CA1206958A - L-alanyl-d-isoglutamine adamantylamide - Google Patents
L-alanyl-d-isoglutamine adamantylamideInfo
- Publication number
- CA1206958A CA1206958A CA000431224A CA431224A CA1206958A CA 1206958 A CA1206958 A CA 1206958A CA 000431224 A CA000431224 A CA 000431224A CA 431224 A CA431224 A CA 431224A CA 1206958 A CA1206958 A CA 1206958A
- Authority
- CA
- Canada
- Prior art keywords
- alanyl
- isoglutamine
- adamantylamide
- residue
- mdp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- NKUHWWPUOXOIIR-CRCLSJGQSA-N (4r)-5-amino-4-[[(2s)-2-azaniumylpropanoyl]amino]-5-oxopentanoate Chemical compound C[C@H](N)C(=O)N[C@@H](C(N)=O)CCC(O)=O NKUHWWPUOXOIIR-CRCLSJGQSA-N 0.000 title claims abstract description 13
- ORILYTVJVMAKLC-UHFFFAOYSA-N adamantane Chemical compound C1C(C2)CC3CC1CC2C3 ORILYTVJVMAKLC-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000000126 substance Substances 0.000 claims description 9
- 125000006239 protecting group Chemical group 0.000 claims description 5
- 108010016626 Dipeptides Proteins 0.000 claims description 4
- -1 nitrophenylthio Chemical group 0.000 claims description 3
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 2
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 claims 1
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 19
- 150000001875 compounds Chemical class 0.000 abstract description 14
- 230000003308 immunostimulating effect Effects 0.000 abstract description 8
- 230000001571 immunoadjuvant effect Effects 0.000 abstract description 6
- 239000002510 pyrogen Substances 0.000 abstract description 3
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 7
- 108010058846 Ovalbumin Proteins 0.000 description 7
- 239000002671 adjuvant Substances 0.000 description 7
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 7
- 229940092253 ovalbumin Drugs 0.000 description 7
- 230000001698 pyrogenic effect Effects 0.000 description 7
- 229940104230 thymidine Drugs 0.000 description 7
- 238000012360 testing method Methods 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- 241000700198 Cavia Species 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 238000001704 evaporation Methods 0.000 description 4
- 230000008020 evaporation Effects 0.000 description 4
- BSOQXXWZTUDTEL-ZUYCGGNHSA-N muramyl dipeptide Chemical compound OC(=O)CC[C@H](C(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)O[C@@H](O)[C@@H]1NC(C)=O BSOQXXWZTUDTEL-ZUYCGGNHSA-N 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- 210000001541 thymus gland Anatomy 0.000 description 4
- 108010042708 Acetylmuramyl-Alanyl-Isoglutamine Proteins 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000000144 pharmacologic effect Effects 0.000 description 3
- 230000004936 stimulating effect Effects 0.000 description 3
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 208000006313 Delayed Hypersensitivity Diseases 0.000 description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- AEFLONBTGZFSGQ-VKHMYHEASA-N L-isoglutamine Chemical compound NC(=O)[C@@H](N)CCC(O)=O AEFLONBTGZFSGQ-VKHMYHEASA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 206010040914 Skin reaction Diseases 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- NWGKJDSIEKMTRX-BFWOXRRGSA-N [(2r)-2-[(3r,4s)-3,4-dihydroxyoxolan-2-yl]-2-hydroxyethyl] (z)-octadec-9-enoate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)C1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-BFWOXRRGSA-N 0.000 description 2
- 238000005903 acid hydrolysis reaction Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 150000001720 carbohydrates Chemical group 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 239000000568 immunological adjuvant Substances 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000002480 mineral oil Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 230000035483 skin reaction Effects 0.000 description 2
- 231100000430 skin reaction Toxicity 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- 201000008827 tuberculosis Diseases 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- JCVDICFLPGDHAT-MLKPTWBGSA-N 1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-methyl-3-(tritritiomethyl)pyrimidine-2,4-dione Chemical compound [3H]C([3H])([3H])n1c(=O)c(C)cn([C@H]2C[C@H](O)[C@@H](CO)O2)c1=O JCVDICFLPGDHAT-MLKPTWBGSA-N 0.000 description 1
- IPDRTIBDOPMMIQ-VAOFZXAKSA-N 1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)-2-methyloxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1[C@]1(C)O[C@H](CO)[C@@H](O)C1 IPDRTIBDOPMMIQ-VAOFZXAKSA-N 0.000 description 1
- XVMSFILGAMDHEY-UHFFFAOYSA-N 6-(4-aminophenyl)sulfonylpyridin-3-amine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)C1=CC=C(N)C=N1 XVMSFILGAMDHEY-UHFFFAOYSA-N 0.000 description 1
- AEFLONBTGZFSGQ-GSVOUGTGSA-N D-isoglutamine Chemical compound NC(=O)[C@H](N)CCC(O)=O AEFLONBTGZFSGQ-GSVOUGTGSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- NJUISRMVIKYYCN-UHFFFAOYSA-N acetic acid;chloroform;methanol;hydrate Chemical compound O.OC.CC(O)=O.ClC(Cl)Cl NJUISRMVIKYYCN-UHFFFAOYSA-N 0.000 description 1
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000000240 adjuvant effect Effects 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 238000007327 hydrogenolysis reaction Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 238000004816 paper chromatography Methods 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000002594 sorbent Substances 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
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Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE:
The invention relates to L-alanyl-D-isoglutamine adamantylamide of the formula
The invention relates to L-alanyl-D-isoglutamine adamantylamide of the formula
Description
12(~69~8 The invention relates to L-alanyl-D-isoglutamine adamantylamide of the formula (I) L D
NH2 - CH - CO - NH - CH - CONH2 ( I ) 5CH3 '7 2)2 CO - NH - Ad wherein Ad represents a residue of adamantane bound in its position 1. This new dipeptide derivative (designated further for briefness as ADP) has a significant immuno-adjuvant and immunostimulatory activity.
As is known, there is a standing lack of compounds - which are capable of enhancing organism defensibility by stimulation of its immune response. For a certaln time bacterial products or fragments of cellular walls were 15 used for this purpose. ~he disadvantages of these compounds include their non-standardizability of effects and serious side effects in clinical use. The situation is similar also in the field of compounds having immuno-adjuvant activity where mainly bacterial products from Mycobacterium tuberculosis are employed, frequently in combination with mineral oils.
Significant progress has been achieved by obtain-ing some active sub-units of bacterial walls, at first enzymatically and thereafter also syntetically (F. Ellouz et al., Biochem. Biophys. Res. Commun. 59, 1317, 1974).
Most of the experimental experience has been made with muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isogl~l-tamine, MDP/ /L. Chedid et al., Progr.Allergy 25, 63 (Karger, Basel 1978); C. Merser et al., Biochem.Biophys.
Res.Commun. 66, 1316, 1975) and with a number of its analogs. In these analogs, biological effects are re-present to different degrees; by the study of various structures it has been found that the peptidic part of the molecule is critical for the adjuvant and immuno-~ .
., lZ06958 stimulatory effect whereas this activity is more or less independent of the carbohydrate residue of the molecule (L. Chedid et al., C. Merser et al., l.c.; K. Masek et al., Experientia 35, 1397, 1979; S.Kotani et al., Bikens's J.
18, 105, 1975; A. Hasegawa et al, Agric. Biol. Chem. 42, /11~, 2187, 1978).
It has also been described that the effect of MDP
and some analogs is higher and more stable when they are administered in the medium of mineral oils or bound to liposomal structures (J. Freud and K. McDermot, Proc. Soc.
Expt.Biol.Med.49, 548, 1942; K.Masek et al,, Experientia ~ 34, 1363, 1978). Provided the hitherto tested, synthe-tically prepared compounds were biologically active, they were simultaneously always more or less pyrogenic; this was the main reason why they were not used in clinical practice. An opinion has been given that the adjuvant effects have a direct connection with pyrogenity (S. Kotani et al., Biken's J. 19, 9, 1976).
A predominant lipophility of the molecule plays a very significant role in its biological manifestation.
This was verified by preparing structural analogs with a higher alkyl radical attached to the oxygen atom of the hydroxyl group in position 5 of the saccharide moiety (S. Kotani et al., cf. the second reference herein above).
A similar effect was observed on introducing an aliphatic lipophilic chain into the isoglutamine molecule (K. Masek et al., cf. the first reference herein above).
In accordance with the present invention a nove]
type of immunostimulatory substances has been prepared by introducing a residue of adamantane into the gamma-carboxamide group of the isoglutamine moiety. The so obtained L-alanyl-D-isoglutamine adamantylamide (further abbreviated ADP~ of the instant application has remark-able immunostimulatory activity without the otherwise common side effects, especially pyrogenity, and is easily ~206958 available by chemical synthesis.
The compound of the invention, of the~formula I
herein above, can be prepared with the use of general methods usual in the preparative chemistry of peptides, preferably by reacting a protected L-alanyl-D-isoglutamine derivative with l-aminoadamantane. Thus, advantageously, l-aminoadamantane is reacted with a dipeptide derivative of the formula II
L D
X - NH - CH - CO - NH - CH - CON~l2 (II), CH3 (CH2)2 wherein X stands for a protective group removable by acidic hydrolysis or by hydrogenation (hydrogenolysis), preferably a tert-butyloxycarbonyl, benzylozycarbonyl or nitrophenylthio group, whereupon said protective group is split off.
Details of the process for preparing the compound of the invention follow from the subsequent example which nevertheless does not limit the scope of the invention.
t-Butyloxycarbonyl-L-alanyl-D-isoglutamine 20.3 g of dicyclohexyl carbodiimide is added to a solution of 19.6 g BOC-Ala and 12.3 g N-hydroxybenztri-azole in 280 ml dioxane at - 10C. The mixture is stirred for 60 minutes at room temperature, filtered and the filtrate is added to a solution of 13.3 g D-isoglutamine in 60 ml water. Stirring is continued overnight. The reaction mixture is filtered and the filtrate is evaporat-ad, to dryness. The evaporation residue is recrystallized from ethylacetate-ether. The yield is 30.9 g of the product (83% of theory) , m.p. 98C , (a)20 -8.2 (c 1, methanol).
t-Butyloxycarbonyl-L-alanyl-D-isoglutamine adamantylamide 1.9 g of l-aminoadamantane hydrochloride base is treated with 2 N sodium hydroxide solution, and the released base is extracted by chloroform. 1.4 g (9 mmoles) of the base obtained by evaporation is dissolved in 30 ml of dimethylformamide and the solution is cooled down to ~10C. This solution is treated with solution of 2.1 g (10 mmoles) dicyclohexyl carbodiimide in 10 ml dimethyl-formamide. After 5 minutes a solution of 1.6 g (5 mmoles) of BOC-L-Ala-D-iGln, 1.3 g N-hydroxybenztriazole, 0.7 ml triethylamine and 5 ml pyridine in 20 ml dimethylEormamide is added. The reaction mixture is stirred at room tempe-rature overnight. Dimethylformamide is evaporated, 100 ml of ethyl acetate is added and the solution is repeatedly extracted with 0.1 N hydrochloric acid and 5 % sodium hydrogen carbonate solution, dried and evaporated. Precip-itation of a methanolic solution of the evaporation residue with water afforded 1.28 g (56 % of theory) of the desired compound; its elementary and amino acid analysis corresponds to the theory.
L-Alanyl-D-isoglutamine adamantylamide t-Butyloxycarbonyl group is split off from the preceding product by treatment with 40 % solution of tri-fluoroacetic acid in methylene chloride. After 60 minutes standing at room temperature the solution is evaporated and the residue is mixed with ether. 0.9 g of product (in the form of trifluoroacetate) is obtained by suction;
the product is immediately dissolved in 5 ml of ethanol, brought on the column of an anion exchanger in OH-cycle and eluted with methanol. 0.8 g (83 % of theory) of foamy product is obtained by evaporation of the eluate.
Analysis of amino acid composition: Ala 1.01, iGln 0.97.
The productpurity was further verified by high-pressure liquid chromatography (HPLC) and by paperelec-lZ(~6958 trophoresis in buffers having pH 2.5 and 5.7.
L-Alanyl-D-isoglutamine adamantylamide was analysed under the following conditions: stationary phase (standard C18 reverse phase sorbent) was formed by treated silica gel with lipophilic groups (column 15x0.3 cm), mobile phase contained 60 to 80 % by volume of organic modifier, preferably methanol, and 40 to 20 % by volume of 0.2 ~ aqueous trifluoroacetic acid. Detection using ultraviolet detector was made at 210 nm. The rate of flow of mobile phase was 20 to 30 ml/hour. For prepa-ration the same stationaryand mobile phase and column - having size 30x2.5 cm were used.
Purity of the product was further verified using this layer chromatography on thin layer of silica gel in the system n-butanol~acetic acid-water (4:1:1) and chloro-form-methanol-acetic acid-water (40:20:10:5).
0.3 g of pure compound (content approximately 95 % by weight as deduced from the peak area) was obtained by HPLC purification of 0.5 g of crude product, which compound was used for pharmacological tests.
Pharmacological properties of I,-alanyl-D-iso-glutamine adamantylamide;
Test No. 1 Immunoadjuvant effect has been tested in guinea-pigs to which it was applied into left back paw 0.2 mlof a mixture of ovalbumin (2.5 mg) and adamantylamide of L-alanyl-D-isoglutamine (ADP) in the amount 100 ug in incomplete Freunds adjuvant (FIA). To the control group of animals only incomplete Freunds adjuvant with ovalbumin was administered. Effects of the compound tested were then compared with hitherto most strongly acting bacterial adjuvant which is known, Mykobacterium tuberculosis. To this group of animals was thus administered a mixture which contained ovalbumin with complete Freunds adjuvant ' 12(~6958 (FCA). The effects of new prepared derivative were compared with the effects of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) as well. Skin reaction in guinea-pigs after administration of 10 and 20 ,ug of ovalbumin was then followed after three weeks after the administra-tion of the test substances. The result of determination of the skin reaction area size in mm are then presented in the Table No. 1.
Table No. 1 Induction of delayed hypersensitivity to ovalbumin in guinea-pigs with synthetic ADP
-Tested Dermatic response after 24 hours Substances Dose (mm) Ovalbumln dose Ovalbumin dose 10 ug 20 ug FIA - 1.0 - 0.48 3.39 - 0.22 FCA 1 mg 3.71- 0.23 / 5.73 - 0.33 MDP 100 ,ug 2.99- 0.23 / 6.07 - 0.21 ADP 100 ug 2.70- 0.21 / 5.82 - 0.27 The numbers represent the average values of two experi-ments at 16 animals +/ statistically significant differences (p ~0.05) Explanatory notes : FIA = Freunds incomplete adjuvant (Bayol + Arlacel 4 : 1 ) FCA = Freunds complete adjuvant (Bayol + Arlacel + 1 mg of mykobacterium tuberculosis) MDP = N-acetyl-muramyl-L-alanyl-D-isoglutamine ADP = L-alanyl-D-isoglutamine adamantylamide ~ .. . .
lZ069S8 From the rèsults of Table 1 it is evident that the adminis-tration of ADP led to enhanced delayed hypersensitivity in guinea-pigs to ovalbumin and the effect in dose used (100 ug) is comparable with the effect of both MDP and FCA.
Test No. 2 Labelled thymidine is most often used for the stNdy of biosynthesie of DNA in vivo. This preformed pyrimidine desoxynucleoside is in a succession of phosphorylation reactions phosphorylated to dTTP (desoxythymidinetriphos-phate) which is the immediate precursor of DNA synthesis ~ in cell nucleus. Enhancement of labelled thymidine incor-poration into DNA represents activation of its biosyn-thesis which precedes cell propagation.
This test is one of the most sensible and reli~
able by which immuno stimulating effects of substances and their effect on cell propagation can be determined. To Wistar male rats weighing 150 to 160 g tested substances in equimolar concentrations were administered, that is MDP in dose 1.00 mg/kg and ADP in dose 0.74 mg/kg. The animals were killed after 16 hours; 2 hours before their killing thymidine (methyl-3H) was administered to them intraperitoneally in the dose 20 ,uCi per animal in 0.3 ml of physiological solution (specific activity 20 Ci/mmole).
Frozen organs were taken out and promptly homo-genized in 0.2 N HC104. After repeated washing of the sediment with 0.2 N HC104 RNA was removed by alkaline hydrolysis. Nucleotide components were released from protein-DNA precipitate by hydrolysis in 1 N HC104.
3p Supernatent was after neutralization evaporated to dry-ness. Nucleotides of DNA were transferred to bases by further acidic hydrolysis in concentrated HC104. Thymine was then isolated in chromatographically pure form by repeated paper chromatography and its radioactivity was 12~69~i8 determined using scintillation spectrometer. Results of the experiment are presented in the Table No. 2.
Table No.2 Utilization of (methyl - 3H) thymidine for synthesis of DNA of liver, kidneys, thymus and spleen of rats after MDP and ADP administration ;
CPM/uml DNA of thymine liver kidneys thymus spleen , Controls 1.750 1.750 940 11.150 dipeptide) 4.100 2.350 1.400 10.900 15ADP (adamantyl- 2.950 2,200 1.550 16.500 The data of Table 2 evidence that already a single admin-istration of ADP in a dose of 0.75 mg/kg has a pronou~c-ed positive effect on the incorporation of methyl-3H
thymidine into the thymus and spleen DNA. The two organs take part to a substantial extent in the development of tumor and cell immunity. The tabulated results show a significant enhancement of thymidine utilization for the synthesis of thymus DNA which is of the same order of magnitude as in the case of MDP, where this effect is known already for a long time. In the case of spleen we can see, whereas the administration of MDP is not accompagnied by enhanced thymidine utilisation, ADP has here also pronounced stimulating effects manifesting themselves by enhanced 3H thymidine utilization.
Presented results as to the effect of MDP on utilization of thymidine for the synthesis of spleen DNA
are in a good agreement with literature data which show lZ(;~695B
that MDP has no effect on this organ, even on isolated spleen cells in vitro. By comparing the effects of the two compounds, i.e., ADP of the invention and MDP of prior art, the new preparation appears much more advantageous due to the fact that it exerts the desired action on both organs playing an important role in the immune reaction.
Test No. 3 Most of the hitherto synthesized MDP analogs have, besides immunoadjuvant and immunostimulative effects, also pro-nounced pyrogenic properties, which represents a substan-tial drawback for clinical practice. This fact hitherto prevented some very promising new substances from clinical use. In the experiments in rabbits, pyrogenic effect of ADP was therefore evaluated ad compared with the effect of MDP. ADP was intravenously administrated to rabbits of 1.8 to 2.0 kg weight in dose 100 ~g/kg and the effects were compared with those of the same dose of MDP. The same volume of isotonic solution (6.2 ml) was then inject-ed~ to control animals in which basal temperature was measured. The temperature was measured for 6 hours using a rectal thermistor thermometer in hour intervals and the results are summarized in the table No. 3.
Table No. 3 Pyrogenic effects of MDP and ADP in rabbits Tested Temperature, C
Sub- Dose 1 2 3 4 5 6 stance hour hours hours hours hours hours Controls (only iso- - -0.1 +0.2 -0.2 -0.1 0 +0.1 tonic solu-tion) ~g/kg +0.3 +0.4 +0.4 +0.4 +0.3 +0.35 ,ug/kg +0.8 +1.7 +1.8 +1.3 +0.7 +0.4 _ g _ lZB6958 The values presented in the Table No. 3 are the average values of 5 animals in a group.
From the results oE the Table No. 3 it is evi-dent that ADP has no pyrogenic effects in doses which come to consideration for clinical application. In the same dose, that is 100 ~g/kg,MDP is already expressively pyrogenic. The fact that ADP is free of any pyrogenic effect with retained immunostimulatory and immunoadjuvant effects represents its substantial advantage and reveals real possibilities for its clinical use.
Presented results of pharmacological evaluation ~ of the compound of the invention allow to suppose that this compound, advantageously in the form of an immuno-stimulatory pharmaceutical preparation in a mixture with usual pharmaceutical auxiliary substances, can find use for stimulating the immune response of organisms, espe-cially for enhancing their resistance in states of dimin-ished antibody formation.
NH2 - CH - CO - NH - CH - CONH2 ( I ) 5CH3 '7 2)2 CO - NH - Ad wherein Ad represents a residue of adamantane bound in its position 1. This new dipeptide derivative (designated further for briefness as ADP) has a significant immuno-adjuvant and immunostimulatory activity.
As is known, there is a standing lack of compounds - which are capable of enhancing organism defensibility by stimulation of its immune response. For a certaln time bacterial products or fragments of cellular walls were 15 used for this purpose. ~he disadvantages of these compounds include their non-standardizability of effects and serious side effects in clinical use. The situation is similar also in the field of compounds having immuno-adjuvant activity where mainly bacterial products from Mycobacterium tuberculosis are employed, frequently in combination with mineral oils.
Significant progress has been achieved by obtain-ing some active sub-units of bacterial walls, at first enzymatically and thereafter also syntetically (F. Ellouz et al., Biochem. Biophys. Res. Commun. 59, 1317, 1974).
Most of the experimental experience has been made with muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isogl~l-tamine, MDP/ /L. Chedid et al., Progr.Allergy 25, 63 (Karger, Basel 1978); C. Merser et al., Biochem.Biophys.
Res.Commun. 66, 1316, 1975) and with a number of its analogs. In these analogs, biological effects are re-present to different degrees; by the study of various structures it has been found that the peptidic part of the molecule is critical for the adjuvant and immuno-~ .
., lZ06958 stimulatory effect whereas this activity is more or less independent of the carbohydrate residue of the molecule (L. Chedid et al., C. Merser et al., l.c.; K. Masek et al., Experientia 35, 1397, 1979; S.Kotani et al., Bikens's J.
18, 105, 1975; A. Hasegawa et al, Agric. Biol. Chem. 42, /11~, 2187, 1978).
It has also been described that the effect of MDP
and some analogs is higher and more stable when they are administered in the medium of mineral oils or bound to liposomal structures (J. Freud and K. McDermot, Proc. Soc.
Expt.Biol.Med.49, 548, 1942; K.Masek et al,, Experientia ~ 34, 1363, 1978). Provided the hitherto tested, synthe-tically prepared compounds were biologically active, they were simultaneously always more or less pyrogenic; this was the main reason why they were not used in clinical practice. An opinion has been given that the adjuvant effects have a direct connection with pyrogenity (S. Kotani et al., Biken's J. 19, 9, 1976).
A predominant lipophility of the molecule plays a very significant role in its biological manifestation.
This was verified by preparing structural analogs with a higher alkyl radical attached to the oxygen atom of the hydroxyl group in position 5 of the saccharide moiety (S. Kotani et al., cf. the second reference herein above).
A similar effect was observed on introducing an aliphatic lipophilic chain into the isoglutamine molecule (K. Masek et al., cf. the first reference herein above).
In accordance with the present invention a nove]
type of immunostimulatory substances has been prepared by introducing a residue of adamantane into the gamma-carboxamide group of the isoglutamine moiety. The so obtained L-alanyl-D-isoglutamine adamantylamide (further abbreviated ADP~ of the instant application has remark-able immunostimulatory activity without the otherwise common side effects, especially pyrogenity, and is easily ~206958 available by chemical synthesis.
The compound of the invention, of the~formula I
herein above, can be prepared with the use of general methods usual in the preparative chemistry of peptides, preferably by reacting a protected L-alanyl-D-isoglutamine derivative with l-aminoadamantane. Thus, advantageously, l-aminoadamantane is reacted with a dipeptide derivative of the formula II
L D
X - NH - CH - CO - NH - CH - CON~l2 (II), CH3 (CH2)2 wherein X stands for a protective group removable by acidic hydrolysis or by hydrogenation (hydrogenolysis), preferably a tert-butyloxycarbonyl, benzylozycarbonyl or nitrophenylthio group, whereupon said protective group is split off.
Details of the process for preparing the compound of the invention follow from the subsequent example which nevertheless does not limit the scope of the invention.
t-Butyloxycarbonyl-L-alanyl-D-isoglutamine 20.3 g of dicyclohexyl carbodiimide is added to a solution of 19.6 g BOC-Ala and 12.3 g N-hydroxybenztri-azole in 280 ml dioxane at - 10C. The mixture is stirred for 60 minutes at room temperature, filtered and the filtrate is added to a solution of 13.3 g D-isoglutamine in 60 ml water. Stirring is continued overnight. The reaction mixture is filtered and the filtrate is evaporat-ad, to dryness. The evaporation residue is recrystallized from ethylacetate-ether. The yield is 30.9 g of the product (83% of theory) , m.p. 98C , (a)20 -8.2 (c 1, methanol).
t-Butyloxycarbonyl-L-alanyl-D-isoglutamine adamantylamide 1.9 g of l-aminoadamantane hydrochloride base is treated with 2 N sodium hydroxide solution, and the released base is extracted by chloroform. 1.4 g (9 mmoles) of the base obtained by evaporation is dissolved in 30 ml of dimethylformamide and the solution is cooled down to ~10C. This solution is treated with solution of 2.1 g (10 mmoles) dicyclohexyl carbodiimide in 10 ml dimethyl-formamide. After 5 minutes a solution of 1.6 g (5 mmoles) of BOC-L-Ala-D-iGln, 1.3 g N-hydroxybenztriazole, 0.7 ml triethylamine and 5 ml pyridine in 20 ml dimethylEormamide is added. The reaction mixture is stirred at room tempe-rature overnight. Dimethylformamide is evaporated, 100 ml of ethyl acetate is added and the solution is repeatedly extracted with 0.1 N hydrochloric acid and 5 % sodium hydrogen carbonate solution, dried and evaporated. Precip-itation of a methanolic solution of the evaporation residue with water afforded 1.28 g (56 % of theory) of the desired compound; its elementary and amino acid analysis corresponds to the theory.
L-Alanyl-D-isoglutamine adamantylamide t-Butyloxycarbonyl group is split off from the preceding product by treatment with 40 % solution of tri-fluoroacetic acid in methylene chloride. After 60 minutes standing at room temperature the solution is evaporated and the residue is mixed with ether. 0.9 g of product (in the form of trifluoroacetate) is obtained by suction;
the product is immediately dissolved in 5 ml of ethanol, brought on the column of an anion exchanger in OH-cycle and eluted with methanol. 0.8 g (83 % of theory) of foamy product is obtained by evaporation of the eluate.
Analysis of amino acid composition: Ala 1.01, iGln 0.97.
The productpurity was further verified by high-pressure liquid chromatography (HPLC) and by paperelec-lZ(~6958 trophoresis in buffers having pH 2.5 and 5.7.
L-Alanyl-D-isoglutamine adamantylamide was analysed under the following conditions: stationary phase (standard C18 reverse phase sorbent) was formed by treated silica gel with lipophilic groups (column 15x0.3 cm), mobile phase contained 60 to 80 % by volume of organic modifier, preferably methanol, and 40 to 20 % by volume of 0.2 ~ aqueous trifluoroacetic acid. Detection using ultraviolet detector was made at 210 nm. The rate of flow of mobile phase was 20 to 30 ml/hour. For prepa-ration the same stationaryand mobile phase and column - having size 30x2.5 cm were used.
Purity of the product was further verified using this layer chromatography on thin layer of silica gel in the system n-butanol~acetic acid-water (4:1:1) and chloro-form-methanol-acetic acid-water (40:20:10:5).
0.3 g of pure compound (content approximately 95 % by weight as deduced from the peak area) was obtained by HPLC purification of 0.5 g of crude product, which compound was used for pharmacological tests.
Pharmacological properties of I,-alanyl-D-iso-glutamine adamantylamide;
Test No. 1 Immunoadjuvant effect has been tested in guinea-pigs to which it was applied into left back paw 0.2 mlof a mixture of ovalbumin (2.5 mg) and adamantylamide of L-alanyl-D-isoglutamine (ADP) in the amount 100 ug in incomplete Freunds adjuvant (FIA). To the control group of animals only incomplete Freunds adjuvant with ovalbumin was administered. Effects of the compound tested were then compared with hitherto most strongly acting bacterial adjuvant which is known, Mykobacterium tuberculosis. To this group of animals was thus administered a mixture which contained ovalbumin with complete Freunds adjuvant ' 12(~6958 (FCA). The effects of new prepared derivative were compared with the effects of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) as well. Skin reaction in guinea-pigs after administration of 10 and 20 ,ug of ovalbumin was then followed after three weeks after the administra-tion of the test substances. The result of determination of the skin reaction area size in mm are then presented in the Table No. 1.
Table No. 1 Induction of delayed hypersensitivity to ovalbumin in guinea-pigs with synthetic ADP
-Tested Dermatic response after 24 hours Substances Dose (mm) Ovalbumln dose Ovalbumin dose 10 ug 20 ug FIA - 1.0 - 0.48 3.39 - 0.22 FCA 1 mg 3.71- 0.23 / 5.73 - 0.33 MDP 100 ,ug 2.99- 0.23 / 6.07 - 0.21 ADP 100 ug 2.70- 0.21 / 5.82 - 0.27 The numbers represent the average values of two experi-ments at 16 animals +/ statistically significant differences (p ~0.05) Explanatory notes : FIA = Freunds incomplete adjuvant (Bayol + Arlacel 4 : 1 ) FCA = Freunds complete adjuvant (Bayol + Arlacel + 1 mg of mykobacterium tuberculosis) MDP = N-acetyl-muramyl-L-alanyl-D-isoglutamine ADP = L-alanyl-D-isoglutamine adamantylamide ~ .. . .
lZ069S8 From the rèsults of Table 1 it is evident that the adminis-tration of ADP led to enhanced delayed hypersensitivity in guinea-pigs to ovalbumin and the effect in dose used (100 ug) is comparable with the effect of both MDP and FCA.
Test No. 2 Labelled thymidine is most often used for the stNdy of biosynthesie of DNA in vivo. This preformed pyrimidine desoxynucleoside is in a succession of phosphorylation reactions phosphorylated to dTTP (desoxythymidinetriphos-phate) which is the immediate precursor of DNA synthesis ~ in cell nucleus. Enhancement of labelled thymidine incor-poration into DNA represents activation of its biosyn-thesis which precedes cell propagation.
This test is one of the most sensible and reli~
able by which immuno stimulating effects of substances and their effect on cell propagation can be determined. To Wistar male rats weighing 150 to 160 g tested substances in equimolar concentrations were administered, that is MDP in dose 1.00 mg/kg and ADP in dose 0.74 mg/kg. The animals were killed after 16 hours; 2 hours before their killing thymidine (methyl-3H) was administered to them intraperitoneally in the dose 20 ,uCi per animal in 0.3 ml of physiological solution (specific activity 20 Ci/mmole).
Frozen organs were taken out and promptly homo-genized in 0.2 N HC104. After repeated washing of the sediment with 0.2 N HC104 RNA was removed by alkaline hydrolysis. Nucleotide components were released from protein-DNA precipitate by hydrolysis in 1 N HC104.
3p Supernatent was after neutralization evaporated to dry-ness. Nucleotides of DNA were transferred to bases by further acidic hydrolysis in concentrated HC104. Thymine was then isolated in chromatographically pure form by repeated paper chromatography and its radioactivity was 12~69~i8 determined using scintillation spectrometer. Results of the experiment are presented in the Table No. 2.
Table No.2 Utilization of (methyl - 3H) thymidine for synthesis of DNA of liver, kidneys, thymus and spleen of rats after MDP and ADP administration ;
CPM/uml DNA of thymine liver kidneys thymus spleen , Controls 1.750 1.750 940 11.150 dipeptide) 4.100 2.350 1.400 10.900 15ADP (adamantyl- 2.950 2,200 1.550 16.500 The data of Table 2 evidence that already a single admin-istration of ADP in a dose of 0.75 mg/kg has a pronou~c-ed positive effect on the incorporation of methyl-3H
thymidine into the thymus and spleen DNA. The two organs take part to a substantial extent in the development of tumor and cell immunity. The tabulated results show a significant enhancement of thymidine utilization for the synthesis of thymus DNA which is of the same order of magnitude as in the case of MDP, where this effect is known already for a long time. In the case of spleen we can see, whereas the administration of MDP is not accompagnied by enhanced thymidine utilisation, ADP has here also pronounced stimulating effects manifesting themselves by enhanced 3H thymidine utilization.
Presented results as to the effect of MDP on utilization of thymidine for the synthesis of spleen DNA
are in a good agreement with literature data which show lZ(;~695B
that MDP has no effect on this organ, even on isolated spleen cells in vitro. By comparing the effects of the two compounds, i.e., ADP of the invention and MDP of prior art, the new preparation appears much more advantageous due to the fact that it exerts the desired action on both organs playing an important role in the immune reaction.
Test No. 3 Most of the hitherto synthesized MDP analogs have, besides immunoadjuvant and immunostimulative effects, also pro-nounced pyrogenic properties, which represents a substan-tial drawback for clinical practice. This fact hitherto prevented some very promising new substances from clinical use. In the experiments in rabbits, pyrogenic effect of ADP was therefore evaluated ad compared with the effect of MDP. ADP was intravenously administrated to rabbits of 1.8 to 2.0 kg weight in dose 100 ~g/kg and the effects were compared with those of the same dose of MDP. The same volume of isotonic solution (6.2 ml) was then inject-ed~ to control animals in which basal temperature was measured. The temperature was measured for 6 hours using a rectal thermistor thermometer in hour intervals and the results are summarized in the table No. 3.
Table No. 3 Pyrogenic effects of MDP and ADP in rabbits Tested Temperature, C
Sub- Dose 1 2 3 4 5 6 stance hour hours hours hours hours hours Controls (only iso- - -0.1 +0.2 -0.2 -0.1 0 +0.1 tonic solu-tion) ~g/kg +0.3 +0.4 +0.4 +0.4 +0.3 +0.35 ,ug/kg +0.8 +1.7 +1.8 +1.3 +0.7 +0.4 _ g _ lZB6958 The values presented in the Table No. 3 are the average values of 5 animals in a group.
From the results oE the Table No. 3 it is evi-dent that ADP has no pyrogenic effects in doses which come to consideration for clinical application. In the same dose, that is 100 ~g/kg,MDP is already expressively pyrogenic. The fact that ADP is free of any pyrogenic effect with retained immunostimulatory and immunoadjuvant effects represents its substantial advantage and reveals real possibilities for its clinical use.
Presented results of pharmacological evaluation ~ of the compound of the invention allow to suppose that this compound, advantageously in the form of an immuno-stimulatory pharmaceutical preparation in a mixture with usual pharmaceutical auxiliary substances, can find use for stimulating the immune response of organisms, espe-cially for enhancing their resistance in states of dimin-ished antibody formation.
Claims (4)
1. A process for producing L-Alanyl-D-iso-glutamine adamantylamide of the formula wherein Ad represents a residue of adamantane bound in its position 1 characterized in that l-aminoadamantane is reacted with a dipeptide derivative of the general formula (II) (II) wherein X stands for an acidically or hydrogenolytically removable protective group, said protective group being thereafter removed to obtain L-Alanyl-D-isoglutamine adamantylamide as defined above.
2. L-Alanyl-D-isoglutamine adamantylamide of the formula wherein Ad represents a residue of adamantane bound in its position 1, whenever obtained by a process as defined in claim 1 or an obvious chemical equivalent thereof.
3. A process as defined in claim 1, charac-terized in that said removable protective group is selected from the class consisting of a tert-butyloxy-carbonyl, benzyloxycarbonyl and nitrophenylthio.
4. L-Alanyl-D-isoglutamine adamantylamide of the formula wherein Ad represents a residue of adamantane bound in its position 1, whenever obtained by a process as defined in claim 3 or an obvious chemical equivalent thereof.
Priority Applications (1)
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|---|---|---|---|
| CA000431224A CA1206958A (en) | 1982-06-29 | 1983-06-27 | L-alanyl-d-isoglutamine adamantylamide |
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|---|---|---|---|
| CSPV4908-82 | 1982-06-29 | ||
| CA000431224A CA1206958A (en) | 1982-06-29 | 1983-06-27 | L-alanyl-d-isoglutamine adamantylamide |
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1983
- 1983-06-27 CA CA000431224A patent/CA1206958A/en not_active Expired
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