US3843629A - Complexs of polyriboinosinic acid and polyribo-2-thiocytidylic acid - Google Patents

Complexs of polyriboinosinic acid and polyribo-2-thiocytidylic acid Download PDF

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US3843629A
US3843629A US00263677A US26367772A US3843629A US 3843629 A US3843629 A US 3843629A US 00263677 A US00263677 A US 00263677A US 26367772 A US26367772 A US 26367772A US 3843629 A US3843629 A US 3843629A
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acid
virus
complex
complexes
polyribo
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K Scheit
O Saiko
P Faerber
K Reuss
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Merck Patent GmbH
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Merck Patent GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

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  • ABSTRACT Two-component complexes consisting of a polyriboinosinic acid of the formula wherein k is an integer from 1 to 3, inclusive; and b is an integer from 1 to 2,000, inclusive; and the metal and ammonium salts thereof, and a polyribo-2- thiocytidylic acid of the formula wherein m is an integer from 1 to 3, inclusive; and n is an integer from 1 to.2,000, inclusive; and the metal and ammonium salts thereof, exhibit antimicrobial, in particular antiviral, activity.
  • This invention relates to novel polynucleotides. It further relates to a method for the production of interferons. It is known that interferons which in the literature are also referred to as virus inhibiting factors are involved in the body defenses of vertebrates against viral infections. They are the only broad spectrum antiviral agents known in the prior art. Interferons are proteins of cellular origin having different molecular weights which can be destructed, and thereby made ineffective, by protein destructing enzymes, e.g., trypsin. They do not inactivate the viruses directly but rather inhibit the virus reproduction by an intracellular mechanism wherein the ribonucleic acid and protein synthesis is involved.
  • protein destructing enzymes e.g., trypsin
  • Interferons are species-specific, i.e., they are effective only in cells of the same or a nearly related species from which they were obtained. They are, however, virus non-specific, i.e., they are effective against different, non-related virus species.
  • a compound which stimulates the production of interferon can be used as a broad spectrum antiviral agent.
  • novel polynucleotides of this invention are twocomponent complexes (hereinafter referred to as AB) consisting of a polyriboinosinic acid (hereinafter referred to as A) of the Formula I wherein k is an integer from 1 to 3, inclusive; and b is an integer from I to 2,000, inclusive; and a polyribo-Z- thiocytidylic acid (hereinafter referred to as B) of the Formula II wherein m is an integer from 1 to 3, inclusive; and n is an integer from I to 2,000, inclusive; and the metal and ammonium salts thereof, i.e., of the A component, of the B component and of both.
  • A polyriboinosinic acid
  • B polyribo-Z- thiocytidylic acid
  • this invention relates to a method of inducing the formation of interferons, thereby inhibiting the growth of viruses, both in vertebrates and in cell cultures of vertebrates, by administering to these organisms or cultures, respectively, a two-' component complex AB according to this invention.
  • the two-component complexes of this invention are useful for protecting cells from virus infections both in vitro and in vivo.
  • administering AB or a metal orammonium salt thereof protects against infections of other disease germs, e.g., bacteria and protozoa. It is believed this effect is due to an increase of the specific as well as the non-specific infection defense mechanisms of the organism. The increase of the specific, i.e., immunological defense mechanisms is due,
  • b. m is 1, preferably wherein both k and m are l;
  • c. b is greater than 2, preferably greater than 10, most preferably greater than d.
  • n is greater than 2, preferably greater than 10,
  • both b and n are greater than 10, more preferably both are greater than 100, especially those of (a) or (b).
  • Preferred salts of A and B and thus of AB are the physiologically acceptable salts, including the alkali metal salts, e.g., lithium, sodium and potassium, the alkaline earth metal salts, e.g., magnesium and calcium, the heavy metal salts, e.g., manganese, zinc and iron, and the unsubstituted and substituted ammonium salts, including the alkyl, dialkyl and trialkyl ammonium salts, wherein alkyl contains one to eight, preferably one to four carbon atoms, and salts of the corresponding amines wherein l, 2, or 3 of the alkyl groups is substituted by a ,8 or y-hydroxy group, e.g., trimethyl-, triethyl-, monoethanol-, diethanol-, triethanoland trin-butylammonium salts.
  • Other salts can be employed for isolation, purification and characterization purposes.
  • the two-component complexes according to this invention are molecular aggregates built up from polyriboinosinic acids A of Formula I and polyribo-2- thiocytidylic acids B of Formula II, or of a salt of one or both of them.
  • the molecular proportions of A and B in these novel complexes may vary considerably, 'as well as the molecular shape of the complexes.
  • An AB complex can be in the form of a double helix, with the two homopolynucleotide strands therein of substantially equal length, i.e., both homopolynucleotides contain substantially the same number of bases.
  • the homopolynucleotides A and B are polymers themselves, consisting of molecules with varying molecular weights, which contain different numbers of bases and are of different length, the complexes AB can, of course, be of largely different shapes.
  • one helix of the two-component complex AB may even contain odd numbers of the complementary bases.
  • One A strand can be combined with two or more shorter or even longer B strands or vice versa. This may even be the case if the overall concentrations of the polyriboinosinic acid and the polyribo-2-thiocytidylic acid are equal.
  • Triple helices may represent other mo- I lecular structures of the two-component complexes AB according to the invention.
  • the reaction mixture contains an excess of one or the other homopolynucleotide which cannot be separated easily from the complex.
  • the antimicrobial activity of the complex is not adversely affected thereby.
  • a particular advantage of the novel two-component complexes according to the invention is their increased stability against the action of heat and body fluids.
  • no T i.e., the temperature where 50 percent of a given amount of the complex AB is dissociated into the components A and B) of aqueous solutions of AB could be determined at temperatures up to 100 C.
  • the two-component complexes AB remain effective after a 24 hour in vitro treatment with human serum.
  • the homopolynucleotides A and B possess a pentose phosphate backbone, the pentose therein being ribose.
  • the nucleic bases contained are hypoxanthin and 2-thiocytosin, respectively.
  • a and B are most frequently prepared using biochemical standard procedures, e.g., treating the corresponding nucleoside dior triphosphates with a polymerizmg enzyme.
  • the preferred method of preparing the new twocomponent complexes AB comprises combining separate aqueous solutions of the homopolynucleotides A and B at a temperature of from 10 to C.
  • This reaction is usually carried out in the presence of one or more inorganic and/or organicsalts, preferably an alkali metal salt, e.g., sodium chloride, in order to maintain a definite ionic strength of preferably from 0.001 to l.
  • the pH value of the solutions of A and B must be from 5.0 to 115, preferably from 6.5 to l 1.0.
  • the homopolynucleotide solutions usually are buffered.
  • Suitable buffer materials are, e.g., organic or inorganic alkali metal salts, preferably sodium salts, e.g., sodium acetate, potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium hydrogen tartrate, sodium citrate and, most preferably, sodium cacodylate.
  • Organic buffer materials can also be used, e.g., tris-(hydroxymethyl)- aminomethane/hydrochloric acid.
  • an organic solvent miscible with water can be added, e.g., a monoor polyvalent alcohol, e.g., methanol, propanol, ethylene glycol and glycerin, or an aprotic dipolar solvent, e.g., dimethyl sulfoxide, formamide and dimethyl formamide.
  • a monoor polyvalent alcohol e.g., methanol, propanol, ethylene glycol and glycerin
  • an aprotic dipolar solvent e.g., dimethyl sulfoxide, formamide and dimethyl formamide.
  • a reaction mixture with thedesired ionic strength favorable for the complex formation is preferably obtained by combining equal volumes of equally concentrated solutions of the components A and B calculated on the base content thereof), both of which are adjusted to the desired ionic strength.
  • the reaction mixture contains euqimolar amounts of the homopolynucleotides A and B (with respect to the bases). It is, however, also possible to use different molar amounts of A and B in the preparation of the two-component complexes according to the invention.
  • the type of the complex formed in a given reaction mixture is nearly indepen-' dent from the molar proportion of its components A and B present during its formation, the type of the complex AB formed depending mainly on the ionic strength and the pH value of the reaction mixture wherein it is formed.
  • Another process for the preparation of AB or its metal or ammonium salts comprises the production of one of A or B in a reaction mixture which already contains the other homopolynucleotide.
  • a and, e.g., polyribo-2,4- dithiouridylic acid the latter homopolynucleotide can be converted to polyribo-4-sulfo-2-thiouridylic acid by treating the polyribo-2,4-dithiouridylic acid with sulfite and/or bisulfite ions, preferably with an alkali metal sulfite and/or bisulfite, e.g., sodium bisulfite, in the presence of an oxidizing agent, e.g., molecular oxygen, at a pH value of 4.5 to 9, preferably about 7.
  • an oxidizing agent e.g., molecular oxygen
  • the polyribo-4-sulfo-2-thiouridylic acid thus obtained is then reacted with ammonia and/or ammonium ions, which preferably are provided in the form of an ammonium halide, e.g., ammonium chloride, at a pH of 7 to 10, preferably about pH 8.5 to produce B, which, with the homopolynucleotide A already present, will form the two-component complex AB according to the invention immediately or after adjusting the pH and the ionic strength of the reaction medium to suitable values.
  • ammonia and/or ammonium ions which preferably are provided in the form of an ammonium halide, e.g., ammonium chloride
  • a variant of the above-described process comprises treating an aqueous solution containing one of the homopolynucleotides A and B, and the monomeric nucleoside dior triphosphate corresponding to the other homopolynucleotide, with a polymerizing enzyme. in this reaction, too, the second component of the complex according to the invention is prepared in situ. It is not important, however, whether or not the monomeric nucleoside phosphates prior to the polymerization are associated with the homopolynucleotide already present, e.g., by hydrogen bonding.
  • Suitable enzymes for the enzymatic synthesis of homopolynucleotides A or B from the corresponding nucleoside phosphates are polynucleotide phosphorylases, e.g., the polynucleotide phosphorylase E.C.2.7.7.8, obtainable, for example, from Escherichia coli.
  • the enzyme can be utilized in the polymerization in the form of crude extracts or in purified form.
  • the polymerization is ordinarily carried out at a pH of from about 5.5 to 9.5, preferably 8 to 9, at a temperature of from 0 to 80 C., preferably to 45 C., especially at about 37 C.
  • Buffer substances e.g., tristhydroxymethyl)-aminomethane tris
  • ammonium carbonate or sodium cacodylate
  • an inorganic salt for example, magnesium chloride, calcium chloride or manganese (ll) chloride.
  • the antimicrobial and interferon inducing activity of a two-component complex according to the invention is not dependent upon the conditions by which it is produced.
  • the desired activity gen- is not dependent upon the conditions by which it is produced.
  • the complexes AB according to the invention can be characterized by physical methods, for example, by the determination of the hyperchromic shift in the ultraviolet absorption spectrum.
  • This hyperchromic shift is determined by comparing the UV spectrum of the complex with a spectrum obtained by the addition of the UV spectra of the components A and B registered at a spectra; of the Svedberg values s of the T values, i.e., the temperatures at which one-half of the complex molecules originally present in a given solution, has been dissociated into A and B; saccharose densitygradient fractionation; and chromatography.
  • the two-component complexes according to the invention are particularly well characterized by a determination of their protective effect against a virus infection in a cell culture.
  • confluent monolayers of secondary rabbit kidney cells are treated with a dilution series of the complex in the cell culture maintenance medium, and are then incubated at 35 to 37 C. for 18 to 24 hours. Thereafter, the liquid is removed from the cell culture vessels, and the cultures are infected with a suitable virus, for example, Herpes Simplex virus.
  • the infected cultures are covered with agar and incubated at 35 to 37 C. until untreated controls exhibit visible holes in the cell layer (hereinafter referred to as plaques) resulting from the destruction of the cells by the virus infection.
  • plaques a suitable virus
  • the cells are stained, and the number of the plaques is determined. From the figures thus obtained, the concentration of the complex AB is determined which brings about 50 percent decrease of the plaques number in the complex treated culture compared with the untreated control.
  • confluent monolayers of, for example, rabbit kidney cells or mouse embryo cells are covered with a solution of AB in the cell culture maintenance medium.
  • the cells are stimulated to produce interferon which according to biochemical standard methods, can be isolated from the supernatant liquid and can be characterized as such.
  • interferon after being treated with trypsin no longer has a protective effect.
  • the dependence of the interferon effect on the protein and RNA synthesis of the cells is shown, for example, by the fact that lar stomatitis virus in such cells which have been I treated with actinomycin D.
  • interferon in vertebrates following the administration of AB, can be shown by injecting a solution of the complex in a physiologically acceptable solvent, for example, Hanks salt solution, intravenously, collecting some blood from the patient 2 to 6 hours later, and isolating the interferon from the serum and characterizing it as described above.
  • a physiologically acceptable solvent for example, Hanks salt solution
  • mice are treated intraperitoneally with a solution of AB in a physiologically acceptable solvent, and one day thereafter are infected with a pathogenic virus,
  • Pneumococcus and by protozoae, for example, Plasmodium Berghei or Eperythrozoon Coccoides, can beshown.
  • a particular advantage of the novel complexes AB is their ability to increase the specific defense mechanisms of the host organism. If, for example, a number of guinea pigs are vaccinated with anti-influenza vaccine, and one-halfthereof additionally with AB, antibodies are earlier detectable in the serum of the animals to which AB is administered. Further, the antibody titer, as determined by the hemagglutination inhibition test or the complement fixation test, becomes higher in the AB-treated than in the AB-untreated vaccinated guinea pigs.
  • the increase of the vaccination protection by additional administration of AB can also be shown directly, for example, by vaccinating mice with an anti-influenza vaccine and also treating one-half of the vaccinated mice with AB and, 14 days after the vaccination, infecting all of the animals with influenza virus. A greater percentage of those animals treated with AB in addition to the vaccination, survived the infection than those which were not treated with AB.
  • the novel complexes When used in the in vitro production of interferons, they are usually added to cell cultures of the vertebrate species to be treated with the thus-produced interferon, preferably in the form of a solution or suspension of AB, in free acid form or as a metal or ammonium salt thereof, in the cell culture maintenance medium. Sterilants or antibiotics can also be added to prevent bacterial contamination of the culture during the incubation.
  • the incubation usually takes place at a temperature near the normal body temperature of the respective vertebrate species. Temperatures of 33 to 40C. are preferred.
  • the culturing time necessary for optimal yields of interferon varies from one species to another. Good results are obtained after culturing times of to 48 hours.
  • interferons thus produced by the cell cultures are isolated using standard procedures for the isolation of proteins, comprising the steps of filtration, precipitation with, for example, ammonium sulfate, and purification by, for example, re-precipitation and/or dialysis.
  • Living organisms can also be used in the production of interferons.
  • a suitable amount of AB is administered to the organism, and after a time necessary for building up at least a considerable interferon level in the serum, blood is collected from the organism and the interferon isolated therefrom in the usual way.
  • the novel complexes of this invention can be employed in mixture with solid, semi-solid, liquid or gaseous excipients as drugs.
  • the AB complexes including their metal and ammonium salts, can be administered enternally, parenterally or topically.
  • a preferred mode of enteral application is oral, e.g., in the form of capsules, syrups or elixiers.
  • the complexes can also be administered rectally, e.g., in the form of suppositories.
  • Parenteral administration can be by the injection of sterile solutions thereof in physiologically acceptable solvents subcutaneously, intramuscularly or intravenously.
  • a particularly well suited mode of application of the novel complexes is the topical administration in the form of solutions, lotions, ointments, creams, powders or aerosol sprays.
  • solutions or sprays . are applied to mucous membranes, e.g., intraorally, in-
  • Suitable excipients for pharmaceutical preparations containing the two-component complexes of this invention are those organic or inorganic substances adapted for enteral, parenteral or topical application and which do not react with the novel complexes, such as, for example, water, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, vaseline, cholesterol, as well as conventional propellants for aerosols, e.g., fluorochlorohydrocarbons.
  • compositions according to this invention can optionally be sterilized or mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, or wetting agents, emulsifiers, salts for influencingthe ionic strength, buffers, coloring, flavoring and- /or aromatous substances.
  • auxiliary substances such as lubricants, preservatives, stabilizers, or wetting agents, emulsifiers, salts for influencingthe ionic strength, buffers, coloring, flavoring and- /or aromatous substances.
  • compositions according to this invention including those which contain AB as the only active ingredient and those which also comprise one or more other active substances, usually contain 0.001 to 200 milligrams of the active ingredient per dosage unit, and preferably 0.01 to 50 milligrams per dosage unit. In combination preparations, preferably at least 10 percent of the active ingredients consists of AB.
  • the theoretically effective doses of the novel twocomponent complexes vary widely from species to species, and depend further on the virus species against which they are used. In mice, the threshold dose is about 0.5 milligrams per kilogram, while in rabbits, it is only about 0.05 micrograms per kilogram.
  • the temperatures are indicated in degrees Centigrade.
  • the s -values were obtained by sedimentation velocity analyses by means of an analytical ultracentrifuge.
  • the figures given for the concentrations of the holopolynucleotides refer always to the monomeric nucleotides which build up the polymeric ones, the molecular weights of which can be calculated from that of the nucleoside monophosphate minus a molecule of water. The slight deviation from actual molecular weight because the end groups are not included is negligible and can be ignored.
  • other conventional media can also be used. The type of the cell culturemedium is only important for the cell culture and does not influence the effect of the two-component complexes according to the invention.
  • polyriboinosinic acid (A) used throughout the following examples is a commercial product of C. F. Boehringer & Soehne G.m.b.I-1., Mannheim-Waldhof, having a s -value of 5.3.
  • tris.HCl tris-(hydroxymethyl)-aminomethane hydrochloride
  • magnesium chloride 0.04 mmol of the disodium salt of 2-thiocytidine-5 diphosphate, 0.04 mmol of dithiothreitol, and 10 enzyme units of polynucleotide phosphorylase (specific activity 0.165 .mmol UDP/hour X mg. protein at 37) are incubated for 4 hours at 37. After removal of the protein, and a 48 hour dialysis of the aqueous phase concentrated to 1.5 ml. at 3 against 0.01 M tris.HCl.(pH 7.0), B having a s -value of 8.3 is obtained.
  • Polynucleotides B having other s -values can also be used with similar success. The same holds for polynucleotides A.
  • UV spectrum shows A 247.5 nm; A 226 nm;
  • B contained in the two-component complex AB is not attacked by polynucleotide phosphorylases.
  • EXAMPLE 2 0.32 mg of A are dissolved in 10 :ml. of a 0.001 M solution of sodium cacodylate (pH 7.0) containing 59 mg. of NaCl. 1 ml. of a 0.001 molar solution of B is added, and the mixture is left standing at room temperature. The complex formation is followed spectrometrically. A solution of AB is obtained whose properties resemble those of the complex AB described in Example 1.
  • EXAMPLE 3 To 5 ml. of an aqueous mixture pH 8.3) containing 0.5 mmol of tris.HCl buffer, 0.01 mmol of MgCl 0.05 mmol of A and 0.16 mg. of disodiu'm 2-thiocytidine-5 diphosphate, 5 enzyme units of polynucleotide phosphorylase (specific activity 0.165 mmol UDP/hour X mg protein at 37) are added, and the mixture is incubated for 4 hours at 37. After removal of the protein by repeated extraction with chloroform-isoamyl alcohol (25:2 parts by volume), the aqueous phase is concentrated to a volume of 2 ml. at 15, and is dialyzed for 48 hours at 3 against 0.01 M tris.HCl buffer. By this procedure, a solution of the complex AB is obtained which exhibits the same properties as that described in Example 1.
  • EXAMPLE 4 To 4 ml. of a 0.001 M sodium cacodylate solution containing 24 mg. of NaCl, 0.1 mmol of polyribo-2,4- dithiouridylic acid, and 0.1 mmol of A, 20 microliter of a sulfite reagent solution consisting of 3 parts by volume of an aqueous 1M Na SO solution and 1 part by volume of an aqueous 1M NaHSO solution, are added, and air is sucked through the reaction mixture. After 1 hour, again 20 microliter of the sullite reagent solution are added, and the air is bubbled through for another hour. Thereafter, 0.5 ml.
  • the infected cultures are layered with agar, incubated for 48 hours at 35, and are then coated with a second agar layer containing a dye for staining the cells. After an additional incubation for 24 hours at 35, the then macrosopically visible virus plaques are counted macroscopically a magnifying lens. From the three values obtained for each concentration of AB, the average value is calculated and is brought in relation to the average value calculated from the untreated controls. From these figures, the 50 percent Plaque Reduction Dose PRD i.e., the dose of AB reducing the plaque member to 50 percent of the untreated control, is determined graphically.
  • EXAMPLE C According to Example 1, a solution was preared which contained 325 micrograms AB/ml. To demonstrate the interferon production in vivo, 23 ml of this solutionwere applicated intravenously to each of two rabbits of conventional breed weighing 2.3 kg, i.e., 325 micrograms AB/kg. After 2 hours blood was taken from the rabbits by heart puncture. The serum was separated and frozen to -70 C. In order to measure the interferon titer, the sera were separated and diluted in 10-fold steps with cell culture maintenance medium. These dilutions were applied to confluent monolayers of sec. rabbit kidney cells in square bottles 10 ml per bottle; three bottles per dilution). The cells were incubated overnight at and then challenged in conventional manner with Herpes Simplex virus. The further procedure and evaluation was as in example A.
  • the interferon titer was measured as described above. A 50 percent reduction of Herpes Simplex virus plaques was obtained with the following dilutions:
  • EXAMPLE D A 45.5 percent reduction of plaques Vaccinia virus was obtained by serum I in a dilution of 1: 1000 under test conditions analogous to those described in Example C.
  • n is greater than 100.

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US00263677A 1971-06-19 1972-06-16 Complexs of polyriboinosinic acid and polyribo-2-thiocytidylic acid Expired - Lifetime US3843629A (en)

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DE19712130544 DE2130544A1 (de) 1971-06-19 1971-06-19 Zweikomponenten-Komplexe mit biologischer Wirkung
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3952097A (en) * 1974-09-27 1976-04-20 The United States Of America As Represented By The Department Of Health, Education And Welfare Nuclease-resistant hydrophilic complex of polyriboinosinic-polyribocytidylic acid
US4024222A (en) * 1973-10-30 1977-05-17 The Johns Hopkins University Nucleic acid complexes
WO1989002921A1 (en) * 1987-09-22 1989-04-06 Temple University Artificial dna base pair analogues
WO2005102278A1 (en) * 2002-07-03 2005-11-03 Oncovir, Inc. Method for preparation of poly-iclc and uses thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62252798A (ja) * 1986-01-06 1987-11-04 Nippon Shinyaku Co Ltd 核酸誘導体
US4882147A (en) * 1987-12-16 1989-11-21 The Research Foundation Of State University Of New York Novel polynucleotide analogs, methods for inhibiting nucleic acid polymerases and methods for inducing synthesis of interferon

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4024222A (en) * 1973-10-30 1977-05-17 The Johns Hopkins University Nucleic acid complexes
US4130641A (en) * 1973-10-30 1978-12-19 Ts O Paul O P Induction of interferon production by modified nucleic acid complexes
US3952097A (en) * 1974-09-27 1976-04-20 The United States Of America As Represented By The Department Of Health, Education And Welfare Nuclease-resistant hydrophilic complex of polyriboinosinic-polyribocytidylic acid
WO1989002921A1 (en) * 1987-09-22 1989-04-06 Temple University Artificial dna base pair analogues
US5126439A (en) * 1987-09-22 1992-06-30 Temple University Artificial dna base pair analogues
WO2005102278A1 (en) * 2002-07-03 2005-11-03 Oncovir, Inc. Method for preparation of poly-iclc and uses thereof

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SE7415908L (en:Method) 1974-12-18
IL39649A (en) 1975-12-31
FR2143060B1 (en:Method) 1975-06-20
GB1364987A (en) 1974-08-29
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FR2143060A1 (en:Method) 1973-02-02
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CH584719A5 (en:Method) 1977-02-15
NL7207984A (en:Method) 1972-12-21

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