0.3%,N:2.39j0.3%, P: 0.28 + 0.03 % (2) Molecular weight: About 100,000 to about 3,000,000 (3) Melting or decomposing point: Melting point indefinite. Carbonized at about 220"C.

(4) Ultraviolet absorption spectrum: As shown in Fig. 1 [determined in 0.1 N NaOH solution] (5) Infrared absorption spectrum: As shown in Fig. 2 (by KBr method) (6) Solubility in various solvents: Soluble in water, readily soluble in aqueous sol utions of sodium hydroxide, potassium hydrox ide and ammonium hydroxide, and substantially insoluble in methanol, ethanol, propanol, butanol, acetone, chloroform and diethyl ether.

(7) Colour reaction: Positive in ninhydrin reaction, phenol/sulfuric acid reaction, Folin's reagent and Dittmer reac tion. Negative in Elson-Morgan reaction.

(8) Nature: Acidic (9) Main chemical constituents: (a) Amino acids: [+OA /O] Oxyproline 15.61 % aspartic threonine 10.90% acid 5.51 % glutamic serine 9.64 % acid 3.96 % proline 4.27 % glycine 9.09 % alanine 8.24 % valine 4.04 % isoleucine 5.59 % leucine 2.56 % phenyl lysine 2.18% alanine 1.32 % tyrosine 1.94% arginine 1.24 % ammonia 12.98% histidine 0.93% about 1,000,000 and preferably possesses an (b) Sugars: [ 0.6 %] Arabinose 9.38 % galactose 20.98 % glucose 64.98 % mannose 3.26 % xylose 1A0% (10) Optical rotation: [(t]D2 = +63"to +69 (+66 in average) (c = OA9 % in 0.1 N NaOH) The molecular weight of the interferon inducing substance of the present invention was determined by ultracentrifugation using a Spinco Model EAnalytical Ultracentrifuge (commercial product of Beckman Instrument Inc., U.S.A.), ultrafiltration using Amicon U Itrafilter with Amicon XM 50,XM 100A and XM 300 membranes (commercial products of Amicon Corpn., U.S.A.) and UK 50, UK 100 and UK 200 membranes (commercial products of Toyo Roshi K.K., Tokyo) and gel filtration using Sephadex G-200 (commercial product of Phar macia Fine Chemicals AB, Sweden) The amino acids present were determined by hyd rolysiswith 6N HCI at 100 for 48 hoursin vacuo, followed by analysis using Technicon Amino Acid Autoanalyzer Type NC-1 (commercial product of Technicon Corpn., U.S.A.) and the sugars present were determined by hydrolysis with 0.1 N sulfuric acid at 80 C for 20 minutes and with 1 N sulfuric acid at 1 00'C for 2 hours respectively, followed by analysis using Technicon Sugar Autoanalyzer Type N-1 (commercial product of Technicon Corpn., U.S.A.).

The substance of the present invention mainly has a molecular weight range of from about 500,000 to about 1,000,000 and prefeably possesses an ultraviolet absorption spectrum in water or in 1 N sodium hydroxide substantially unchanged from that shown in Fig.

Biological characteristics: 1) IF inducing activity: Samples of the iF inducer of this invention were used to induce IF in the cell and serum of test animals, and the activity of the resultant IF was determined by the method hereinafter described in Experiment 1. The results are shown in Tables 1 and 2 which indicate that the IF inducing activity is positive.

TABLE 1 Concentration of sample ( g/ml) 10 1.0 0.1 0.01 Activity (in vitro) > 100 > 100 96 < 10 TABLE2 Activity Time of collection of blood (in vivo) after administration (hours) 0 1 2 4 6 Rabbit 1 15 80 470 175 50 Rabbit 2 15 90 500 280 70 Table 2 indicates that the results obtained by the method hereinafter described in Experiment 1 using two rabbits, from which it is apparent that the IF activity reaches its maximum 2 hours afteradministration. It was also found that by the method of Experiment 1, IF was induced in the body of the test animal by the action of the IF inducer of this invention.

2) Stability of the IF inducing activity: Samples (each 1 mg) of the IF inducer of this invention were respectively dissolved in water (each 1 ml) and heated at 100'Cforagiven time or art a given temperature for one hour. Each sample was then treated in a similar manner to that described in Experiment 1 (in vitro method) to obtain the results shown in Tables 3 and 4 indicating that the IF inducer' of this invention was highly stable to heat.

TABLE3 Heating IF activity temperature (concentration of sample) ( C) 10 1.0 0.1 0.01 ,ag/ml Untreated > 100 > 100 90 < 10 37 > 100 > 100 95 < 10 60 > 100 > 100 90 < 10 80 > 100 > 100 92 < 10 100 > 100 > 100 93 < 10 Heating time: one hour TABLE4 IF activity Heating time (concentration of sample) (hour) 10 1.0 0.1 0.01 ,ag/ml Untreated > 100 > 100 92 < 10 37 > 100 > 100 95 < 10 60 > 100 > 100 93 < 10 80 > 100 > 100 65 < 10 100 > 100 75 35 < 10 Heatingtemperature:lOO"C 3) Acute toxicity: Male and female mice (ddy-strain; 5 weeks old; weight 20 + 1 g; each group consisting of 10 mice) were used as test animals.A physiological solution of sodium chloride containing the IF inducer of this invention was administered to the mice (ip. or orally) in order to ascertain the LD,, values of > 1.5 g/kg (ip.) or > 10 g/kg (oral). No significant difference was observed between the male and female mice.

4) 4) Anti-tumour activity: Mice (ddy-strain; 5 weeks old; weight 20 + 1 g; each group consisting of 10 mice) were used as test animals. A sample of S-180 Sarcoma solid tumour (2 x 2 x 2 mm) or Ehrlich ascites tumour (2.5 x 106 cells) was inoculated into each of the test animals under the armpit. After 24 hours, the IF inducer of this invention (0.2 mg) dissolved in water was orally administered to each mouse. The administration was effected once daily and continued for 14 days. A significant anti-tumour effect was observed.

From the above-mentioned characteristics, it has been found that the IF inducer of this invention is a complex of protein and sugar containing phosphoric acid and has a molecular weight of about 100,000 to about 3,000,000 (mainly about 500,000 to 1,000,000).

Also this substance apparently conforms to the widely recognized definition of any IF inducer because it induces IF in animal cell or serum in vivo orin vitro, which is inactivated with 0.08 %trypsin at 37"C for2 hours, and moreover, the activity of the substance induced is specific with respect to animal species and non-specific with respect to viral species. It is therefore believed that the substance of this invention is not only a new IF inducer but also a new substanceperse because no substance having the same physico-chemical and biological characteristics as those of the IF inducerofthis invention has, to our knowledge, ever been reported in the art.

For example, mitogenic agents having IF inducing activity (e.g. phytohemagglutinin, pokeweed mitogen and concanavalin A) described in the literatures are types of protein having molecular weights of greater than 100,000 and their IF inducing activity is very weak, which is inactivated on heating at 56"C for 5 hours. On the contrary, the IF inducer of this invention has different chemical constituents, high heat stability even at 100"C for several hours and good IF inducing activity. The known IF inducer isolated from the root of Toki (Angellica acutiloba Kitagawa) is also high molecular (more than 100,000) and its IF inducing activity is not inactivated when heated at 100'C for one hour.However, its chemical constituents (hex9se: 48 %, uronic acid: 40 %, protein: 5 %) and infrared absorption spectrum are different from those of the IF inducer of this invention. The known IF inducers isolated from the peeling of the mulberry root contain as main con stituenta 1-3 bonded glucose (hexose:96%) and has a molecular weight of more than 20,000 (mainly more than 60,000), and thus this IF inducer is also different from the IF inducer of this invention.

Moreover, the mitogenic activity which is found in known IF inducers originating from bacterial endotoxin and higher plants is very low in the IF inducer of this invention.

Various plants belonging to the genus Carthamus which may be used as a source of the product of the IF inducer of the present invention contain, for example, carthamine, carthamone, neocarthamine and the like, all of which are different from the IF inducer of this invention with respect to the physico-chemical and biological characteristics and have no IF inducing activity. The physico-chemical characteristics of the known IF inducers disclosed in US Patents 3,773,924 and 3,884,845 and Japanese PatentApplication as laid open to public inspection as Kokai Koho 121919us are different from those of the IF inducer of this invention.

The IF inducer of this invention is of potential interest as a medicament for preventing and treating various viral diseases of humans and animals, caused by viruses such as animal tumour type viruses because its IF inducing activity is excellent when compared with known IF inducers isolated from plant tissues and is active against animal tumour type viruses. Moreover, its acute toxicity is very low when administered orally to humans and animals.

According to a further feature of this invention, there is provided a process for the preparation of a substance having interferon inducing activity, which process comprises extracting the said active substance from a plant of the genus Carthamus or a variant thereof containing the said substance and recovering the said substance from the extract thereby obtained.

The plants which may be used for the process of this invention are both grown abundantly in the wild and are cultured in various countries of the world and are liable to form various variants such as mutants and hybrids naturally or artificially. For example, Carthamus tinctoris Linne (safflower) and variants thereof have been cultured and used over many years for their yellow and red pigments and nowadays are cultured mainly for their oil in the achenes in various countries. Moreover, although the flower has long been used as a folk cure in Japan and China, the presence of a substance in the tissues of the plant which has IF inducing activity has never, we believe, been reported in the art.

If desired the plants of the genus Carthamus may, it is believed, be obtained from the Germplasm reservoir maintained by the United States Department of Agriculture at the Plant Introduction Station, Washington State University, Pullman, Washington, U.S.A.

Any and all plants belonging to the genus Carthamus and containing the substance of the present invention may be used as the starting material in the process of the present invention. The following plants are merely indicated by way of example: Carthamus tinctorius Linne; Carthamus lank tuts Linne; Carthamus arborescens Linne; and variants thereof such as C. baeticus Nyman or a variant thereof.

The botanical names referred to in this specification are designated with reference to "Yakuyo Shokubutsu Dai Jiten", edited by Kariyone and Kimura and published by Hirokawa Shoten, Tokyo (1974); "Saishin Yakuyo Shokubutsu", by Kariyone and Kimura and published by Hirokawa Shoten, Tokyo (1978); "Flora Europaea", vol.4, published by Cambridge University Press, London (1976); and "Illustrated Flora of The Pacific States", vol. IV by Stanford University Press, CA. (1965).

In general, among the tissues of the plant, the flower is particularly rich in the active substance of the present invention.

For better preservation and extraction, it is preferred to use the dried plant, although the fresh material may be used if desired. The drying method is optional e.g. natural drying, drying in hot air and the like. If desired, the material may be washed with water before use.

The extraction may be effected with water at any convenient temperature e.g. from ambient to the boiling point of the extraction mixture. Because the active substance of this invention is particularly soluble in water under alkaline conditions (e.g. pH 7-10), it is preferred to adjust the pH of water before use, for example, by using a suitable buffer solution, sodium hydroxide, potassium hydroxide or ammonium hydroxide. The extraction may be effected over any convenient period of time, usually 1-5 days at room temperature, which time may be shortened if the extraction temperature is raised.

Thus, for example, extraction may be effected for 30 minutes to 6 hours at40-100 C. According to this process, it is possible to extract a major portion of the active substance contained in the starting material (in some cases, more than 90%). However, the use of an excessively high extraction temperature should be avoided because the quantity of undesired impurities such as pigments, low molecular substances etc. appearing in the extract may thereby increase. It is also possible to add, if desired, a suit able antiseptic agent to the extracting water. The extraction may be effected continuously or intermittently, and any convenient ratio of the extracting water to the raw material may be used.

It is alternatively possible to extract the active sub stance of th is invention from the plant tissue with a hydrophilic organic solvent (such as e.g. methanol, ethanol, propanol, butanol, acetone etc.) in any con venient amount (e.g.20-80%). In this case, the extraction time and temperature may be convenient (e.g. for4 hours to 3 days at 40 to 80 C). Although extraction with water is simpler, safer and cheaper in operation, extraction using such hydrophilic organic solvent may be effected, in spite of the insolubility of the pure product of the invention in such solvent, when the extracted solution contains a mixture or complex of substances such as fatty acids, steroids, proteins, saponine, mono- or polysaccharides, and the like.Whilst we do not wish to be bound by theoretical considerations, it is believed that extrac tion using such organic solvents is made possible by buffer action.

The residue of the plant is removed from the extracted solution in conventional manner, for example, by filtration, pressing, centrifugation and the like. After this, undesired impurities such as pigments and low molecular weight substances are removed from the resultant supernatant in order to allow recovery of the active fraction. Preferred methods which may be used for this purpose are exemplified as follows.

A) The supernatant is fractionated by ultrafiltra tion e.g. using a suitable membrane for fractionating substances having a molecular weight of more than 100,000 because the active substance of this inven tion is present in fractions having a molecular weight of about 100,000 to about 3,000,000 (mainly about 500,000 to 1,000,000). The ultrafiltration may be effected under suitable pressure (e.g. 0.1 to 5 kg/cm2) by using a membrane capable of retaining sub stances having a molecular weight of more than 100,000 or 200,000. The active fractions are collected and combined, and the combined fractions are freeze-dried to obtain brown powders.

i B) The supernatant is concentrated, if desired, under reduced pressure and is treated with a hyd rophilic organic solvent (e.g. methanol, ethanol, propanol, n-butanol, acetone and the like) at a con venient concentration (e.g. 40 to 70 %, w/v) so as to form a precipitate containing the active substance, which is then freeze-dried to obtain brown powders.

C) Instead of the organic solvent, it is also poss ible to add to the supernatant an ammonium salt (e.g. ammonium chloride, ammonium sulfate, cetylmethylammonium-bromide and the like) or an inorganic metalic salt (e.g. zinc chloride, copper chloride and the like, at a convenient concentration (e.g. 20 to 50 %, wlv) so as to form a precipitate. The precipitate is desalted for example by using a dialyz ing membrane or ultrafilter capable of retaining sub stances having a molecular weight of 5000-10,000 and freeze-dried to obtain brown powders.

It is possible to recover the major portion of the active substance contained in the starting material (in some cases, more than 90 %). However, the quan tity of impurities contained in the crude powder is lowest in the case of method (A) and also method (A) may be effected simply. Moreover, it has been con firmed that any significant side effect may be avoided even when a large amount of the crude powder obtained by (A) is orally administered to humans and animals, and thus this crude powder may be used for oral administration without any further purification. In this respect, it is noted that for example safflower Carthamus tinctorius L.) has been used over many years throughout the world, for example, as a folk cure (in Japan and China) and as a raw material for rouge and edible oil.

If desired, the crude powder thus-obtained may be further purified, for example, by column chromatography using a suitable agent for gel filtration or an ion exchanger. In the former case, the elution may be effected with water, although it is possible to use a suitable buffer solution. In the latter case, the elution may be effected with a suitable buffer solution.

Preferred agents for gel filtration are exemplified by Sephadex G-50 to G-200, Sephadex 2B to 6B, Sephacryl S-200 or S-300 (commercial products of Pharmacia Fine Chemicals AB, Sweden), Bio-Gel P-30 to P.300, Bio-Gel A (commercial products of Bio-Rad Laboratories Ltd., U.S.A.), Sagavac (commercial product of Saravac Laboratories Ltd., U.K.) and the like, and preferred agents for ion exchange treatment are exemplified by QAE-Sephadex A-25 and A-50 (Cl- form), CM-Sephadex C-25 and C-50 (Na+ form), DEAE-Sephacel (Cl-form), DEAE Sepharose CL-6B(CI- form), CM-Sepharose CL-6B(Na+ form) (commercial products of Pharmacia Fine Chemicals AB, Sweden) and the like.It is also possible to use a suitable anion or cation ion exchange cellulose for the purification. The product thus-obtained may contain certain impurities, although its IF inducing activity is sufficient for practical purpose. If desired, the amounts of impurities may be further reduced by combining these treatments.

According to a still further feature of this invention, there is provided a pharmaceutical composition comprising as active ingredient a substance of the present invention as hereinbefore defined in association with a pharmaceutical carrier or excipient. The composition may be presented in a form suitable for oral, rectal or parenteral administration. Thus, for example, compositions for oral administration may be solid or liquid and may be in the form of granules, tablets, coated tablets, capsules, syrups, emulsions, suspensions or drops, such composition comprising carriers or excipients conventionally used in the pharmaceutical art. Thus, for example, suitable tabletting excipients include lactose, potato and soluble starches and magnesium stearate.

For parenteral administration, the carrier may be a sterile, parenterally acceptable liquid such as sterile water, or a pharmaceutically acceptable oil e.g.

arachis oil, contained in ampoules. Composition for rectal administration may take the form of suppositories, the carrier comprising a suppository base.

Advantageously, the composition may be formulated as dosage units, each being adapted to supply a fixed dose of the active ingredient. Tablets, coated tablets, capsules, suppositories and ampoules are examples of suitable dosage unit forms.

The present invention also provides a substance as defined herein when used as a medicament, for example, for the treatment and/or prevention of virus infections or as an anti-tumour agent.

In the accompanying drawings Fig. 1 shows the ultraviolet absorption spectrum of the active substance of the present invention in 0.1 N sodium hydroxide solution, the spectrum showing shoulders at about 250 and 280 m,u; and Fig 2 shows the infrared absorption spectrum of the substance of the present invention using the KBr method.

The following non-limiting Examples illustrate the invention.

Example 1 In the first step, dried flowers of Carthamus tinctorius Linne (2 kg) were washed with water and allowed to stand in water (40 1) at room temperature for 3 days to effect extraction, followed by centrifugation (6000 r.p.m.) for 20 minutes to remove the residue which was washed twice with water (each 10 I). The washing liquid was combined with the supernatant. The total solid content in the combined solution was 562.037 g (dry basis). The combined solutions were fractionated by ultrafiltration using an ultrafilter (Model UD-6, commercial product of Bio Engineering K.K., Tokyo) with a UK-200 membrane (commercial product of Toyo Roshi K.K., Tokyo) for retaining substances having a molecular weight of more than 200,000 at a pressure of 3 kg/cm2 to give a residue which was then freeze-dried to obtain a brown powder (89.46 g).For comparison, a similar treatment was effected by using XM1OOA membrane (commercial product of Amicon Corpn., U.S.A. > for retaining substances having a molecular weight of 100,000 to give a brown powder (93.50 g).

In the second step (purification), the first brown powder (2 g) was dissolved in water (5 ml) and transferredto a column (4.5 x 70 cm) packed with Sephadex G-200 (an agent for gel filtration). The elution was effected using water (600 ml) and the effluent was divided into fractions (each 3 ml). Fraction Nos. 25 to 55 were collected and combined and the combined fractions were then freeze-dried to obtain a whitish powder (155.04 mg).

In the third step (further purification), this powder (100 mg) was dissolved in a 0.1 M tris-HCI buffer solution (5ml; pH 7.0; 1 = 0.01) and transferred to a column (2.5 x 70 cm) packed with DEAE-Sephadex A-50 (an ion exchanger) and was eluted with a 0.1 M tris-HCI buffer solution (300 ml; pH 9.0; containing 0.5M NaCI). The effluent was divided into fractions (each 3 ml) and Fraction Nos. 16 to 27 were collected and combined. The combined fractions were freeze-dried to obtain a whitish amorphous powder (72.3 mg) containing smaller amounts of impurities and having substantially the same IF inducing activity to that of the first whitish powder. The final product had the physico-chemical characteristics as herein before described and its high purity was confirmed by ultracentrifugation and electrophoresis.

For comparison purposes, the IF inducing activities of the substances obtained by respective steps in this example were determined in a similar manner to that hereinafter described in Experiment 1 (in vitro method) to give the following results.

TABLE 5 Sample Activity at a concentration Step collected after of sample (,ag/ml) 10 1.0 0.1 1 Extraction > 100 < 10 < 10 1 Ultrafiltration > 100 95 < 10 2 Gel filtration > 100 > 100 96 3 lon exchange > 100 > 100 > 100 S treatment (final product) Example 2 A similar treatment to that described in Example 1 was carried out with the exception that Carthamus lanatus Linne was extracted by allowing the flower to stand in water at room temperature for 2 hours, followed byfurtherextraction at100 Cfor2 hours.

The physico-chemical characteristics of the final product (70.3 mg) were substantially the same as those of the final product of Example 1.

Experiment 1 Determination of IF induction with IF inducer and IF assay: [Reference: Y. Kojima's report in Kitasato Arch.

Exp. Med.,43:35 (1970)] (a) IF induction (in vitrd): A rabbit (weight about 1 kg; New Zealand White; SPF) was sacrificed by cardiac puncture and its spleen, bone marrow and lymph node cells were collected and combined together to prepare a cell suspension containing the mixed cells[lO7ceIls] which was divided into samples (each 1 ml). Four samples were independently added with 10, 1.0,0.1 or 0.01 ,xg/ml of a final product prepared in a similar manner to that described in Example 1 and were cultured at 25"C for 24 hours, followed by centrifugation to obtain supernatants, each of which was used as a sample to determine the IF activity induced.

(b) IF induction (in vivo): The final product of Example 1(1 mg) was dissolved in water (2 ml) and injected into the auricular vein of a rabbit (weight about 1 kg; New Zealand White; SPF). 1,2,4 and 6 hoursaftertheadministra- tion, a 2 ml sample of blood was removed on each occasion from the test animals and the serum of each sample was isolated from the blood and used as a sample for determining the activity of the IF induced.

(c) Determination of IF activity: In both methods (a) and (b), Vesicular stomatitis virus was used as the challenge virus in order to determine the activity of the IF induced in the following manner. A monolayer culture of the lined cells RK-13 of rabbit was put in a dish and added with a predetermined amount of the sample of the solution obtained by the above-mentioned (a) or (b). The culturewas incubated at37 C overnight with addition of Vesicular stomatitis virus used as the challenge virus. The IF activity was determined in reliance with the reduction ratio of plaques. The unit of the IF activity is expressed by the reciprocal number of the highest dilution of the sample required for reducing the numbers of plaques to 50 %.

Experiment 2 Definition of IF inducer: It has been confirmed that the samples prepared by the methods (a) and (b) inhibit the growth of Ves icular virus and Vaccinia virus in the RK-13 lined cells obtained from a rabbit of the same animal species, but do not inhibit the growth of Vaccinia stomatitis virus in Cells obtained from mice (i.e. of a different animal species). Moreover, their IF activities are inactivated when treated with 0.08 % trypsin at 37"C for 2 hours. These facts show that the active substance of the present invention represents an IF inducer.

Experiment3: In Example 1, the electrophoresis was effected at 4"C in the usual manner using a commercially available device (Model AE-2, commercial product of Toyo Kagaku Sangyo K.K., Tokyo), a polyacrylamide gel plate (thickness 3 mm) and a 0.3M boric acid buffer solution (pH 8.4). The resultant single band showed that the tested final product had high purity.

Claims (28)

1. A substance having interferon inducing activity, which is stable in the form of an amorphous whitish powder and which in substantially pure form possesses the following physico-chemical characteristics: (1) Elemental analysis: H: 6.54 i 0.3%, C: 41.9 + 0.3%, N: 2.39 + 0.3%, P: 0.28 + 0.03% (2) Molecular weight: About 100,000 to about 3,000,000 (3) Melting point or decomposing point: Melting point indefinite. Carbonized at about 220eC.

(4) Ultraviolet absorption spectrum: As shown in Fig. 1 [determined in 0.1 N NaOH solution] (5) Infrared absorption spectrum: As shown in Fig. 2 (by KBr method) (6) Solubility in various solvents: Soluble in water, readily soluble in aqueous solutions of sodium hydroxide, potassium hydroxide and ammonium hydroxide, and substantially insoluble in methanol, ethanol, propanol, butanol, acetone, chloroform and diethyl ether.

(7) Colour reaction: Positive in ninhydrin reaction, phenol/sulfuric acid reaction, Folin's reagent and Dittmer reac tion and negative in Elson-Morgan reaction.

(8) Nature: Acidic (9) Main chemical constituents: (a) Amino acids: [~0.4 /O] Oxyproline 15.61% asparticacid 5.51% threonine 10.90% serine 9.64% glutamic acid 3.96% proline 4.27% glycine 9.09% alanine 8.24% valine 4.04% isoleucine 5.59% leucine 2.56% phenylalanine 1.32% lysine 2.18% arginine 1.24% tyrosine 1.94% histidine 0.93% ammonia 12.98% (b) Sugars: [r0.60/6] Arabinose 9.38% galactose 20.98% glucose 64.98% mannose 3.26% xylose 1.40% (10) Optical rotation: [a]26D = +63 to +69 (+660 in average) (c = 0.49 /O in 0.1 N NaOH)

2. A substance as claimed in claim 1 having a molecular weight of about 500,000 to 1,000,000.

3. A substance as claimed in claim 1 or claim 2 in the form of an amorphous powder.

4. A substance as claimed in any one of the preceding claims in substantially pure form.

5. A substance as claimed in claim 1 as herein specifically described.

6. A process for the preparation of a substance having interferon inducing activity, which comprises extracting the said substance from a plant of the genus Carthamus or a variant thereof containing the said substance and recovering the said substance from the extract thereby obtained.

7. A process as claimed in claim 6 wherein the plant is Carthamus tinctorius Linne or a variant thereof.

8. A process as claimed in claim 6 wherein the plant is Carthamus lanatus Linne or a variant thereof.

9. A process as claimed in claim 6 wherein the plant is Carthamus arborescens Linne or a variant thereof.

10. A process as claimed in claim 6 wherein the plant is Carthamus baeticus Hyman or a variant thereof.

11. A process as claimed in any one of claims 6 to 10 wherein the extraction is effected using the flower of the said plant.

12. A process as claimed in any one of claims 6 to 11 wherein the extraction is effected by the use of water.

13. A process as claimed in claim 12 wherein the extraction is effected by the use of an aqueous solution of an alkali.

14. A process as claimed in claim 13 wherein the aqueous solution has a pH of from 7 to 10.

15. A process as claimed in any one of claims 6 to 14 wherein the extraction is effected at a temperature of from ambient temperature to the boiling temperature of the extraction mixture.

16. A process as claimed in claim 15 wherein the extraction is effected at a temperature of from 40 to 100"C.

17. A process as claimed in any one of claims 6 to 16 wherein the extraction is effected by the use of a hydrophilic organic solvent.

18. A process as claimed in claim 17 wherein the organic solvent comprises methanol, ethanol, propanol, butanol or acetone.

19. A process as claimed in claim 17 or claim lu wherein the extraction is effected at a temperature of from 40-80"C for from 4 hours to 3 days.

20. A process as claimed in any one of claims 6 to 19 wherein the recovery is effected by formation of a supernatant which is fractionated by ultrafiltration to yield fractions containing the substance having interferon-inducing activity.

21. A process as claimed in claim 20 wherein the ultrafiltration is effected using a membrane capable of retaining substances having a molecular weight of more than 100,000.

22. A process as claimed in claim 6 substantially as herein described.

23. A process for the preparation of a substance having interferon-inducing activity substantially as herein described in Example 1 or Example 2.

24. A substance having interferon-inducing activity when prepared by a process as claimed in any one of claims 6 to 23.

25. A pharmaceutical composition which comprises a substance as defined in claim 1 or claim 24 as active ingredient in association with a pharmaceutical carrier or excipient.

26. The substance as defined in claim 1 or claim 24 when used as a medicament.

27. The substance as defined in claim 1 or claim 24 when used as a medicament in the treatment and/or prevention of virus infections.

28. The substance as defined in claim 1 or claim 24 when used as an anti-tumour agent.