CA1156145A - Pharmaceutical composition comprising modified polyriboinosinic-polyribocytidylic acid, for induction of interferon in primates - Google Patents
Pharmaceutical composition comprising modified polyriboinosinic-polyribocytidylic acid, for induction of interferon in primatesInfo
- Publication number
- CA1156145A CA1156145A CA000360530A CA360530A CA1156145A CA 1156145 A CA1156145 A CA 1156145A CA 000360530 A CA000360530 A CA 000360530A CA 360530 A CA360530 A CA 360530A CA 1156145 A CA1156145 A CA 1156145A
- Authority
- CA
- Canada
- Prior art keywords
- poly
- lysine
- interferon
- solution
- prepared
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
TITLE OF THE INVENTION
Pharmaceutical Composition Comprising Modified Polyriboinosinic-Polyribocytidylic Acid, for Induction of Interferon in Primates.
ABSTRACT OF THE INVENTION
A complex of polyriboinosinic-polyribocytidylic acid (poly I:C) with poly-L-lysine hydrobromide ([lysine.HBr]n) having a defined molecular weight is prepared which is about 5-15 times as resistant to hydrolysis by pancreatic ribonuclease as the parent poly I:C. A pharmaceutical solution, con-taining a relatively high concentration of the complex, prepared as described, induces significant antiviral levels of serum interferon in monkeys under conditions in which poly I:C itself induces little or no interferon. An important feature of this invention is that the product is a soluble material requiring no special solubilizing agent, thereby facilitating the preparation of solutions having the desired concentration. The product will hereinafter be designated as poly I:C/poly-L-lysine.
Pharmaceutical Composition Comprising Modified Polyriboinosinic-Polyribocytidylic Acid, for Induction of Interferon in Primates.
ABSTRACT OF THE INVENTION
A complex of polyriboinosinic-polyribocytidylic acid (poly I:C) with poly-L-lysine hydrobromide ([lysine.HBr]n) having a defined molecular weight is prepared which is about 5-15 times as resistant to hydrolysis by pancreatic ribonuclease as the parent poly I:C. A pharmaceutical solution, con-taining a relatively high concentration of the complex, prepared as described, induces significant antiviral levels of serum interferon in monkeys under conditions in which poly I:C itself induces little or no interferon. An important feature of this invention is that the product is a soluble material requiring no special solubilizing agent, thereby facilitating the preparation of solutions having the desired concentration. The product will hereinafter be designated as poly I:C/poly-L-lysine.
Description
11~6~45 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
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
- 2 - 16336IA
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 _ 3 _ 16336IA
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.
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 11~6~
_ 5 _ 16336IA
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
~ here is same degree of variability in the chemical characteristics of the components, see the following table of ranges:
oo oo o~
~o .~ ~ o ~1 o I I ,~ o I I I ~o O ~rl O
~ a) o ~ n ~ 3 o ~ ~
~o O a) o ô
N O
N r--
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 _ 3 _ 16336IA
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.
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 11~6~
_ 5 _ 16336IA
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
~ here is same degree of variability in the chemical characteristics of the components, see the following table of ranges:
oo oo o~
~o .~ ~ o ~1 o I I ,~ o I I I ~o O ~rl O
~ a) o ~ n ~ 3 o ~ ~
~o O a) o ô
N O
N r--
3 ~ ~
u~ ~
oo o ~n c~ co o u~
o~o , .
,~
H C~
>, O O O
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
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 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.
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 11~6~
_ 9 _ 16336IA
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 11~6~
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.
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.
CO LO~7 ~o ~ ~
~n '~ C~
~r Y
H
~ 10 ' ~i I
~
~ .
U~ ~ O
1 1~6 1~5 ~ ~ ~ o ~ ~ o o o~
~ ~ ~ O ~ In ~
~ co o Q O ~ I O O O O ~D O
~ ~ ~ ~ In ~ ~ I O n ~ o O t` ~ O Lr~ O O O N ~ I` Ul ~ o o n Ln Ln o o o o o ~ ~ C) ~ ~D CO O O ~ 'n ~ ~ D O ~r O
~ ~.~ O O t~ ~ ~ 1-l coI ~1 ~
~ ii~ ,~ ,t ~
~ ~ ' U~ ~ O O O O O O O O O O O
~ I In u~ o o o o o o o ' I O ~ In ~ O O O O ~ O O
~1 I _I ~ ~r Lt ) O u~ ~ o N O O
~: I ~n o In t` r`
u~ ~
oo o ~n c~ co o u~
o~o , .
,~
H C~
>, O O O
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
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 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.
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 11~6~
_ 9 _ 16336IA
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 11~6~
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.
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.
CO LO~7 ~o ~ ~
~n '~ C~
~r Y
H
~ 10 ' ~i I
~
~ .
U~ ~ O
1 1~6 1~5 ~ ~ ~ o ~ ~ o o o~
~ ~ ~ O ~ In ~
~ co o Q O ~ I O O O O ~D O
~ ~ ~ ~ In ~ ~ I O n ~ o O t` ~ O Lr~ O O O N ~ I` Ul ~ o o n Ln Ln o o o o o ~ ~ C) ~ ~D CO O O ~ 'n ~ ~ D O ~r O
~ ~.~ O O t~ ~ ~ 1-l coI ~1 ~
~ ii~ ,~ ,t ~
~ ~ ' U~ ~ O O O O O O O O O O O
~ I In u~ o o o o o o o ' I O ~ In ~ O O O O ~ O O
~1 I _I ~ ~r Lt ) O u~ ~ o N O O
~: I ~n o In t` r`
Claims (12)
1. A method for preparing an interferon-inducing solution of a complex of poly I:C/poly-L-lysine.
hydrochloride or hydrobromide which comprises mixing a solution of poly I:C with a solution of poly-L-lysine hydrobromide or hydrochloride and recovering a homogene-ous solution of the desired complex, the concentration of the poly I:C being from about 1 to about 2 mg/ml, the ratio by dry weight of poly I:C to the poly-L-lysine hydrobromide or hydrochloride being about 1 to about 0.3 or a molar ratio of 2:1, the poly I having a Sw20° of 11-20, and an El% at 248 nm of 240-280; the poly C
having a Sw20° of 5-7 and an E1% at 248 nm of 175-185, and the poly-L-lysine having a molecular weight of from about 10,000 to about 70,000 daltons.
hydrochloride or hydrobromide which comprises mixing a solution of poly I:C with a solution of poly-L-lysine hydrobromide or hydrochloride and recovering a homogene-ous solution of the desired complex, the concentration of the poly I:C being from about 1 to about 2 mg/ml, the ratio by dry weight of poly I:C to the poly-L-lysine hydrobromide or hydrochloride being about 1 to about 0.3 or a molar ratio of 2:1, the poly I having a Sw20° of 11-20, and an El% at 248 nm of 240-280; the poly C
having a Sw20° of 5-7 and an E1% at 248 nm of 175-185, and the poly-L-lysine having a molecular weight of from about 10,000 to about 70,000 daltons.
2. The method of Claim 1 in which the poly I:C is about 1 mg/ml.
3. The method of Claim 1 in which the poly-L-lysine is in the hydrobromide form.
4. The method of Claim 3 in which the poly-L-lysine hydrobromide is from about 10,000 to about 35,000 daltons.
5. The method of Claim 1 in which the poly I
has a Sw20° of about 19 and an E1% at 248 nm of 240.
has a Sw20° of about 19 and an E1% at 248 nm of 240.
6. The method of Claim 1 in which poly C has a Sw20° of about 5 and an E1% at 248 of 177.
7. An interferon-inducing solution of a complex of poly I:C/poly-L-lysine hydrobromide or hydro-chloride, the concentration in solution of the poly I:C
being from about 1 to about 2 mg/ml, the ratio by dry weight of the poly I:C to the poly-L-lysine hydrobromide or hydrochloride being about 1 to about 0.3, or a molar ratio of 2:1; the poly I having a Sw20° of 11-20, and an E1% at 248 nm of 240-280; the poly C having a Sw20° of 5-7 and an El% at 248 nm of 175-185; and the poly-L-lysine having a molecular weight of from about 10,000 to about 70,000 daltons, when prepared by the process defined in Claim 1 or by an obvious chemical equivalent.
being from about 1 to about 2 mg/ml, the ratio by dry weight of the poly I:C to the poly-L-lysine hydrobromide or hydrochloride being about 1 to about 0.3, or a molar ratio of 2:1; the poly I having a Sw20° of 11-20, and an E1% at 248 nm of 240-280; the poly C having a Sw20° of 5-7 and an El% at 248 nm of 175-185; and the poly-L-lysine having a molecular weight of from about 10,000 to about 70,000 daltons, when prepared by the process defined in Claim 1 or by an obvious chemical equivalent.
8. The solution of Claim 7 in which the poly I;C concentration is about 1 mg/ml, when prepared by the process defined in Claim 2 or by an obvious chemical equivalent.
9. The solution of Claim 7 in which the poly-L-lysine is in the hydrobromide form, when prepared by the process defined in Claim 3 or by an obvious chemical equivalent.
10. The solution of Claim 9 in which the molecular weight of the poly-L-lysine hydrobromide is from about 10,000 to about 35,000 daltons, when prepared by the process defined in Claim 4 or by an obvious chemical equivalent.
11. The solution of Claim 7, wherein the poly I has a Sw20° of about 19 and an E1% at 248 nm of 240, when prepared by the process defined in Claim 5 or by an obvious chemical equivalent.
12. The solution of Claim 7, wherein poly C
has a Sw20° of about 5 and an E1% at 248 of 177, when prepared by the process defined in Claim 6 or by an obvious chemical equivalent.
has a Sw20° of about 5 and an E1% at 248 of 177, when prepared by the process defined in Claim 6 or by an obvious chemical equivalent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US7600479A | 1979-09-17 | 1979-09-17 | |
| US76,004 | 1979-09-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1156145A true CA1156145A (en) | 1983-11-01 |
Family
ID=22129313
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000360530A Expired CA1156145A (en) | 1979-09-17 | 1980-09-17 | Pharmaceutical composition comprising modified polyriboinosinic-polyribocytidylic acid, for induction of interferon in primates |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JPS5653621A (en) |
| CA (1) | CA1156145A (en) |
| ZA (1) | ZA805716B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0621469Y2 (en) * | 1991-04-25 | 1994-06-08 | パラマウントベッド株式会社 | Floor structure in bed |
| CN1166366C (en) * | 1998-05-25 | 2004-09-15 | 日本新药株式会社 | Process for producing composite preparation containing nucleic acid |
-
1980
- 1980-09-16 ZA ZA00805716A patent/ZA805716B/en unknown
- 1980-09-17 JP JP12902480A patent/JPS5653621A/en active Granted
- 1980-09-17 CA CA000360530A patent/CA1156145A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5653621A (en) | 1981-05-13 |
| JPH0223531B2 (en) | 1990-05-24 |
| ZA805716B (en) | 1982-04-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4130641A (en) | Induction of interferon production by modified nucleic acid complexes | |
| US4388306A (en) | Pharmaceutical composition comprising modified polyriboinosinic-polyribocytidylic acid, for induction of interferon in primates | |
| Carter et al. | Structural requirements of the rIn· rCn complex for Induction of human interferon | |
| Levy et al. | A modified polyriboinosinic-polyribocytidylic acid complex that induces interferon in primates | |
| Field et al. | Inducers of interferon and host resistance. II. Multistranded synthetic polynucleotide complexes. | |
| AU725195B2 (en) | Interferon conjugates | |
| US3931397A (en) | Biologically active material | |
| US4349538A (en) | Nuclease-resistant hydrophilic complex of polyriboinosinic-polyribocytidylic acid | |
| KR20150013526A (en) | The use of chitosan polymer in the treatment and prevention of infections caused by coronaviruses | |
| US4163780A (en) | Ks-2-a | |
| EP0025766B1 (en) | Pharmaceutical composition comprising modified polyriboinosinic-polyribocytidylic acid, for induction of interferon in primates | |
| US4124702A (en) | Polynucleotides active as inducers of interferon production in living animal cells | |
| CA1139306A (en) | Interferon stabilization | |
| CA1156145A (en) | Pharmaceutical composition comprising modified polyriboinosinic-polyribocytidylic acid, for induction of interferon in primates | |
| US4389395A (en) | Low molecular weight complex of polyriboinosinic-polyribocytidylic acid and method of inducing interferon | |
| Guggenheim et al. | Clinical Studies of an Interferon Inducer, Polyriboinosinic-Polyribocytidylic Acid [Poly (I)• Poly (C)], in Children | |
| LAMPSON et al. | Poly I: C/poly-L-lysine: potent inducer of interferons in primates | |
| EP0084953B1 (en) | Interferon induction method | |
| Blevy | [34] Induction of interferon in vivo and in vitro by polynucleotides and derivatives, and preparation of derivatives | |
| Reuss et al. | Induction of interferon by polyribonucleotides containing thiopyrimidines | |
| Rice et al. | Dose responsiveness and variation among inbred strains of mice in production of interferon after treatment with poly (I)· poly (C)[poly-D-lysine] complexes | |
| Levy et al. | Interferon induction in primates by stabilized polyriboinosinic acid-polyribocytidylic acid: effect of component size | |
| Merigan et al. | Influence of increasing 2′-O-methylation on the interferon stimulating capacity of poly (rI)· poly (rC) | |
| Elslager et al. | Repository drugs. V. 4', 4'''-[p-Phenylenebis (methylidyneimino-p-phenylenesulfonyl)] bisacetanilide (PSBA) and related 4', 4'''-[bis (imino-p-phenylenesulfonyl)] bisanilides; a novel class of long-acting antimalarial and antileprotic agents | |
| EP0344643B1 (en) | Antiviral pharmaceutical composition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry | ||
| MKEX | Expiry |
Effective date: 20001101 |