Immunosuppressant

TECHNICAL FIELD
This invention relates to the new uses of the extracts from a certain kind of microorganisms including genus Isaria and their analogues, and their related novel compounds.
BACKGROUND ART AND DISCLOSURE OF THE INVENTION
As an immunosuppressant known heretofore, ciclosporin can be mentioned. Ciclosporin has been used for suppression of rejection in transplantation of the kidney and possesses an excellent immunosuppressive effect.
Ciclosporin, however, has a drawback that it causes side effects (e.g. renal disturbances, hepatic disturbances).
Therefore, there has been demanded immunosuppressants which have potent immunosuppressive activities and the lowest possible side effects.
From such a viewpoint, the present inventors have conducted extensive studies to find that the compounds of the formula (I) mentioned below which include novel compound and their lactone possessed excellent immunosuppressive activities while having lower side effects, and further studies have led to the completion of the present invention.
That is, this invention relates to immunosuppressive agents which comprise at least one compound selected from the compounds of formula ##STR3## wherein R represents a hydrogen atom or an acyl, Y represents carbonyl or hydroxymethylene and bond represents a single bond or a double bond [hereinafter referred to as compounds (I)] and their lactones, in an effective amount and a pharmaceutically acceptable carrier, to a method for suppressing rejection or a method of prophylaxis and therapy for autoimmune diseases which comprises administering at least one compound selected from compounds (I) and their lactones in an effective amount, and to a compound of the formula (I-1) ##STR4## and its lactone.
Referring to R in the present specification, mention is made of, for example, alkanoyls having 2 to 5 carbon atoms such as acetyl, propionyl, butyryl, pivaloyl, etc. and aromatic acyls having 7 to 11 carbon atoms such as benzoyl, phenylacetyl, etc as the acyl.
Preferred compounds (I) and their lactones are as shown below: ##STR5##
Among the above-mentioned compounds, the last two compounds, namely, the compound of the formula (I-1) and its lactone are novel compounds.
The compound of the formula (I) wherein R is a hydrogen atom, is a double bond and Y is carbonyl which is the compound obtained in accordance with Example 1 mentioned hereafter, namely, ISP-I is known as Myriocin or Thermozymocidin [See The Journal of Antibiotics, vol. XXV No. 2, 109-115 (1972), The Journal of Organic Chemistry, 38(7), 1253-1260 (1973), J. Chem. Soc. Perkin Trans. I, 1613-1619 (1983), J. Chem. Soc., Chem. Commun., 488-490 (1982) etc.], with its action being an antifungal action.
Compounds (I) and their lactones can be produced by fermentation method or synthesis method in accordance with, for example, the following Production Methods 1-5.
The production method for ISP-I has been disclosed in the above literatures as well.
Production Method 1 (Fermentation Method)
ISP-I can be usually produced by fermenting an ISP-I-producing microorganism and collecting ISP-I from the culture. As the microorganisms to be used, mention is made of, for example, those belonging to Ascomycotina and Deuteromycotina, more specifically, genus Isaria and genus Mycelia belonging to Deuteromycotina and genus Myriococcus (Thielavia) belonging to Ascomycotina, which are respectively deposited at American Type Culture Collection as Isaria sinclairii ATCC No. 74121, Myriococcum albomyces ATCC No. 74120 and Mycelia sterilia ATCC No. 74122, under terms of the Budapest Treaty.
ISP-I can also be produced by a mutant which can be obtained by modification of a strain mentioned above by way of a conventional artificial mutating means such as ultraviolet rays, microwave radioactive rays and chemicals.
ISP-I producing microorganisms can be cultivated in various culture-media comprising usual nutrient sources for fungi. For example, there can be suitably added glucose, starches, glycerine, sugar millet jelly, dextrin, molasses, maltose, xylose, and the like as carbon sources; inorganic or organic nitrogen compounds such as corn steep liquor, peptone, yeast extract, potato extract, meat extract, soy bean meal, wheat germ, potassium nitrate, sodium nitrate, ammonium sulfate, casein, gluten meal, cotton seed meal, feather meal as nitrogen sources; other conventional inorganic salts; and conventional additives for cultivation, such as organic and inorganic substances and antifoaming agents which help growth of microorganisms and can promote the production of ISP-I.
Though there is no particular limitation to the cultivation method thereof, aerobic submerged cultivation is more advantageous. The preferable culture temperature in the case of strains belonging to genus Isaria is in the range from 20.degree. C. to 35.degree. C., more preferably 25.degree. C. to 30.degree. C., and that in the case of strains of genus Myriococcum or Mycelia is in the range from 30.degree. C. to 50.degree. C., preferably 35.degree. C. to 45.degree. C.
The ISP-I produced in the culture can be harvested from the culture by conventional procedures such as extraction, adsorption or by combination of conventional procedures. For example, in the case of strains such as Isaria sinclairii belonging to genus Isaria, insoluble matters such as cells are removed by a separation method such as filtration or centrifugation, and the resulting culture filtrate is put in contact with Amberlite XAD-2 to adsorb the ISP-I for harvesting. The thus-obtained ISP-I is dissolved in methanol, and the dissolved objective ISP-I is subjected to reverse phase chromatography for fractionation to obtain highly purified ISP-I. In the case of strains such as Myriococcum albomyces and Mycelia sterilia which belong to genus Myriococcum or genus Mycelia, cells are removed from the culture by a separation method such as filtration and centrifugation, and the culture filtrate is subjected to the same procedure as that in the case of strains of genus Isaria. Meanwhile, ISP-I is extracted from the separated cells with methanol and the extract is subjected to Amberlite XAD-2 as for the filtrate, followed by further purification such as chromatography or recrystallization to give ISP-I.
The compounds (I) and their lactones other than ISP-I can be produced, for example, by the following methods.
Production Method 2 (Synthesis Method)
The lactones of the compounds (I) can be produced by 1 treating the corresponding compounds (I) including ISP-I with an inorganic acid such as hydrochloric acid or an organic acid such as acetic acid or 2 treating them with a tertiary alcohol such as tert-amyl alcohol.
The reaction 1 can be conducted usually in the presence of a solvent inert to the reaction (e.g. an alcohol such as methanol or ethanol). The reaction temperature is usually in the range from 0.degree. C. to 50.degree. C., preferably about room temperature, and the reaction time is usually 10 to 30 hours, preferably about 20 hours.
The reaction temperature in the reaction 2 is usually in the range from 80.degree. C. to 150.degree. C., preferably 100.degree. C. to 120.degree. C. The reaction time is usually 10 to 30 hours, preferably about 20 hours.
Production Method 3 (Synthesis Method)
Among the compounds (I) including ISP-I, the compounds (I) wherein is a single bond and their lactones can be produced by hydrogenating the corresponding compounds (I) wherein is a double bond or their lactones.
Such hydrogenation is conducted in the presence of a conventional catalyst such as a palladium compound, a nickel compound or a platinum compound. The reaction is usually conducted in the presence of a solvent (e.g. an alcohol such as methanol or ethanol). The reaction temperature usually ranges from 0.degree. C. to 50.degree. C., preferably about room temperature, and the reaction time is usually 1 to 10 hours, preferably about several hours.
Production Method 4 (Synthesis Method)
The compounds (I) wherein Y is hydroxymethylene and their lactones can be produced by reducing the corresponding compounds (I) wherein Y is carbonyl or their lactones.
The reduction reaction can be conducted by a metal hydrogen complex compound such as sodium borohydride or lithium aluminum hydride.
The reaction is conducted usually in the presence of a solvent (e.g. an alcohol such as methanol or ethanol). The reaction temperature is usually 0.degree.-50.degree. C., preferably about room temperature, and the reaction time is usually 0.5-4 hours, preferably about 1 hour.
Production Method 5 (Synthesis Method)
The compounds (I) wherein R is an acyl or their lactones can be produced by acylating the corresponding compounds (I) wherein R is a hydrogen atom or their lactones by a per se known means.
As the acylating agent to be used for the acylation, mention is made of, for example, acid anhydrides, acid halides, active esters and the like.
The acylation reaction can be carried out under per se known conditions.
The compounds (I) and their lactones possess excellent immunosuppressive actions, and therefore are usable as a suppressive agent for rejection in organ or marrow transplantation, as a prophylactic or therapeutic agent for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, multiple sclerosis, grave myasthenia, I-type diabetes, endocrine ophthalmopathy, primary biliary cirrhosis, Crohn's diseases, glomerulonephritis, sarcoidosis, psoriasis, pemphigus, hypoplastic anemia, idiopathic thrombocytopenic purpura and allergy, or as a medical and pharmaceutical reagent to humans, cattles, horses, dogs, mice, rats and so on.
The compounds (I) or their lactones are admixed with carriers, excipients, diluents and the like to be formulated into dosage forms such as powders, capsules, tablets, injections for administration to patients. They may also be lyophilized into a pharmaceutical composition by a per se known means.
While the dosage of the compounds (I) or their lactones varies depending on diseases, symptoms, body weight, sex, age and so on, for the suppression in kidney transplantation, for example, they can be usually administered at the daily dosage per adult of 0.1-10 mg (potency), in one to several divided doses.

EXAMPLES
The following examples will illustrate this invention in more detail, though this invention should not be restricted to these examples as far as it comes within the scope of this invention. Immunosuppressive activities were assayed by the following methods.
Said activities are assayed based on various immune reactions using mouse, rat and human lymphocytes; for example, immunosuppressive activities are assayed with high sensitivity by using mouse, rat or human allogenic mixed lymphocyte reactions (allogenic MLR). Allogenic MLR is blastogenesis of lymphocytes induced by mixed culture of lymphocytes derived from two individuals that are allogenic but different in their major histocompatibility antigens, such as spleen cells, lymphnode cells and peripheral blood lymphocytes. This allogenic MLR is a reaction representing a phenomenon which reflects the difference in the major histocompatibility antigens among the donors; for example, blastogenesis of lymphocytes cannot be observed by the mixed culture of lymphocytes from monozygotic twins. Therefore, allogenic MLR is widely used for selection of the donor and the recipient in organ transplantation.
It is usual for allogenic MLR that the lymphocytes from one of the two donors are used as stimulator cells after treatment with X-ray irradiation or with mitomycin C to inhibit their mitotic proliferation, while blastogenesis of the lymphocytes from the other donor (responder cells) is measured (one way-MLR).
Immunosuppressive activities can be determined also by measuring the activities to suppress the induction of major histocompatibility antigens-restricted cytotoxic T cells in allogenic MLR.
In addition, immunosuppressive activities can be evaluated also as the activities to suppress blastogenesis of lymphocytes induced by stimulation with various mitogens (e.g. concanavalin A, phytohemagglutinin, pokeweed mitogen, etc.), or as the activities to suppress the mitotic proliferation or induction of functions of lymphocytes induced by cytokines (e.g. interleukin 1, 2, 3 ,4 ,5, 6, etc.) which enhance the mitotic proliferation or promote differentiation of lymphocytes such as T cells and B cells. Immunosuppressive activities can also be evaluated as the activities to suppress the production of such cytokines from T cells, macrophages, etc.
Immunosuppressive activities can also be evaluated as the activities to suppress the induction of plasma cells producing the anti-xenogenic red blood cell antibodies induced within mouse spleen cells which have been immunized in advance with xenogenic red blood cells, etc. by intraperitoneal, oral, intravenous or intradermal injection to mice, or as the activities to suppress rejection in organ transplantation from allogenic mice, graft-versus-host reaction, delayed allergy, adjuvant arthritis, etc.
Furthermore, immunosuppressive activities can be evaluated as the suppression of production of anti-DNA antibody, production of rheumatoid factor, nephritis, abnormal proliferation of lymphocytes, or as the life-prolonging effect in MRL/lpr mice, NZB/WF.sub.1 mice or BXSB mice, which are the model mice of autoimmune diseases, by the administration of the compounds (I) or their lactones.
EXAMPLE 1
(i) Jar cultivation of Isaria sinclairii
One hundred ml of the GPY medium (30 g of glucose, 5 g of peptone, 3 g of yeast extract, 0.3 g of KH.sub.2 PO.sub.4, 0.3 g of K.sub.2 HPO.sub.4 and 0.3 g of MgSO.sub.4 .multidot.7H.sub.2 O in one liter, pH 5.5) was placed into each of two 500 ml-long-neck shaking flasks, and autoclaved at 121.degree. C. for 20 minutes, followed by inoculation of about 1 cm.sup.2 of mycelia of Isaria sinclairii ATCC No.74121 grown on the potato dextrose agar medium, which was then incubated at 25.degree. C. for 6 days in a reciprocal shaker (145 rpm, amplitude: 8 cm). The resultant culture was inoculated as the seed into the 10 1-fermentation jar in which 5 1 of the GPY medium described above had been placed, which was then subjected to aerobic spinner culture (1 VVM, 300 rpm) at 25.degree. C. for 10 days.
(ii) Collection of ISP-I from the culture of Isaria sinclairii
From 4.5 l of the culture obtained in (i) cells and insoluble matters were removed by filtration to give 4.0 l of culture filtrate. The obtained culture filtrate was allowed to pass through a column of Amberlite XAD-2 (.phi. mm.times.750 mm) so that ISP-I could be adsorbed. The column was washed with 4.0 l of water. Then 6 l of methanol was allowed to pass to elute ISP-I. The eluate was concentrated under reduced pressure, dissolved in 200 ml of ethyl acetate, and extracted 3 times each with 200 ml of water in a separatory funnel.
The extract with water and that with ethyl acetate were separately concentrated under reduced pressure, and freeze-dried to give 2.23 g and 0.34 g, respectively, of ISP-I.
(iii) Purification of ISP-I
ISP-I (2.23 g) obtained in (ii) by freeze-drying of the water extract was dissolved in 5 ml of water and fed to the column for reverse phase chromatography (ODS DM-1020T manufactured by Fuji-Devison Chemicals, Co.) (.phi. mm.times.h85 mm). Elution was begun with water, and fractionation was carried out by gradient elution with increasing methanol concentration. The fractions eluted with 70% methanol were concentrated to dryness under reduced pressure, dissolved in a small amount of hot methanol, and allowed to cool to give crystals of ISP-I. The crystals were dissolved again in hot methanol for recrystallization to give 40 mg of pure ISP-I.
EXAMPLE 2 (i) Jar cultivation of Myriococcum albomyces
One hundred ml of the GCY medium (20 g of glucose, 5 g of corn steep liquor, 3 g of yeast extract and 0.5 g of MgSO.sub.4 .multidot.7H.sub.2 O in one liter, pH 6) was placed into each of two 500 ml-long-neck shaking flasks, and autoclaved at 121.degree. C. for 20 minutes, followed by inoculation of about 1 cm.sup.2 of mycelia of Myriococcum albomyces ATCC No. 74120 grown on the potato dextrose agar medium, which was then incubated at 40.degree. C. for 3 days in a reciprocal shaker (145 rpm, amplitude: 8 cm). The resultant culture was inoculated as the seed into the 10 l -fermentation jar in which the GCY medium described above and 1 g of an antifoaming agent (F-18 manufactured by Dow Coning Co.) had been placed, which was then subjected to aerobic spinner culture (0.5 VVM, 300 rpm) at 40.degree. C. for 7 days.
(ii) Collection of ISP-I from the culture of Myriococcum albomyces
From 4.5 l of the culture obtained in (i) cells were removed and the culture filtrate was obtained. The culture filtrate (4 l) was allowed to pass through a column of Amberlite XAD-2 (.phi. 40mm.times.h750 mm) so that ISP-I could be adsorbed. The column was washed with 1 l of water. Then 1 l of 30% methanol, 1 l of 50% methanol and 3 l of 80% methanol were allowed to pass in this order, and the eluate with 80% methanol containing ISP-I was collected.
Separately, the cells were extracted 3 times with methanol of an amount about 5 times that of the wet weight of the cells, and water was added to the extract to give a 30% methanol solution, which was then allowed to flow through a column of Amberlite XAD-2 (.phi.40 mm.times.h750 mm) so that ISP-I could be adsorbed. One liter of 30% methanol, 1 l of 50% methanol and 3 l of 80% methanol were allowed to flow in this order, and the eluate with 80% methanol containing ISP-I was collected.
The fractions eluted with 80% methanol from the culture filtrate and from the cells obtained as described above were combined, concentrated under reduced pressure, and freeze-dried to give 0.5 g of powders containing ISP-I.
(iii) Purification of ISP-I
The powders (0.5 g) containing ISP-I obtained in (ii) was washed with ethyl acetate and then with hot water (60.degree. C.), dissolved in hot methanol, and allowed to cool to give crystals of ISP-I. Repeated recrystallization from methanol gave 250 mg of ISP-I.
EXAMPLE 3
(i) Jar cultivation of Mycelia sterilia
Mycelia sterilia ATCC No. 74122 was cultivated in the same manner as in Example 2 (i).
(ii) Collection of ISP-I from the culture of Mycelia sterilia
In the same way as in Example 2 (ii), 1 g of powders containing ISP-I was obtained.
(iii) Purification of ISP-I
In the same way as in Example 2 (iii), 600 mg of ISP-I was obtained.
The physical properties of the ISP-I obtained in Examples 1 to 3 are as follows. ##STR6##
melting point: 172.degree.-177.degree. C.
.sup.1 H-NMR .delta.(ppm CD.sub.3 OD) : 5.52(m), 5.39(m), 3.99(d), 3.87(d), 3.83(m), 3.78(d)
IR .nu.(KBr) : 3400, 1710, 1670, 1605, 970 cm.sup.-1
EXAMPLE 4
One hundred mg of ISP-I was dissolved in 20 ml of methanol, to which 0.4 ml of 44% methanolic hydrochloric acid was added, and kept standing overnight at room temperature, and thereafter the solvent was evaporated off under reduced pressure. The residue was purified by chromatography on silica gel (10 g) using a mixture of chroloform and methanol (9:1), followed by recrystallization from chloroform-petroleum ether to give 75 mg of the compound having the following structure formula. ##STR7## The physical properties of the compound thus obtained are as follows:
melting point: 75.degree.-76.degree. C.
.sup.1 H-NMR .delta.(ppm CDCl.sub.3) : 5.62(m), 5.43(m), 4.48(m), 4.14(d), 3.75(d), 3.66(d)
IR .nu.(CHCl.sub.3) 3400, 1770, 1705, 975 cm.sup.-1
EXAMPLE 5
Fifty mg of ISP-I was dissolved in 15 ml of methanol and the solution was subjected to hydrogenation using 5% palladium carbon (40 mg) as the catalyst. Palladium carbon was filtrated off, the solvent was evaporated under reduced pressure, and repeated recrystallization from methanol gave 38 mg of the compound having the following structural formula. ##STR8## The physical properties of the compound are as follows:
melting point: 154.degree.-155.5.degree. C.
.sup.1 H-NMR .delta.(ppm CD.sub.3 OD) : 3.99(d), 3.87(d), 3.81(m), 3.87(d)
IR .nu.(KBr) : 3400, 1715, 1670 cm.sup.-1
EXAMPLE 6
Fifty mg of ISP-I was dissolved in 15 ml of methanol, to which sodium borohydride (20 mg) was added little by little. Thirty minutes later, 1 ml of a saturated ammonium chloride solution was added to stop the reaction, followed by extraction with chloroform and recrystallization from methanol to give 46 mg of the compound having the following structural formula. ##STR9## The physical properties of the compound thus obtained are as follows:
melting point: 162.degree.-165.degree. C.
.sup.1 H-NMR .delta.(ppm CD.sub.3 OD) : 5.52(m), 5.38(m), 3.99(d) 3.87(d), 3.82(m), 3.78(s), 3.48(m)
IR .nu.(KBr) : 3300, 1665, 1635, 970 cm.sup.-1
EXAMPLE 7
The compound obtained in Example 5 was treated in the same way as in Example 4 and the compound having the following structural formula was obtained. ##STR10## The physical properties of the compound are as follows: melting point: 96.degree.-98.degree. C.
.sup.1 H-NMR .delta.(ppm CDCl.sub.3) 4.49(t), 4.13(s), 3.72(m)
IR .nu.(KBr) : 3400, 1770, 1720 cm.sup.-1
EXAMPLE 8
The compound obtained in Example 6 was treated in the same way as in Example 4 and the compound having the following structural formula was obtained. ##STR11## The physical properties of the compound are as follows: melting point: 55.degree.-56.degree. C.
.sup.1 H-NMR .delta.(ppm CDCl.sub.3) 5.61(m), 5.44(m), 4.48(m), 4.14(d), 3.72(d), 3.66(d), 3.57(m)
IR .nu.KBr) : 3350, 1760, 975 cm.sup.-1
EXAMPLE 9
The compound obtained in Example 5 was treated in the same way as in Example 6 and the compound having the following structural formula was obtained. ##STR12##
The physical properties of the compound are as follows: melting point: 161.degree.-162.degree. C.
.sup.1 H-NMR .delta.(ppm CD.sub.3 OD) : 4.00(d), 3.88(d), 3.81(m), 3.74(s), 3.49(m)
IR .nu.KBr) : 3300, 1630 cm.sup.-1
EXAMPLE 10
The compound obtained in Example 9 was treated in the same way as in Example 4 and the compound having the following structural formula was obtained. ##STR13## The physical properties of the compound are as follows: melting point: 71.5.degree.-72.5.degree. C.
.sup.1 H-NMR .delta.(ppm CDCl.sub.3) 4.49(m), 4.11(d), 3.74(d), 3.69(d), 3.58(m)
IR .nu.KBr) : 3350, 1760 cm.sup.-1
EXAMPLE 11
One hundred mg of ISP-I was dissolved in 30 ml of methanol, to which 10 ml of acetic anhydride was added, kept standing overnight at room temperature. Water was added to decompose acetic anhydride, and the solvent was evaporated off under reduced pressure. The residue was purified by chromatography on silica gel (10 g) using chloroform-methanol (9:1) to give 60 mg of the compound having the following structural formula. ##STR14## The physical properties of the compound are as follows: melting point: 105.5.degree.-107.degree. C.
.sup.1 H-NMR .delta.(ppm CDCl.sub.3) : 6.59(s), 5.62(m), 5.42(m), 4.66(m), 4.60(m)
IR .nu.KBr) : 3300, 1760, 1710, 1650, 975 cm.sup.-1
EXPERIMENTAL EXAMPLE
Experimental Example 1 (Immunosuppressive activity of immunosuppressive compounds)
The immunosuppressive activity of the compounds (I) and their lactones was assayed by using mouse allogenic mixed lymphocyte reaction (hereinafter sometimes abbreviated as MLR). Mouse allogenic MLR was carried out by mixed culture of the BALB/c mouse (H-2.sup.d) spleen cells as the responder cells and the mitomycin C-treated C57BL/6 (H-2.sup.b) spleen cells as the stimulator cells in equal amounts.
The responder cells were prepared as follows: The spleen was resected from 5- to 6-week-old male BALB/c mice, and single cell suspension of spleen cells was prepared by using the RPMI1640 culture medium (containing 60 .mu.g/ml of kanamycin sulfate, 2 mM of L-glutamine, 10 mM of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (HEPES) and 0.1% sodium hydrogencarbonate) to which heat-inactivated fetal calf serum (hereinafter sometimes abbreviated as FCS) had been added to 5%. After hemolytic treatment, the cell suspension was adjusted to a concentration of 10.sup.7 cells/ml by using the RPMI1640 culture medium supplemented with 10.sup.-4 M 2-mercaptoethanol and 20% FCS, and was used as the responder cell suspension.
The stimulator cells were prepared as follows:
The spleen was resected from 5- to 6-week-old male C57BL/6 mice, and single cell suspension of spleen cells was prepared by using the RPMI1640 culture medium. After hemolytic treatment, the cells were treated with 40 .mu.g/ml of mitomycin C at 37.degree. C. for 60 minutes. After washing three times, the concentration of the cell suspension was adjusted to 10.sup.7 cells/ ml by using the RPMI1640 culture medium supplemented with 10.sup.-4 M 2-mercaptoethanol and 20% FCS, and was used as the stimulator cell suspension.
Fifty .mu.l of the responder cell suspension, 50.mu.l of the stimulator cell suspension prepared by the above method, and 100 .mu.l of the test substance were placed in 96-well microculture plates, and were cultured at 37.degree. C. in an atmosphere of 5% carbon dioxide for 4 days.
The blastogenesis of lymphocytes was assayed by a method of .sup.3 H-thymidine uptake as an index. That is, after termination of the culture, 0.5 .mu.Ci/well of .sup.3 H-thymidine was added and cultured for 4 hours. Cells were harvested by a cell-harvester, and the radioactivity incorporated into the cells was determined by a liquid scintillation counter to obtain the index of blastogenesis of lymphocytes in the mouse allogenic MLR. The suppression of the mouse allogenic MLR was evaluated by calculating the percent suppression by the following formula. The results are summarized in Tables 1A to 1D. ##EQU1##
TABLE 1A______________________________________ .sup.3 H-thy- Sup-Res- Stim- midine pres-ponder ulator Test Dose uptake sioncell cell substance (.mu.g/ml) (cpm) (%)______________________________________BALB/c -- -- -- 963 ---- C57BL/6 -- -- 124 --BALB/c C57BL/6 -- -- 14375 --BALB/c C57BL/6 compound of 0.001 14760 0 Example 1 compound of 0.01 6436 59.2 Example 1 compound of 0.1 686 100.0 Example 1 compound of 1 698 100.0 Example 1______________________________________
TABLE 1B______________________________________ .sup.3 H-thy- Sup-Res- Stim- midine pres-ponder ulator Test Dose uptake sioncell cell substance (.mu.g/ml) (cpm) (%)______________________________________BALB/c -- -- -- 2125 ---- C57BL/6 -- -- 491 --BALB/c C57BL/6 -- -- 29716 --BALB/c C57BL/6 compound of 0.001 29598 0.4 Example 4 compound of 0.01 10394 70.0 Example 4 compound of 0.1 1414 100.0 Example 4______________________________________
TABLE 1C______________________________________ .sup.3 H-thy- Sup-Res- Stim- midine pres-ponder mulator Test Dose uptake sioncell cell substance (.mu.g/ml) (cpm) (%)______________________________________BALB/c -- -- -- 6195 ---- C57BL/6 -- -- 55 --BALB/c C57BL/6 -- -- 27267 --BALB/c C57BL/6 compound of 0.001 22321 23.5 Example 5 compound of 0.01 8182 90.6 Example 5 compound of 0.1 1952 100.0 Example 5 compound of 0.001 16237 52.3 Example 6 compound of 0.01 9276 85.4 Example 6 compound of 0.1 3017 100.0 Example 6 compound of 0.001 24446 13.4 Example 7 compound of 0.01 34378 0 Example 7 compound of 0.1 3546 100.0 Example 7 compound of 0.001 22815 21.1 Example 8 compound of 0.01 3081 100.0 Example 8 compound of 0.1 1586 100.0 Example 8 compound of 0.001 25682 7.5 Example 9 compound of 0.01 23668 17.1 Example 9 compound of 0.1 3167 100.0 Example 9 compound of 0.001 19595 36.4 Example 10 compound of 0.01 19019 39.1 Example 10 compound of 0.1 2405 100.0 Example 10 compound of 0.001 32597 0 Example 11 compound of 0.01 30777 0 Example 11 compound of 0.1 23430 18.2 Example 11______________________________________
The test substance was dissolved in methanol or suspended in a mixture of methanol and acetic acid, and then diluted with the RPMI1640 culture medium. Methanol and acetic acid were used at a concentration of less than 0.01%, and they did not affect the allogenic MLR at all.
The compounds (1) and their lactones in the final concentration range of 1 .mu.g/ml to 0.001 .mu.g/ml were examined for their suppressive activity for blastogenic response of lymphocytes in mouse allogenic MLR. As shown in Tables 1A to D, ISP-I was suppressive in mouse allogenic MLR, with the 50%-inhibition concentration (IC.sub.50) being 7.1.times.10.sup.-3 .mu.g/ml. The activities of other compounds (I) and their lactones were comparable to that of ISP-I, with the IC.sub.50 values being about 1/10 that of cyclosporin A or less. However, these compounds even at the concentration of 10 .mu.g/ml were not cytotoxic to mouse L929 cells (IC.sub.50 was 10 .mu.g/ml or more).
Blastogenesis of lymphocytes can be evaluated also by the following colorimetry using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT).
.circleincircle. Colorimetry using MTT
The supernatant (100 .mu.l) is removed from each well after termination of the culture, and 20 .mu.l of the 5 mg/ml MTT solution is added to each well, which is cultured at 37.degree. C. for 4 hours. Thereafter, 100 .mu.l of a 0.01 N hydrochloric acid solution containing 10% sodium dodecylsulfate is added thereto and cultured at 37.degree. C. overnight to dissolve the resultant purple crystals of formazan. The absorbancy at 550 nm is measured using a microplate absorption spectrophotometer as an index of blastogenic response of lymphocytes in mouse allogenic MLR. Suppression of mouse allogenic MLR is evaluated by calculating the percent suppression by the following formula: ##EQU2##
Experimental Example 2 (Human allogenic MLR-suppressive activity)
The human allogenic MLR-suppressive activity was assayed as follows:
Human peripheral blood lymphocytes obtained by Ficoll-Paque density gradient centrifugation of normal human peripheral blood were suspended in the RPMI1640 medium supplemented with 10% FCS, placed in plastic dishes, and incubated at 37.degree. C. in an atmosphere of 5% carbon dioxide for 2 hours. After termination of incubation, the supernatant after gentle pipetting was harvested and centrifuged (1000 rpm, for 5 minutes) to obtain plastic-nonadherent cells. The plastic-nonadherent cells were allowed to pass through a nylon-wool column to give nylon-nonadherent cells, and the concentration of the cell suspension was adjusted to 4.times.10.sup.6 cells/ml by using the RPMI1640 culture medium supplemented with 10% FCS, and used as the responder cell suspension.
The plastic-adherent cells were removed from the plastic dish by vigorous pipetting after addition of phosphate buffered saline supplemented with 5% FCS and 0.02% disodium ethylenediamine-tetraacetic acid (EDTA). The plastic-adherent cells were treated with 40 .mu.g/ml of mitomycin C at 37.degree. C. for 60 minutes, washed three times, and suspended to the concentration of 4.times.10.sup.6 cells/ml in the RPMI1640 culture medium supplemented with 10% FCS. The resultant suspension was used as the stimulator cell suspension. Fifty .mu.l of the responder cell suspension from the donor A or C was mixed with 50 .mu.l of the stimulator cell suspension from the donor B or D, to which 100 .mu.l of the test substance was added, and cultured at 37.degree. C. in the an atmosphere 5% carbon dioxide for 5 days.
After termination of the culture, 1.0 .mu.Ci/well of .sup.3 H-thymidine was added, and after 18 hours of culture, the cells were harvested by a cell-harvester. The radioactivity incorporated into the cells was measured by a liquid scintillation counter as the index of blastogenic response of lymphocytes in human allogenic MLR. The suppression of human allogenic MLR was evaluated by calculating the percent suppression by the following formula. ##EQU3##
The compounds (1) and their lactones in the final concentration range of 10 .mu.g/ml to 10.sup.-5 .mu.g/ml were examined for their suppressive activity for blastogenic response of lymphocytes in human allogenic MLR. As shown in Tables 2A and 2B, the 50%-inhibition concentration (IC.sub.50) of ISP-I in Example 1 of this invention in the human allogenic MLR was 1.0.times.10.sup.-4 .mu.g/ml, IC.sub.50 of the compound in Example 4 was 7.9.times.10.sup.-4 .mu.g/ml, IC.sub.50 of the compound in Example 6 was 2.2.times.10.sup.-4 .mu.g/ml, and IC 50 of the compound in Example 5 was 3.5.times.10.sup.-4 .mu.g/ml.
Based on the results shown in Tables 2A and 2B, the IC.sub.50 values of the compounds (I) and their lactones in human allogenic MLR were found to be lower than that of ciclosporin A.
TABLE 2A______________________________________ .sup.3 H-thy- Sup-Res- Stim- midine pres-ponder ulator Test Dose uptake sioncell cell substance (.mu.g/ml) (cpm) (%)______________________________________Donor A -- -- -- 2408 ---- Donor B -- -- 118 --Donor A Donor B -- -- 23891 --Donor A Donor B compound of 0.0001 12769 51.8 Example 1 compound of 0.001 7190 77.7 Example 1 compound of 0.01 8138 73.3 Example 1 compound of 0.1 6922 79.0 Example 1 compound of 1 6690 80.1 Example 1 compound of 10 1082 100.0 Example 1 compound of 0.0001 16963 32.3 Example 4 compound of 0.001 13715 47.4 Example 4 compound of 0.01 9754 65.8 Example 4 compound of 0.1 4734 89.2 Example 4 compound of 1 5954 83.5 Example 4 compound of 10 5332 86.4 Example 4 compound of 0.0001 20789 14.4 Example 5 compound of 0.001 14690 42.8 Example 5 compound of 0.01 11130 59.4 Example 5 compound of 0.1 5181 87.1 Example 5 compound of 1 5548 85.4 Example 5 compound of 10 2439 99.8 Example 5 compound of 0.0001 14191 45.2 Example 6 compound of 0.001 11753 56.2 Example 6 compound of 0.01 6335 81.7 Example 6 compound of 0.1 6418 81.3 Example 6 compound of 1 5877 83.9 Example 6 compound of 10 4564 90.0 Example 6______________________________________
TABLE 2B______________________________________ .sup.3 H-thy- Sup-Res- Stim- midine pres-ponder ulator Test Dose uptake sioncell cell substance (.mu.g/ml) (cpm) (%)______________________________________Donor C -- -- -- 89 ---- Donor D -- -- 56 --Donor C Donor D -- -- 17427 --Donor C Donor D compound of 0.00001 10896 37.7 Example 1 compound of 0.0001 9806 44.0 Example 1 compound of 0.001 5646 67.9 Example 1 compound of 0.01 4460 74.8 Example 1 compound of 0.1 3613 79.2 Example 1 compound of 1 4167 76.5 Example 1 compound of 10 2018 88.8 Example 1 compound of 0.00001 7746 55.8 Example 4 compound of 0.0001 6700 61.9 Example 4 compound of 0.001 7278 58.5 Example 4 compound of 0.01 3417 80.8 Example 4 compound of 0.1 2708 84.9 Example 4 compound of 1 3703 79.2 Example 4 compound of 10 Example 4 compound of 0.00001 11827 32.3 Example 5 compound of 0.0001 7941 54.7 Example 5 compound of 0.001 9057 48.3 Example 5 compound of 0.01 7346 58.1 Example 5 compound of 0.1 2746 84.7 Example 5 compound of 1 3466 80.5 Example 5 compound of 10 Example 5 compound of 0.00001 16241 6.8 Example 6 compound of 0.0001 14996 14.0 Example 6 compound of 0.001 8468 51.7 Example 6 compound of 0.01 4082 77.0 Example 6 compound of 0.1 3501 80.3 Example 6 compound of 1 2448 86.4 Example 6______________________________________
Experimental Example 3 [Suppression of induction of alloreactive cytotoxic T cells in mouse allogenic mixed lymphocyte culture (MLC)]
The spleen cells suspension, 0.5 ml, (2.times.10.sup.7 cells/ml) of BALB/c mouse (H-2.sup.d) prepared in the same way as in Experimental Example 1, 0.5 ml of a suspension of mitomycin C-treated C57BL/6 mouse (H-2.sup.b) spleen cells (2.times.10.sup.7 cell/ml) and 1.0 ml of the test substance were added to 24-well multidishes, and cultured at 37.degree. C. in an atmosphere of 5% carbon dioxide for 6 days.
After termination of the culture, the cells were harvested by centrifugation, and the concentration of the cell suspension was adjusted to 5.times.10.sup.6 -6.25.times.10.sup.5 cells/ml by using the RPMI1640 culture medium supplemented with 10% FCS, and used as the effector cell suspension.
The target cells used were leukemia cells EL4 from the syngenic (H-2.sup.b) C57BL/b mouse as used for preparation of the stimulator cells. By incubating 10.sup.6 EL4 cells in the presence of 100 .mu.Ci of Na.sub.2.sup.51 CrO.sub.4 (1 mCi/ml) at 37.degree. C. for 1 hour to incorporate .sup.51 Cr into the cytoplasm. The cell were washed, adjusted to the concentration of 10.sup.4 cells/ml and used as the target cell suspension.
For the assay of the cytotoxic activity, 0.1 ml of the effector cell suspension and 0.1 ml of the target cell suspension were added to 96-well flat-bottomed plates, and cultured at 37.degree. C. for 4 hours. The amount of .sup.51 Cr released into the supernatant was determined and the cytotoxic activity was calculated by the following formula. ##EQU4##
The cytotoxic T cells induced by the method described above exhibited strong cytotoxic activity to the EL4 cells (H-2.sup.b) which are syngenic with the stimulator cells (H-2.sup.b) whereas they were not cytotoxic to the allogenic Meth A cells (H-2.sup.d), and thus it was suggested that they were H-2.sup.b -restricted allo-reactive cytotoxic T cells.
As shown in Table 3, addition of the compound (I) or a lactone thereof markedly inhibited the induction of the allo-reactive cytotoxic T cells and at the same time cytotoxic activity was hardly observed.
TABLE 3______________________________________ Amount Cyto- of .sup.51 Cr toxic Test Dose release activity substance (.mu.g/ml) (cpm) (%)______________________________________Total radioactivity -- -- 2179 --of target cellsTarget cells alone -- -- 788 0Effector cells + -- -- 1392 43.4target cells(100:1)(50:1) -- -- 983 14.0(25:1) -- -- 996 15.0Effector cells + compound of 0.01 808 1.4target cells Example 1(100:1)(50:1) 0.01 783 0(25:1) 0.01 816 2.0Effector cells + compound of 0.1 771 0target cells Example 1(100:1)(50:1) 0.1 773 0(25:1) 0.1 764 0Effector cells + compound of 0.1 761 0target cells Example 4(100:1)(50:1) 0.1 780 0(25:1) 0.1 767 0______________________________________
Experimental Example 4 (Suppression of blastogenic response of mouse spleen cells by mitogen stimulation)
The effect on the mouse blastogenic response of mouse spleen cells stimulated with phytohemagglutinin (PHA) or with pokeweed mitogen (PWM) was examined as follows:
The spleen was resected from 5- to 8-week-old male spleen cells BALB/c mice, and single cell suspension of spleen cells was prepared by using the RPMI1640 culture medium supplemented with 5% FCS. After hemolytic treatment, the concentration of the suspension was adjusted to 5.times.10.sup.6 cells/ml by using the RPMI1640 culture medium supplemented with 10.sup.-4 M 2-mercaptoethanol and 20% FCS, to which PHA or PWM was added. One hundred .mu.l of the cell suspension was added to each well of 96-well microculture plates to which 100 .mu.l per well of the test solution had been added (5.times.10.sup.5 mouse spleen cells per well). After culturing at 37.degree. C. in an atmosphere of 5% carbon dioxide for 72 hours, 0.5 .mu.Ci/well of .sup.3 H-thymidine was added and cultured under the same conditions for further 4 hours. After termination of the culture, the cells were harvested by a cell harvester, and the radioactivity incorporated into the cells was measured by a liquid scintillation counter, which was used as the index of blastogenic response of mouse spleen cells. The results are summarized in Table 4.
TABLE 4______________________________________ .sup.3 H-thy- Test Dose midine up- Suppres-Mitogen substance (.mu.g/ml) take (cpm) sion (%)______________________________________ -- -- -- 1109 --PHA(1/100) -- -- 8693 --PHA(1/100) + compound of 0.01 3238 71.9 Example 1 0.1 3624 66.8 1 1686 92.4PHA(1/100) + compound of 0.01 6074 34.5 Example 4 0.1 4312 57.8 1 3308 71.0-- -- -- 1916 --PWM(1/100) -- -- 31646 --PWM(1/100) + compound of 0.01 13499 61.0 Example 1 0.1 9895 73.2 1 6529 84.5PWM(1/100) + compound of 0.01 19335 41.4 Example 4 0.1 9325 75.1 1 8712 77.1______________________________________
As shown in Table 4, the compounds (I) and their lactones strongly inhibited the incorporation of .sup.3 H-thymidine induced by PHA or PWM as compared with the control without the compounds.
Experimental Example 5 (Suppression of the interleukin 1 (IL1) response of mouse thymocytes)
The thymus was resected from 7-week-old male C3H/HeN mice, and single cell suspension was prepared using the serum-free RPMI1640 culture medium. After three times washing with the medium, the cells were suspended to a concentration of 1.5.times.10.sup.7 cells/ml in the RPMI1640 culture medium supplemented with 20% fetal calf serum, 5.times.10.sup.-5 M 2-mercaptoethanol, 2.times.10.sup.-3 M L-glutamine, 1.times.10.sup.-3 M sodium pyruvate, 1 .mu.g/ml of phytohemagglutinin (PHA, Wellcome Co., HA16/17) and 2 units/ml of human ultrapure interleukin 1 (Genzyme Co., GUPi-1). One hundred .mu.l of this cell suspension and 100 .mu.l of the solution containing ISP-I were mixed in each well of the 96-well flat-bottomed microculture plate, cultured at 37.degree. C. in an atmosphere of 5% carbon dioxide for 66 hours, and cultured for further 6 hours after addition of 0.5 .mu.Ci/well of .sup.3 H-thymidine. After termination of the culture, the cells in each well were harvested onto a filter by a multiple cell harvester, and the radioactivity incorporated into the cells was measured by liquid scintillation method using a toluene-base scintillator.
The results obtained are summarized in Table 5. In the table, SD means standard deviation. The percent suppression (%) was calculated by the following formulation. ##EQU5##
TABLE 5______________________________________ .sup.3 H-thymidineTest Dose uptake Suppres-substance (.mu.g/ml) (cpm .+-. SD) sion (%)______________________________________-- -- 1389 .+-. 42 --PHA -- 3270 .+-. 316 --PHA + IL1 + -- 11803 .+-. 1740 0compound of Example 1 2 3973 .+-. 39 91.8 0.2 4646 .+-. 826 83.9______________________________________
As shown in Table 5, it is evident that the compound (I) and the lactones thereof show suppression of the IL1 response in a dose-dependent manner.
Experimental Example 6 (Suppression of IL2 production in mouse allogenic mixed lymphocyte culture (MLC) and in PHA-stimulated mouse spleen cells)
Mouse allogenic MLC was carried out as follows:
The respondor cell suspension and the stimulator cell suspension (0.5 ml each) prepared in the same way as in Experimental Example 1 together with 1 ml of test substance were added to 24-well multidishes, and cultured at 37.degree. C. in an atmosphere of 5% carbon dioxide for 2 days. After termination of the culture, the supernatant was collected and used as the supernatant of mouse allogenic MLC.
Culture of PHA-stimulated mouse spleen cells was carried out as follows: PHA (1/100 dilution) was added to the BALB/c mouse spleen cells suspension prepared in the same way as in Experimental Example 4. The cell suspension (1 ml) and 1 ml of the test substance were added to 24-well multidishes, and cultured at 37.degree. C. in an atmosphere of 5% carbon dioxide for 24 hours. After termination of the culture, the supernatant was collected and used as the supernatant of PHA-stimulated cultures.
The IL2 activity in the supernatants of mouse allogenic MLC and the supernatants of PHA-stimulated mouse spleen cells culture was assayed as follows: IL2-dependent mouse cell line CTLL-2 cells were suspended in the RPMI1640 culture medium supplemented with 30% FCS to the concentration of 10.sup.5 cells/ml, and 100 .mu.l of the suspension was added to each well of 96-well microculture plates in which 100 .mu.l of the supernatant of the MLC described above had been placed. After culturing at 37.degree. C. in an atmosphere of 5% carbon dioxide for 20 hours, 0.5 .mu.Ci/well of .sup.3 H-thymidine was added, and incubated for 4 hours under the same conditions. After termination of the culture, the cells were harvested by a cell harvester, and the radioactivity incorporated into the cells was measured by a liquid scintillation counter. IL2 activity was expressed in U/ml as the titer at various concentrations at which the .sup.3 H-thymidine incorporation was 50% of the maximum. The results are summarized in Table 6.
TABLE 6______________________________________ IL2 activity Suppres-Sample (U/ml) sion (%)______________________________________culture supernatant of responder <1 --cells aloneCulture supernatant of Untreated 9.8 --MLCSupernatant of MLC treated withcompound of Example 1(0.01 .mu.g/ml) 4.0 59.2(0.1 .mu.g/ml) 2.0 79.6(1 .mu.g/ml) 1.1 88.8(10 .mu.g/ml) <1 >90Supernatant of MLC treated withcompound of Example 4(0.01 .mu.g/ml) 4.0 59.2(0.1 .mu.g/ml) 1.7 82.7(1 .mu.g/ml) <1 >90(10 .mu.g/ml) <1 >90Unstimulated culture supernatant <1 --Culture supernatant stimulated 12.1 --with PHAPHA-stimulated culture supernatanttreated with compound ofExample 1(0.01 .mu.g/ml) 6.5 46.3(0.1 .mu.g/ml) 2.5 79.3(1 .mu.g/ml) 1.1 90.9(10 .mu.g/ml) <1 >92PHA-stimulated culture supernatanttreated with compound ofExample 4(0.01 .mu.g/ml) 4.9 59.5(0.1 .mu.g/ml) 2.6 78.5(1 .mu.g/ml) <1 >92(10 .mu.g/ml) <1 >92______________________________________
As shown in Table 6, it was suggested that the compounds (I) and their lactones suppress the production of IL2 in mouse allogenic MLC and in the PHA-stimulated mouse spleen cells.
Experimental Example 7 [Suppression of IL-2 induced .sup.3 H-thymidine incorporation of IL2-dependent mouse cell line CTLL-2]
IL2-dependent mouse cell line CTLL-2 cells were suspended in the RPMI1640 culture medium supplemented with 30% FCS to a concentration of 2.times.10.sup.5 cells/ml. The suspension (50 .mu.l) and 50 .mu.l of concanavalin A-stimulated rat spleen cell culture supernatants containing IL2 were added to each well of 96-well microculture plates in which 100 .mu.l of the test substance had been placed. After culturing at 37.degree. C. in an atmosphere of 5% carbon dioxide for 20, 44, or 68 hours, 0.5 .mu.Ci/well of .sup.3 H-thymidine was added, and cultured for 4 hours under the same conditions. After termination of the culture, the cells were harvested by a cell harvester, and the radioactivity incorporated into the cells was measured by a liquid scintillation counter. The results are summarized in Table 7.
TABLE 7______________________________________Incuba- .sup.3 H-tion Test Dose thymidine Suppres-time IL2 substance (.mu.g/ml) uptake (cpm) sion (%)______________________________________24 hrs. - -- -- 185 -- + -- -- 6671 -- + compound 0.01 6003 10.3 of Example 1 + 0.1 3722 45.5 + 1 3977 41.5 + 10 1968 72.5 + compound 0.01 5907 11.8 of Example 4 + 0.1 4240 37.5 + 1 5400 19.6 + 10 2602 62.748 hrs. - -- -- 436 -- + -- -- 25216 -- + compound 0.01 19592 22.7 of Example 1 + 0.1 7324 72.2 + 1 4794 82.4 + 10 1649 95.1 + compound 0.01 21930 13.3 of Example 4 + 0.1 9202 64.6 + 1 7202 72.7 + 10 7465 71.672 hrs. - -- -- 683 -- + -- -- 78515 -- + compound 0.01 56688 28.0 of Example 1 + 0.1 2436 97.7 + 1 846 99.8 + 10 478 100.0 + compound 0.01 69355 11.8 of Example 4 + 0.1 16705 79.4 + 1 4088 95.6 + 10 11806 85.772 hrs. - -- -- 56 -- + -- -- 30168 -- + compound 0.1 26108 13.5 of Example 5 + 1 1532 95.1 + compound 0.1 7237 76.2 of Example 6 + 1 1346 95.7 + compound 0.1 37960 0 of Example 7 + 1 2088 93.3 + compound 0.1 7262 76.1 of Example 8 + 1 1591 94.9 + compound 0.1 33835 0 of Example 9 + 1 2247 92.7 + compound 0.1 28083 6.9 of Example 10 + 1 1844 94.1 + compound 0.1 33948 0 of Example 11 + 1 4876 84.0______________________________________
As shown in Table 7, the compounds (I) and their lactones strongly suppressed IL2-induced .sup.3 H-thymidine incorporation of CTLL-2 cells.
Experimental Example 8 (Suppression of induction of interleukin 2 receptor (IL-2R, Tac) expression in mouse allogenic MLC and PHA-stimulated mouse spleen cells)
Mouse allogenic MLC and PHA-stimulated mouse spleen cells were prepared in the same as in Experimental Example 3.
The IL-2R (Tac) induced in mouse allogenic MLC and PHA-stimulated mouse spleen cells was assayed as follows:
After culturing the mouse allogenic MLC and the PHA-stimulated mouse spleen cells at 37.degree. C. in an atmosphere of 5% carbon dioxide for 24 hours, the cells were collected by centrifugation (1000 rpm, 5 minutes, 4.degree. C.), and about 10.sup.6 cells were cultured in 10 .mu.l of phosphate buffered saline (PBS), to which 0.02% sodium azide-containing rat anti-mouse IL-2R monoclonal antibody (manufactured by Boehringer Co., 40 .mu.g/ml) had been added, for 30 minutes with ice-cooling. After washing three times with ice-cooled PBS containing 0.02% sodium azide, 50 .mu.l of fluorescein isothiocyanate (FITC)-labeled goat anti-rat immunoglobulin G antibody was added and cultured for 30 minutes with ice-cooling. To the cell pellet after washing three times with ice-cooled 0.02% sodium azide-added PBS was added, a few drops of PBS containing 0.2% sodium azide and 50% glycerin. Prepared specimens on non-fluorescent slide glasses were made and the IL-2R positive cells were counted under a fluorescent microscope to determine the ratio of numbers of IL-2R positive cells in the total numbers of the cells.
TABLE 8______________________________________ IL-2R positive Suppres- cells (%) sion (%)______________________________________Respondor cells 0 --Untreated MLC cells 24.4 --MLC cells treated with compoundof Example 1(0.001 .mu.g/ml) 22.4 8.2(0.01 .mu.g/ml) 17.6 27.9(0.1 .mu.g/ml) 13.1 44.3(1 .mu.g/ml) 11.4 53.3Unstimulated spleen cells 0 --Untreated PHA-stimulated 46.2 --spleen cellsPHA-stimulated spleen cellstreated with compoundof Example 1(0.1 .mu.g/ml) 36.6 20.8(1 .mu.g/ml) 26.6 42.4(10 .mu.g/ml) 13.4 71.0______________________________________
As shown in Table 8, it was suggested that the compounds (I) and their lactones suppress the expression of IL-2R induced by stimulation with the alloantigen and with PHA in a concentration-dependent manner.
Experimental Example 9 (Suppression of IL3 production in mouse allogenic MLC and PHA-stimulated mouse spleen cells)
The supernatant of mouse allogenic MLC and the supernatant of PHA-stimulated mouse spleen cells culture were prepared in the same manner as in Experimental Example 6.
The IL3 activity in the supernatant described above was assayed as follows: IL3-dependent mouse cell line FDC-P2 cells were suspended in the RPMI1640 culture medium supplemented with 10% FCS to the concentration of 10.sup.5 cells/ml, and 100 .mu.l of the suspension was added to each well of 96-well microculture plates in which 100 .mu.l per well of the two-fold serial dilutions of the supernatant described above had been placed. After culturing at 37.degree. C. in an atmosphere of 5% carbon dioxide for 20 hours, 0.5 .mu.Ci/well of .sup.3 H-thymidine was added, and cultured for further 4 hours under the same conditions. After termination of the culture, the cells were harvested by a cell harvester, and the radioactivity incorporated into the cells was measured by a liquid scintillation counter. The IL3 activity was expressed in U/ml as the titer at the concentrations at which the amount of .sup.3 H-thymidine incorporated was 50% of the maximum. The results are summarized in Table 9.
TABLE 9______________________________________ IL3 activity Suppres-Sample (U/ml) sion (%)______________________________________culture supernatant of responder <2 --cells aloneCulture supernatant of Untreated MLC 36.8 --Supernatant of MLC treated withcompound of Example 1(0.01 .mu.g/ml) 13.9 62.2(0.1 .mu.g/ml) 12.1 67.1(1 .mu.g/ml) 9.8 73.4(10 .mu.g/ml) <2 >95Supernatant of MLC treated withcompound of Example 4(0.01 .mu.g/ml) 26.0 29.3(0.1 .mu.g/ml) 16.0 56.5(1 .mu.g/ml) 8.6 76.6(10 .mu.g/ml) 6.1 83.4Unstimulated culture supernatant <2 --Culture supernatant stimulated 17.1 --with PHAPHA-stimulated culture supernatanttreated with compound ofExample 1(0.01 .mu.g/ml) 4.0 76.6(0.1 .mu.g/ml) <2 >90(1 .mu.g/ml) <2 >90(10 .mu.g/ml) <2 >90PHA-stimulated culture supernatanttreated with compound ofExample 4(0.01 .mu.g/ml) 9.1 46.8(0.1 .mu.g/ml) 2.8 83.6(1 .mu.g/ml) 2.3 86.5(10 .mu.g/ml) <2 >90______________________________________
As shown in Table 9, it was suggested that the compounds (I) and their lactones suppress the IL3 production in mouse allogenic MLC and PHA-stimulated mouse spleen cells.
Experimental Example 10 [Suppression of proliferation of IL3-dependent mouse cell line FDC-P2 induced by interluekin 3 (IL3)]
IL3-dependent mouse cell line FDC-P2 cells were suspended in the RPMI1640 culture medium supplemented with 10% FCS to the concentration of 2.times.10.sup.5 cells/ml. One hundred .mu.l of the suspension and 50 .mu.l of the supernatant of the culture of mouse luekemia cells WEHI3 containing IL3 were added to each well of 96-well microculture plates in which 100 .mu.l of the test substance had been placed. After culturing at 37.degree. C. in an atmosphere of 5% carbon dioxide for 20 hours, 0.5 .mu.Ci/well of .sup.3 H-thymidine was added, and cultured for 4 hours under the same conditions. After termination of the culture, the cells were harvested by a cell harvester, and the radioactivity incorporated into the cells was determined by a liquid scintillation counter, and was used as an index of the IL3-dependent proliferation.
As shown in Table 10, the compounds (I) and their lactones suppressed the increase of IL3-induced .sup.3 H-thymidine incorporation into FDC-P2 cells. It was thus suggested that the immunosuppressive compounds of this invention have the activity to suppress the IL3-dependent proliferation.
TABLE 10______________________________________ Test Dose .sup.3 H-thymidine Suppres-IL3 substance (.mu.g/ml) uptake (cpm) sion (%)______________________________________- -- -- 1540 --+ -- -- 8315 --+ compound of 0.001 6858 21.5 Example 1+ 0.01 6572 25.7+ 0.1 5203 45.9+ 1 5029 48.5+ 10 2750 82.1+ compound of 0.001 7754 8.3 Example 4+ 0.01 6557 25.9+ 0.1 5861 36.2+ 1 4922 50.1+ 10 2902 79.9______________________________________
(In the table, the marks `+` and `-` mean the presence of IL3 and the absence of IL3, respectively.)
Experimental Example 11 (Suppression of interleukin 6 (IL6) response of mouse spleen cells)
The spleen was resected from 8-week-old male BALB/c mice, and single cell suspension in the serum-free RPMI1640 culture medium was prepared, and thereafter cell pellets were obtained by centrifugation (1000 rpm, 5 minutes) of the suspension. Then a mixture of 9 parts of a 0.16 M ammonium chloride solution and 1 part of a 0.17 M Tris solution (pH 7.65) was added to lyse red blood cells, and the suspension was washed three times with the serum-free RPMI1640 culture medium. The obtained cells were suspended to the concentration of 5.times.10.sup.6 cells/ml in the RPMI1640 culture medium supplemented with 20% FCS, 5.times.10.sup.-5 M 2-mercaptoethanol, 2.times.10.sup.-3 M L glutamine, 1.times.10.sup.-3 sodium pyruvate and 25% of the supernatant of the culture of T24 human bladder carcinoma cell line as a source of interluekin 6. One hundred .mu.l of this cell suspension and 100 .mu.l of the test substance were mixed in each well of 96-well microculture plates, and cultured at 37.degree. C. in an atmosphere of 5% carbon dioxide for 72 hours. After termination of the culture, 50 .mu.l of the cell suspension was collected from each well. Three hundred .mu.l of the RPMI1640 culture medium supplemented with 0.7% agarose gel, 20 .mu.l of physiological saline containing 40% protein A-bound sheep red blood cells and 20 .mu.l of 300-fold dilution of anti-mouse IgG anti-serum diluted in physiological saline were mixed in a test tube, and extended uniformly on Rohduck plates (Falcon Co. 1034). After gel formation by keeping still at room temperature for a few minutes, the plates were cultured at 37.degree. C. in an atmosphere of 5% carbon dioxide for about 4 hours.
Three hundred .mu.l of a 40-fold dilution of guinea pig complement in the serum-free RPMI1640 culture medium was added to each plate, which was then incubated for further 2 hours. The number of plaques formed was counted under stereoscopic microscope.
The results of this procedure are summarized in Table 11. SD in the Table means standard deviation. The percent suppression (%) was calculated by the following formula. ##EQU6##
TABLE 11______________________________________ Number of plaque Dose (plaque/1 .times. 10.sup.6 SuppressionTest substance (.mu. g/ml) cells .+-. SD) (%)______________________________________-- -- 12 .+-. 2 --IL6 -- 206 .+-. 12 0IL6 + compound 2 60 .+-. 8 75.3of Example 1 0.2 78 .+-. 6 66.0 0.02 108 .+-. 10 50.5 0.002 130 .+-. 4 39.2______________________________________
As shown in Table 11, it is evident that the compounds (I) and their lactones have the suppressive effect of the IL6 response does-dependent.
Experimental Example 12 (Suppression for mouse anti-sheep red blood cell antibody production)
Males 5- to 7-week-old BALB/c mice were immunized with sheep red blood cells (SRBC), and the spleen was resected three or four days later. By counting the plaque forming cells (PFC), suppressive effect on the production of the anti-sheep red blood cell antibody was examined as follows.
Experiment 1:
BALB/c mice were immunized with SRBC (1.times.10.sup.7 cells/mouse, intravenous administration), and ISP-I in the Example 1 was administered intraperitoneally for 4 consecutive days from the day of immunization. Four days after immunization (the next day of the final administration), the spleen was resected and the anti-sheep red blood cell antibody-producing cells were determined by the direct plaque method. At the same time the body weight of the mouse, wet weight of the thymus and the spleen, and numbers of spleen cells were also determined.
Experiment 2:
BALB/c mice were immunized with SRBC (5.times.10.sup.7 cells/mouse, intravenous administration), and the compound in the Example 5 was administered intraperitoneally for 4 consecutive days from the day of immunization. Four days after immunization (the next day of the final administration), the spleen was resected, and the anti-sheep red blood cell antibody-producing cells were determined by the direct plaque method. At the same time, the body weight of the mouse, wet weight of the thymus and the spleen, and numbers of spleen cells were also determined.
TABLE 12B__________________________________________________________________________Test Body Number ofsub- Dose weight Wet weight (mg) spleen cells Number of PFCstance (mg/kg/day) (g) thymus spleen (.times. 10.sup.8 cell) .times. 10.sup.3 /1 .times. 10.sup.7 cell .times. 10.sup.4 /spleen__________________________________________________________________________-- -- 10 .+-. 1 47 .+-. 6 134 .+-. 6 0.9 .+-. 0.1 17.0 .+-. 8.5 15.4 .+-. 7.7Compound of 10 18 .+-. 1 25 .+-. 9 103 .+-. 30 1.3 .+-. 0.2 6.7 .+-. 1.2 8.5 .+-. 1.9Example 8__________________________________________________________________________
TABLE 12A__________________________________________________________________________Test Body Number ofsub- Dose weight Wet weight (mg) spleen cells Number of PFCstance (mg/kg/day) (g) thymus spleen (.times. 10.sup.8 cell) .times. 10.sup.3 /1 .times. 10.sup.7 cell .times. 10.sup.4 /spleen__________________________________________________________________________Experiment 1-- -- 25 .+-. 1 48 .+-. 12 237 .+-. 28 1.4 .+-. 0.2 2.2 .+-. 1.6 1.4 .+-. 0.8Compound of 0.3 23 .+-. 2 48 .+-. 5 202 .+-. 4 1.4 .+-. 0.3 1.4 .+-. 0.4 0.9 .+-. 0.3Example 1 1 23 .+-. 1 50 .+-. 1 206 .+-. 32 1.3 .+-. 0.4 0.5 .+-. 0.2 0.3 .+-. 0.1 3 19 .+-. 1 25 .+-. 14 137 .+-. 17 1.3 .+-. 0.2 0.2 .+-. 0.1 0.1 .+-. 0.0Experiment 2-- -- 24 .+-. 1 58 .+-. 10 295 .+-. 18 2.4 .+-. 0.4 9.4 .+-. 3.9 21.6 .+-. 8.5Compound of 10 24 .+-. 1 52 .+-. 5 239 .+-. 40 2.3 .+-. 0.3 5.6 .+-. 1.7 12.9 .+-. 3.9Example 5__________________________________________________________________________
As shown in Table 12A and B, it is evident that the compound (I) and the lactones thereof have a suppressive effect on anti-sheep red blood cell antibody production, that is, they decrease PFC numbers per unit spleen cells numbers (1.times.10.sup.7 cells) and PFC number per total spleen cells numbers.
Experimental Example 13 (Suppression of allo-reactive cytotoxic T cells induced by immunization of mouse with allogenic cells)
Male BALB/c mice were immunized with spleen cells of C57BL/6 mice (5.times.10.sup.7 cells/mouse, intraperitoneal administration), and ISP-I in Example 1 was administered intraperitoneally 6 times at the doses of 0.1 mg/kg/day from the next day of immunization. Eight days after immunization, the spleen was resected to prepare effector cells, with which the .sup.51 Cr release test was carried out as in Experimental Example 3, using EL4 cells as the target cells to measure the cytotoxic activity.
As shown in Table 13, it is evident that administration of the compound (I) or a lactone thereof suppresses the induction of allo-reactive cytotoxic T cells.
TABLE 13______________________________________ Amount of Cytotoxic .sup.51 Cr activity Test substance release (cpm) (%)______________________________________Total radioactivity -- 7235 --of target cellsTarget cells alone -- 720 --Effector cells +target cells(100:1) -- 2618 29(50:1) -- 2340 25(25:1) -- 1436 11Effector cells +target cells(100:1) compound of 1543 13 Example 1(50:1) 1258 8(25:1) 1046 5______________________________________
Experimental Example 14 (Cytotoxicity to mouse L929 cells)
Cytotoxicity to mouse L929 cells was assayed as follows:
L929 cells were suspended to the concentration of 1.5.times.10.sup.5 cells/ml in the F12 culture medium supplemented with 10% FCS. One hundred .mu.l of the suspension was added to each well of 96-well microculture plates, and cultured at 37.degree. C. in an atmosphere of 5% carbon dioxide for 24 hours, to which 100 .mu.l of the test solution was added and cultured for further 48 hours. After the culture, 100 .mu.l of the supernatant was removed and the absorbancy at 550 nm was measured in the same way as the colormetry using MTT in Experimental Example 1. The percent suppression was calculated by the following formula used as the index of cytotoxicity. ##EQU7## The results are summarized in Table 14A and B.
TABLE 14A______________________________________ Dose SuppressionTest substance (.mu.g/ml) Absorbancy (%)______________________________________-- -- 0.669 --Compound of Example 1 1 0.699 0 10 0.693 0 100 0.321 52.0______________________________________
TABLE 14B______________________________________ Dose SuppressionTest substance (.mu.g/ml) Absorbancy (%)______________________________________-- -- 0.573 --Compound of Example 5 0.1 0.775 0 1 0.805 0 10 0.749 0Compound of Example 6 0.1 0.732 0 1 0.714 0 10 0.733 0Compound of Example 7 0.1 0.799 0 1 0.811 0 10 0.696 0Compound of Example 8 1 0.817 0 10 0.738 0 100 0.023 96.0Compound of Example 9 1 0.749 0 10 0.747 0 100 0.567 1.0Compound of Example 10 1 0.722 0 10 0.661 0 100 0.023 96.0Compound of Example 11 1 0.710 0 10 0.669 0 100 0.047 91.8______________________________________
As shown in Tables 14A and B, it is evident that the compounds (I) and their lactones show very low cytotoxicity to mouse L929 cells.
Experimental Example 15 (Cytotoxicity to various tumor cell lines)
Cytotoxicity to human tumor cell lines was examined as follows:
Cells of human cell lines, K562, MOLT4, U937, HL60, KATOIII, KB, PC-6, PC-14 and CCRF-CEM were separately suspended to the concentration of 2.times.10.sup.5 cells/ml in the RPMI1640 culture medium supplemented with 20% FCS. Fifty .mu.l of this suspension was added to each well of 96-well microculture plates to which 50 .mu.l of the test solution had been added. After culturing at 37.degree. C. in an atmosphere of 5% carbon dioxide for 72 hours, the absorbancy at 550 nm was measured in the same way as the colorimetry using MTT in Experimental Example 1, and the percent suppression was calculated in the same manner as in Experimental Example 14, which was used as the index of cytotoxicity. As shown in Tables 15A to 15F showing the results of the calculation, the cytotoxicity of the compounds (I) and their lactones to the various cultured human tumor cell lines is weak, with the concentration of 50% inhibition (IC.sub.50) being 10 .mu.g/ml or more.
TABLE 15A______________________________________Compound of Example 1 Dose SuppressionCell lines (.mu.g/ml) Absorbancy (%)______________________________________K562 0 0.990 -- 0.1 0.886 10.6 1.0 0.905 8.6 10.0 0.929 6.2 100.0 0.583 41.1MOLT4 0 0.618 -- 0.1 0.560 9.4 1.0 0.559 9.5 10.0 0.499 19.3 100.0 0.074 88.0U937 0 0.642 -- 0.1 0.619 3.6 1.0 0.567 11.7 10.0 0.497 22.6 100.0 0.156 75.7HL60 0 0.631 -- 0.1 0.402 36.3 1.0 0.390 38.2 10.0 0.377 40.3 100.0 0.101 84.0KATOIII 0 0.961 -- 0.01 0.990 0 0.1 0.972 0 1.0 0.961 0 10.0 0.869 9.6 100.0 0.041 95.7______________________________________
TABLE 15B______________________________________Compound of Example 1 Dose SuppressionCell lines (.mu.g/ml) Absorbancy (%)______________________________________KB 0 0.888 -- 0.01 0.898 0 0.1 0.865 2.6 1.0 0.879 1.0 10.0 0.886 0.2 100.0 0.025 97.2PC-6 0 0.272 -- 0.01 0.244 10.3 0.1 0.245 9.9 1.0 0.209 23.2 10.0 0.202 25.7 100.0 0.000 100.0PC-14 0 0.787 -- 0.01 0.801 0 0.1 0.770 2.2 1.0 0.732 7.0 10.0 0.751 4.6 100.0 0.074 90.6CCRF-CEM 0 0.708 -- 0.01 0.678 4.2 0.1 0.709 0 1.0 0.660 6.8 10.0 0.637 10.0 100.0 0.013 98.2______________________________________
TABLE 15C______________________________________Compound of Example 4 Dose SuppressionCell lines (.mu.g/ml) Absorbancy (%)______________________________________K562 0 0.614 -- 0.1 0.524 14.7 1.0 0.563 8.3 10.0 0.525 14.5 100.0 0.007 98.9MOLT4 0 0.408 -- 0.1 0.351 14.0 1.0 0.350 14.2 10.0 0.315 22.8 100.0 0.012 97.1U937 0 0.373 -- 0.1 0.313 16.1 1.0 0.384 0 10.0 0.362 2.9 100.0 0.004 99.0HL60 0 0.346 -- 0.1 0.265 23.4 1.0 0.300 13.3 10.0 0.273 21.1 100.0 0.008 97.7KATOIII 0 0.430 -- 0.1 0.407 5.3 1.0 0.401 6.7 10.0 0.319 25.8 100.0 0.009 97.9______________________________________
TABLE 15D______________________________________Compound of Example 4 Dose SuppressionCell lines (.mu.g/ml) Absorbancy (%)______________________________________KB 0 1.092 -- 0.1 0.884 19.0 1.0 1.065 2.5 10.0 0.726 33.5 100.0 0.012 99.0PC-6 0 0.291 -- 0.1 0.247 15.1 1.0 0.273 6.2 10.0 0.271 6.9 100.0 0.014 95.2PC-14 0 0.633 -- 0.1 0.656 0 1.0 0.679 0 10.0 0.657 0 100.0 0.030 95.3CCRF-CEM 0 0.636 -- 0.1 0.564 11.3 1.0 0.556 12.6 10.0 0.389 37.4 100.0 0.004 99.4______________________________________
TABLE 15E______________________________________Cell Dose Absorb- Suppressionlines Test substance (.mu.g/ml) ancy (%)______________________________________K562 -- -- 0.836 -- Compound of 0.1 0.823 1.6 Example 5 1.0 0.810 3.1 10.0 0.780 6.7 Compound of 0.1 0.823 1.6 Example 6 1.0 0.798 4.5 10.0 0.811 3.0MOLT4 -- 0.620 -- Compound of 0.1 0.591 4.7 Example 5 1.0 0.588 5.2 10.0 0.508 18.1 Compound of 0.1 0.615 0.8 Example 6 1.0 0.599 3.4 10.0 0.567 8.5U937 -- 0.484 -- Compound of 0.1 0.462 4.5 Example 5 1.0 0.485 0 10.0 0.416 0 Compound of 0.1 0.439 14.0 Example 6 1.0 0.436 9.9 10.0 0.435 10.1HL60 -- 0.746 -- Compound of 0.1 0.593 20.5 Example 5 1.0 0.657 11.9 10.0 0.590 20.9 Compound of 0.1 0.619 16.0 Example 6 1.0 0.589 21.0 10.0 0.627 17.0CCRF-CEM -- 0.503 -- Compound of 0.1 0.485 3.6 Example 5 1.0 0.466 7.4 10.0 0.394 21.7 Compound of 0.1 0.463 8.0 Example 6 1.0 0.457 9.1 10.0 0.475 5.6KB -- 0.730 -- Compound of 0.1 0.761 0 Example 5 1.0 0.734 0 10.0 0.711 2.6 Compound of 0.1 0.771 0 Example 6 1.0 0.728 0.3 10.0 0.707 3.2PC-14 -- 0.474 -- Compound of 0.1 0.420 11.4 Example 5 1.0 0.396 16.5 10.0 0.352 25.7 Compound of 0.1 0.372 21.5 Example 6 1.0 0.405 14.6 10.0 0.351 25.9______________________________________
TABLE 15F______________________________________Cell Dose Absorb- Suppressionlines Test substance (.mu.g/ml) ancy (%)______________________________________K562 -- 0.385 -- Compound of 0.1 0.387 0 Example 7 1.0 0.332 13.8 10.0 0.302 21.6 Compound of 0.1 0.327 15.1 Example 8 1.0 0.321 16.6 10.0 0.298 22.6 Compound of 0.1 0.378 1.8 Example 9 1.0 0.364 5.5 10.0 0.317 17.7 Compound of 0.1 0.390 0 Example 10 1.0 0.343 10.9 10.0 0.311 19.2 Compound of 0.1 0.317 17.7 Example 11 1.0 0.337 12.5 10.0 0.257 34.5MOLT4 -- 0.145 -- Compound of 0.1 0.107 26.2 Example 7 1.0 0.123 15.2 10.0 0.100 31.0 Compound of 0.1 0.142 2.1 Example 8 1.0 0.137 5.5 10.0 0.133 8.3 Compound of 0.1 0.111 23.4 Example 9 1.0 0.132 9.0 10.0 0.142 2.1 Compound of 0.1 0.148 0 Example 10 1.0 0.159 0 10.0 0.170 0 Compound of 0.1 0.182 0 Example 11 1.0 0.150 0 10.0 0.128 11.7U937 -- 0.583 -- Compound of 0.1 0.588 0 Example 7 1.0 0.593 0 10.0 0.551 5.5 Compound of 0.1 0.590 0 Example 8 1.0 0.580 0.5 10.0 0.513 12.0 Compound of 0.1 0.640 0 Example 9 1.0 0.592 0 10.0 0.584 0 Compound of 0.1 0.545 6.5 Example 10 1.0 0.541 7.2 10.0 0.517 11.3 Compound of 0.1 0.598 0 Example 11 1.0 0.583 0 10.0 0.449 23.0HL60 -- 0.522 -- Compound of 0.1 0.432 17.2 Example 7 1.0 0.427 18.2 10.0 0.428 18.0 Compound of 0.1 0.434 16.9 Example 8 1.0 0.445 14.8 10.0 0.418 19.9 Compound of 0.1 0.450 13.8 Example 9 1.0 0.435 16.7 10.0 0.413 20.9 Compound of 0.1 0.447 14.4 Example 10 1.0 0.415 20.5 10.0 0.406 22.3 Compound of 0.1 0.475 9.0 Example 11 1.0 0.456 12.6 10.0 0.381 27.0KB -- 0.730 -- Compound of 0.1 0.726 0.5 Example 7 1.0 0.695 4.8 10.0 0.493 32.5 Compound of 0.1 0.741 0 Example 8 1.0 0.749 0 10.0 0.706 3.3 Compound of 0.1 0.778 0 Example 9 1.0 0.762 0 10.0 0.696 4.7 Compound of 0.1 0.743 0 Example 10 1.0 0.735 0 10.0 0.610 16.4 Compound of 0.1 0.679 7.0 Example 11 1.0 0.688 5.8 10.0 0.510 30.1PC-14 -- 0.474 -- Compound of 0.1 0.446 5.9 Example 7 1.0 0.424 10.5 10.0 0.315 33.5 Compound of 0.1 0.452 4.6 Example 8 1.0 0.425 10.3 10.0 0.326 31.2 Compound of 0.1 0.404 14.8 Example 9 1.0 0.441 7.0 10.0 0.374 21.1 Compound of 0.1 0.433 8.6 Example 10 1.0 0.411 13.3 10.0 0.317 33.1 Compound of 0.1 0.443 6.5 Example 11 1.0 0.413 12.7 10.0 0.284 40.0CCRF-CEM -- Compound of 0.1 0.681 1.6 Example 7 1.0 0.653 5.6 10.0 0.587 15.2 Compound of 0.1 0.720 0 Example 8 1.0 0.694 0 10.0 0.665 3.9 Compound of 0.1 0.702 0 Example 9 1.0 0.696 0 10.0 0.680 1.7 Compound of 0.1 0.672 2.9 Example 10 1.0 0.647 6.5 10.0 0.645 6.8 Compound of 0.1 0.694 0 Example 11 1.0 0.663 4.2 10.0 0.603 12.9______________________________________
Formulation Example
______________________________________(1) Soft capsules (in one capsule)______________________________________compound of Example 1 30 mgpolyethyleneglycol-300 300 mgpolysorbate 80 20 mgtotal 350 mg______________________________________
Procedure of preparation
Polyethyleneglycol-300 and polysorbate 80 are added to the compound of Example 1, and the mixture is filled in soft capsules.
______________________________________(2) Injections (in one ampoule, 10 ml)______________________________________compound of Example 1 0.3%polyethyleneglycol-300 20%ethanol 60%______________________________________
A sufficient quantity of distilled water is added to make the total amount 10 ml.
Procedure of preparation
Ethanol and polyethyleneglycol-300 are added to the compound of Example 1 for dissolution and a sufficient quantity of distilled water is added thereto to make the whole volume.
Thus, an injection containing 30 ml of the compound of Example 1 in 10 ml in an ampoule is obtained.

Number	Name	Date
3758529	Craveri et al.	Sep 1973
4375475	Willard et al.	Mar 1983
4857546	Duggen et al.	Aug 1989

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