IL33659A - Nucleotides as inducers of interferon - Google Patents

Description

¥ Nucleotides as inducers of interferon MERCK & CO. INC. , G. 31704 1 This Invention Is concerned with the induction of 2 the formation* in vivo and In vitro by the use of complexed 3 polymers, of substances which inhibit viral growth, for example, interferons as well as with the Induction of. re- 5 sistance to viral infection in vivo and in vitro by the ad- 6 ministration of said complexed polymers. 7 This invention more particularly is concerned with 8 the induction of interferon production and the induction of 9 resistance to viral infection by administration of complexed polymers formed by an admixture composed of two homopoly- 11 nucleotides, or of one homopolynucleotide and one homooli- 12 gonucleotide. 13 In the specification and claims the term interim ferons shall be used and shall be understood to apply to the same active principle identified in the literature by various 16 terms such as viral inhibiting factor and viral inhibitory 17 substance. In the following discussion and claims, the term 18 homopolynucleotide will be used to Include not only the homo- 19 polynucleotides but also the homooligonuleotides, the diff- 20 erence being only in the length of the polymer; that is, it 21 will embrace known polymers having from two to thousands of 22 nucleotide base residue units. These known polymers are pre- 23 pared by enzy atically treating nucleotide diphosphate 2^ H vitro to form the desired polynucleotide in varying numbers of nucleotide units, the number of which cannot be known 26 with precision. 27 In virus chemotherapy research, the hope has been 28 to find a broad spectrum chemical antiviral agent. To date, 29 the only broad spectrum agents known are the interferons which appear in the blood and tissues of animals and man in - 2 - 35659/2 the course of viral and other infections and appear to provide a possible mechanism for arrest of viral dleease which is separate and distinct from the specific immunologic mechanisms. However, production of interferon in practical quantities for use in chemotherapy has not been achieved.

Another approach to virus chemotherapy has been the eearch for a chemically defined, non-toxic, non-antigenio, non-replicating, and readily available substance that will stimulate animal cells to produce interferon, which would then confer protection against virus infection.

Numerous interferon inducers have been reported including viable as well as inactivated viruses, endotoxin, j' phytohemagglutinin, trachoma and Inclusion conjunctivitis agents (TRIC agents), Brucella abortus, and others. However, for each, very serious inadequacies, such as toxicity, antigenicity and/or replicability have limited their practicability for treatment of virus disease.

It ie already known that the complex of po¾.y(ribo-inosinio acid)-pol (ribocytidylio acid) is active in induoing the resistance to viral infection in a variety of cell cultures (including rabbit, dog and calf kidney and mouse embryo). Suoh resistance was broad-spectrum with respect to various kinds of viruses (vesicular stomatitis, vaccinia, herpes simplex, and rhinovirus), but the amount of the complex required for induction varied considerably for different cells and was not always consistent for a particular kind of cell.

It has now been found that high concentrations Interferons can be Induced in a host animal by administra - 3 - 33659/2 either parenterally , such as subcutaneously , intradermally , intraperitoneally , intravenously, intramuscularly, orally, or topioally, preferably on a mucous membrane such as intra-nasally and into the respiratory tract. These complexed polymers are produced on admixture of two different homo-polynucleotides which themselves are synthetic and oommerloally available, and can be made by established procedures.

The complexed polymers according to the present invention; are complexed polymers being double-stranded polynucleotide complexes composed of two different synthetic complementary homopolynucleotides and consisting of (a) polyribonucleotide alternating copolymers, or (b) complexes of two homopolyribonucleotides containing halogen or alkyl ' substituted nucleotide components, or (o) hybrids between a polydeoxyribonuoleotide and a polyribonucleotide.

These synthetic double-stranded polynucleotides are,for example, the complex formed between two homopolyribonucleotides (rlsrBC), complexes formed by self-complexing of alternating polyribonucleotide copolymers (rid, rIBO, rAU), or those obtained by complexing between a polydeoxyribonuoleotide and a polyribonucleotide (Fl DNA-RNA). The explanation of these symbols is the following: rlsrScf - complex οί' pol rlboinoslnio . acid and polyribo- '.' bromocytidylic acid rIC - alternating copolymer of rlboinosinic and ribo- ., i cytidylic acids i rIBC - alternating copolymer of rlboinosinic and bromo- ribocytldyllc acids rAU - alternating copolymer of riboadenylic and ribo- if urldylic acids .

Fl DNA-RNA - oomplex between Fl bacteriophage DNA and RNA ( of complimentary nucleotide sequence.

The dash over the BC lndioates brominatlon and - A - 33659/2 _ The1 synthetic employed in the, preparation of the complexed polymers have a pentose-phOB-phare skeleton, preferably wherein the pentose is ribose or deoxyriboee, as well as a opcclfic Identifiable pyrimldite or purine base such as adenine, inosine, cytosine, uraoll, . guanine and the like. The prior art teaches that mixing of -certain homopolynucleotldes in aqueous solution results in the formation of a complexed polymer identifiable through various physical testa and which la different from either of the two homopolynucleotldes from which the complexed polymer is formed. The ratio in which the two homopoly- . nucleotides are mixed is normally not controlling as to the ratio of the two homopolynucleotldes Incorporated in the complex. A mixture in n 1:1 molar ratio can result in a complexed polymer containing the two nitrogenous bases in a 1:1, 1:2, and/or 2:1 ratio or some other ratio of small whole numbers, that io, the resultant ratio is not a function o the ratio in which the components ore mixed but io determined by some natural propensity to so combine whloh is peouliur to the particular homopolynucleotldes employed.

The complexed polymers are those formed by mixing one homopolymer with another homopolymer, the seleoted pair being complementary; for example, a homopolymer of adenyllo. ,, acid mixed with a homopolymer of uridylic acid at least one of them. being substituted such as bromlnated or alkylated derivative thereof. Or the mixed pair may be inosinio and polycytidylio acids one of them being abrominated or alkyl substituted derivative.:. It Is to be understood i that for the guanyllo acid may be substituted inoeinic acid ι because it in a derivative. The binding of these pairs ; is by hydrogen binding as is evidenced by hypochromlclty in the ultraviolet refiion. in addition, one of the components of the oomplex must be n purine polymer and the other coin- * The complexed polymers employed as Interferon Inducers in the present Invention are prepared by the synthesis procedures, among other Journal references of Thach, R. E. , and P. Doty, 19β5, Science l47i 1310, Krakow, J.S. and yra Karstadt, PNAS 8, 2094 . ( 1967 ) , Milman G. , Mangridge, R. , M.J. Chamberlin, PNAS 7, l804-l8l0 ( 1967 ) , "The Structure of a DNA-RNA Hybrid". For example, the complexed polymers are prepared by mixing two dissimilar homopolynucleotldes in a 1:1 molar ratio with respect to their bases for a few minutes at ambient temperature in an aqueous buffer system having a broad pH range, i.e., between about 5.0-10·0, and an ionic strength of between about 0.001-1.0. Ratios other than 1:1 can be used as indicated above. Buffer systems that have been found useful are Ο.ΟΟβΜ sodium phosphate in 0.85·;'^ sodium chloride solution and 0.01M glycylclycine in 0.59^ sodium chloride. Any non- toxic buffer can be used. Indeed, no buffer need be used, since it has been found that the buffer capacity of the physiological system of the animal to which the mixture of homopolynucleotldes is to be administered is satisfactory to promote the desired complex formation after administration and provide the same induction of interferon production and protection from viral infection as when the two homopoly- nucleotides are pre-complexed. The invention also contemplates the stimulation of iitorferon formation in animal hosts by /the administration to the. animal host, inc-ludlng huroano, of the nucleotides which will form in their bodies the complexed polymers which serve as interferon inducers.

Another method for the preparation of complexed polymers that can be employed is to treat a mixture of two nucleotide-diphosphates or deoxynucleotide-triphosphates in Such treatment results In polymerization of the two monomers to two homopolynucleotides with concomitant formation of the complexed polymer.

The complexed polymers ormed in vitro can be characterized by hypochromic shift in the ultraviolet absorp-tlon spect/G¾m, sucrose density gradient fractionation, chromatography, and the capacity to induce the production of interferon, an activity which is lacking in either of the homopolynucleotides from which the complex is formed. The production of interferon serves most significantly to chara-cterize the complexed polymers used in the process of this invention since physical methods often lack sufficient sensi-tivity.

The production of interferon by administration of the complexed polymers is demonstrated by the protection of host animals as well as cell cultures from virus challenge. The interferon so produced also can be characterized by iso-lation of the induced interferon by known methods followed by in vitro determination of its viral inhibiting properties and characterization by host specificity, trypsin sensitivity, isoelectric point and molecular weight determination.

The induction of interferon formation iri vivo and/or the induction of resistance to viral infection, accord' ing to this invention is achieved by administration, of a complexed polymer, prepared as described above, to a host animal such a3 a rabbit, mouse, or other animal. The admini-stration can be parenteral or topical, particularly to a mucous membrane, such as intranasal, or in the respiratory tract. The effective dose depends on the host. species and to some extent on the virus against which the protection is there are no overt signs of toxicity either locally at the site of Injection or generally in the well-being of the whole animal.

The effectiveness of the complexed polymers in inducing Interferon formation in the host animal ca be determined, inter alia, by parenteral administration of the complexed polymer to an animal and after about 1 to hours taking blood samples. The serum is separated from clotted samples of the blood, sterilized, and titrated at several dilutions in culture tubes containing cells from the .same animal species as that used for host. After incubation at 35° C. for about 18 to hours the cell cultures are challenged with any one of the known cytopathic viruses and again incubated at 35° C. for about 3 days. The cultures then are examined for cytopathic effects and the interferon titer is determined as the reciprocal of the dilution at which 50$ of the tubes show no such cytopathic effects.

The effectiveness of the complexed polymers to induce Interferon production and host resistance in a large variety of living cell systems, both in vivo and in vitro is shown by the considerable number of tissue cell cultures and hosts in which interferon can be elicited. Repre.senta-tive of these are chick embryo, chick chorioallantoic membrane which may be In ovo as well as In vitro . monke -kidney cell culture, rabbit skin and rabbit testes in vito. calf-kidney cell culture, rabbit-kidney cell culture, human-ammion cell culture, intrallantoic in ovo, MCN cell culture, mouse-fIbroblast cell culture, human foreskin, chick-embryo cell culture, KB cell culture, mouse-lung in vivo, adult mouse brain In vivo, chick-fibroblast cell culture, human adult 1 thyroid gland or embryo-lung or embryo kidney cell culture, 2 human leukocytes, mouse-embryo-cell lines (3B, ME 29) in 3 cell culture and primary mouse-embryo cell culture. Prom ^ thi3 it can be expected that any living cell whether in the host or in a culture medium and which l3 capable of eliciting 6 interferon will be induced to produce interferon under 7 the influence of the complexed polymers in accordance with 8 this invention. 9 The interferons produced by the above method are 0 shown to be species specific using a plaque reduction inter-1 feron assay which involves incubation of an aliquot of the interferon-containlng serum with individual cell cultures 3 of various species followed by challenge with a virus, such ^ as vesicular stomatitis virus or other known cytopathogenic 5 virus, and Incubation to allow virus plaque formation. Plaque numbers on interferon treated cultures are compared to those 7 in untreated virus-infected control cultures. It is ob- served from such tests that interferon activity is demon- strated only in those cases wherein the cell cultures are of the same animal species as that from which the inter- feron-containing serum was Isolated.

The interferons induced by the method of the pre- sent invention employing complexed polymers can be. shown to be trypsin sensitive using a plaque reduction interferon assay, that I3, the interferon activity is destroyed by trypsin treatment.

The interferon induced as hereinbefore described is further characterized by known methods as to Isoelectric point and molecular weight as described in the examples.

The following discussion illustrates the prepara- tion of the complexed polymers that are used in the method - 9 - 33659/2 SUBSTANCE I Preparation of deoxyrlbohomopolymera The process for synthesizing deoxyrlbohomopolymera IB deecrlbed by R. 13. Inmon and R. L. Baldwin In J. Mol. Biol, ( 1964 ) Q> I52 .

SUBSlANCE.II Preparation of 0 complex in which one of the components 13 chemically altered e.g. rI;rBC The complexed polymers rl.rBC ia made by a proceee - described by Ώ. R. Davles and A.J. Rich in J, Amer. Chem.

Soc.,. 80, 1003 (1950) for the preparation of rirrC excep that 5-bromocyto3lne-i5' triphosphate wa3 used in the synthesis, of rBC. The complex han n Tm of 89.5eC in an ionic trenfith buffer of 0.1. This same procedure can be followed to obtain other complexes from other bromlnated or halogenated poly- nucleotides such as rA:rBU\ rl : rCIC, etc. In like "inanner the bnsca may have been chemically altered by nlkylation in known manner..

SUBSTANCE III Preparation of a complex of alternating poly- ribonucleotide copolymer, rIC.rIC .

This alternating copolymer is made by the process described by J.S. Krakow et al, in the above-mentioned journal article. The complex has a Tm of 6, -65°C in a 0.1 ionic strength buffer. 1 These alternating complexes are synthesized, as' 2 stated in the article, by using an enzyme prepared from 3 micro-organisms and is called R A polymerase. The polymers ^ are in a 1:1 ratio and are double-stranded about themselves by hydrogen bonding of the alternating complimentary bases. 6 These materials can be made by using this enzyme in the 7 presence of a template which can be dAT or heat denatured 8 DNA. It is, of course preferable to use dAT. These 9 alternating copolymers are not to be confined to regularly alternating copolymers but also include irregularly alterna- 11 ting copolymers as long as the components are in a definite 12 ratio. Other alternating copolymers are similarly made, 13 such as rAU:rAU, etc.

IV Ik SUBSTANCE V¾ . Preparation of a complex of alternating ribonucleotide copolymer using a halo- 16 genated ribonucleotide 17 An example of this is rIBC.rlBC made by the pro- 18 cedure of the J.S. Krakow et al article. It has a Tm of 19 91°C in 0.1 ionic strength buffer. Other examples are rABU:rABtJ, rIcTC:rIClC, etc.

V 21 SUBSTANCE V¾¾J Preparation of a complex between a poly- 2 deoxyribonucleotide and a polyribonucleo- 3 tide l\- An example of this double-stranded complex is 5 PI DNA-RNA which is a complex between Fl bacteriophage DNA 6 and an RNA of complimentary nucleotide sequences. The Fl 7 bacteriophage DNA is here used as a template for enzymatLc 8 RNA synthesis. The procedure for making this and like 9 double-stranded hybrids is described by G. Mllman, et al in 0 the journal article described above. The complex Fl DNA-RNA 1 has a Tm of 82° in 0.1 ionic strength buffer. 1 The following examples are included to demonstrate 2 the induction of interferon by the administration of certain 3 of the homopolynucleotide complexes, such as those described '•I- above, both in living host animals and in isolated cell cultures and it should not be inferred therefrom that other 6 complexed polymers contemplated b this invention though 7 not exemplified will not stimulate interferon production in ϋ a like manner. 9 . EXAMPLE 1 0 Induction of Interferon in Rabbits 1 The complexed polymers described above are separately 2 administered as 0. 5 ml. aliquots to 4. 5 to 5.0 pound rabbits 3 by Intravenous injection. After about 2 hours, blood samples are taken from each rabbit by cardiac puncture. Serum is 5 . separated from each of the clotted blood samples and separately o sterilized by exposure to ultraviolet Irradiation. Aliquots 7 of these sterilized samples are employed in the following; 8 tests. 9 Determination of Interferon Titers 0 The sterilized rabbit serum from each rabbit Is' 1 titrated separately by serial two-fold dilutions from 2 1 : 5-1:640 using cell culture growth medium as diluent. A 3 one ml. sample of each dilution is added to each of four 4 tube cultures of rabbit kidney cells which have been drained 5 of spent growth medium. After overnight incubation at 35°C., the tube cultures are again drained and infected with 10 to 7 100 TCIDp-Q (Tissue culture infectious dose of vesicular stomatitis virus contained in 1 ml. of growth medium. Each 9 tube culture Is incubated at 35° C. for an additional 3 days, then observed for evidence of viral cytopathic effects and - 12 - 33659/2 ^ 1 scored (+) for positive evidence of such effects or (O).for lack of evidence of cytopathic effects. The Interferon titer 3 for each serum sample i3 determined as the reciprocal of H the Berum dilution at which 50'/. of the tubes show no cyto- 5 pathic effects. Serum from untreated animals (i.e., normal. 6 control animals) is titrated in each experiment to evaluate normal serum faotors.

In an assay test based on Example 1 it was found that the following amounts of the complexes, when injeoted intravenously per rabbit induced production of deteotable concentrations of serum interferon in two hours, 10.5 MS of rlrrBC, 10 jag of rICirIC, 12 μ& of rIBC:rIBC and 5 μ& of Fl DNA-RNA. - 13 33659/2 KXAMPLT''. 2 ! In Vitro Activity oC Complexed Polymers r as Vlr.il Inhibiting Substances ϋ Each of the cell cultures identified in Table V . 9 is incubated overnight with one of the complexed polymers Q diluted in the growth medium known to be required by the cell 1 culture used. After removal of the complexed polymer-con- . 12 taining eolations, each culture is infeotcd with vesicular 13 stomatitis virus suspensions, incubated, and observed for ..' i plaque formation described for the test demonstrating species specificity. 16 TABLE ί 17 )xg, Dose of Complexed Polymer/ml, required 18 to Induce Resistance to VSV Plaque Formation 1 19 Cell Culture Poly I l- C Poly A . H U 20 (rlsrC) (rA:rU) 21 Primary Rabbit Kidney < 0.00125 0.0015 22 Primary Human Amnion 0.04 26.25 23 Primary Human Embryonic Kidney 1.25 2Ί Primary Chick Embryo 0.33 >100 .

Primary Bovine Kidney 5.00 26 Primary Dog Kidney 1.25 27 Primary Mouse Embryo >5.25 >100 : 8 The relative effective eoo of the oomplexeo compared 29 to their uncomplex nucleotides la shown in Table Va, usln/j; thiB same test. of vesicular stomatitis virus suspension and the cultures relncubated at 35" C. for an additional 1.5 hours. An overlay of 5 ml. of maintenance culture medium containing methylcellulose as a solidifying agent is added to each flaek and inoubation continued for an additional 3-'* doye a 35° C. to. allow virus plaque formation. The overlay medium then is removed and the cells stained with carbol fuchsin. Plaque numbers on interferon treated monolayers are compared to those in untreated virus infected control monolayers. The reciprocal dilution of interferon givin at least a 50£ reduction in plaque number is considered the interferon titer of that sample. The titers thus determined illustrate species specificity, that is, inter' feron induced in an animal of a given species is active only in cells derived from an animal of that same species j . . ( ) ■ ■ 35659/2 1 Demonstration of Trypsin Sensitivity of Induced Interferon f> Interferon-containin?, serum from animals Induced with a complexed polymer 1B Isolated by chromatography on Ί . CM-Sephadex (a cation-exchange material obtained by the 3 introduction of cnrboxymethyl groups into Sephadex"). A 6 sample of the isolated interferon is treated with crystal- V line trypsin solution ( 50 ug/ml final concentration) for 8 hours at 35° C. Λ similar untreated interferon sample 9 is also incubated for '< hours at 35° C. After the 4 hour intubation period, soybean trypsin inhibitor is added to 11 each sample, including the control. The samples are 12 titrated for interferon activity by the plaque reduotion i . ' . 13 method. · Trypsin solution to which soybean trypsin inhibitor l hap been added is also titrated for antiviral aotivity.

! Determination of Molecular eigh of Induced Interferon 8 ■ The molecular weights of complexed polymer in- 9 duced interferon are determined according to the following 0 method. Columns of 2 x 35 cm. size are paoked with hydrated 1 Sephadex 0-200 bends and slowly percolated for 2 to 3 days - 18 - 33659/2 The above description concerning the preparation i| of the complexed polymers and the examples illustrating the method of this invention specifically describe certain as- 6 pects of this invention. It is not to be considered limit- 7 ing as the invented method can be carried out with complexed 8 polymers made from known homopolynucleotides, homooligo- 9 nucleotides or their deoxy analogs administered parenterally 0 , or topically as hereinbefore described, 1 EXAMPLE 3 2 Oral-nasal preparation 3 As a propylactic intranasal preparation against 4 . respiratory viruses such as the common cold, a dose of from .01 tolO milligrams of complexed polymer.-^IrrO- would 1 be applied to the nasal and oral membranes every 2 to 3 days, , 17 This period of administration is due to the prolonged dura-.; 18 tion of the induced interferon. 19 The application could be by way of an aqueous suspension in an aspirator spray so that one or two sprays 21 would deliver about.01 to 10 milligrams. Or, an aeroeol pre-. 22 paratlon using conventional lubrochlorohydrocarbons as pro- . 23 pellants could be prepared and used. Additionally, the 2'[ preparation could be applied as a nose drop pharmaceutical liquid. ' , ' 26 This preparation would also be used therapeutically 2 / about every 2-3 days in case of actual respiratory infection, 28 An observing physician may decide that a more frequent (such 29 as daily) or a less frequent (as every ^th day) may be lndi- 30 cated. - 19 - 33569/2 " : 0.,.' , ; .- ■ » ■ 12530 EXAMPLE 41 Eye Preparation For prophylactic and therapeutic use In the eyes, a conventional eye drop preparation such as sterile peanut oil is made up so that a single drop would contain from 0.01 to 10 milligrams of the complex polymer- Λ single drop would be applied . to the eye every 2 to 3 days.

The drops would be applied for its therapeutio activity to an eye infected with H rpes, Adenovirus and Vaooinia and also infected with Trachoma. It would be applied to the noninfected eye as a prophylaotio protection ■·. at the same time. , . ' A conventional eye ointment would be prepared so that the small amount ordinarily applied. would contain compiexed polymer, from 0.01 to 10 milligrams of the ■ This ointment, oould be a polyethylene-liquid petrolatum gel, for instance.

Cutaneous preparation For application to the skin in abraded areas, the interferon inducer can be added to conventional bases for ointments, creams, lotlona or liquid preparations so that : compiexed. polymer one gram contains from 0.1 to 100 milligrams ofj for example. It would be applied every ? to 3 days, | EXAMPLE .6' ! Sterile injection Representative of a preparation for* parenteral administration is a conventional sterile oily or aqueous solution or suspension, such as physiological saline. One oo of this would contain from 0.1 to 100 milligrams of the seleoted interferon inducer of this invention, and It would be' injected every 2 to 3 days in cases of viral infeotion, 33659/2

Claims (7)

1. Complexed polymers being double-stranded polynucleotide complexes composed of two different synthetic complementary homopolynucleotides and consisting of* (a) polyribonucleotide alternatin copolymers, or (b) complexes of two homopolyribonucleotides containing halogen or alkyl substituted nucleotide components, or (c) hybrids between a polydeoxyribonucleotide and a polyribonucleotide.

2. Complexed polymers according to Claim 1 composed of complexes of two different homopolynucleotides containing adenylic, inosinio or cytidylic acids of which at least one is halogen or alkyl substituted.

3. · A complexed polymer according to Claim 2, wherei the two complementary homopolynucleotides are polyribaiaoslnic acid and polyribobromocytidylie acid (rI:rBC).

4. A complexed polymer acoording to Claim 1 being an alternating copolymer e^ rlboinosinic and ribocytid lic acid (riCzrIC).

5. · A complexed polymer according to Claim 1 being an alternating copolymer of rlboinosinic and bromoribocytidylic acid. (rIBCirlBO).

6. A complexed polymer according to Claim 1, being a comple between PI bacteriophage DHA and RNA of complementary nucleotide sequence (PI DNA-R A).

7. Complexed polymers according to Claim 1, substantially as described herein with reference to the Examples. 8· A method of preparing a complexed polymer according to 33659/2 9., Pharmaceuti -cVal com-positions . comprising a. pharmace·u*tical carrier and a complex according to any one of Claims 1 to 7. 10· A method of stimulating the formation of interferon in the cells of living hoeta (other than man) in order to make them resistant to germicidal infection which comprises administering to the host a composition according to Claim 9· 11· Λ method of producing an interferon containing preparation comprising preparing a" culture of human or animal cells in a nutrient culture" medium and adding to the culture a completed olymer according to any of laims 1 to 7. For the Applicants DR.REINHOU) W AND PARTNERS By: