Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
Pharmaceutical Composition Comprising Modified
Polyriboinosinic-Polyribocytidylic Acid, for
Induction of Interferon in Primates.
BACKGROUND OF T~E INVENTION
-
The synthetic double-stranded RNA, polyribo-
inosinic-polyribocytidylic acid (poly I:C), is
effective as an interferon inducer in rodents and
rabbits and as such provides protection against a
variety of RNA and DNA viruses in these species
[A.K. Field et al., Proc. Natl. Acad. Sci. U.S.A.,
_, pp. 1004-1010, (1967)]. However, poly I:C shows
only a very weak capacity to induce interferon in
man and non-human primates.
It has been stated that enzymatic inactivation
of poly I:C is the reason for its poor capacity to
induce interferon in primates [H.B. Levy et al.,
J. Inf. Dis., 132, p. 434 (1975)]. These authors
have reported the preparation of a solubili~ed
formulation of poly I:C and poly-L-lysine (molecular
ds 5
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weight 2000 to 5000) in carboxymethylcellulose (CMC)
as the solubilizing agent [see also Levy, U.S.
Patent 3,952,097 (1976)]. This complex is relatively
resistant to ribonuclease and induces significant
quantities of interferon in rhesus monkeys, chim-
panzees and man.
However, certain disadvantages are associated
with CMC. It is a polysaccharide which is at best
poorly biodegradable and on repeated injection may
cause deposition of residues which may cause
irritations (pathology). In addition, CMC ha~
been considered to be a potential carcinogen. Both
the sodium and ammonium salts of CMC appear on the
NIOSH Suspected Carcinogen List, U.S. Environmental
Protection Agency, Office of Toxic Substances,
March, 1976. The references upon which these
listings were made are: A.L. Walpole, Morphol.
Precursors Cancer, Proc. Inter. Conf. Perugia,
:
Italy, pp. 83-88, 1961 (Publ. 1962) and Rev.
Canad~ Biol. (Mont.), 20, pp. 701 (1961).
SUMMARY OF THE INVENTION
In the present invention a combination of poly
I:C (having a high molecular weight) with poly-L-
lysine hydrobromide (having a high molecular weight)
have been prepared in such a molar ratio as to
preclude the need for a solubilizing agent in a
pharmaceutical solution of relatively high concentra-
tion. Our preparations of poly I:C/poly-L-lysine
hydrobromide are superior inducers of antiviral
levels of interferon in primates. The combination
is prepared so that the molar ratio of poly I:C ~o
poly-L-lysine hydrobromide (or poly-L-lysine hydro-
chloride) is about 2 to 1 (calculated as ratio of
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nucleotide residue to lysine residue). Since the
peptide hydrobromide is generally hygroscopic, on a
moisture-free basis (of the peptide) the best
complex may be prepared by mixing by weigh~ one
part of poly I:C with 0.31 parts of poly-L-lysine
hydrobromide. Viscosity and solubility considera-
tions limit the concentration of ingredients per
ml that may be prepared. In practice the concentra-
tion of poly I:C per ml is used at from about 1-2
mg/ml. This requires a theoretical amount of from
about 0.31 to 0.62 mg of dry poly-L-lysine hydro-
bromide, respectively. [In practice, the ratio of
poly I:C to poly-L-lysine hydrobromide is such
as to yield a complex at the limit of solubility.]
The vehicle used is isotonic, pyrogen-free phosphate
buffered saline tpH 7.2) although any suitable
physiologically acceptable vehicle may be used.
Although the hydrobromide and hydrochloride salts of
the peptide are described, other suitable acids can
be used.
The combination of poly I:C and poly-L-lysine
hydrobromide is prepared by mixing a solution of
poly I:C of known concentration and suitable molecu-
lar weight with a solution of poly-L-lysine hydro-
bromide of known concentration and suitable molecularweight. The solutions areeach prepared in phosphate
buffered saline. When the two solutions are mixed a
precipitate appears which will go into solution
(become soluble) with mixing at room temperature
or at 2-8C in approximately 72 hours, although
a small amount of undissolved solids (less than 2%
of the ingredients) may be present. The solution
is clarified by filtration through suitable glass
or membrane filters to yield a homogeneous solution.
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The solution of poly I:C is prepared by mixing
solutions of equimolar quantities of the individual
homopolynucleotides, poly I and poly C, to a final
concentration of about 2 mg/ml. The complex is
characterized prior to addition of the poly-L-
lysine.HBr.
The poly I used has the following properties:
Ultraviolet spectrum .................. Satisfactory
Absorption maximum ..................... 248 nm
10 Absorption minimum ..................... 225 nm
Extinction coefficient
(E1%) at 248 nm .................. 240
Nucleotide content .................... 2.4 ~M/mg
Sedimentation coefficient (Sw, 20) .... 19.2
15 The poly C used has the following properties:
Ultraviolet spectrum .................. Satisfactory
Absorption maximum .................... 268 nm
Absorption minimum .................... 249 nm
Extinction coefficient
(El%) at 248 nm .................. 177
Nucleotide content .................... 2.95 ~M/mg
Sedimentation coefficient (Sw, 20) .... 5.0
The poly-L-lysine.HBr used has the following
properties:
25 Molecular weight ...................... 32,500
Degree of polymerization (D.P.) ....... 155 residues
Lysine content (% of theoretical) .... ..................................89.7
(of net weight)
Bromide content (% of theoretical) .... ..................................87.2
(of net weight)
Other amino acids ..................... None
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The poly I:C, prepared from the above poly I
and poly C, has the following properties:
Ultraviolet spectrum ................. Satisfactory
Absorption maximum ................... 248, 265 nm
5 Absorption minimum .................... 228 nm
Extinction coefficient
(El%) at 265 nm ................. 145
Hypochromicity at 248 nm ............. 35.5%
Thermal denaturation midpoint (Tm) ... 64C
10 Hyperchromicity on thermal
denaturation .................... 77.8%
Sedimentation coefficient (Sw, 20) .. 11.0
Relative viscosity ................... 1.59
The poly I:C/poly-L-lysine prepared from the
above poly I, poly C, and poly-L-lysine, has the
following properties:
Ultraviolet spectrum ................. Satisfactory
Absorption maximum ................... 248, 265 nm
Absorption minimum ................... 230 nm
20 Extinction coefficient
(El%) at 265 nm ................. 140
Thermal denaturation midpoint (Tm) ... 83C
Hyperchromicity on thermal
denaturation .................... 109% at 83C,
<10 at 64C
Sedimentation coefficient (Sw, 20) .. 11.5
Relative viscosity ................... 1.65
Ribonuclease resistance compared
to that of poly I:C ............. 14.0%
Conductivity ......................... 7.1 millimhos
Nominal poly I:C concentration ....... 1.0 mg/ml
Actual measured poly I:C concentration 1.0 mg/ml
Nominal poly-L-lysine concentration . 0.310 mg/ml
Actual measured poly-L~lysine
concentration ................... 0.330 mg/ml
115~il4S
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~ here is same degree of variability in the
chemical characteristics of the components, see
the following table of ranges:
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The compositions prepared by this invention
are useful in inducing antiviral levels of inter-
feron in mammalian and other animal systems where
uncomplexed poly I:C is not an efficient inducer of
interferon. For example, in grivet monkeys, poly
I:C/poly-L-lysine injected intravenously at one
milligram per kilogram weight equivalent of poly I:C
results in the induction of high titered circulating
interferon. At a level of 0.25 mg equivalent of
poly I:C substantial circulating interferon titers
are achieved. Uncomplexed poly I:C injected at
these dosage levels stimulates low or no detectable
serum interferon.
Although the complex of this invention has
not yet been administered to humans, by analo~y
to other similar compositions, the preferred
dosage range for humans of the poly I:C/poly-L-
lysine complex may be as high as 0.3-0.4 mg/kg
body weight, administered as for instance on a
daily basis by IV injection. Initial doses can be
as low as 10 ~g/kg body weight daily.
PREFERRED EMBODIMENTS
EXAMPLE 1
Preparation of Solutions of Poly I:C and
Poly-L-lysine Hydrobromide (Lot 327)
From the calculated extinction coefficient
a solution containing 2.16 mg/ml of polyriboinosinic
acid (poly I) as defined above in phosphate buffered
saline, was obtained by heating in an 80C water
bath. The heated solution was sterilized by filtra-
tion through a 0.~5 ~ membrane.
1 15~'145
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From the calculated extinction coefficient
a solution containing 2 mg/ml of polyribocytidylic
acid (poly C), as defined above in phosphate buffered
saline, was obtained by stirring at ambient tempera-
ture. The poly C solution was sterilized byfiltration through a 0.45 ~ filter membrane.
Equal volumes of poly I and poly C were mixed
with stirring in an 80C water bath until a clear
solution was obtained. The mixture was then allowed
to cool slowly at ambient temperature in order to
anneal the poly I and poly C with the formation of
poly I:C.
A solution of poly-L-lysine hydrobromide was
prepared to contain on a dry weight basis 0.62 mg/ml
in phosphate buffered saline by stirring into
solution at ambient temperature. The clear solution
was sterilized by filtration through a 0.2 ~ membrane.
The preparation of poly-L-lysine which was used was
demonstrated by appropriate analytical procedures to
contain equimolar content of L-lysine and bromine
~the poly-L-lysine hydrobromide should be construed
to have a composition as follows: [lysine.HBr]n ~
where n is the degree of polymerization), and a
molecular weight as defined.
EXAMPLE 2
Preparation of Poly I:C/Poly-L-lysine (lot 827)
Equal volumes of poly I:C (2.08 mg/ml)
prepared as set forth in Example 1 and poly-L-
lysine hydrobromide (0.62 mg/ml) prepared as set
forth in Example 1 were mixed with stirring. Stirring
was continued for 48-72 hours at 2-8~C until only a
trace of undissolved material remained. The viscous
solution was clarified by filtration through a
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sterile clarlfying membrane~ The filtered solution
was dispensed into ampoules and kept at 2-8C untll
used. The filtered solution was demonstrated by
appropriate analytical procedures to contain greater
than 98% of the nominal concentration of complexed
poly I:C/poly-L-lysine hydrobromide. All of the
poly-L-lysine was demonstrated by appropriate sedi-
mentation experiments to be bound to the poly I:C.
The ribonuclease resistance of the poly I:C in the
complex was increased 5-15 fold over the parent
poly I:C. The thermal transition mid-point (Tm)
was increased from 64C (for the poly I:C alone) to
82-83C for the complex when measured in 0.15 molar
NaCl. The measurements were made by appropriate
standard spectrophotometric measurements of hyper-
chromicity.
The following summarizes the physical charac-
teristics of this preparation of poly I:C/poly-L-
lysine, Lot 827:
20 Ultraviolet spectrum .................. Satisfactory
Absorption maximum .................... 248, 265 nm
Absorption minimum .................... 230 nm
Extinction coefficient
~El%) at 265 nm ................. 140
25 Thermal denaturation point (Tm) ....... 83C
Hyperchromicity on thermal
denaturatlon at 64C ............. <10%
at 83C ............. 109%
Sedimentation coefficient (Sw, 20) ... 11.5
Relative viscosity ................... 1.65
Nominal measure poly I:C
concentration ................... 1.0 mg/ml
Nominal poly-L-lysine
concentration ................... 0.310 mg/ml
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EXAMPLE 3
Interferon Induction in Grivet Monkeys
(Cercopithecus aethiops)
Poly I:C alone or combined with poly-L-lysine
as set forth in Examples 1 and 2 were prepared and
injected intravenously at 1.0 mg (as poly I:C)/kg
body weight into grivet monkeys. Blood samples were
obtained from animals prior to injection (prebled
at time O hours) and at intervals thereafter.
Interferon titers were determined by assay of
serial dilutions of serum samples for reduction of
infection of cell cultures by vesicular stomatitis
virus. Peak interferon titers were obtained at
approximately eight hours after injection. Charac-
1~ terization of the poly I:C complexes includedmeasurement of the resistance to hydrolysis by
pancreatic ribonuclease and the thermal transition
midpoint (Tm) (e.g, the temperature at which half
of the poly I:C has separated into the individual
polynucleotides t poly I and poly C) as evidenced by
hyperchromicity. Data are presented in Table 1.
Peak interferon titers at least 10-100 fold greater
than those obtained from monkeys induced with poly
I:C alone, were obtained from monkeys induced with
poly I:C complexed with poly-L-lysine.
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EXAMPLE 4
Comparison of Interferon Induction in Grivet
Monkeys Using poly I:C Complexed with poly-L-
lysines of Various Molecular Weiahts
Complexes of poly I:C/poly-L-lysine.HBr were
prepared using samples of poly-L-lysine.HBr of
defined molecular weight ranges. These complexes
were compared for their capacity to induce inter-
feron in grivet monkeys as described in Example 3.
Ribonuclease sensitivity and thermal transition
midpoints were determined for each of the com-
plexes where feasible. All monkeys were injected
intravenously with 1 mg (as poly I~C)/kg body
weight. Samples of blood were taken immediately
prior to and 8 hours post injection. Serum
prepared from these samples was assayed for inter-
feron. Resistance to ribonuclease was significantly
increased in all samples measured including the
molecular weight range from 1050 through approxi-
mately 9000-10,000. The thermal transition midpoint
was signficantly raised at all molecular weights
of poly-L-lysine. Interferon induction was
enhanced only marginally (geometric mean titer
range 50-200) with poly-L-lysines of molecular
weights up through approximately 10,000. At
molecular weights above 10,000 there was a sharp
rise in enhancement of interferon induction with
a range of geometric mean interferon titers of 52g
to 1988. These data are presented in Table 2.
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