U.S. patent number 3,843,629 [Application Number 05/263,677] was granted by the patent office on 1974-10-22 for complexs of polyriboinosinic acid and polyribo-2-thiocytidylic acid.
This patent grant is currently assigned to Merk Patent Gesellschaft mit beschrankter Haftung. Invention is credited to Peter Faerber, Karl Reuss, Otto Saiko, Karl-Heinz Scheit.
United States Patent |
3,843,629 |
Scheit , et al. |
October 22, 1974 |
COMPLEXS OF POLYRIBOINOSINIC ACID AND POLYRIBO-2-THIOCYTIDYLIC
ACID
Abstract
Two-component complexes consisting of a polyriboinosinic acid of
the formula ##SPC1## 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 ##SPC2## 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.
Inventors: |
Scheit; Karl-Heinz (Darmstadt,
DT), Faerber; Peter (Darmstadt, DT), Reuss;
Karl (Darmstadt, DT), Saiko; Otto (Darmstadt,
DT) |
Assignee: |
Merk Patent Gesellschaft mit
beschrankter Haftung (Darmstadt, DT)
|
Family
ID: |
25761303 |
Appl.
No.: |
05/263,677 |
Filed: |
June 16, 1972 |
Foreign Application Priority Data
|
|
|
|
|
Jun 19, 1971 [DT] |
|
|
2130544 |
Apr 14, 1972 [DT] |
|
|
2218057 |
|
Current U.S.
Class: |
536/25.5;
536/28.5; 536/27.8; 435/5; 514/889 |
Current CPC
Class: |
C07H
21/00 (20130101); Y10S 514/889 (20130101) |
Current International
Class: |
C07H
21/00 (20060101); C07d 051/52 (); C07d
051/54 () |
Field of
Search: |
;260/211.5R |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Brown; Johnnie R.
Attorney, Agent or Firm: Millen, Raptes & White
Claims
What is claimed is:
1. A two-component complex of a polyriboinosinic acid of the
formula ##SPC5##
wherein k is an integer from 1 to 3, inclusive; and b is an integer
from 1 to 2,000, inclusive; and of a polyribo-2-thiocytidylic acid
of the formula ##SPC6##
wherein m is an integer from 1 to 3, inclusive; and n is an integer
from 1 to 2,000, inclusive; and the physiologically acceptable
metal and ammonium salts thereof.
2. A two-component complex according to claim 1 wherein k is 1.
3. A two-component complex according to claim 1 wherein m is 1.
4. A two-component complex according to claim 1 wherein both k and
m are 1.
5. A two-component complex according to claim 1 wherein b is
greater than 10.
6. A two-component complex according to claim 1 wherein n is
greater than 10.
7. A two-component complex according to claim 1 wherein b is
greater tha 100.
8. A two-component complex according to claim 1 wherein n is
greater than 100.
9. A two-component complex according to claim 1 wherein both b and
n are greater than 100.
10. A two-component complex according to claim 4 wherein both b and
n are greater than 10.
11. A two-component complex according to claim 4 wherein both of b
and n are greater than 100.
Description
BACKGROUND OF THE INVENTION
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. 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.
Thus, a compound which stimulates the production of interferon can
be used as a broad spectrum antiviral agent.
It is an object of this invention to provide novel antiviral
polynucleotides, a process for their preparation, and a method for
the treatment and prevention of viral, bacterial and protozoal
infections by administering these polynucleotides. Another object
is to provide a process for the preparation of interferons which
comprises treating cell cultures with the polynucleotides according
to the invention and isolating the interferon thus produced. Other
objects will be apparent to those skilled in the art to which this
invention pertains.
SUMMARY OF THE INVENTION
The novel polynucleotides of this invention are two-component
complexes (hereinafter referred to as AB) consisting of a
polyriboinosinic acid (hereinafter referred to as A) of the Formula
I ##SPC3##
wherein k is an integer from 1 to 3, inclusive; and b is an integer
from 1 to 2,000, inclusive; and a polyribo-2-thiocytidylic acid
(hereinafter referred to as B) of the Formula II ##SPC4##
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, i.e., of the A component, of the B component and of
both.
In its process aspect, this invention relates to a process for the
preparation of the two-component complexes AB according to the
invention which comprises (a) admixing an aqueous solution of the
homopolynucleotide A and an aqueous solution of the
homopolynucleotide B at a temperature of from 10.degree. to
95.degree. C., or (b) producing one of the homopolynucleotides A or
B in a reaction mixture containing the other homopolynucleotide, or
(c) treating an aqueous solution containing one of A and B in the
homopolynucleotide form, and the other in the form of their
monomeric nucleoside diphosphate or triphosphates, with a
polymerizing enzyme.
In a method of use aspect, 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. Thus, the two-component complexes of this invention are
useful for protecting cells from virus infections both in vitro and
in vivo. Further, administering AB or a metal or ammonium 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, for example, to an
increased production of antibodies. This effect can be used to
improve the results of vaccination with vaccines which do not
contain reproducible germs or viruses. The production of antibodies
triggered by a vaccine can be magnified by simultaneous
administration of AB or a metal or ammonium salt thereof. Thus, a
satisfactory vaccination can be achieved with a smaller than usual
amount of vaccine. Vaccination with the usual amount of vaccine
will result in an increased production of antibodies.
DETAILED DISCUSSION
Of the two-component complexes AB of this invention, including the
metal and ammonium salts thereof, preferred are those wherein:
a. k is 1;
b. m is 1, preferably wherein both k and m are 1;
c. b is greater than 2, preferably greater than 10, most preferably
greater than 100;
d. n is greater than 2, preferably greater than 10, most preferably
greater than 100;
e. 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 1, 2, or 3 of the
alkyl groups is substituted by a .beta.- or .gamma.-hydroxy group,
e.g., trimethyl-, triethyl-, monoethanol-, diethanol-, triethanol-
and tri-n-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. However, because
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. Thus,
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 molecular structures of
the two-component complexes AB according to the invention. As the
stoichiometric composition of the active complex molecule is not
precisely known, it may well be that 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, however, 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. Thus, no T.sub.m (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.degree. C. Further,
the two-component complexes AB remain effective after a 24 hour in
vitro treatment with human serum.
In the preparation of the new two-component complexes usually at
least one of the homopolynucleotides A and B is used as starting
material. 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 di- or
triphosphates with a polymerizing enzyme.
The preferred method of preparing the new two-component complexes
AB comprises combining separate aqueous solutions of the
homopolynucleotides A and B at a temperature of from 10.degree. to
95.degree. C. This reaction is usually carried out in the presence
of one or more inorganic and/or organic salts, preferably an alkali
metal salt, e.g., sodium chloride, in order to maintain a definite
ionic strength of preferably from 0.001 to 1. The pH value of the
solutions of A and B must be from 5.0 to 11.5, preferably from 6.5
to 11.0. In order to maintain a constant pH value, 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.
Optionally, an organic solvent miscible with water can be added,
e.g., a mono- or polyvalent alcohol, e.g., methanol, propanol,
ethylene glycol and glycerin, or an aprotic dipolar solvent, e.g.,
dimethyl sulfoxide, formamide and dimethyl formamide.
A reaction mixture with the desired 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. Of course, it is also
possible to combine odd volumes of solutions which are different in
the concentrations of A and B, and in their ionic strengths, in
such manner that the reaction mixture eventually has the desired
ionic strength. In this case, it is also preferred that 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 independent 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. Thus,
in an aqueous solution containing 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. 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.
A variant of the above-described process comprises treating an
aqueous solution containing one of the homopolynucleotides A and B,
and the monomeric nucleoside di- or 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.degree. to 80.degree. C., preferably 20.degree. to
45.degree. C., especially at about 37.degree. C. Buffer substances,
e.g., tris(hydroxymethyl)-aminomethane ("tris"), ammonium
carbonate, or sodium cacodylate, are advantageously added. It is
also advantageous to add an inorganic salt during the
polymerization, for example, magnesium chloride, calcium chloride
or manganese (II) chloride. The polymerization is normally
terminated after about 1-72 hours.
Homopolynucleotides A or B with quite narrow mol weight ranges are
obtained e.g., by gel filtration. By selecting appropriate
fractions of the filtrate the polymers with b or n greater than 100
are separated from those with smaller b or n values.
Within a wide range, 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 generally is due to that structure of the complex
which is the most stable under the physiological conditions in the
organism of the host, i.e., the buffer capacity and the salt
content of the physiological system of the particular vertebrate
treated, which determine a definite pH value and ionic strength. An
AB complex produced under non-physiological conditions may possibly
be rearranged to form the active complex in the host organism, or
in the cell culture grown under physiological conditions. Thus, it
is theoretically possible to bring about a protective effect by
separate administration of A and B, if the two components are
administered in a manner whereby the active complex AB is formed in
situ in the organism or cell culture, respectively.
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 concentration of
50 percent of that of the complex. The presence of AB is preferably
proven by the determination of the hyperchromic shift at some
different wavelengths. Other physical methods for the
characterization of AB include the determination of the ORD
spectra; of the Svedberg values s.sub.20,w ; of the T.sub.m 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 density-gradient fractionation; and
chromatography.
It is, however, preferred to establish the presence of the new
complexes AB of this invention by biological methods, e.g., by
their ability to induce the production of interferon. This is the
most important method of characterization of the new complexes
because its sensitivity is greater than the physical methods
mentioned above. It has been established that neither of the
homopolynucleotides A and B separately exhibit interferon inducing
activity under the test conditions described in the examples.
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.
Thus, 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.degree. to
37.degree. 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.degree. to
37.degree. 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. At this state, 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.
In analogously conducted experiments using different types of cells
of different animal species, and also other viruses, it was proven
that the protective effect of the novel complex is neither
associated exclusively with a particular kind of cell nor limited
to a particular species of virus.
In order to confirm the interferon induction by the complex AB,
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. Thereby, 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.
In order to confirm the virus inhibiting activity of the thus
produced interferon, cell layers of rabbit kidney cells are
incubated overnight in the presence of rabbit interferon, and are
then infected with a virus. The further conduction and evaluation
of these experiments are the same as in the plaque reduction test
described above. The interferon content of the liquids tested can
thus be determined. By using different species of virus, e.g.,
Herpes Simplex, Vaccinia, or Vesicular Stomatitis virus, the virus
non-specificity of the interferon is shown. The specificity of the
interferon with respect to the animal species is, for example,
proven by treating mouse embryo cells with rabbit interferon, which
does not inhibit virus reproduction therein. It can also be shown
that the 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 interferon exhibits no protective effect against
vesicular stomatitis virus in such cells which have been treated
with actinomycin D.
The production of 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.
The protective effect of AB against virus infections can also be
shown directly by animal tests. Thus, for example, mice are treated
intraperitoneally with a solution of AB in a physiologically
acceptable solvent, and one day thereafter are infected with a
pathogenic virus, e.g., Herpes Simplex. Depending on the dose of AB
administered, the treatment results in an increased survival time
or even full survival of the infection, while control mice which
have not been treated with AB, are killed by the infection.
By similar experiments the protective effect of the complexes
according to the invention against a great variety of viruses
including Vaccinia virus, Vesicular-Stomatitis virus, and Influenza
virus can be shown.
Analogously, the protective effect of AB against other infections,
e.g., infections by yeasts, for example, Cryptococcus Neoformans,
by bacteria, for example, Pneumococcus, and by protozoae, for
example, Plasmodium Berghei or Eperythrozoon Coccoides, can be
shown.
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-half thereof 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.
When the novel complexes are 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.degree. to 40.degree. C. 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 5 to 48 hours. The 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.
For that purpose, 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.
In experiments conducted on laboratory animals, AB has displayed
anti-tumor activity, in mice which were inoculated with Ehrlich
ascites tumor cells, the mice which were treated with AB survived
considerably longer than untreated ones.
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. Preferably, solutions or sprays are applied to
mucous membranes, e.g., intraorally, intranasally or
conjunctivally.
Suitable excipients for pharmaceutical preparations containing the
two-component complexes of this invention, including their metal or
ammonium salts, 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. The pharmaceutical 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 influencing
the ionic strength, buffers, coloring, flavoring and/or aromatous
substances.
Pharmaceutical 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 two-component
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.
Without further elaboration, it is believed that one skilled in the
art can, using the preceding description, utilize the present
invention to its fullest extent. The following preferred specific
embodiments are, therefore, to be construed as merely illustrative
and not limitative of the remainder of the disclosure in any way
whatsoever.
In the examples set forth below, the temperatures are indicated in
degrees Centigrade. The s.sub.20,w -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. In place of the cell culture medium used in the
experiments described below, other conventional media can also be
used. The type of the cell culture medium is only important for the
cell culture and does not influence the effect of the two-component
complexes according to the invention.
The polyriboinosinic acid (A) used throughout the following
examples is a commercial product of C. F. Boehringer & Soehne
G.m.b.H., Mannheim-Waldhof, having a s.sub.20,w -value of 5.3.
PREPARATION
The starting polyribo-2-thiocytidylic acid B was prepared employing
the method described in Example 1(b) of the copending application
filed Aug. 20, 1971, Ser. No. 173,656, as follows:
4 ml. of an aqueous mixture (pH 8.3) containing 0.4 mmol of
tris-(hydroxymethyl)-aminomethane hydrochloride (hereinafter
referred to as tris.HCl), 0.008 mmol of 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 .times. mg.
protein at 37.degree.) are incubated for 4 hours at 37.degree..
After removal of the protein, and a 48 hour dialysis of the aqueous
phase concentrated to 1.5 ml. at 3.degree. against 0.01 M
tris.HCl.(pH 7.0), B having a s.sub.20,w -value of 8.3 is
obtained.
Polynucleotides B having other s.sub.20,w -values can also be used
with similar success. The same holds for polynucleotides A.
EXAMPLE 1
To 0.53 ml of a 0.01 M tris.HCl buffer solution (pH 7.0) containing
0.19 micromol of the polyribo-2-thiocytidylic acid described in the
Preparation above per ml., 1.37 ml. of Hanks' salt solution (cf. J.
M. Hoskins, Virological Procedures, London 1967, page 313) is
added, and the combined solution is mixed with 0.10 ml. of a Hanks'
salt solution containing 1 micromol of the commercially available
polyriboinosinic acid A per ml. This mixture, thus containing 0.1
micromol of each of A and B, is set aside for 1 hour to form a
solution of AB exhibiting the following properties:
Hyperchromicity of the UV spectrum in comparison with a spectrum
obtained by the addition of the UV-spectra of each of A and B
alone, as shown in the following table: Wavelength Hyperchromicity
(nm) (%) ______________________________________ 250 18 260 18.2 270
20.8 280 17 ______________________________________
The UV spectrum shows .lambda..sub.max 247.5 nm; .lambda..sub.min
226 nm;
[M].sub.340 = -1.2 10.sup.3, [M].sub.320 = 0, [M].sub.303 = 1.4
10.sup.3,
[m].sub.297 = 0, [m].sub.262 = 21.8 10.sup.3, [m].sub.242 = 0,
[m].sub.232 = 18.8 10.sup.3 ;
apparent pK value 11.5; isobestic point 230 nm.
In aqueous solution, no T.sub.m of AB can be determined between
0.degree. and 100.degree. C. In an aqueous mixture containing 30
percent of ethylene glycol and 0.05 M Na.sup.+ ions, a sharp
transition is shown with a T.sub.m -value of 77.degree..
The average s.sub.20,w -value of AB is 12.4; the integral
distribution of the s.sub.20,w -values of AB is shown in the
following table (the values were determined in a 0.1 M phosphate
buffer at pH 7.0, after 97 minutes at 20,000 rpm):
s.sub.20,w C'/C. ______________________________________ 5.5 0.02
6.5 0.04 7.5 0.08 8.5 0.13 9.7 0.20 10.5 0.28 11.5 0.39 12.5 0.50
13.5 0.83 14.5 0.93 15.5 1.00
______________________________________
Contrary to the free polyribo-2-thiocytidylic acid (B), 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.sub.2, 0.05 mmol of A and 0.16
mg. of disodium 2-thiocytidine-5'-diphosphate, 5 enzyme units of
polynucleotide phosphorylase (specific activity 0.165 mmol UDP/hour
.times. mg protein at 37.degree.) are added, and the mixture is
incubated for 4 hours at 37.degree.. 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.degree., and is dialyzed for 48 hours at 3.degree. 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.sub.2 SO.sub.3 solution and 1
part by volume of an aqueous 1M NaHSO.sub.3 solution, are added,
and air is sucked through the reaction mixture. After 1 hour, again
20 microliter of the sulfite reagent solution are added, and the
air is bubbled through for another hour. Thereafter, 0.5 ml. of an
aqueous 0.2M NH.sub.4 Cl solution are added and the mixture is
adjusted to pH 8.5 by adding aqueous ammonia. After standing for 1
hour at room temperature, the reaction mixture is concentrated to 2
ml., and is dialyzed for 60 hours at 3.degree. against 0.01 M
tris.HCl buffer. A solution of AB having the same properties as
that described in Example 1 is thus obtained.
EXAMPLE A
From an AB solution obtained according to Example 1, a standard
solution is prepared by adding 18 ml. of a cell culture maintenance
medium. This medium is prepared from a commercial tissue culture
medium (TCM 199 manufactured by Wellcome Reagents Limited,
Beckenham) by adding a 0.168 percent NAHCO.sub.3 solution as well
as 100 IU of penicillin and 100 microgram of streptomycin per ml.
By repeated diluting this standard solution with the
above-described cell culture maintenance medium in a ratio of 1:10,
a dilution series is prepared. 10 Ml. of each of these solutions
are used to layer 7 days old primary rabbit kidney cell cultures
grown in square bottles. Three bottles are used for each
concentration of AB, and three similar bottles are layered with the
above-described cell culture preserving medium alone. All cultures
are incubated overnight at 35.degree., and then infected with
Herpes Simplex virus. The infected cultures are layered with agar,
incubated for 48 hours at 35.degree., 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.degree., 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.sub.50), i.e., the dose of AB reducing the plaque member to 50
percent of the untreated control, is determined graphically.
In further experiments with the same AB standard solution, dilution
series in a ratio of 1:2 and 1:4 were tested. Moreover, in other
experiments secondary instead of primary rabbit kidney cells were
used, as well as Vaccinia virus instead of Herpes Simplex virus.
The results are shown in the following table:
Test Cell Culture Used Virus PRD.sub.50 No. (microgram/ml)
______________________________________ 1 prim. rabbit kidney Herpes
Simplex 0.0035 2 prim. rabbit kidney Herpes Simplex 0.0065 3 sec.
rabbit kidney Vaccinia 0.0030 4 sec. rabbit kidney Herpes Simplex
0.0061 ______________________________________
The same tests were also conducted with A and B alone. Under no
circumstances was a reduction of the number of plaques observed at
concentrations of up to 0.61 microgram/ml.
EXAMPLE B
In order to determine the biological activity of the complex AB
obtained according to Example 2, plaque reduction tests were
conducted as described in the foregoing Example A. Unlike Example
A, in this test secondary rabbit kidney cells were used in all
experiments, and further, during the experiments referred to as No.
3 and No. 4 in the following table, no streptomycin was added to
the cell culture maintenance medium.
______________________________________ Experiment PRD.sub.50 No.
Virus microgram/ml. ______________________________________ 1 Herpes
Simplex 0.013 2 Vaccinia 0.00067 3 Herpes Simplex 0.0015 4 Herpes
Simplex 0.0057 ______________________________________
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 solution were 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.degree. 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 35.degree. and
then challenged in conventional manner with Herpes Simplex virus.
The further procedure and evaluation was as in example A.
A 50 percent reduction of Herpes Simplex virus plaques was obtained
by the following serum dilutions:
Serum Dilution ______________________________________ I 1:1800 II
1:1050 ______________________________________
(Sera which were taken from the rabbits before treating them with
AB had no effect under the test conditions).
In a second experiment 0,1 ml of serum I were diluted with 2.7 ml
of Hanks' salt solution without Ca.sup.2.sup.+ and Mg.sup.2.sup.+
(pH 7.4) and 0.4 ml of a solution containing 1 mg of twice
crystallized bovine trypsin (obtained from SERVA Feinbiochemica
GmbH & Co, Heidelberg) per ml Hanks' salt solution were added.
The mixture was incubated for 3 hours at 37.degree., then 0.8 ml of
a solution containing 1 mg soybean trypsin inhibitor (obtained from
SERVA Feinbiochemica GmbH & Co, Heidelberg) per ml Hanks' salt
solution and the mixture set aside for 1 hour at 22.degree..
A control was run with the respective solvents only, i.e., without
trypsin and inhibitor.
The interferon titer was measured as described above. A 50 percent
reduction of Herpes Simplex virus plaques was obtained with the
following dilutions:
Solution Dilution ______________________________________ Serum I +
trypsin 1:180 Serum I (as control 1:1450 without trypsin)
______________________________________
From this it can be seen that the residual interferon activity of
serum I after treatment with trypsin was only 12 percent.
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.
The preceding examples can be repeated with similar success by
substituting the generically or specifically described reactants
and/or operating conditions of this invention for those used in the
preceding examples.
From the foregoing description, one skilled in the art can easily
ascertain the essential characteristics of this invention, and
without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions.
* * * * *