US20100160413A1 - Double-stranded ribonucleic acids with rugged physico-chemical structure and highly specific biologic activity - Google Patents

Double-stranded ribonucleic acids with rugged physico-chemical structure and highly specific biologic activity Download PDF

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US20100160413A1
US20100160413A1 US12/591,270 US59127009A US2010160413A1 US 20100160413 A1 US20100160413 A1 US 20100160413A1 US 59127009 A US59127009 A US 59127009A US 2010160413 A1 US2010160413 A1 US 2010160413A1
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poly
dsrna
tlr3
ribo
acid
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William A. Carter
David Strayer
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AIM Immunotech Inc
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Hemispherx Biopharma Inc
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Priority claimed from PCT/US2009/005797 external-priority patent/WO2010047835A2/fr
Priority to US12/591,270 priority Critical patent/US20100160413A1/en
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Assigned to HEMISPHERX BIOPHARMA, INC. reassignment HEMISPHERX BIOPHARMA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CARTER, WILLIAM A., STRAYER, DAVID
Publication of US20100160413A1 publication Critical patent/US20100160413A1/en
Priority to PCT/US2010/002970 priority patent/WO2011059505A2/fr
Priority to CA2780723A priority patent/CA2780723A1/fr
Priority to EP10830330.6A priority patent/EP2499247A4/fr
Priority to US13/077,742 priority patent/US8722874B2/en
Priority to US13/758,930 priority patent/US20140170191A1/en
Priority to US14/176,360 priority patent/US9315538B2/en
Priority to US14/275,754 priority patent/US20140335112A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/331—Universal or degenerate base
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/533—Physical structure partially self-complementary or closed having a mismatch or nick in at least one of the strands

Definitions

  • the invention relates to our discovery of a novel double-stranded ribonucleic acid (dsRNA) having specific biological activities, which includes acting as a selective agonist for activation of Toll-like receptor 3 (TLR3).
  • dsRNA double-stranded ribonucleic acid
  • TLR3 Toll-like receptor 3
  • Its “rugged” molecular structure as measured by physico-chemical techniques is resistant to molecular unfolding (i.e., denaturation). This structure appears to be responsible for increased efficacy of dsRNA in therapeutic applications and improved biological activity (e.g., used as an immunoregulatory agent).
  • AMPLIGEN® (rintatolimod) poly(I):poly(C 12 U) was developed as a synthetic double-stranded ribonucleic acid (dsRNA) for therapeutic applications based on an understanding of both the beneficial and adverse effects induced by poly(I):poly(C) on the physiology of a subject.
  • poly(I):poly(C 12 U) was developed by us to preserve the beneficial aspects of dsRNA without the adverse effects of poly(I):poly(C) by modifying the latter's structure with the occasional introduction of uridylate into the poly(C) strand to produce duplexes containing specifically-configured regions which are not base paired (i.e., “mismatched”) at the position of the modification. These regions accelerate dsRNA hydrolysis and lessen toxicity (Greene, 1984).
  • the ability to induce interferon synthesis was retained as long as the modified dsRNA persisted for a half life of at least five minutes and the frequency of random insertion into the poly(ribocytidylic acid) strand was not greater than each 0.5 to 1.0 helical turn of perfectly base-paired dsRNA (Brodsky, 1987).
  • poly(I):poly(C 12 U) While poly(I):poly(C 12 U) is stable in solution, it is susceptible to hydrolysis like all other conventional nucleic acids. The hydrolysis is highly dependent on nucleic acid structure, as well as on the presence of nuclease and divalent cations, pH, and temperature. RNA is more susceptible to hydrolysis than DNA because of the 2′-OH group present in the former that facilitates hydrolysis. Moreover, poly(I):poly(C 12 U) was designed to degrade more rapidly than other dsRNA in a nuclease-containing environment, such as blood and other tissue fluids. Nucleic acids are initially stable in physiological salt buffers at room temperature, but gradually begin to degrade with time. This hydrolysis rate is temperature dependent, increasing greatly at higher temperatures.
  • CD Circular dichroism
  • CD measurement with a combination of scanning and thermal stress modes also can provide precise characterization of the critical double-helical structure. Indeed, minor changes in second-order structure of polynucleotides have been measured by CD (Gray, 1995), including the effects of ligand binding (Sumita, 2005).
  • the half life of poly(I):poly(C 12 U) was reduced to a safe level of about 4 to 5 minutes by precise substitution of the poly(C) strand, specifically the cytidine to uridine ratio (U.S. Pat. No. 5,258,369).
  • Introduction of the unpaired base uracil into the poly(C) strand at a ratio of 1:12 resulted in a minimum base-paired length of about one helical turn, which is required for the interaction of dsRNA with its bioactive receptor.
  • placing a maximum size limitation of about 350 repeat units on the dsRNA resulted in a half life of about 4 to 5 minutes (Greene, 1978; Pitha, 1972).
  • dsRNA double-stranded ribonucleic acid
  • a “rugged” molecule resistant to unfolding (i.e., denaturation) of its helical structure has improved dsRNA activity as a selective agonist of Toll-like receptor 3 (TLR3).
  • TLR3 Toll-like receptor 3
  • At least partial purification of rugged dsRNA from other dsRNA present after synthesis is expected to increase specificity in its use as a medicament and thereby reduce adverse effects attributable to the dsRNA that is not rugged.
  • rugged dsRNA may be provided.
  • Specifically-configured dsRNA may be of the general formula ribo(I n ).ribo(C 4-29 U) n , ribo(I n ).ribo(C 11-14 U) n , or ribo(I n ).ribo(C 12 U) n , wherein the strands are comprised of ribonucleotides (ribo) and n is an integer from about 40 to about 40,000 repeats.
  • a strand comprised of poly(ribocytosinic 4-29 uracilic acid), poly(ribocytosinic 11-14 uracilic acid), or poly(ribocytosinic 12 uracilic acid) may be partially hybridized to an opposite strand comprised of poly(riboinosinic acid) such that the two strands form an RNA double helix that is not paired at the uracil base (i.e., mismatch).
  • rugged dsRNA may be isolated by at least subjecting the partially hybridized strands of a population of dsRNA to conditions that denature most dsRNA (at least 50 mol %, at least 80 mol %, at least 90 mol %, or at least 95 mol %) in the population, and then selection negatively or positively (or both) for dsRNA that remain partially hybridized.
  • the purity of rugged dsRNA may thus be increased from less than about 0.1-10 mol % (e.g., less than about 5 mol %) relative to all RNA in the population after synthesis.
  • the rugged dsRNA be more than about 80-98 mol % relative to all RNA present in the same mixture with the rugged dsRNA (at least 80 mol %, at least 90 mol %, at least 95 mol %, or at least 98 mol %) after selection.
  • the denaturing conditions to unfold at least partially hybridized strands of dsRNA may comprise appropriate choice of buffer salts, pH, solvent, temperature, or any combination thereof. Conditions may be empirically determined by observation of the unfolding or melting of the duplex strands of ribonucleic acid.
  • the yield of rugged dsRNA may be improved by partial hydrolysis of longer strands of ribonucleic acid, then selection of (partially) hybridized stands of appropriate size and resistance to denaturation.
  • the molecular weight of rugged dsRNA may be from about 250 Kda to about 320 Kda, or from about 270 Kda to about 300 Kda. Lengths of a single or both strands of rugged dsRNA may be from about 380 bases to about 450 bases, or from about 400 bases to about 430 bases. The number of helical turns made by duplexed RNA strands of rugged dsRNA may be from about 30 to about 38, or from about 32 to about 36.
  • At least one or more different rugged dsRNA may be administered to a subject (e.g., human patient or animal) in need of such treatment.
  • Rugged dsRNA may be administered at a dosage of from about 0.5 mg to about 60 mg/dose. This dosage may be administered once per week or month, or two or more doses per week or month.
  • Each dose e.g., from about 0.5 mg to about 60 mg, from about 5 mg to about 40 mg, or from about 10 mg to about 20 mg
  • the rugged dsRNA may act specifically through a TLR3 receptor.
  • the function and phenotype of dendritic cells may be normalized in a subject (e.g., human patient or animal).
  • Administering at least an effective amount of one or more rugged dsRNA to a subject (e.g., human patient or animal) may thereby decrease the number or reduce the severity of symptoms when the subject is afflicted by a disease or other pathological condition.
  • Use of rugged dsRNA may correct dendritic cell maturation abnormalities in the subject without the hazard of inducing a cytokine storm.
  • Antigen presenting cells e.g., dendritic cells, macrophages, B cells
  • mucosal tissues e.g., gastric or respiratory epithelium
  • One or more antigens may be presented to cells of the immune system, and the antigen(s) should be susceptible to the action of the rugged dsRNA acting selectively as a TLR3 agonist.
  • Cells of the immune system, microbes, cancer cells, or other transformed cells may be susceptible to specific cytokine response patterns activated by rugged dsRNA acting selectively as a TLR3 agonist.
  • the rugged dsRNA is preferably administered by intravenous infusion; intradermal, subcutaneous, or intramuscular injection; intranasal or intratracheal inhalation; or oropharyngeal or sublingual application.
  • a medicament is provided as a pharmaceutical composition.
  • One or more different rugged dsRNA may be used for their beneficial effect(s) on a subject's health, as selective TLR3 agonist(s), to treat a disease or other pathological condition, or to manufacture medicaments or pharmaceutical compositions to treat a disease or other pathological condition.
  • Optional inert ingredients of the composition include excipients and a vehicle (e.g., saline buffer or water) as a single dose or a multi-dose package (e.g., an injection vial or vials), and instructions for their use.
  • a vehicle e.g., saline buffer or water
  • a multi-dose package e.g., an injection vial or vials
  • one or more different rugged dsRNA may be formulated at a concentration from about 0.05 mg/mL to about 0.25 mg/mL (e.g., 10 mg dissolved in 4 mL or 20 mg dissolved in 8 mL) in physiological phosphate-buffered saline and stored at from 2° C. to 8° C. in a refrigerator under aseptic conditions.
  • FIG. 1A shows an HPLC chromatogram for poly(I):poly(C 12 U).
  • the minor peak (not integrated) centered at a retention time of about 5.00 min is duplexed poly(I):poly(C 12 U).
  • the first major peak centered at a retention time of about 7.58 min is the single-stranded poly(C 12 U).
  • the second major peak centered at a retention time of about 10.05 min is the single-stranded poly(I).
  • the molecular identity of each peak was determined by photodiode array (PDA) analysis.
  • PDA photodiode array
  • FIG. 1B is an HPLC chromatogram for lyophilized poly(I):poly(C 12 U) showing aggregates lyophilization can produce. Note that aggregation does not occur in the solution process of FIG. 1C , which avoids lyophilization.
  • FIG. 1C shows a HPLC chromatogram of a sterile solution of poly I:poly(C 12 U) also showing a novel 5 minute peak.
  • FIG. 2 shows PDA analyses of the three HPLC peaks.
  • Acetonitrile which is used as a solvent, is responsible for the strong absorbance at 230 nm.
  • Absorbance at 245 nm indicates the presence of poly(I); absorbance at 265 nm indicates the presence of poly(C 12 U).
  • FIG. 2A is PDA analysis of the peak centered at a retention time of about 5.01 min, which contains both poly(I) and poly(C 12 U) character.
  • FIG. 2B is PDA analysis of the peak centered at a retention time of about 7.58 min, which contains poly(C 12 U).
  • FIG. 2C is PDA analysis of the peak centered at a retention time of about 10.05 min, which contains poly(I).
  • FIG. 3 is a circular dichroism (CD) of the dsRNA.
  • the melting point of 64° C. represents the condition of 1 ⁇ 2 double stranded structure.
  • FIG. 4 is the CD wavelength scan of dsRNA.
  • the double stranded structure is characterized by two peaks at 245 nm and 278 nm, representing two chiral centers normally present in fully double stranded poly I:poly(C 12 U). These centers represent chirality due to base pair structure (278 nm) and the base stacking which is associated with the formation of duplex double helix.
  • FIG. 5 shows the circular dichroism of poly(I):poly(C 12 U) with the characteristic chiral peaks at 245 nm and 278 nm
  • FIG. 6 shows a plot of the derivative of the thermal melt of poly(I):poly(C 12 U). Integrity of the structure is characterized by the melting point and the 1 ⁇ 2 width of this derivative profile, both expressed as degrees C.
  • FIG. 7 shows by HPLC that preparation with heating abolishes all double strand structure as reflected by loss of 245 nm peak since ( FIG. 4 ) the 245 nm peak is due to chiral base stacking.
  • analysis by circular dichroism shows that, as a product of thermal stress, the 5 minute peak maintains both double helix configuration and chiral centers in the backbone.
  • FIG. 8 shows a CD plot of a thermal melt of single stranded poly(I).
  • the chiral center for inosine provides a weak signal at about 252 nm.
  • FIG. 9 shows a plot of the derivative of the thermal melt of single stranded poly(I) There is no evidence of intra molecular base stacking at thermal condition which would otherwise disrupt a double helix.
  • FIG. 10 shows a CD plot of a thermal melt of single stranded poly(C 12 U).
  • a strong signal is apparent due to the chirality of cytidine.
  • the absence of a second peak at 245 nm shows that intra molecular base stacking of Poly C 12 U does not occur.
  • FIG. 11 shows a plot of the derivative of the thermal melt of single stranded poly(C 12 U). There is no evidence of intra molecular base stacking at thermal condition which would otherwise disrupt a double helix.
  • FIG. 12 shows a CD plot of a thermal melt of poly(I):poly(C 12 U) and poly(I):poly(C). Base stacking is evident in both compounds as indicated by the peak at 245 nm.
  • FIG. 13 shows a plot of the derivative of the thermal melt of poly(I):poly(C 12 U) and poly(I):poly(C). Both compounds exhibit the critical melting point for disruption of the double helix. However, the lower melting point of Poly I:Poly C 12 U illustrates a more labile character which in turn affords the advantageous safety profile of the uridine substituted compound.
  • FIG. 14 shows a CD wavelength scan of poly(I):poly(C 12 U) and poly(A):poly(U).
  • a very weak and shifted single structure may be associated with the propensity for chiral aggregation of poly(A):poly(U)
  • FIG. 15 shows a plot of the derivative of the thermal melt of poly(I):poly(C 12 U) and poly(A):poly(U). Somewhat high melting point is likely related to the aggregation tendency of poly(A):poly(U) noted in FIG. 14 .
  • FIG. 16 shows the derivative of a thermal melt of single stranded poly(I):poly(C 10 U).
  • the greater degree of Uridine substation (compare poly(I):poly(C 12 U, FIG. 6 ) has compromised the double helical structure.
  • the 1:12 ratio of U:C is optimal, providing one interruption per helical turn.
  • FIG. 17 shows a CD plot of thermal melt of single stranded poly(I):poly(C 10 ). Consistent with the lack of thermal melt behavior ( FIG. 16 ), the greater degree of Uridine substation (1:10 ratio of U:C, cf. 1:12 in poly(I):poly(C 12 U) has abolished the base stacking signal at 245 nm.
  • FIG. 18 shows size exclusion chromatography of complexes of TLR3-ECD and poly(I):poly(C 12 U) ( FIG. 18A ), the receptor TLR3-ECD only ( FIG. 18B ), and the ligand poly(I):poly(C 12 U) only ( FIG. 18C ).
  • FIG. 19 shows the effect of thermal stress (40° C.) on the size of dsRNA as measured by analytical centrifugation.
  • the decrease in sedimentation coefficient (S 20,w ) reflects a loss of size due to hydrolysis.
  • FIG. 20 shows the effect of thermal stress (40° C.) upon the component strands of dsRNA (7 minute and 10 minute peaks) and the rugged dsRNA as measured by high performance liquid chromatography (HPLC). Whereas the larger poly(I) and poly(C 12 U) strands hydrolyze at 40° C., the quantity of rugged dsRNA peak increases.
  • FIG. 21 shows the relative size of AMPLIGEN vs new rugged dsRNA (peak 5 minutes).
  • FIG. 22 Partial view of poly(I):poly(C 12 U) partially hybridized strands and the interaction of bases of individual poly(I) and the poly(C 12 U) strands. Molecular weight 1,100,000 da.
  • FIG. 23 Partial view of poly(I):poly(C 12 U) partially hybridized strands and the interaction of bases of individual poly(I) and the poly(C 12 U) strands. Molecular weight 286,000 da.
  • dsRNA double-stranded ribonucleic acid
  • the invention may be used to treat a subject (e.g., human or animal, especially birds, fishes, or mammals) with an incipient or established microbial infection, to treat a subject for other pathological conditions marked by abnormal cell proliferation (e.g., neoplasm or tumor), or for use as an immunostimulant to treat the subject for a disease or other pathological condition caused by at least infection, abnormal cell proliferation, chronic fatigue syndrome, or cell damage from autoimmunity or neurodegeneration. It is preferred that the amount of rugged dsRNA used is sufficient to bind Toll-Like Receptor 3 (TLR3) on immune cells of the subject. Innate or adaptive immunity may be triggered thereby.
  • TLR3 Toll-Like Receptor 3
  • rugged dsRNA may be used to activate TLR3 selectively without activating other Toll-like receptors like TLR4 or an RNA helicase like RIG-I or mda-5, or without inducing an excessive pro-inflammatory response as seen with the nonselective TLR3 agonist poly(I):poly(C) in a phenomenon known as “cytokine storm” in the art.
  • the subject may be infected with at least one or more bacteria, protozoa, or viruses.
  • a pharmaceutical composition which is comprised of rugged dsRNA in an amount sufficient to bind to TLR3 is administered to the subject. Infection of the subject is reduced or eliminated thereby as assayed by decreased recovery time, increased immunity (e.g., increase in antibody titer, lymphocyte proliferation, killing of infected cells, or natural killer cell activity), decreased division or growth of the microbe, or any combination thereof as compared to the subject not treated with the rugged dsRNA.
  • the immunity induced by treatment is preferably specific for the microbe, although inducing innate immunity may also be efficacious.
  • microbe An infection by a microbe may be treated.
  • the microbe may infect a human or animal subject.
  • the infection may be incipient or established.
  • the microbe may be a bacterium, protozoan, or virus; especially those that cause disease (i.e., pathogenic microbes).
  • pathogenic microbes i.e., pathogenic microbes.
  • the terms “microbe” and “micro-organism” are used interchangeably.
  • the bacterium may be a species of the genus Bacillus (e.g., B. anthracis, B. cereus ), Bartonella ( B. henselae ), Bordetella (e.g., B. pertussis ), Borrelia (e.g., B. burgdorferi ), Brucella (e.g., B. abortus ), Campylobacter (e.g., C. jejuni ), Chlamydia (e.g., C. pneumoniae ), Clostridium (e.g., C. botulinum, C. difficile, C. perfringens, C. tetani ), Corynbacterium (e.g., C.
  • M. genitalium e.g., M. genitalium, M.
  • Neisseria e.g., N. gonorrheae, N. meningitidis
  • Pneumocystis e.g., P. carinii
  • Pseudomonas P. aeruginosa
  • Rickettsia e.g., R. rickettsia, R. typhi
  • Salmonella e.g., S. enterica
  • Shigella e.g., S. dysenteriae
  • Staphylococcus e.g., S. aureus, S. epidermidis
  • Streptococcus e.g., S. pneumoniae, S.
  • Treponema e.g., T. pallidum
  • Vibrio e.g., V. cholerae, V. vulnificus
  • Yersinia e.g., Y. pestis
  • These include Gram-negative or Gram-positive bacteria, chlamydia, spirochetes, mycobacteria, and mycoplasmas.
  • the protozoan may be a species of the genus Cryptosporidium (e.g., C. hominis, C. parvum ), Entamoeba (e.g., E. histolytica ), Giardia (e.g., G. intestinalis, G. lamblia ), Leishmania (e.g., L. amazonensis, L. braziliensi, L. donovani, L. mexicana, L. tropica ), Plasmodium (e.g., P. falciparum, P. vivax ), Toxoplasma (e.g., T. gondii ), or Trypanosome (e.g., T. bruci, T. cruzi ).
  • Cryptosporidium e.g., C. hominis, C. parvum
  • Entamoeba e.g., E. histolytica
  • Giardia e.g., G. intestinalis, G. lamb
  • the virus may be a DNA or RNA virus that infects humans and animals.
  • DNA viruses include those belonging to the Adenoviridae, Iridoviridae, Papillomaviridae, Polyomavirididae, and Poxyiridae families (Group I double-stranded DNA viruses); the Parvoviridae family (Group II single-stranded DNA viruses).
  • RNA viruses include those belonging to the Birnaviridae and Reoviridae families (Group III double-stranded RNA viruses); the Arteriviridae, Astroviridae, Caliciviridae, Hepeviridae, and Roniviridae families (Group IV positive single-stranded RNA viruses); and the Arenaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae, and Rhabdoviridae families (Group V negative single-stranded RNA viruses).
  • Rugged dsRNA may also be used to treat infection by DNA viruses from the Herpesviridae family and RNA viruses from the Flaviviridae, Hepadnaviridae, Orthomyxoviridae, Picornaviridae, Retroviridae, and Togaviridae families.
  • the subject may be afflicted by a disease or pathological condition that is characterized by abnormal cell proliferation (e.g., neoplasm or tumor, other transformed cells).
  • a pharmaceutical composition which is comprised of rugged dsRNA in an amount sufficient to bind to TLR3 is administered to the subject.
  • Disease, symptoms thereof, their number, or their severity in the subject may be reduced or eliminated thereby as assayed by improved morbidity or mortality, increased immunity (e.g., increase in antibody titer, lymphocyte proliferation, killing proliferating or transformed cells, or NK cell activity), decreased division or growth of proliferating or transformed cells, or any combination thereof as compared to the condition of a subject not treated with rugged dsRNA.
  • the subject's cells undergoing the abnormal proliferation may be a neoplasm or tumor (e.g., carcinoma, sarcoma, leukemia, lymphoma), especially cells transformed by a tumor virus (e.g., DNA or RNA virus carrying a trans-forming gene or oncogene) or otherwise infected by a virus associated with cancer.
  • a tumor virus e.g., DNA or RNA virus carrying a trans-forming gene or oncogene
  • Epstein-Barr virus is associated with nasopharyngeal cancer, Hodgkin's lymphoma, Burkitt's lymphoma, and other B lymphomas; human hepatitis B and C viruses (HBV and HCV) are associated with liver cancer; human herpesvirus 8 (HHV8) is associated with Kaposi's sarcoma; human papillomaviruses (e.g., HPV6, HPV11, HPV16, HPV18, or combination thereof) are associated with cervical cancer, anal cancer, and genital warts; and human T-lymphotrophic virus (HTLV) is associated with T-cell leukemia and lymphoma.
  • Cancers include those originating from the gastrointestinal (e.g., esophagus, colon, intestine, ileum, rectum, anus, liver, pancreas, stomach), genitourinary (e.g., bladder, kidney, prostate), musculoskeletal, nervous, pulmonary (e.g., lung), or reproductive (e.g., cervix, ovary, testicle) organ systems.
  • gastrointestinal e.g., esophagus, colon, intestine, ileum, rectum, anus, liver, pancreas, stomach
  • genitourinary e.g., bladder, kidney, prostate
  • musculoskeletal, nervous pulmonary
  • pulmonary e.g., lung
  • reproductive e.g., cervix, ovary, testicle
  • Dendritic cell maturation may be induced in the subject.
  • Immature dendritic cells which are capable of antigen uptake, may be induced to differentiate into more mature dendritic cells, which are capable of antigen presentation and priming an adaptive immune response (e.g., antigen-specific T cells).
  • an adaptive immune response e.g., antigen-specific T cells.
  • they may at least change cell-surface expression of major histocompatibility complex (MHC) molecules, costimulatory molecules, adhesion molecules, or chemokine receptors; decrease antigen uptake; increase secretion of chemokines, cytokines, or proteases; grow dendritic processes; reorganize their cytoskeleton; or any combination thereof.
  • MHC major histocompatibility complex
  • the subject may be vaccinated against at least infection or cancer.
  • both infection and cancer may be treated.
  • a medicament or pharmaceutical composition which is comprised of rugged dsRNA in an amount sufficient to bind to TLR3 is administered to the subject.
  • the immune response to a vaccine or dendritic cell preparation is stimulated thereby.
  • the vaccine or dendritic cell preparation may be comprised of killed, fixed, or attenuated whole microbes or cells (e.g., proliferating or transformed); a lysate or purified fraction of microbes or cells (e.g., proliferating or transformed); one or more isolated microbial antigens (e.g., native, chemically synthesized, or recombinantly produced); or one or more isolated tumor antigens (e.g., native, chemically synthesized, or recombinantly produced).
  • In situ vaccination may be accomplished by the subject's production of antigen at a site or circulation thereto (e.g., produced in a natural infection or cell growth, or shed antigen), and rugged dsRNA acting as an adjuvant thereon.
  • Specifically-configured dsRNA may be of the general formula ribo(I n ).ribo(C 4-29 U) n , ribo(I n ).ribo(C 11-14 U) n , or ribo(I n ).ribo(C 12 U) n , wherein strands are comprised of ribonucleotides (ribo) and n is an integer from about 40 to about 40,000 repeats.
  • a poly(riboinosinic acid) strand may be partially hybridized to poly(ribocytosinic 4-29 uracilic acid), poly(ribocytosinic 11-14 uracilic acid), or poly(ribocytosinic 12 uracilic acid) strand such that the two strands do not form a duplex at the position of the uracil base (i.e., no base pairing at the mismatched position).
  • Specifically-configured dsRNA include: ribo(I).ribo(C 4 , U), ribo(I).ribo(C 11 , U), ribo(I).ribo(C 13 , U), ribo(I).ribo(C 18 , U), ribo(I).ribo(C 20 , U), ribo(I).ribo(C 24 , G), and ribo(I).ribo(C 29 , G).
  • dsRNA are based on copolynucleotides such as poly(C m U) and poly(C m G) in which m is an integer from about 4 to about 29, or analogs of poly(riboinosinic acid) and poly(ribocytidilic acid) formed by modifying the ribo(I n ).ribo(C n ) to incorporate unpaired bases (uracil or guanine) in the polyribocytidylate r(C m ) strand.
  • copolynucleotides such as poly(C m U) and poly(C m G) in which m is an integer from about 4 to about 29, or analogs of poly(riboinosinic acid) and poly(ribocytidilic acid) formed by modifying the ribo(I n ).ribo(C n ) to incorporate unpaired bases (uracil or guanine) in the polyribocytidylate
  • specifically-configured dsRNA may be derived from ribo(I).ribo(C) dsRNA by modifying the ribosyl backbone of poly(riboinosinic acid) ribo(I n ), e.g., by including 2′-O-methyl ribosyl residues.
  • Specifically-configured dsRNA may also be modified at the molecule's ends to add a hinge(s) to prevent slippage of the base pairs, thereby conferring a specific bioactivity in solvents or aqueous environments that exist in human biological fluids.
  • 4,024,222; 4,130,641; and 5,258,369 are generally suitable for use according to the present invention after selection for rugged dsRNA.
  • One or more different rugged dsRNA may be complexed with a stabilizing polymer such as polylysine, polylysine plus carboxymethylcellulose, polyarginine, polyarginine plus carboxymethylcellulose, or any combination thereof.
  • Rugged dsRNA as at least a portion of a medicament or formulated with other compatible components in a pharmaceutical composition may be administered to a subject (e.g., human patient or animal, especially birds, fishes, or mammals) by any local or systemic route known in the art including enteral (e.g., oral, feeding tube, enema), topical (e.g., device such as a nebulizer for inhalation through the respiratory system, skin patch acting epicutaneously or transdermally, suppository acting in the rectum or vagina), and parenteral (e.g., subcutaneous, intravenous, intramuscular, intradermal, or intraperitoneal injection; buccal, sublingual, or transmucosal; inhalation or instillation intranasally or intratracheally).
  • enteral e.g., oral, feeding tube, enema
  • topical e.g., device such as a nebulizer for inhalation through the respiratory system, skin patch acting epi
  • the rugged dsRNA may be micronized by milling or grinding solid material, dissolved in a vehicle (e.g., sterile buffered saline or water) for injection or instillation (e.g., spray), topically applied, or encapsulated in a liposome or other carrier for targeted delivery. Dissolving the rugged dsRNA in water for injection (WFI) and injection of the composition into the subject are preferred.
  • a carrier may be used to target the rugged dsRNA to the TLR3 receptor on antigen presenting cells and epithelium. For example, immature dendritic cells may be contacted in skin, mucosa, or lymphoid tissues. It will be appreciated that the preferred route may vary with the age, condition, gender, or health status of the subject; the nature of disease or other pathological condition, including the number and severity of symptoms; and the chosen active ingredient.
  • Formulations for administration may include aqueous solutions, syrups, elixirs, powders, granules, tablets, and capsules which typically contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, wetting agents, suspending agents, emulsifying agents, preservatives, buffer salts, flavoring, coloring, and/or sweetening agents.
  • excipients such as binding agents, fillers, lubricants, disintegrants, wetting agents, suspending agents, emulsifying agents, preservatives, buffer salts, flavoring, coloring, and/or sweetening agents.
  • the preferred formulation may vary with the age, condition, gender, or health status of the subject; the nature of disease or other pathological condition, including the number and severity of symptoms; and the chosen active ingredient.
  • Rugged dsRNA may be dosed at from about 0.5 mg to about 60 mg, from about 5 mg to about 40 mg, or from about 10 mg to about 20 mg in a subject (e.g., body mass of about 70-80 Kg for a human patient) on a schedule of once to thrice weekly (preferably twice weekly), albeit the dose amount and/or frequency may be varied by the physician or veterinarian in response to the subject's symptoms.
  • Nucleic acid in solid form may be dissolved in physiological phosphate-buffered saline and then infused intravenously.
  • Cells or tissues that express TLR3 are preferred sites in the subject for delivering the nucleic acid, especially antigen presenting cells (e.g., dendritic cells, macrophages, B lymphocytes) and endothelium (e.g., endothelial cells of the respiratory and gastric systems).
  • antigen presenting cells e.g., dendritic cells, macrophages, B lymphocytes
  • endothelium e.g., endothelial cells of the respiratory and gastric systems.
  • the preferred dosage may vary with the age, condition, gender, or health status of the subject; the nature of disease or other pathological condition, including the number and severity of symptoms; and the chosen active ingredient.
  • TLRs Toll-like receptors
  • dsRNA is a selective agonist of TLR3.
  • Rugged dsRNA may be used as a selective agent for activation of TLR3.
  • Dysfunction in co-stimulatory molecule e.g., CD80, CD83, CD86
  • signaling in dendritic cells may be associated with the disease or other pathological condition to be treated. This abnormality may be normalized by using rugged dsRNA as a selective TLR3 agonist.
  • the effects of rugged dsRNA may be inhibited or blocked by mutation of the TLR3 gene (e.g., deletion), down regulating its expression (e.g., siRNA), binding with a competitor for TLR3's ligand-binding site (e.g., neutralizing antibody) or a receptor antagonist, or interfering with a downstream component of the TLR3 signaling pathway (e.g., MyD88 or TRIF).
  • mutation of the TLR3 gene e.g., deletion
  • down regulating its expression e.g., siRNA
  • binding with a competitor for TLR3's ligand-binding site e.g., neutralizing antibody
  • a receptor antagonist e.g., a receptor antagonist
  • Circular dichroism is a physico-chemical technique for characterizing the conformation of specifically-configured dsRNA. It can also be used as a surrogate for binding of AMPLIGEN® (rintatolimod) poly(I):poly(C 12 U) as a receptor agonist to its receptor TLR3. Furthermore, the helical structure of rugged dsRNA and the structural requirements for binding of specifically-configured dsRNA to TLR3 can be precisely characterized by CD.
  • physico-chemical techniques that may be used to characterize rugged dsRNA are reverse phase chromatography, PDA (photodiode array) analysis, gas pressure chromatography (GPC), specific ligand binding to TLR3 receptor, and sedimentation velocity measured by ultracentrifugation.
  • PDA photodiode array
  • GPC gas pressure chromatography
  • Rugged dsRNA provides a selective agent for dissecting out the effects of TLR3 activation on the immune system that was not previously available with such potency.
  • Other agents like TLR adapters MyD88 and TRIF mediate signaling by all TLR or TLR3/TLR4, respectively. Thus, activation or inhibition of signaling through MyD88 or TRIF would not restrict the biological effects to those mediated by TLR3.
  • TLR3 and its signaling is a requirement for AMPLIGEN® (rintatolimod) poly(I):poly(C 12 U) to act as a receptor agonist
  • Such confirmation of TLR3 activity can be performed before, during, or after administration of the agonist.
  • the agonist can be used to restrict the immune response to activation of TLR3 without activating other Toll-like receptors or RNA helicases.
  • abnormal cytokine e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , TNF- ⁇ , IL-6, IL-10, IL-12
  • co-stimulatory molecule e.g., CD80, CD83, CD86
  • This abnormality may be remodulated by using rugged dsRNA as a selective agonist of TLR3.
  • Antigen presentation may be improved by conjugating the antigen (or a peptide analog thereof) to a ligand (or a receptor) that specifically binds to the cell surface (especially a component of the endosomephagosome internalizing pathway) of one or more antigen presenting cells.
  • the specific binding molecule may be an antibody to a cell surface molecule, or a derivative thereof (e.g., Fab, scFv).
  • CD80, CD83, and CD86 may be analyzed by flow cytometry using fluorescently-labeled antibodies. Following overnight shipment, blood samples are stained within one hour of receipt. Conventional techniques are used for lysis of red blood cells and cell marker analyses by flow cytometry. Dendritic cells are identified based on low level expression of lymphocyte, monocyte, and NK cell markers along with high HLA-DR expression. Dendritic cells may also characterized according to CD11c and CD123 expression. Monocytes are identified by side scatter analysis and expression of a monocyte lineage marker. Analyses of CD80, CD83, and CD86 expression are performed after cell type identification. Measurements from healthy volunteers serve as controls, and they would indicate normal distribution and levels of marker expression for mature dendritic cells such as CD80, CD83, and CD86.
  • Synthesis of single-stranded poly(I) and poly(C 12 U) began with enzymatic polynucleotide synthesis of the polynucleotides from the respective mononucleotide starting materials: inosine for poly(I); cytidine (C) and uridine (U) for poly(C 12 U). Then repetitive extraction and precipitation steps were used to remove residual impurities.
  • the reaction solutions containing the products were concentrated by ultrafiltration and extracted with phenol four times. The concentrated and extracted solutions were precipitated, dissolved, and re-precipitated from aqueous ethanol (50:50).
  • Enzymatic Synthesis The enzymatic synthesis used in the manufacturing process is dependent on the enzyme polynucleotide phosphorylase to synthesize polyinosinic acid and polycytidilic 12 uridilic acid from their respective starting materials: cytidine 5′-diphosphate, trisodium salt (CDP.Na 3 ), uridine 5′-diphosphate, disodium salt (UDP.Na 2 ) and inosine 5′ diphosphate, trisodium salt (IDP.Na 3 ).
  • the enzyme catalyzes polynucleotide formation in a reversible reaction using Mg ++ as a co-factor and ATP as a source of energy.
  • Polynucleotides were synthesized in the 5′ to 3′ direction with concurrent liberation of inorganic phosphate. Maximum yield was limited by the equilibrium between synthesis and reverse rates, degradative reaction (phosphorolysis). The progress of the reaction was followed by measuring the consumption of CDP or IDP. Viscosity of the reaction solution was also monitored. Purified water was filtered into the tank.
  • TRIS hydroxymethyl aminomethane
  • urea magnesium chloride hexahydrate
  • edetate ethylenediaminetetraacetic acid
  • EDTA.Na 2 disodium salt
  • the lower phenol waste phase is then pumped into containers for disposal.
  • the location of the phenol cut was important in order to effectively separate phenol and protein from the upper, product phase, which contains poly(C 12 U) or poly(I).
  • the phenol phase and an intermediate “rag” layer which contains denatured protein solids, were discarded by visually observing the liquid flowing through the site glass at the tank outlet. When the phenol and rag layer disappeared and only product phase was observed, the outlet valve was closed and the phenol cut is considered complete.
  • Poly(I) Poly(C 12 U), Sterile Solution, for Intravenous Infusion.
  • Poly(I) and poly(C 12 U) were dissolved in phosphate-buffered saline. Equal molar amounts were mixed in an annealing step, and cooled to room temperature. The solutions were sterile filtered.
  • the formulated bulk was sterile filtered in-line into a steam sterilized surge vessel.
  • the filling operation was performed. After each vial was filled, a sterile stopper is used to stopper the vial. Stoppered vials were then conveyed from the aseptic processing area where they were sealed.
  • Rugged dsRNA was isolated from the annealed poly(I):poly(C 12 U), which was prepared according to the above, by either analytical or preparative high performance liquid chromatography (HPLC) as a substantially purified and pharmaceutically-active molecule. Its molecular weight is about 286 Kda and is about 413 base pairs in length with about 34 complete turns of the RNA helix. It is only from about 1 mol % to about 4 mol % of an unfractionated AMPLIGEN® (rintatolimod) composition.
  • dsRNA (about 96 mol % to about 99 mol %) after synthesis has a molecular weight of about 1.2 Mda and is about 2000 base pairs in length with about 166 complete turns of the RNA helix.
  • the rugged dsRNA in the 5 min HPLC peak is about 4.9 times smaller than the bulk of the dsRNA, and more closely fits the ligand binding site of its cell surface receptor (TLR3).
  • rugged dsRNA Due to its structure, rugged dsRNA is unusually resistant to disruption of its RNA double helix and molecular unfolding. Thus, rugged dsRNA under the assay conditions described herein has about 100- to about 1,000-fold greater bioactivity than the same weight of unselected AMPLIGEN® (rintatolimod) poly(I):poly(C 12 U).
  • TLR3 Activation is Linked to Expression of IFN- ⁇ / ⁇ , IL-6, or IL-12.
  • the relationship between IFN expression through TLR3 activation by dsRNA was established by Alexopoulou (2001) using 293T cells that express different Toll-like receptors (human TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, or TLR9). Only those cells containing human TLR3 showed marked expression of IFN- ⁇ / ⁇ , IL-6 or IL-12 when stimulated with poly(I):poly(C).
  • Poly(I):Poly(C 12 U) Induces Host Defense Gene Modulation through Highly Selective Activation of TLR3.
  • TLR3-dependent innate immune response To understand the relationship of the TLR3-dependent innate immune response to viral protection, Gowen (2007), subjected TLR3-deficient mice to dsRNA and measured expression of IFN- ⁇ / ⁇ , IL-6, and IL-12. The mice were also subsequently challenged by exposure to Punta Toro virus (PTV). Protection from the viral challenge was extraordinarly sensitive to treatment with poly(I):poly(C 12 U). Viral protection conferred by poly(I):poly(C 12 U) was completely abolished for the case of TLR3-deficient mice.
  • PTV Punta Toro virus
  • This selective targeting of the TLR3 signaling pathway represents a significant advantage for therapeutic applications of poly(I):poly(C 12 U) as compared to other possible cytosolic mechanisms such as, for example, the use of unsubstituted dsRNA poly(I):poly(C) to stimulate cytokine production through RNA helicases such as MDA-5 and RIG-1 (Pichlmair, 2006).
  • TLR3 Binding Site Studying the structure of native TLR3 crystals, Choe (2005) found that TLR3 is a large horseshoe-shaped, right-handed, solenoid structure comprised of 23 leucine-rich repeats. The glycosylated, convex surface and negatively-charged concave surfaces are unlikely binding sites for dsRNA. Consequently, they proposed that dsRNA binding occurs at positively-charged patches located on the lateral face.
  • Circular dichroism provides detailed information concerning the secondary, helical structures of dsRNA or alterations thereof which accompany ligand binding; as well as structural changes caused by enzymatic hydrolysis and addition of metal ions. Also, in the thermal stress mode, conformational information imparted by CD provides valuable insights to explain RNA stability.
  • a double-stranded RNA composition may be analyzed by high performance liquid chromatography (HPLC) as shown in FIGS. 1A , 1 B and 1 C.
  • HPLC high performance liquid chromatography
  • Analysis of a representative lot of AMPLIGEN® (rintatolimod) poly(I):poly(C 12 U) resulted in two distinct peaks: one with retention times from 9.85 to 10.35 min corresponding to the poly(I) strand and from 7.30 to 7.80 min corresponding to the poly(C 12 U) strand.
  • Rugged dsRNA is found at a retention time of about 5 min representing a molecular species uniquely resistant to denaturation and unfolding. Denaturing conditions would eliminate biological activity exclusively due to TLR3 receptor binding.
  • This analytical method may also be used as a stability indicating assay and, in particular, it may be used to show that the rugged dsRNA is unusually resistant to disruption of its double helix and to molecular unfolding.
  • each peak is determined by analysis with a photodiode array (PDA) detector as shown in FIGS. 2A , 2 B and 2 C.
  • PDA photodiode array
  • a UV absorption scan of wavelengths from 200 nm to 360 nm was obtained.
  • Duplex poly(I):poly(C 12 U) and individual poly(I) and poly(C 12 U) strands have their own specific peak absorption wavelengths.
  • Absorption peaks centered at both 248 nm and 265 nm indicate the presence of rugged dsRNA (about 286,000 daltons) having poly(I) and poly(C 12 U), respectively ( FIG. 2A ).
  • Peak absorption centered at about 265 nm indicates the presence of the poly(C 12 U) strand ( FIG.
  • Peak absorption centered at about 248 nm indicates the presence of the poly(I) strand ( FIG. 2C ).
  • Absorption centered at about 230 nm is due to acetonitrile used as solvent. Because of the relative scarcity of rugged dsRNA, the signal at 230 nm was subtracted from FIG. 2A .
  • FIG. 21 shows the relative size of AMPLIGEN vs new rugged dsRNA (peak 5 minutes)
  • FIG. 22 Shown in FIG. 22 are a partial view of poly(I):poly(C 12 U) partially hybridized strands and the interaction of bases of individual poly(I) and the poly(C 12 U) strands.
  • Single inosine bases bind to cytosine bases, but not to the uridine base.
  • the poly(inosinic acid) is hydrogen bonded (dashed lines between bases) to poly(cytidylic acid), with uridylic acid substitution occurring on an average of every 12-13 bases.
  • the number of repeat units (n) corresponding to the size of poly(I):poly(C 12 U) of approximately 1.2 Mda is 2000 base pairs or 166 full helical turns.
  • FIG. 23 Shown in FIG. 23 are a partial view of poly(I):poly(C 12 U) partially hybridized strands and the interaction of bases of individual poly(I) and the poly(C 12 U) strands.
  • Single inosine bases bind to cytosine bases, but not to the uridine base.
  • the poly(inosinic acid) is hydrogen bonded (dashed lines between bases) to poly(cytidylic acid), with uridylic acid substitution occurring on an average of every 12-13 bases. This is “rugged” dsRNA.
  • the number of repeat units (n) corresponding to the size range of new variant, also termed rugged dsRNA (also termed peak 5 min on HPLC) is 286 Kda having 413 base pairs representing 34 complete turns of RNA helix and is resistant to disassembly of hydrogen-bonded strands under elevated thermal or abnormal ionic conditions.
  • Circular dichroism has been used to measure secondary structure (duplexed helices) of biological and synthetic polymers, including proteins and nucleic acids.
  • CD is the measurement of absorption of right- or left-circular polarized light, at a specific wavelength, by chiral molecules. Chemical chirality is the property of a molecule being nonsuperimposable on its mirror image. An atom that makes its molecule chiral is called a chiral atom or, more commonly, a chiral center.
  • Poly(I):poly(C 12 U) has a number of chiral centers because of its primary and secondary structures.
  • Chiral centers are found in the nucleotide bases, which form the two primary structures for the two individual RNA strands (ssRNA) of poly(I):poly(C 12 U). Additional chiral centers come from hybridizing each ssRNA to the other through hydrogen bonding of their complementary bases. Hydrophobic bonding between adjacent bases of dsRNA is known as base stacking and produces a flexible, linear symmetrical, helical secondary structure of defined shape and size.
  • CD spectra for AMPLIGEN® (rintatolimod) poly(I):poly(C 12 U) which is dependent on the wavelength, are observed to be a function reflecting the Gaussian absorption for each chiral center. Therefore, the CD spectrum for a dsRNA such as poly(I):poly(C 12 U) is dependent on the complementary base pairing of double-stranded structures and the complex chirality of the resultant helical structure.
  • the specific ellipticity measured in a wavelength scan provides a quantitative parameter, which is calculated as the ellipticity ratio at certain “critical” wavelengths.
  • the value of this structural parameter, the ratio CD 278 /CD 245 is unique to poly(I):poly(C 12 U).
  • ellipticity is measured during heating. As poly(I):poly(C 12 U) is heated and thermally denatured, the individual poly(I) and poly(C 12 U) strands unwind due to the breakdown of hydrogen bonding between complementary base pairs.
  • the minimum derivative value corresponds to melting temperature, defined as the point where 50% of the double-stranded conformation is unwound.
  • the width at half-height of the peak a measure of structural uniformity, also becomes an indication of its integrity. Taken together, these thermal indices provide a measure of the strength of the dsRNA helixes.
  • the wavelength scan detects two peaks: a first peak at 245 nm corresponding to the doubled stranded helix of the poly(I):poly(C 12 U) and a second peak at 278 nm corresponding to the stacking of the nucleic acid's base pairs.
  • dsRNA affords separate peaks in the CD wavelength scan, at 245 and 278 nm, the former peak associated with base stacking attribute of helical structure. Accordingly, the ratio of peak heights at 278/245 is typically within 0.69-0.79 for dsRNA but much higher in the absence of double helical structure.
  • the reversed phase HPLC assay is utilized to distinguish “rugged” dsRNA (5.0 minute peak) from the separated, component strands of poly(I):poly(C 12 U): 7.0 (poly C 12 U) and 10.0 minute peaks (poly I). It is clear from the 278/245 ratio that only the 5.0 minute, rugged dsRNA fraction retains helical structure, in contrast to the separated, component strands of Poly I:Poly C 12 U.
  • CD assay of AMPLIGEN® (rintatolimod) poly(I):poly(C 12 U) acts in a precise manner during thermal analysis for the determination of T M and width at half height of the first derivative of the thermal melt curve and during the CD scan analysis for determination of the ratio of CD at 278 nm to CD at 245 nm.
  • This CD method for characterizing poly(I):poly(C 12 U) is also specific because it can between differentiate duplexed nucleic acids and single-stranded nucleic acids, or other similar double-stranded nucleic acids that do not meet the manufacturing and release specifications for AMPLIGEN® (rintatolimod) poly(I):poly(C 12 U).
  • the scans of double-stranded molecules such as poly(I):poly(C 12 U), poly(I):poly(C), and poly(A):poly(U) differed significantly from those obtained during analysis of single-stranded molecules such as poly(I) and poly(C 12 U). See FIGS. 8-17 . Furthermore, each of the CD scans was unique for the molecular species being assayed.
  • the specificity of the assay was also investigated to assess, unequivocally, the ability to detect compounds of closely related structure.
  • Double-stranded ribonucleic acids of different nucleotide base composition such as poly(I):poly(C 12 U), poly(I):poly(C), and poly(A):poly(U). ( FIGS. 10 , 12 and 14 ).
  • (c) Double-stranded ribonucleic acid formulated from poly(I) and poly(C x U y ) strands with a cytidine to uridine base ratio of 11-14 to 1 ( FIGS. 16 and 17 ) (C:U ratio 11:1 to 14:1).
  • the CD method is specific for detection of poly(I):poly(C 12 U) formulated from polymers not meeting the aforementioned specifications for size.
  • the results from the CD analysis of these molecules do not meet specifications for AMPLIGEN® (rintatolimod) in regards to T M and width at half-height of the first derivative of the thermal melt curve.
  • the failure to meet specifications for these CD parameters is observed with these formulations even when the ⁇ 1.5 S size differential specification is satisfied.
  • the CD 278 /CD 245 ratio determinations were less specific. CD scans alone did not differentiate between poly(I):poly(C 12 U) and non-poly(I):poly(C 12 U) formulations that did not meet manufacturing and/or release specifications for polymer size.
  • CD analysis is sensitive to the size of the single-stranded polymer strands.
  • size difference between the complementary single-stranded polymer components, poly(I) and poly(C 12 U) is 2.4 S or greater, the CD thermal melt analyses will differentiate poly(I):poly(C 12 U) from similar molecules not meeting the specification for the complementary polymer size differential.
  • CD analysis can distinguish between poly(I):poly(C 12 U) and similar molecules that do not meet specifications for the amount of double strandedness or base pairing between the complementary poly(I) and poly(C 12 U) strands.
  • the amount of base pairing is dependent on the relative proportion of cytidylic acid to uridylic acid (C:U ratio) of the poly(C x U y ) polymer.
  • C:U ratio cytidylic acid to uridylic acid
  • the ratio of cytidine to uridine in the poly(C x U y ) polymer affects the melting temperature (T M ) as well as the width at half height of the first derivative of the melting curve.
  • Increasing the cytidine to uridine ratio of the poly(C x U y ) strand increases the base pairing between the complementary strands of the helix and, therefore, increases the observed T M and decreases the observed width at half-height of the first derivative of the thermal curve.
  • the CD 278 /CD 245 ratio determinations were demonstrated to be less sensitive to differences in the C:U ratio in AMPLIGEN® (rintatolimod) formulations.
  • CD method is an important analytical tool for characterization of poly(I):poly(C 12 U).
  • CD scans and determinations of the CD 278 /CD 245 ratio are less specific than the thermal melt analysis determinations of T M and width at half-height of the first derivative of the melt curve, all three CD parameters may be used in combination for the thorough characterization and identification of poly(I):poly(C 12 U).
  • Bioactivity of dsRNA and poly(I):poly(C 12 U) were measured, and then compared utilizing a ligand-binding assay. Stability was measured using the product release test, reverse phase HPLC assay.
  • TLR Toll-like receptors
  • PAMP pathogen-associated molecular patterns
  • TLR3 recognizes dsRNA, the genomic structure of some viruses, and also an intermediate generated during viral RNA replication. dsRNA is also produced intracellularly by stem-loop forming or with siRNA-aligned mRNAs.
  • AMPLIGEN® Rostatolimod
  • TLR3 binding and downstream signaling events are signaling molecules recognizing pathogen-associated molecular patterns (PAMP) and activating innate immune defense mechanisms.
  • dsRNA the genomic structure of some viruses, and also an intermediate generated during viral RNA replication.
  • dsRNA is also produced intracellularly by stem-loop forming or with siRNA-aligned mRNAs.
  • AMPLIGEN® rintatolimod
  • poly(I):poly(C) signaling has alternate routes
  • the poly(I):poly(C 12 U) pathway acts exclusively through TLR3 binding as AMPLIGEN® (rintatolimod) treatment protects TLR3 ++ but not TLR3 ⁇ / ⁇ mice from Punta Toro virus infection.
  • TLR3 ⁇ / ⁇ cells do not produce IFN upon poly(I):poly(C 12 U) treatment while IFN is induced by poly(I):poly(C) in TLR3 knockout cells.
  • TLR3 molecule ectodomain (ECD) conformation and its relation to binding of dsRNA is well characterized, including the prospective binding site.
  • Amino acids H539 and N541 are involved in the interaction with the double helix. Mutational analysis of these amino acids at the binding site further strengthens the argument.
  • Inosine 30 I 30
  • C 30 poly(I):Cytosine 30
  • IFN induced interferon
  • I 20 :C 20 , I 30 :C 30 , and I 40 :C 40 were ineffective IFN inducers. Therefore, characterizing AMPLIGEN® (rintatolimod) by its TLR3 binding capacity is a biomarker to predict its biological activity.
  • TLR3-ECD A range of ratios of TLR3-ECD to unselected Ampligen® (rintatolimod) or rugged dsRNA are reacted by the method of Leonard (2008).
  • the components are separated by the size-exclusion chromatographic method described below. From the peak quantities of free TLR3-ECD and the ligand-receptor complex, the ratio of TLR3-ECD that is required for saturation of either Ampligen® (rintatolimod) or rugged dsRNA is determined.
  • This threshold TLR3-ECD/dsRNA ratio provides a direct indication of the strength of the ligand-receptor binding and, therefore, of bioactivity.
  • TLR3-ECD (1.12 ⁇ 10 2 Kda) and poly(I):poly(C 12 U) (0.2-2 ⁇ 10 3 Kda) have different elution patterns, they can be separated from each other by size-exclusion chromatography (SEC). According to results obtained from poly(I):poly(C) using a SUPERDEX 200 PC 3.2/30 column and collecting 80 ⁇ l fractions, most of the poly(I):poly(C) appears in fractions 3-5 while TLR3-ECD is eluted in fractions 9-12 (Bell, 2005).
  • SEC size-exclusion chromatography
  • TLR3-ECD binding of TLR3-ECD to poly(I):poly(C) or poly(I):poly(C 12 U) creates a complex that is larger in size than either of the initial components.
  • the later eluting free TLR3-ECD is separated from the complex. Optimization of the separation identified that the SUPEROSE 200 PC column afforded superior binding by reducing tailing, due to absence of nonspecific interactions with dsRNA.
  • FIG. 18 shows the resulting chromatograms obtained from the reacted mixture of TLR3-ECD/poly(I):poly(C 12 U) compared to component injections of TLR3-ECD and poly(I):poly(C 12 U) alone, respectively.
  • TLR3-ECD is a His tag-containing recombinant protein.
  • a capture anti-His tag antibody immobilizes TLR3-ECD in a microplate well.
  • a second, biotinylated primary antibody quantitatively binds to the immobilized TLR3-ECD.
  • This secondary antibody is selected to have an epitope distal from the dsRNA binding site on the TLR3-ECD molecule and also from the epitope recognized by the capture antibody.
  • HRP-conjugated streptavidin recognizes the biotinylated second primary antibody. The appropriate substrate metabolized by HRP produces a soluble color suitable for quantitative measurement of TLR3-ECD.
  • AMPLIGEN® (rintatolimod) concentration in, the size-exclusion chromatography fractions is measured by fluorescence using standard dilutions and chromatography fractions in a quantitative riboGreen test. This assay permits testing of AMPLIGEN® (rintatolimod) out-of-the-bottle (i.e., not selected for rugged dsRNA) without further processing, preparation, or extraction, thereby maintaining its condition as a pharmaceutical.
  • Binding of TLR3-ECD to rugged dsRNA is more effective than binding of TLR3-ECD to unselected AMPLIGEN® (rintatolimod).
  • An approximately 2-fold greater ratio of TLR3-ECD is required to “unsaturate” rugged dsRNA ( ⁇ 0.50:1) as compared to AMPLIGEN® (rintatolimod) (0.25:1).
  • the binding profile at various ratios shows a much sharper endpoint for saturation for the case of rugged dsRNA which may reflect greater structural uniformity for this more compact dsRNA.
  • TLR3 binding of rugged dsRNA is 2-fold better than receptor binding of unselected AMPLIGEN® (rintatolimod).
  • Free TLR3 (area>10%) appears at a TLR3:dsRNA ratio of 0.25:1 for unselected AMPLIGEN® (rintatolimod) as compared to a 0.50:1 for rugged dsRNA
  • Stability of Rugged dsRNA Stability of poly(I):poly(C 12 U) was measured at an accelerated temperature condition of 40° C. as compared to the long-term storage temperature of from 2° C. to 8° C. As shown in FIG. 19 , the size of poly(I):poly(C 12 U) decays at this temperature as measured by analytical ultracentrifugation (S 20,w ). Decrease in size is due to unfolding of the double helix (loss of hydrogen bonds) and concurrent hydrolysis of the phosphodiester bonds.
  • the bioactivity of dsRNA requires a sedimentation coefficient from about 10.0 to about 15.0 S( 20,w ), whereas the size of poly(I):poly(C 12 U) at more than 180 days indicates a loss of bioactivity at about 8.0 S( 20,w ).
  • FIG. 20 shows the results of a second stability indicating parameter, the reversed phase HPLC assay, previously described, that separates poly(I):poly(C 12 U) into its individual strands. It is clearly evident that hydrolysis begins with the poly(I) strand followed by the poly(C 12 U) strand. HPLC results show that loss of size does not begin until commencement of the hydrolysis of the second strand poly(C 12 U); the RNA molecule retains double-stranded structure when only one of the strands undergoes hydrolysis. This loss of size at about 90 days occurs with the hydrolysis of both poly(I) and poly(C 12 U) strands.
  • the rugged dsRNA (5 min) peak is entirely unaffected by thermal stress. In fact, it increases in relation to the poly(I) and poly(C 12 U) strands. This conclusively shows that rugged dsRNA is not only “rugged” but can form spontaneously from smaller strands of degraded poly(I):poly(C 12 U).

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EP10830330.6A EP2499247A4 (fr) 2009-11-13 2010-11-12 Nouveaux acides ribonucléiques double brin à structure physico-chimique accidentée et à activité biologique hautement spécifique
PCT/US2010/002970 WO2011059505A2 (fr) 2009-11-13 2010-11-12 Nouveaux acides ribonucléiques double brin à structure physico-chimique accidentée et à activité biologique hautement spécifique
CA2780723A CA2780723A1 (fr) 2009-11-13 2010-11-12 Nouveaux acides ribonucleiques double brin a structure physico-chimique accidentee et a activite biologique hautement specifique
US13/077,742 US8722874B2 (en) 2008-10-23 2011-03-31 Double-stranded ribonucleic acids with rugged physico-chemical structure and highly specific biologic activity
US13/758,930 US20140170191A1 (en) 2008-10-23 2013-02-04 Novel double-stranded ribonucleic acids with rugged physico-chemical structure and highly specific biologic activity
US14/176,360 US9315538B2 (en) 2008-10-23 2014-02-10 Double-stranded ribonucleic acids with rugged physico-chemical structure and highly specific biologic activity
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US20110196020A1 (en) * 2008-10-10 2011-08-11 Carter William A Treatment of chronic fatigue syndrome using selective agonists of toll-like receptor 3 (tlr3)
US20130297023A1 (en) * 2012-05-07 2013-11-07 Hee-Jeong Im Sampen Methods and Devices For Treating Intervertebral Disc Disease
WO2017081703A1 (fr) * 2015-11-12 2017-05-18 Council Of Scientific & Industrial Research Procédé in-silico pour identifier des protéines combinatoires comme stimulateurs immunitaires contre la leishmaniose

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100310600A1 (en) * 2008-02-15 2010-12-09 Carter William A Selective agonist of toll-like receptor 3
US20110196020A1 (en) * 2008-10-10 2011-08-11 Carter William A Treatment of chronic fatigue syndrome using selective agonists of toll-like receptor 3 (tlr3)
US20130297023A1 (en) * 2012-05-07 2013-11-07 Hee-Jeong Im Sampen Methods and Devices For Treating Intervertebral Disc Disease
WO2017081703A1 (fr) * 2015-11-12 2017-05-18 Council Of Scientific & Industrial Research Procédé in-silico pour identifier des protéines combinatoires comme stimulateurs immunitaires contre la leishmaniose

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