DE2001734A1 - Chemical processes and products - Google Patents

Description

Patent attorneys Dr. Ing. Walter Abitz Dr. Dieter R Morf Dr. Hans-A. Brauns 8MOOGiMn 86, Pi «nzensuerstr. 28

January 15, I97O 12530

MERCK & CO. INC. Rahway, New Jersey 07065 / USA

Cheoieohe processes and products

The present invention relates to the induction of the formation of substances which inhibit viral waobetun, for example of interferons in vivo and in vitro Use of complex polymers and the induction of resistance to viral infection in vivo and

009830/1828

in vitro by administering these complex polymers.

More particularly, the present invention relates to inducing interferon production and inducing resistance to viral infection by administration of complex polymers that are formed by a mixture consisting of two homopolynucleotides or composed of a homopolynuoleotide and a homooligonuoleotide.

In describing the present invention, the term interferons will be applied to the same active material "used in the literature with different expressions, how "virus-inhibiting factor" and "virus-inhibiting substance" are identified. The term homopolynucleotide is used in describing the present invention in the sense that it not only includes the homopolynuoleotides, but also includes the "homooligonucleotides", the only difference between which is the length of the polymer. That is to say, this term includes known polymers which have two to thousands of nucleotide base radical units. These known polymers are transformed into aur by the enzymatic treatment of nucleotide phosphate in vitro Formation of the usual polynucleotide with varying Number of Nuoleotideinheit manufacturedι the number of which is nioht can be specified with accuracy

In the chemotherapeutic virus research there is Hope to be an ohemlsohes remedy for viruses with broad Spectrum to be found «To date, the only known means with a broad spectrum are the Interferpne, dl · in blood and in the tissues of animals and men in the course of Viral infections and other infections occurred and an essential ingredient for the inhibition of viral diseases

009930/1828

seem to deliver that of the special immunological Mechanisms is separate and different from them However, no production of interferon has been achieved in practical amounts for use in chemotherapy. Another attempt at viral chemotherapy was the search for a chemically defined, non-toxic, non-antigenic, non-replicating, and readily available substance that animal cells can use to produce interferon stimulated, which then gives protection against viral infection «

Numerous interferon inducers have been described, including viable and inactivated viruses, Endotoxin, Phytohemagglutinin, Trachoma and Inclusion Conjunctivitis agents (TRIC agents), Brucella abortus, and others include · However, each of them has very serious drawbacks such as toxicity, formation of antigens and / or replicability, their practical suitability for the Treatment of viral diseases' limited.

It has now been found that high concentrations of interferons can be induced in a host animal by administering complex polymers which chemically defined, non-toxic, non-antigenic, non-replicative synthetic agents that are easily made available homopolynucleotides can be produced, the administration of the complex polymers can either parenterally, such as subcutaneous, intradermal, intraperitoneal, be intravenous, intramuscular, oral, or topical, preferably on a mucosal membrane such as intranasal and in the respiratory tract. These complex polymers are made as a mixture made from two different homopolynucleotides which are themselves synthetic and commercially available and can be produced by fixed working methods

009830/1828

12530

These synthetic polynucleotides are double stranded
(double-stranded) and represent the following:
(a) Complex product, formed between two homopolyribonucleotides (rI: rC, rA: rU, tI: tSÜ), self-complexing or alternating polyribonucleotide copolymers
(rIC, TiBG ₉ rAU) or complex product between a polyribonucleotide and a polyribonucleotide (Fl DNA-RHA) The following explanations apply to these symbols:

rltrC rAzrU rl + CpO

rIO

rlS? T rAU

- Complex of polyriboinosinic acid and polyribocytidylic acid

- Complex of polyriboadenylic acid and polyribouridylic acid

- Mixture of polyriboinosinic acid and
Cytidylyl cytidine

- Complex of polyriboinosinic acid and polyribobromocytidylic acid

- alternating copolymer of riboinosinic and ribocytidylic acid

- alternating copolymer of riboinosin- and bromo-ribooytidylic acid

- alternating copolymer of riboadenylic and ribouridylic acid

Yl DHA-RNA - complex between Fl-bacteriophage DKA and RXA

of complementary nucleotide sequence "

The bar on BO indicates Bronierung and changes ähnlioh

009830/1828

Halogenated components as well as alkylated components are encompassed by the present invention. Halogenation or short-chain alkylation of a component of the alternating Ribomiechpolymerisate or the complex Ribohomopolymerisate does not reduce the ability to induce interferon and host bed resistance "

The synthetic homopolynucleotides used in the production of the complex polymers have a Pentose phosphate skeleton, in which the pentose is preferably Ribose or deoxyribose is ^ and contain a specific identifiable pyrimidine or purine base, such as Adenine, Inοsine, Cytosine, Uraeil, Guanine and the like It is already known that mixing certain homopolynucleotides in aqueous solution to form a complex polymer leads, which can be identified by means of various physical tests and from each of the the two homopolynucleotides from which the complex polymer is formed is different. The ratio in which the two homopolynucelotides are mixed is usually not determinant of the ratio of the two Homopolynucleotides built into the complex · A mixture in a molar ratio of 1: 1 can result in a complex Lead polymer that the two nitrogen-containing bases in a ratio of 1: 1, 1: 2 and / or 2: 1 or contains in another ratio with small integers, i.e. the resulting ratio is not a function the ratio in which the components are mixed, but is determined by a certain natural tendency to unite, as is peculiar to the particular homopolynuoleotides used.

Complex polymers are formed by mixing one homo with another homopolymer, whereby

009130/1828

when the selected pair is complementary, for example a Homopolymer of adenylic acid mixed with a homopolymer of uridylic acid or a substituted, for example brominated, derivative thereof. Bas mixed Pair can also consist of inosinic acid and polycytidylic acid or their brominated or otherwise substituted derivatives exist · It is clear that substituted for guanylic acid Inosinic acid can be present because it is a derivative. These pairs are bonded by hydrogen bonding, as can be seen from the hypochromicity in the ultraviolet range. In addition, one of the components of the complex must be a purine polymer and the other component must be be a pyrimidine polymer to create an active complex.

The complex polymers used as interferon inducers in the present invention are prepared by synthetic methods such as those in Thaoh, RE, and P.Doty, 1965, Soienoe 147 t 1310, Krakow, JS and Myra Karstadt, PNAS £ 8, 2094 (1967) »Nilman G., Mangridge, R., HJ Chamber Hn ₁ PNAS £ 2, 1804-1810 (1967)," The Structure of a DNA-RHA Hybrid ". are written. The complex polymers are prepared by adding two dissimilar homopolynuoleotides in a molar ratio of 1i1 with respect to their bases for a few minutes at ambient temperature in an aqueous buffer system with a broad pH range, for example Ewioohen about 5.0 and 10.0, and an ionic strength between about 0.001 and 1.0 can be mixed.Other ratios ale 11 can, as already mentioned above, be used.Buffer eysteoses, which have all been found useful, are 0.006 mol sodium phosphate in 0.85 J ^ igtr, sodium chloride solution and 0 , 01 KoI Olyoylolyoin in Oi59 Jtig · »Natriuaohlorid. Xe everyone can nioht-toxieoh ·

009130/1828

Buffers are used. In fact, no buffer need be used as it has been found that the buffering capacity of the physiological system of the animal, the the mixture of homopolynucleotides is to be administered is sufficient to promote the desired complex formation after administration and to provide the same induction of interferon production and protection against viral infection as when the two homopolynucleotides are previously complexed. The present The invention accordingly also comprises the administration of the nucleotides to the host animal, including humans, which form the complex polymers in the respective body that serve as interferon inducers.

Another way of making the complex polymers that can be used is treatment a mixture of two nucleotide diphosphates or deoxynucleotide triphosphates, in a phosphate buffer with about, pH 7 with the corresponding ribonucleic acid polymerase or deoxyribonucleic acid polymerase. Such treatment leads to the polymerization of the two monomers two homopolynucleotides with simultaneous formation of the complex polymer.

The complex polymers formed in vitro can be characterized by hypochromic shift in the ultraviolet absorption spectrum, by sucrose density gradient precipitation, chromatography and the ability will induce the production of interferon, an activity that any of the homopolynucleotides lack which the complex is formed. The generation of interferon is the most important possibility for the characterization of the used in the method according to the invention complex polymers, since physical methods are often used

- 7 -009830/1828

have insufficient sensitivity

The generation of interferon by administering the complex polymers is shown by the protection of host animals and cell cultures from virus attack. The interferon generated in this way can also be obtained by isolating the induced interferon by known working methods and then determining in vitro its virus-inhibiting properties and characterizing it by host specificity, Trypsin sensitivity, isoelectric point and molecular weight determination can be characterized.

The induction of interferon formation in vivo according to the invention and / or the induction of resistance to viral infection is achieved by administering one of the above described manner produced complex polymers to a host animal, for example a rabbit, a mouse or another animal. The administration can be carried out parenterally or topically, in particular on a Mucous membrane, such as intranasally, or in the airways. The effective dose depends on the host species and, to some extent, on the virus against which protection is sought. In mice the threshold dose is about 0.5 mg / kg, while in rabbits it is about 0.05 / µg / kg · In these In doses there are no visible signs of toxicity, either locally or generally at the injection site Well-being of the entire animal.

The effectiveness of the complex polymers in terms of inducing interferon formation in the host animal can be determined, among other things, by parenteral administration of the complex polymer to an animal and taking blood samples after about 1 to 5 hours

- 8 -009830/1828

lized and titrated at several dilutions in culture tubes containing cells from the same animal species as used as the host. After incubation at 35 ⁰ C for about 18 to 24 hours, the cell cultures are exposed to attack by any of the known cytopathic viruses and again incubated for about 3 days * 'at 35 ⁰ C. The cultures are then examined for cytopathogenic effects and the interferon titer is determined as the reciprocal value of the dilution at which 50 % of the tubes show no such cytopathogenic effects.

The effectiveness of the complex polymers in inducing interferon production and host stability in a wide variety of living cell systems both in vivo and in vitro is evident from the considerable number of tissue cell cultures and hosts in which interferon can be produced. Representative for this are ^J chicken embryo, chicken chorioallantoic skin (chiok chorioallantoio membrane), which can be present in the egg as well as in vitro, monkey kidney cell culture, rabbit skin and rabbit testis in vitro, calf kidney cell culture, rabbit kidney cell culture, human annion cell culture, intraallantoic cell culture, MCN in the egg (MCN cell aulture), mouse fibroblast cell culture, i

human foreskin, chicken embryo cell culture, KB cell culture, mouse lung in vivo, adult mouse brain in vivo, chicken pibroblast cell culture, adult human thyroid or embryo lung or embryonic kidney cell culture, menechliobic leukocytes, mouse embryo lines 29 (3B cells) in Cell culture and primary mouse embryo cell culture can therefore be expected to occur in each case, either in the host or in a culture which is able to induce interftron transmission,

009130/1028

under the influence of the complex polymers according to the invention is induced to produce interferon.

Since the interferons generated according to the above method belong to a specific species, it is shown using an interferon stain reduction test, the incubation of an aliquot of the interferon-containing serum with individual cell cultures of different species, subsequent attack by a virus such as. vesicular stomatitis virus or other known m. cytopathogenic virus, and incubation to allow virus staining. The stain count on interferon-treated cultures is compared to that on untreated virus-infected control cultures. In the type tests it is observed that interferon activity occurs only in those cases in which the cell cultures jamming of the gleiohen Tierspeoies from which the inter feron-containing serum let been isolated. ',

That the interferons induced by the methods according to the invention using complex polymers are susceptible to trypsin, can be shown using an interferon fleece reduction test, ie "dl" interferon activity is detected by trypein treatment.

The interferon induced in the manner described above is also used as described in the examples is characterized by known methods in terms of the electrical point and the molecular weight.

The production of the la according to the invention is described below Process used co-plex polyaerizates veransohaulioht.

- 10 -

009130/1821

JJ

Production of complex polymers

SUBSTANCE It production of a complex from polyinosinic acid and polycytidylic acid, "Poly (I f C)"

A solution of polyinosinic acid (I) is prepared containing 550 µg / ml in a buffer which is 0.01 molar in glycylglycine and 0.1 molar in sodium chloride. Prepare a solution of polycytidylic acid (C) containing 500 / Ug / ml in the same buffer. The solution of the polyinosinic acid and the solution of the polycytidylic acid are then mixed so that there is a ratio of 1x1 with respect to the bases.The resulting solution is used directly as the source of the complex polymers, poly (I + C), also known as rlxrC, used. It has a melting transition midpoint (Tm) of 60.5 ⁰ , the ion strength is 0.1.

SUBSTANCE II: Preparation of a complex from polyadenylic acid and polyurldylic acid, "Poly (A + U)"

In accordance with the procedure described above for the preparation of substance I, but using equimolar amounts (with respect to the Baeen) of polyadenylic acid (A) and polyuridylic acid (U) instead of the polyinosinic acid and polycytidylic acid used there, the complex polymer poly (A + U) , which is also called ale rAtrU. It has a Tm of 56 ⁰ C, the ionic strength is 0.1.

- 11 -

009830/1828

12530

SUBSTANCE III: Preparation of a complex from polyinosine

acid and cytidylylcytidine "Poly (I + CpC)"

If the procedure described above with regard to the preparation of substance I is used, but the one there used polycytidylic acid by an equimolar amount replaced by cytidylylcytidine, an oligonucleotide, and a buffer system made up of 0.006 moles of sodium phosphate in 0.85 # saline solution, is used with pH 7.0, the complex polymer, poly (I + CpC), which is also called rI: rCpC, is formed.

If the procedure used for the preparation of substance I is used, but the polyinosinic acid and polycytidylic acid are replaced by equivalent amounts of other known homopolynucleotides, the following complexes poly (A + I), poly (G + C), poly (A + DHU), poly (A + 8U), poly (U + 8A) formed, where A, I, C and U have the meanings given above, (J polyguanylic acid, DHU polydihydrouridylic acid and 8A or # 8U polyadenyl or Mean polyuridylic acid, each containing eight nucleotide units per molecule. These same complexes can also be identified as rA: rI, ^ rG: rC , and so on.

SUBSTANCE IV: Manufacture of deoxyribohomopolymers

The process for the preparation of deoxyribohomopolymers has been described by RB Inman and RL Baldwin, in J. Mol. Biol. (1964) 8, 452.

- 12 -

009830/1828

Jfe

SUBSTANCE V: making a complex in which one of the

Components are chemically changed, for example rl; r! 5ci

The complex polymers rI: rSO * are according to one of DR Davies and AJ Rieh, in J. Amer. Chem. Soc ^ eo, 1003 (1958), prepared for the preparation of rl: rC procedure described, with the exception that in the synthesis of rB "(T 5-bromocytosine-5'-triphosphate is used. The complex has a . Tm of 89.5 ⁰ C in a buffer having an ionic strength of 0.1 This same procedure can be used, brominated or other complexes from other to obtain halogenated polynucleotides, for example rA. r5TT, rltrüTc *, etc. Similarly, the bases may have been chemically changed by alkylation in a known manner

SUBSTANCE VI: Preparation of a complex from alternating polyribonucleotide copolymer, rIC: rI0

The alternating copolymer is made according to the method described by JS Krakow et al. produced in the above-mentioned magazine article ■ described procedure. The complex has a Tm of 64 to 65 ⁰ C in a buffer with an ionic strength of 0.1.

As noted in the referenced article, these alternating complexes are synthesized using an enzyme made from microorganisms called RNA polymerase. The polymers are in a ratio of 111 and are double-ranked due to the hydrogen bond of the alternating complementary bases . These materials can be made using

- 13 -009830/1828

12530

this enzyme can be produced in the presence of a template, which may be dAT or heat-denatured DNA, of course, dAT is preferably used. These alternating Copolymers are not limited to regularly alternating copolymers, but include also irregularly alternating copolymers, as long as the components are present in a certain ratio. Other alternating copolymers are produced similarly, for example rAUxrAU and the like.

SUBSTANCE VII: Preparation of a complex from alternating ribonucleotide copolymer using a halogenated ribonucleotide

An example of this is rl5?: RlBl ?, made according to the procedure in the article by JS Krakow et al. ^{It has a Tm of 91 0} O in a buffer with an ionic strength of 0.1. Other examples are rABl?: RAW, rlClCsrluIB 'and the like.

SUBSTANCE VIII: Making a complex between a

Polydeoxyribonucleotide and a nucleotide Polyribo

W An example of this double-stranded complex is P1 DttA-RNA, which is a complex between F1 bacteriophage DNA and an RNA with complementary nucleotide sequences. The PI bacteriophage DNA is used here as a template for enzymatic RNA synthesis. The process for making these and similar double-stranded hybrids is described in the above-mentioned journal article by O. Milman • t al •. The complex Pl DNA-RNA has a Tm of 82 ° in a buffer with an ionic strength of 0.1

- 14 -

009130/1828

12530

The following examples are intended to demonstrate the induction of interferon by administration of certain of the above-described Show homopolynucleotide complexes both in living host animals and in isolated cell cultures, However, other complex according to the invention, poly. merisate can be used to stimulate interferon production in a similar way

example Induction of interferon in rabbits

The complex polymers described above are separated as 0.5 ml aliquots by intravenous Injection in rabbits weighing 2.04 to 2.27 kg (4.5 to 5.0 ' pounds). Kach about 2 hours will be from each Blood samples drawn from rabbits by cardiac puncture. Serum is separated from each of the clotted blood samples and separately sterilized by exposure to ultraviolet radiation. Rope aliquots from these sterilized specimens are used in the following tests.

Determination of interferon titers

The sterilized rabbit serum from each rabbit is titrated separately by serial two-fold dilutions from 1: 5 to 1: 640 using cell culture growth medium as the diluent. A 1 ml sample of each dilution is added to each of four renal cell tube cultures from rabbits of spent guard tumsmedium

- 15 009830/1828

have been drained. After overnight incubation at 35 ⁰ C, the tube cultures are again flow ge blank and that in 1 ml of growth medium is contained at 10 to 100 TCID ^ q (tissue culture infecting dose γλ) vesicular stomatitis virus infected. Each tube culture is incubated for an additional 3 days at 35 ⁰ C and then analyzed in regard to the occurrence of cytopathic viruses effects and counted with (+) with positive occurrence of such effects or with (0) in the absence of the occurrence of cytopathic effects. The interferon titer for each serum sample is determined as the reeiproke value of the serum dilution at which 50 # of the tubes show no cytopathogenic effects. Serum from untreated animals (ie, from normal control animals) is titrated in each experiment to determine normal serum factors. The titers thus determined are shown in Table I.

- 16 -

009830/1828

12530

TABLE I. Interferon titers, determined in rabbit kidney cell cultures

chemical agent

Dosia / animal

I + C (rI: rC) 2 / Ug Il 0.5 / Ug Il 0.25 / Ug Poly I. 25th / Ug Poly C. 25th / Ug normal Comparative samples no

Interferon titre

> 640 20-40

<5

<5

<5

<5

A + π (rA: rTJ)

Polyadenylic acid
Polyuridylic acid
normal comparison sample

200 / Ug 100 / Ug 50 / Ug • 25. / Ug 12.5 / Ug 6.25 / Ug 200 / Ug 200 / Ug

no

10-20 10-20 40-80 20-40 5-10 5-10 <5 5 <5

Poly 1 + CpC (rlirCpO)
η

normal comparison sample

100 / Ug 10 / Ug

1 / ug

50 / Ug

none > 640 <5 <5

<5

< 5

- 17 -

009830/1828

In a test based on example 1 it is found that the following amounts of the complexes, when injected intravenously per rabbit, will generate induce detectable concentrations of serum interferon in two hours:

• O | 5 / Ug rI: rC, 25 / Ug rA: rU, 100 / Ug rI: rCpC, 10.5 / Ug rI: rSc \ 10 / Ug rIC: rIC, 12 / ug rlEc ": rIB" c "and 5 / Ug Pl DNA-RNA. For some of these substances the quantities given for induction are not the minimum quantities

Example 2

Induction of interferon in mice

A solution of poly (I + C) (substance I) as well as solutions of folyinosinic acid and polycytidylic acid are administered separately in 0.2 ml aliquots by intravenous injection to mice of 16-18 g. Each solution is thus administered to 25 mice administered. After about 2 hours, blood samples are drawn from each mouse. From each of the | Serum is separated from clotted blood samples. Sera from those animals injected with an aliquot of the same polynucleotide or the same complex polymer solution are pooled and sterilized by exposure to ultraviolet radiation and titrated against attack by veeicular stomatitis virus by the stain reduction method on mouse embryo cells. Combined · Stra from untreated animals are titrated tbeneo.

- 18 -

009130/1128

! EABELlE II

Stimulation of interferon production in mice, determined by the peck reduction method

Chemical agent dose / animal interferon titer

Poly I 55.0 / µg <8

Poly C 50.0 / µg <8

Poly I + C (rI: rC) 52.5 / Ug 256

normal buffered
Comparative diluent - <8

example

Induced Resistance to Columbia SK Virus Infection in Mice

Infection by Columbia SK has in mice, the symptoms of a ruffled fur, lethargy and weak paralysis result, the majority of the animals 3 dies until days after the injection · To determine the effectiveness of the complex polymers with regard to the induction of a protective The following experimental procedure is used for the amount of interferon:

0.5 ml of the test solution of the complex polymers of the homopolynucleotide identified in Table III are

- 19 -009830/1828

12530

administered peritoneally 18 hours before infection to 15 homes each, each weighing between 14 and 16 g. Then, an amount of Columbia SK virus, sufficient 90 i "of the mice to kill 5 days after infection, injected subcutaneously in a 0.5 τώΙ aufmachenden aliquot and each Ha-us then with 0.5 al of Test solution treated _? ά: e ir, tracer ;, toncal 3 hours after infection injirie: - '■■■■: .. ■ ■■ _: Zv, "· o: /:"!e.\ciien Y / r; ire rait complexem Polymer! υ α ;, ', ο 3 "■' :: ■ Ο; ■■ λ. ■ ■. Vox ^ d bh

j e 1 ". '.: ■ 5 However not in Hi ηr I '. ·? ;; on d η ο : ' ^: '"'(· - ■"I' .: ·: " ^ • -; c Toxinitä i-f ^; -

brob

.: '<on yours

IU V:

The ZoI '.: wirö t ^: -;, ' \ obacht et

Mejr-i XiT ■■; üie. "ahj. of the dead animals 7> ± e animals are loaded for 0 days

- 20 -

009830/1828

12530

TABLE ^TTT

XXi.

Induced resistance or resistance to Columbia SK virus infection in mice

Chemical agent

Total dose ° J> per day by average animals living per animal surviving animals

Pho sphat buffer «Te mg 3.3 4 ₃ 7 Polyinosinic acid 0.91 Sg 0.0 4.5 Polycytidylic acid 1.00 mg 0.0 4.2 Poly I + C (rI: rC) 1.05 rag 40.0 8.5 Poly I + C (rI: rC) 0.53 ag 20.0 7.1 Poly I + C (rI: rC) 0.26 6.7 6.0

If the same working method is used : ■ wiiv * ^ iν with the exception that the post-infection uosis wi ^ elaBer ;. results are obtained which are comparable to the results given in Table III wi? = · ^.

In other tests it is found that oe3 Kliuac * a: & er t -. 'Legends Combination against the Columbia SK virus gc-achutr.-i; will?

rlirC, intranasal and intranasal virus attack,

rI: rC, administered intraperitoneally and Yirysaiicrifi intraperitoneally,

rltrOf administered intraperitoneÄl νινί Yi.iUB & K ^ r-iff ir.-fcra-

009830/1828

In a similar test, a dose of 16 mg rI: rC administered intraperitoneally per mouse was found to protect it against Sendai virus.

Example 4

Induced resistance to Pneumonia Virus (PVM) Infection in mice

Infection of mice by intranasal inoculation from mouse pneumonia virus (PVM) has a viral infection of the respiratory tract, which culminates in pneumonia, with 6 to 7 in the majority of animals Death occurs days after infection.

Solutions of the complex polymers and of hemopolynucleotides are tested for their ability to provide prophylactic protection against PVM infection by pretreating 20 mice with 8 to 10 g intranasally with 0.03 ml of the solution containing the test material given in Table IV, before inoculating with the virus intranasally. There is plenty of used virus kill by 75 i »the Mäusepopulstiön 6 to 7 day after inoculation. At the end of the experiment (after H days) 59 of the 60 infected but untreated mice died of the virus infection.

The number of living and the number of dead animals is determined daily. The animals throw H days observed *

- 22 -

TABLES

Induced Persistence vsn i! Är ~ * ng eg entity tion slit Ma

Chemical β middle!

Poly I f C (rj.:r·.

It is also found - "a2 K-urr- ßdrovi an attack by Vaccinia Virus; .-: - jüist cLnc a prophylactic treatment either or subcutaneously rlsrC is administered.

- 23 -

009830/1828

12530

example

In vitro activity of complex polymers as viruses inhibiting substances

Each of the cell cultures identified in Table V is flattens with a complex of polymers, diluted in the Wa:; hstumsmedium, white from the one that for the verv ^f i = -, is required AETE cell culture incubated. After the complex polymer-containing solutions have been removed , each culture is infected with vesicular stomatitis virus suspensions, incubated and observed with regard to the “yeast formation” described for the test beforehand. that shows kind specificity

TABLE V

/ ug dose of the complex polymers / ml that is required let to induce resistance to VSV staining

Cell culture

primary rabbit kidney primary human amnion primary human embryonic kidney primary chick embryo primary bovine kidney primary canine kidney primary β mouse embryo

Poly I + C Poly A + U > 100 - (rI: rC) (rA: rU) <0.00125 0.0015 0.04 26.25 1.25 - 0.33 5.00 1.25

> 100

- 24 -

009830/1828

12530 5

The relative effectiveness of the complexes compared to their uncomplexed nucleotides is shown in Table Va using this same test.

TABLE Va

Concentration of complex polymer that is required is to induce resistance to VSV staining in primary rabbit kidney cell cultures

Polymer / µg / rol

Poly I> 11

Poly C> 10

Poly I + 0 (rI: rC) 0.091-5

Poly A ':

Poly U ': -10

Poly A + U (rAsrU) 0.0015

rI: rB "C * 0.08

(not the minimum quantity)

rIC: rIC 0.003

rTSffjrTBc * 0.04

rAUirAU 0.04

PI DHA RNA 0.6-1.25

009830/1828

12530

If the procedures described in Examples 1 to 5 are used, however, substance IV or the complexes described after the description of substance III If polymers are used for substances I, II and III used here, similar results are obtained obtain·

example

Evidence of the possible therapeutic effect of the intranasally administered interferon inducer rI: rC against intranasal attack by mouse pneumonia virus

Mice are injected by the intranasal route with Pneumonia virus and subsequently, 48, 72 and 96 hours after the attack be 24 33 Mg (rl: rC) intranasally administered · ¹ for each day of the attack control animals are kept. The results are shown in Table VI, from which it can be seen that the interferon inducer rI: rO has a significant effect as a therapeutic agent for about 72 hours after attack by Pneumonia · * virus.

- 26 -

12530 3Λ

TABLE YI

Therapeutic efficacy de ¹ :: rlirO-Iiid / L ... 1 .-- i * ·.;: .. ■. ■ on virtues resistance in mice against-.u. PYM-Arigr '■. fi!

: g kit rlsrO * according to. 5a I ¹ I he j

24 stubborn.
46 hours

72 hours
95 St "ut: d yn

* rIirO

A.E / rrir

Example

Demonstration in animal experiments of the duration of the snow from Mice against the attack by Pneumonia viruses - I one single intranasal dose of interferon inclusion? - by means of

All animals receive a dose of 32 / ag rI: rC intranasally. Equal animals are kept separate for each period of attack. Animals are challenged with mouse pneumonia virus 3 hours, 4 days, 5 days, 6 days, 7 days and 8 days after the inducer. The results are shown in Table VIX. It can be seen that a single dose of 32 / ug per home for a

- 27 -

009830/1328

provides excellent protection against virus attacks over a span of 6 days.

TABLE YII

Duration of the protective effect of inducing host stable
ability in mice against intranasal attack by PVM virus io survivors versus
Comparison animals Attack time according to rI: rC * 100 3 hours 71 4 days 100 5 days 88 6 days 24 7 days j 24 8 days

* 32 / ug rlsrC ι All animals received 1 dose intranasally 3 hours before the first attack.

- 28 -

009830/1828

B is ρ ie 1 8

A test for overcoming the ferocity of the virus attack, if rI: rC, administered intranasally in mice, induces resistance or resistance is

All animals treated in this experiment are administered 16 / Ug rI: rC 3 hours prior to attack with measured doses of pneumonia virus. The attack ranges from 31 to 10,000 LDcQ · The attack is also intranasal. The results are given in Table VIII and it can be seen that a dose of 16 / µg rI: rC administered intranasally 3 hours prior to attack induces excellent resistance to attack of at least 10,000 LDc ₀ ·

- 29 -

009130/1828

12530 "J Q

Table VIII

Determination of the degree of effectiveness of host stability against PVM induced in mice by rI: rC

Survivors

attack in I »Dcq 10 000 3 150 1 000 315 100 31.5

93 73 87

100 93

100

* Mouse Pneumonia Virus, all animals receive 16 / Ug rI: rC 3 hours before the attack.

Identification of interferons

The interferons are identified as relatively low molecular weight protein molecules that have the ability to Interfering with virus replication only in cells of the same species from which the interferons are generated. For characterization of the interferons one uses the proof of the species specificity, r the antivirus activity, the trypsin sensitivity (a test for proteins), the isoelectric point and molecular weight determinations.

The following tests are used to identify the active ones Virus-inhibiting substances as interferons.

About the species specificity of induced interferon

Samples of 3 ml of two- fold serial dilutions in the culture medium of the interferon samples from Beisr'tl 1, the duroh exposure

- 30 -

009830/182

sterilized by ultraviolet radiation are added to monolayers of different cell types grown separately in 30 ml tissue culture flasks. After overnight incubation, the serum samples are withdrawn and replaced by 0.5 ml veeikuläre Stromatitisvirussuspension and the cultures are again for v / urther 1 ^ 5 hours at 35 ⁰ C. Then a top coat is in front. 5 ml Aufrechterhai tung culture medium (maintenance culture ^ ediuro) containing methyl cellulose as a solidifying agent eiitMlt, in; each flask and the incubation is i'u ^ _ν: ⁴ tere 3 to 4 days at 35 ⁰ C continued to Yirusfieo ^! ?. allow education au. The overlay medium is then entered \ mä the cells are stained with carbolfuohsin, the Ηθίΐο treated with interferon. ·· .αίο ^ν ^ number of spots of untreated Viruv-IiK.

monoechichteri compared «Oie reciprocal In the one at least 50 i: .. f: 3 Verruinderun ?, -I results, as the Ititerferon titer the?

The titers determined in this way show species specificity that interferon induced in a certain species is only active in cells which come from an animal of the same species . These titers are shown in Table IX.

j .. _s ::: i <: eri ^ ahleTi de ή one f; "" - 'we . : '; ϋ j / •-I ... it ; r <rmng t; ■■ £ ■ '■ , "^ - : crö \ ' Ah]. <■> ■ ' JL-'' i-s.iaur _-mind. f. ₍ > - ,. " in ät, d f

- 31 -

009830/1828

12530

Table IX

Species specificity of the induced interferons in tests with an attack by vesicular stomatitis virus

Serum-
source Interferon titer, determined atff
Culture <16 Rabbits
kidney the line chemical
middle Chicken mice
embryo embryo 128 > 2048 BSC *
monkey Rabbits _ <8 <16 <16 I + C mouse <8 <8 512 - I + C Rabbits <32 <16 > 2048; - A + U Rabbits - <16 - I + CpC Rabbits - <16 untreated
delt

♦ BSC: A detected cell line produced by kidney cells from African green monkey.

Evidence of the trypsin sensitivity of the induced interferon8

An interferon-containing serum from animals which have been induced with a complex polymer is isolated by chromatography on CM-Sephadex (a cation exchange material which is obtained by introducing carboxymethyl groups into Sephadex). A sample of the isolated interferon is treated with a crystalline trypsin (50 / ug / ml final concentration) for 4 hours at 35 ⁰ C. A similar non-treated interferon sample is just the case for 4 hours at 35 ⁰ C. After the 4 hour incubation period, a soybean trypsin inhibitor is added to each sample including the control sample. The samples are naoh for their interferon activity

- 32 -

009830/1828

titrated using the stain reduction method. The trypsin solution, to which the soybean trypsin inhibitor has been added, is also titrated for its antiviral activity. The results of the trypsin susceptibility test are summarized in Table X.

T a b e 1 1 e X *

Trypsin sensitivity of the induced interferon

Treatment of interferon titers

Poly (l + C) induced interferon + trypsin 16 Poly (I + C) induced interferon 256

Trypsin comparison sample 16

Poly (I + CpC) induced interferon + trypsin 8 PoIy (I-J-CpC) induced interferon 1024

Trypsin comparison sample,. . <8,

Determination of the molecular weight of the induced interferon

The molecular weights of the interferon induced by the complex polymers are determined using the following method. Columns 2 35 cm in size are packed with hydrated Sephadex G-200 spheres and a 0.006 M sodium phosphate / 0.15 M NaCl buffer (pH 7) is slowly trickled through for 2 to 3 days to achieve equilibrium (Sephadex is a hydrophilic insoluble substance formed by crosslinking polysaccharide dextran. The designation "G-200" refers to the extent of crosslinking and therefore the porosity of the hydrated gel). 1 ml amounts of the serum which contain induced interferon, which has been prepared according to Example 1, is applied to the columns. The flow velocity in the column

τ 33 -

009830/1828

is adjusted to 20-25 ml per hour, with fractions being collected in 3 ml amounts. The fractions are examined for their interferon content by the ^{stain reduction method, the molecular weight of each sample being calculated according to the formula M 1} ^ ⁵ = 146 [1.480 - V / Vo ¹ ^ ⁵ ], where "M" stands for the molecular weight, "V ^{n is} the elution volume of the material tested and" Vo "represents the empty volume of the column. This method gives values that are within 10 $ of the molecular weights reported when tested with purified proteins.

The results of the molecular weight determinations are like | follows:

Rabbit interferon molecular weight poly (I + C), induced 49,000 to 52,000

Determination of the isoelectric point of interferon

The serum samples which contain interferon, which has been prepared according to the method described in Example 1, are dialyzed overnight against a 0.1 M sodium phosphate buffer (pH 6 or simply diluted 1: 3 with buffer). 20 ml of each sample are applied to a 1.5 x 10 cm CM-Sephafc dex column which has been brought to an equilibrium state using the same buffer. The interferon is eluted by successive addition of 5 ml amounts of 0.1 M sodium phosphate with increasing pH value by 0.2 units. The effluent is collected in 5 ml fractions, and the pH and interferon activity of each fraction are measured. The pH of the peak activity fraction is recorded and the isoelectric point is calculated by adding 0.4 pH unit . The electrical point determined in this way for interferons for the administration of in a rabbit

- 34 -

008130/1828

₁₂₅ 3o ■

\ ~? ply (I + C) has been determined, contributing 6.8 to 6.9.

The above description of the manufacture of the complex Polymers and the examples illustrate certain features of the present invention. The inventive method is but not limited to these examples. Rather, it can also be carried out with complex polymers consisting of known homopolynucleotides, homooligonucleotides or their deeoxy analogs have been prepared and described in the above Administered parenterally or topically.

Example 9 Oral-nasal preparation

As a prophylactic intranasal preparation against viruses of the Respiratory like the common cold, a dose of 0.01 to 10 mg of the complex polymer rI: rG every 2 to Applied for 3 days to the nasal and oral parts of the skin. These Administration period is based on the prolonged duration of the induced interferon.

The application can be carried out by means of an aqueous suspension in an aspirator spray, so that one or two sprays release about 0.01 to 10 mg. It can also be aerosolized using conventional chlorofluorocarbons materials are produced and used as propellants. In addition, the preparation can be used as a pharmaceutical nasal drop liquid.

This preparation is also used therapeutically about every 2 to 3 days in the event of an actual infection of the respiratory tract. It is up to the doctor to decide whether more frequent (e.g. daily) or less frequent (such as every 4th day) administration is indicated.

- 35 -

009830/1828

One of the other inducers according to the invention can replace the rlrrC of the present example, their relative activity discussed above is to be taken into account.

Example 10

Eye preparation "*

For prophylactic and therapeutic use in the eyes, a conventional 'eye drop preparation, such as sterile peanut oil, prepared in such a way that a single drop of 0.01 to 10 mg of the complex polymer rlrrC or another of the contains the above inducers. A single drop is applied to the eye every 2 to 3 days.

^ Drops are used because of their therapeutic activity applied to an eye infected with herpes, adenovirus and vaccinia and also with trachoma. It will be at the same time applied as a prophylactic protection to the non-infected eye.

A conventional eye ointment is prepared so that the small amount usually used contains 0.01 to 10 mg of the rI: rC. This ointment can for example be a polyethylene / liquid petrolatum gel. _:

Example 11 Skin preparation

For application to the skin in damaged areas, the interferon can InduzierungBmittel conventional bases for ointments, creams, or liquid preparations frötions derjärt 'are BUgesetzt that 1 g of 0.1 to 100 mg, for example, contains rlirC. Eb is applied every 2 to 3 days.

- 36 -

009830/1828

Example 12 Sterile injection preparation

A conventional sterile solution or suspension-on oil- is representative of a preparation for parenteral administration.

or water-based, such as physiological saline solution. A ■
■ 5

cnr thereof contains 0.1 to 100 mg of the selected interferon inducer according to the invention and is injected every 2 to 3 days in cases of viral infection.

- 37 -

001830/1828

Claims (1)

12530 January 15 2 # 8 1 7 3 A PATENT CLAIMS 1. A method for inducing the formation of interferon in living cells, characterized in that one of the Cells administered a complex polymer, which under polynucleotide-alternating Copolymers, complexes of two homopolynucleotides, the substituted nucleotide components contain, and hybrids between a polydeoxyribonucleotide and a polyribonucleotide, the substituted nucleotide components is selected. 2. The method according to claim 1, characterized in that the bonding of the pairs takes place by hydrogen bonding, such as which shows Hypochrom! quote in the ultraviolet range. .3. A method according to claim 1, characterized in that the cells are in a living host. ·, 4 * The method according to claim 1, characterized in that the cells are present in a living host and that the 'complex Polymer is administered systemically. 5. The method according to claim 1, characterized in that the cells are in a living host "and the complex Polymer is administered topically. 6. The method naoh claim 1, characterized in that the cells have been separated from a living host and are in a culture medium. 7. The method according to Anspruoh 1, characterized in that the complex polymer consists of complexes of two different homopolynucleotides which are substituted · Nuoleotidein- - 38 - OFUGWAL INSPECTED 001130/1828 contain units selected from substituted adenylic acid, substituted uridylic acid, substituted inosinic acid and substituted ytidylic acid. 8. The method according to claim 1, characterized in that only one of the polynucleotides is chemically altered. 9. The method according to claim 1, characterized in that the complex polymer is rI: rBC. 10. The method according to claim 1, characterized in that the complex polymer is rICrrIC. 11. The method according to claim 1, characterized in that the complex polymer is rIBCYrIBC. 12. The method according to claim 1, characterized in that the complex polymer Pl is DNA-RNA. 13. Pharmaceutical agent, characterized in that When administered to living cells, it induces the formation of interferon in the cells and is a pharmaceutical Carrier and a complex polymer comprises, among polynucleotide-alternating copolymers, complexes of two homopolynucleotides containing substituted nucleotide components and hybrids between a polydeeoxyribo- " nucleotide and a polyribonucleotide containing substituted nucleotide components is selected. . ■ 1.4. Mittel.nach claim 13 »characterized in that the complex polymer consists of complexes of two different homopolynucleotides which contain substituted '' ~~ .. nucleotide units, selected from substituted adenylic acid, substituted uridylic acid, substituted inoic acid and substituted cytidylic acid · - 39 - 009830/1828 HO 15. Composition according to claim 13, characterized in that only one of the polynucleotides is chemically changed. 16. Composition according to claim 13, characterized in that the complex polymer is rI: rBC. 17. Agent according to claim 13, characterized in that the complex polymer is rIC: rIC. 18. Agent according to claim 13, characterized in that the complex polymer is rIBC: rIBC. 19. Agent according to claim 13, characterized in that the complex polymer P1 is DNA-RNA . - 40 - 009830/1828