U.S. patent application number 12/591270 was filed with the patent office on 2010-06-24 for double-stranded ribonucleic acids with rugged physico-chemical structure and highly specific biologic activity.
This patent application is currently assigned to HEMISPHERx BIOPHARMA, INC.. Invention is credited to William A. Carter, David Strayer.
Application Number | 20100160413 12/591270 |
Document ID | / |
Family ID | 43992616 |
Filed Date | 2010-06-24 |
United States Patent
Application |
20100160413 |
Kind Code |
A1 |
Carter; William A. ; et
al. |
June 24, 2010 |
Double-stranded ribonucleic acids with rugged physico-chemical
structure and highly specific biologic activity
Abstract
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 the 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). Medicaments, processes for their
manufacture, and methods for their use are provided herein.
Inventors: |
Carter; William A.; (Spring
City, PA) ; Strayer; David; (Bryn Mawr, PA) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
HEMISPHERx BIOPHARMA, INC.
Philadelphia
PA
|
Family ID: |
43992616 |
Appl. No.: |
12/591270 |
Filed: |
November 13, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2009/005797 |
Oct 23, 2009 |
|
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12591270 |
|
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61193030 |
Oct 23, 2008 |
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Current U.S.
Class: |
514/44A ;
536/23.1 |
Current CPC
Class: |
A61K 31/7105 20130101;
C12N 2310/331 20130101; C07H 21/02 20130101; C12N 2310/17 20130101;
C12N 2310/533 20130101; A61K 31/713 20130101; C12N 15/117 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
514/44.A ;
536/23.1 |
International
Class: |
C07H 21/02 20060101
C07H021/02; A61K 31/7105 20060101 A61K031/7105 |
Claims
1. An isolated double-stranded ribonucleic acid (dsRNA) which is
resistant to denaturation under conditions that are able to
separate hybridized poly(riboinosinic acid) and poly(ribocytosinic
acid) strands.
2. The dsRNA of claim 1 which contains only partially hybridized
strands.
3. The dsRNA of claim 1, wherein only a single strand of said dsRNA
comprises one or more uracil or guanine bases that are not based
paired to an opposite strand.
4. The dsRNA of claim 3, wherein said single strand is partially
hybridized to an opposite strand comprised of poly(riboinosinic
acid).
5. The dsRNA of claim 3, wherein said single strand is comprised of
poly(ribocytosinic.sub.4-29uracilic acid).
6. The dsRNA of claim 5, wherein said single strand is partially
hybridized to an opposite strand comprised of poly(riboinosinic
acid).
7. The dsRNA of claim 1, wherein both strands of said dsRNA
comprise one or more uracil or guanine bases that are not based
paired to an opposite strand.
8. The dsRNA of claim 1, wherein a strand of said dsRNA is
comprised of ribo(I.sub.n).ribo(C.sub.4-29U).sub.n, in which ribo
is a ribonucleotide and n is an integer from 40 to 40,000.
9. The dsRNA of claim 8, wherein a strand of said dsRNA is
comprised of ribo(I.sub.n).ribo(C.sub.11-14U).sub.n, in which ribo
is a ribonucleotide and n is an integer from 40 to 40,000.
10. The dsRNA of claim 9, wherein a strand of said dsRNA is
comprised of ribo(I.sub.n).ribo(C.sub.12U).sub.n, in which ribo is
a ribonucleotide and n is an integer from 40 to 40,000.
11. The dsRNA of claim 1 which has a molecular weight from about
250 Kda to about 320 Kda.
12. The dsRNA of claim 1 which has at least one strand of a length
from about 380 bases to about 450 bases.
13. The dsRNA of claim 1 which has from about 30 to about 38
helical turns of duplexed RNA strands.
14. A composition comprising one or more different dsRNAs as
defined in claim 1.
15. An isolated double-stranded ribonucleic acid (dsRNA) which is
resistant to denaturation under conditions that are able to
separate hybridized poly(riboinosinic acid) and poly(ribocytosinic
acid) strands, wherein the isolated dsRNA: has an HPLC chromatogram
substantially the same as the 5 minute peak of FIG. 1B or FIG. 1C;
is stable to exposure to thermal stress at 40.degree. C.; and has
an increased bioactivity as evidenced by binding to receptor
TLR3-ECD as compared to unselected poly(I):poly(C.sub.12U).
16. The dsRNA of claim 15 in which stability over time following
thermal stress exposure at 40.degree. C. substantially as shown in
the HPLC purity peaks of FIG. 20.
17. A method of treating a subject, said method comprising
administration to the subject of at least the dsRNA defined in
claim 1 in a therapeutic amount.
18. The method according to claim 17, wherein the therapeutic
amount of at least said dsRNA or said composition is infused
intravenously.
19. The method according to claim 17, wherein the therapeutic
amount is injected intradermally, subcutaneously, or
intramuscularly; inhaled intranasally or intratracheally; or
applied intranasally, intratracheally, oropharyngeally, or
sublingually.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Application No. PCT/US2009/005797, filed Oct. 23, 2009 which claims
priority benefit of U.S. provisional application Ser. No.
61/193,030, filed Oct. 23, 2008 the disclosures of which are hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] 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). 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).
BACKGROUND OF THE INVENTION
[0003] AMPLIGEN.RTM. (rintatolimod) poly(I):poly(C.sub.12U) 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. Acting on the hypothesis that the
nucleotide sequence requirements for beneficial and adverse effects
are different, poly(I):poly(C.sub.12U) 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). On the other hand, 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).
[0004] While poly(I):poly(C.sub.12U) 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.sub.12U) 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.
[0005] Properties of poly(I):poly(C.sub.12U) are characterized by
physico-chemical assays as shown in Table 1. Circular dichroism
(CD) (e.g., ellipticity, melting behavior) is used to characterize
the double-helical RNA structure, which is critical to potency.
Briefly, Toll-like receptor 3 (TLR3) is activated by dsRNA
(Alexopoulou, 2001), which leads to a host defense recruitment
sequence, ultimately producing type I interferons (Schroeder,
2005). Initiation of interferon production by dsRNA binding to TLR3
requires RNA helical structure (Bell, 2006). Although X-ray
diffraction and NMR alone are the definitive techniques to
determine RNA second-order structure, 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).
TABLE-US-00001 TABLE 1 Biological Activity and Measured Attributes.
Measured Property Identity Attribute Activity Attribute
Conformation: Second Degree CD: Ellipticity Double-Stranded RNA:
binding to TLR3 integrity of helix CD: Melting Behavior:
Double-Stranded RNA: binding to TLR3 Melting Point 1/2 Width
integrity and uniformity of helix Composition and Size Maximum Size
No. of Repeat Units Half Life: safety C:U Ratio identity Half Life:
safety
Therefore, circular dichroism can be employed to characterize the
therapeutic potency of specifically-configured dsRNA including
poly(I):poly(C.sub.12U).
[0006] As regards adverse toxic effects, the half life of
poly(I):poly(C.sub.12U) 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 (Greene, 1978) resulted in a minimum base-paired
length of about one helical turn, which is required for the
interaction of dsRNA with its bioactive receptor. Furthermore,
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).
[0007] It was our objective to identify a new family of improved
dsRNA having specific physico-chemical structure and highly
specific biological activities, which includes acting as a
selective agonist for TLR3. Its rugged structure as measured by
physico-chemical techniques is resistant to molecular unfolding
(i.e., denaturation). Improvement in at least one or more
biological activities may result from the rugged structure of this
particular form of poly(I):poly(C.sub.12U). Other advantages and
improvements are described below, or would be apparent from the
disclosure herein.
SUMMARY OF THE INVENTION
[0008] It is an objective of the invention to provide improved
forms of double-stranded ribonucleic acid (dsRNA). Their
physico-chemical structure and biological activities are described
herein. 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). 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.
[0009] In one aspect, rugged dsRNA may be provided.
Specifically-configured dsRNA may be of the general formula
ribo(I.sub.n).ribo(C.sub.4-29U).sub.n,
ribo(I.sub.n).ribo(C.sub.11-14U).sub.n, or
ribo(I.sub.n).ribo(C.sub.12U).sub.n, wherein the strands are
comprised of ribonucleotides (ribo) and n is an integer from about
40 to about 40,000 repeats. For example, a strand comprised of
poly(ribocytosinic.sub.4-29uracilic acid),
poly(ribocytosinic.sub.11-14uracilic acid), or
poly(ribocytosinic.sub.12uracilic 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).
[0010] After synthesis, 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. It is preferred that 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.
[0011] 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.
[0012] In another aspect, 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) may be provided to the subject without limitation to the
formulation of the pharmaceutical composition, or its route of
administration (although intravenous infusion is preferred). Use of
an effective amount of rugged dsRNA to achieve a feeling of
improved health and may be continued until at least one symptom is
improved. The effective amount required to obtain such improvement
may be identical to or higher than the amount required for
maintenance of the effect(s).
[0013] 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.
[0014] Antigen presenting cells (e.g., dendritic cells,
macrophages, B cells) and mucosal tissues (e.g., gastric or
respiratory epithelium) are preferred targets in the body for
rugged dsRNA. 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.
[0015] In another aspect, 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. Processes for making and using the
pharmaceutical composition (medicament) are also provided. For
example, 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.degree. C. to
8.degree. C. in a refrigerator under aseptic conditions.
[0016] Further aspects of the invention will be apparent from our
description of specific embodiments and the appended claims, and
generalizations thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1A shows an HPLC chromatogram for
poly(I):poly(C.sub.12U). The minor peak (not integrated) centered
at a retention time of about 5.00 min is duplexed
poly(I):poly(C.sub.12U). The first major peak centered at a
retention time of about 7.58 min is the single-stranded
poly(C.sub.12U). 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.
[0018] FIG. 1B is an HPLC chromatogram for lyophilized
poly(I):poly(C.sub.12U) showing aggregates lyophilization can
produce. Note that aggregation does not occur in the solution
process of FIG. 1C, which avoids lyophilization.
[0019] FIG. 1C shows a HPLC chromatogram of a sterile solution of
poly I:poly(C.sub.12U) also showing a novel 5 minute peak.
[0020] 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.sub.12U). 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.sub.12U) character. FIG. 2B is PDA analysis of the peak
centered at a retention time of about 7.58 min, which contains
poly(C.sub.12U). FIG. 2C is PDA analysis of the peak centered at a
retention time of about 10.05 min, which contains poly(I).
[0021] FIG. 3 is a circular dichroism (CD) of the dsRNA. The
melting point of 64.degree. C. represents the condition of 1/2
double stranded structure.
[0022] 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.sub.12U). 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.
[0023] FIG. 5 shows the circular dichroism of
poly(I):poly(C.sub.12U) with the characteristic chiral peaks at 245
nm and 278 nm
[0024] FIG. 6 shows a plot of the derivative of the thermal melt of
poly(I):poly(C.sub.12U). Integrity of the structure is
characterized by the melting point and the 1/2 width of this
derivative profile, both expressed as degrees C.
[0025] 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.
However, 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.
[0026] 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.
[0027] 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.
[0028] FIG. 10 shows a CD plot of a thermal melt of single stranded
poly(C.sub.12U). A strong signal is apparent due to the chirality
of cytidine. However, the absence of a second peak at 245 nm shows
that intra molecular base stacking of Poly C.sub.12U does not
occur.
[0029] FIG. 11 shows a plot of the derivative of the thermal melt
of single stranded poly(C.sub.12U). There is no evidence of intra
molecular base stacking at thermal condition which would otherwise
disrupt a double helix.
[0030] FIG. 12 shows a CD plot of a thermal melt of
poly(I):poly(C.sub.12U) and poly(I):poly(C). Base stacking is
evident in both compounds as indicated by the peak at 245 nm.
[0031] FIG. 13 shows a plot of the derivative of the thermal melt
of poly(I):poly(C.sub.12U) 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.sub.12U
illustrates a more labile character which in turn affords the
advantageous safety profile of the uridine substituted
compound.
[0032] FIG. 14 shows a CD wavelength scan of
poly(I):poly(C.sub.12U) 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)
[0033] FIG. 15 shows a plot of the derivative of the thermal melt
of poly(I):poly(C.sub.12U) 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.
[0034] FIG. 16 shows the derivative of a thermal melt of single
stranded poly(I):poly(C.sub.10U). The greater degree of Uridine
substation (compare poly(I):poly(C.sub.12U, FIG. 6) has compromised
the double helical structure. The 1:12 ratio of U:C is optimal,
providing one interruption per helical turn.
[0035] FIG. 17 shows a CD plot of thermal melt of single stranded
poly(I):poly(C.sub.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.sub.12U) has abolished the
base stacking signal at 245 nm.
[0036] FIG. 18 shows size exclusion chromatography of complexes of
TLR3-ECD and poly(I):poly(C.sub.12U) (FIG. 18A), the receptor
TLR3-ECD only (FIG. 18B), and the ligand poly(I):poly(C.sub.12U)
only (FIG. 18C).
[0037] FIG. 19 shows the effect of thermal stress (40.degree. C.)
on the size of dsRNA as measured by analytical centrifugation. The
decrease in sedimentation coefficient (S.sub.20,w) reflects a loss
of size due to hydrolysis.
[0038] FIG. 20 shows the effect of thermal stress (40.degree. 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.sub.12U) strands hydrolyze at 40.degree. C., the quantity of
rugged dsRNA peak increases.
[0039] FIG. 21 shows the relative size of AMPLIGEN vs new rugged
dsRNA (peak 5 minutes).
[0040] FIG. 22. Partial view of poly(I):poly(C.sub.12U) partially
hybridized strands and the interaction of bases of individual
poly(I) and the poly(C.sub.12U) strands. Molecular weight 1,100,000
da.
[0041] FIG. 23. Partial view of poly(I):poly(C.sub.12U) partially
hybridized strands and the interaction of bases of individual
poly(I) and the poly(C.sub.12U) strands. Molecular weight 286,000
da.
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0042] Many uses of double-stranded ribonucleic acid (dsRNA) are
known. Efficacy of such treatments, which includes a decrease in
the number and/or a reduction in the severity of adverse effects of
nonselected populations of dsRNA, is improved by the use of at
least partially purified, rugged dsRNA. 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. Preferably, 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.
[0043] 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.
[0044] 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).
Here, the terms "microbe" and "micro-organism" are used
interchangeably.
[0045] 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.
amycolatum, C. diphtheriae), Escherichia (e.g., E. coli O175:H7),
Haemophilus (e.g., H. influenzae), Heliobacter (e.g., H. pylori),
Klebsiella (K. pneumoniae), Legionella (e.g., L. pneumophila),
Listeria (e.g., L. monocytogenes), Mycobacterium (e.g., M. avium,
M. bovis, M. branderi, M. leprae, M. tuberculosis), Mycoplasma
(e.g., M. genitalium, M. pneumoniae), 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. pyogenes), Treponema (e.g.,
T. pallidum), Vibrio (e.g., V. cholerae, V. vulnificus), or
Yersinia (e.g., Y. pestis). These include Gram-negative or
Gram-positive bacteria, chlamydia, spirochetes, mycobacteria, and
mycoplasmas.
[0046] 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).
[0047] 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.
[0048] 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.
[0049] 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. For example,
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.
[0050] 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). During their conversion
from immature to mature dendritic 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. They may
be induced to migrate to sites of inflammation or lymphoid tissue
through blood or lymph to bring microbes, neoplastic or tumor
cells, or other transformed cells into proximity.
[0051] The subject may be vaccinated against at least infection or
cancer. In some cases, e.g., virus-induced cancers, both infection
and cancer may be treated. Immediately before, during, or
immediately after vaccination (e.g., within 10 days of
vaccination), 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.
[0052] Specifically-configured dsRNA may be of the general formula
ribo(I.sub.n).ribo(C.sub.4-29U).sub.n,
ribo(I.sub.n).ribo(C.sub.11-14U).sub.n, or
ribo(I.sub.n).ribo(C.sub.12U).sub.n, wherein strands are comprised
of ribonucleotides (ribo) and n is an integer from about 40 to
about 40,000 repeats. For example, a poly(riboinosinic acid) strand
may be partially hybridized to poly(ribocytosinic.sub.4-29uracilic
acid), poly(ribocytosinic.sub.11-14 uracilic acid), or
poly(ribocytosinic.sub.12uracilic 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.sub.4, U),
ribo(I).ribo(C.sub.11, U), ribo(I).ribo(C.sub.13, U),
ribo(I).ribo(C.sub.18, U), ribo(I).ribo(C.sub.20, U),
ribo(I).ribo(C.sub.24, G), and ribo(I).ribo(C.sub.29, G). Other
specifically-configured dsRNA are based on copolynucleotides such
as poly(C.sub.mU) and poly(C.sub.mG) 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.sub.n).ribo(C.sub.n) to incorporate unpaired bases (uracil
or guanine) in the polyribocytidylate r(C.sub.m) strand.
Alternatively, specifically-configured dsRNA may be derived from
ribo(I).ribo(C) dsRNA by modifying the ribosyl backbone of
poly(riboinosinic acid) ribo(I.sub.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. The specifically-configured dsRNA described in
U.S. Pat. Nos. 4,024,222; 4,130,641; and 5,258,369 (incorporated by
reference) 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.
[0053] 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). 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.
[0054] Formulations for administration (i.e., pharmaceutical
compositions) 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. It will be appreciated that 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.
[0055] The recommended dosage of rugged dsRNA will depend on the
clinical status of the subject and the physician's or
veterinarian's experience treating the disease or other
pathological condition. 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). It will be appreciated that 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.
[0056] Dendritic cells which act as sentinel cells possess
molecular surface structures that recognize pathogen-associated
molecular patterns (PAMPs). These PAMPs include a set of Toll-like
receptors (TLRs) that specifically recognize all dsRNA. In
particular, 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).
[0057] Circular dichroism (CD) 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.RTM.
(rintatolimod) poly(I):poly(C.sub.12U) 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.
[0058] Other 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.
[0059] 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. Since the presence of TLR3 and its signaling is a
requirement for AMPLIGEN.RTM. (rintatolimod)
poly(I):poly(C.sub.12U) to act as a receptor agonist, one could
assay for the absence of TLR3 mutations, the presence of TLR3
protein, intact TLR3-mediated signaling, or any combination thereof
in the cell or tissue of a subject prior to administration of the
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. For
example, abnormal cytokine (e.g., IFN-.alpha., IFN-.beta.,
IFN-.gamma., TNF-.alpha., IL-6, IL-10, IL-12) production or
co-stimulatory molecule (e.g., CD80, CD83, CD86) signaling may have
resulted from at least infection by the microbe, abnormal cell
proliferation, autoimmune damage, or neurodegeneration. 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).
[0060] Expression of 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.
EXAMPLES
[0061] Synthesis of single-stranded poly(I) and poly(C.sub.12U)
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.sub.12U). 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). Whereas precipitated poly(I) was separated
by centrifugation, the supernatant (waste) liquid phase of adherent
poly(C.sub.12U) was simply removed by aspiration. The precipitated
pastes were redissolved, then concentrated, diafiltered, and
further concentrated. The final bulk solutions containing
polynucleotide was filtered. The filtered solution was freeze dried
and the raw materials were stored frozen.
[0062] Enzymatic Synthesis. The enzymatic synthesis used in the
manufacturing process is dependent on the enzyme polynucleotide
phosphorylase to synthesize polyinosinic acid and
polycytidilic.sub.12uridilic acid from their respective starting
materials: cytidine 5'-diphosphate, trisodium salt (CDP.Na.sub.3),
uridine 5'-diphosphate, disodium salt (UDP.Na.sub.2) and inosine 5'
diphosphate, trisodium salt (IDP.Na.sub.3).
[0063] The enzyme catalyzes polynucleotide formation in a
reversible reaction using Mg.sup.++ 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. The following
ingredients were added to the tank one at a time with mixing: TRIS
(hydroxymethyl) aminomethane, urea, magnesium chloride hexahydrate
(MgCl.6H.sub.2O), and ethylenediaminetetraacetic acid (edetate),
disodium salt (EDTA.Na.sub.2). Raw material mononucleotides were
also added.
[0064] Each ingredient was dissolved before the next one was added.
After all of the ingredients were added, the solution was mixed for
a minimum of 10 minutes. The mixture was then adjusted and purified
water was added to obtain a final batch volume. This pre-enzyme
reaction mixture was sampled for initial CDP or IDP concentration.
The enzyme polynucleotide phosphorylase was added with mixing,
whereupon the synthesis of polynucleotide commenced. Also, the
viscosity profile at the optimal enzyme concentration must exhibit
the usual increase in viscosity over time without significant
decrease at the conclusion of the batch reaction; significant
decrease in viscosity would indicate undesired degradation of
polynucleotide. After the optimized amount of enzyme was added to
the production batch, enzymatic synthesis progressed under
constant, controlled agitation. The consumption of CDP or IDP was
monitored approximately every hour. The reaction was terminated by
the addition of a stop solution. Viscosity was also monitored, for
information only, during the process.
[0065] Concentration of Reaction Solution. To minimize the required
volume of phenol for extractions, the reaction product solution was
concentrated.
[0066] Extraction of Poly(I) and Poly(C.sub.12U). Residual enzyme
was removed predominately by phenol extraction. The concentrated
poly(C.sub.12U) or poly(I) reaction product solutions was
transferred into the extraction tank and 2M TRIS and sodium dodecyl
sulfate (SDS) were added. After at least 5 minutes of mixing,
liquefied phenol was added and the two phase solution was mixed to
disperse the phenol phase in the aqueous phase. SDS was employed as
a surface-active agent to facilitate dissolution of denatured
protein into the phenol phase; TRIS was required to buffer the
solution at an optimal pH for polynucleotide stability. The
extraction mixture stands without mixing for pre-determined
settling times to afford coalescence of the phases. 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.sub.12U) 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.
[0067] Precipitation of Poly(C.sub.12U) or Poly(I). Contaminating
phenol, SDS, and other salts remaining in solution were removed by
precipitation with denatured ethyl alcohol. The poly(C.sub.12U) or
poly(I) concentrated solution was pumped into the precipitation
tank. The denatured alcohol was added and after mixing the
precipitate was separated.
[0068] Concentration and Diafiltration. Remaining bulk salts, a
small amount of unreacted mononucleotide, and phenol were removed
by diafiltration against water. The precipitate was dissolved in
the original precipitation vessel with gentle mixing and heating.
After dissolving, the solution was then concentrated and
diafiltered against water for injection (WFI). The solution was
filtered prior to freeze drying.
[0069] Freeze Drying. The filtered poly(C.sub.12U) or poly(I)
material was loaded into a freeze drier. The material was frozen,
and a vacuum was then applied. The product was considered dry when
the programmed cycle was complete.
[0070] Manufacture of Poly(I):Poly(C.sub.12U), Sterile Solution,
for Intravenous Infusion. Poly(I) and poly(C.sub.12U) 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.
[0071] Preparation of Buffer Vehicle, Excipient Solution. WFI was
Added to the tank. The excipients were added to the tank, and
mixed. After mixing, the batch was sampled for pH and osmolality.
Quality control must be within in-process limits prior to use for
formulating the solutions containing poly(I) and
poly(C.sub.12U).
[0072] Formulating Poly(I) and Poly(C.sub.12U) solutions. An
initial quantity of buffer solution was subdivided according to the
batch formula and was filtered into the tank. Poly(I) or
poly(C.sub.12U) was added to the buffer solution, and dissolved by
mixing. The temperature of the solution was increased and
maintained with mixing. The solution is then recirculated.
[0073] Annealing of Poly I:Poly C.sub.12U Strands. Equivalent
quantities of poly(I) and poly(C.sub.12U) were transferred to the
tank. With continual mixing, the temperature of the solution was
increased. Samples were removed and tested for potency, and pH.
[0074] Sterile Filtration. The formulated bulk was sterile filtered
in-line into a steam sterilized surge vessel.
[0075] Filling Operations. 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.
[0076] Rugged dsRNA was isolated from the annealed
poly(I):poly(C.sub.12U), 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.RTM. (rintatolimod)
composition. Most 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).
[0077] 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.RTM. (rintatolimod)
poly(I):poly(C.sub.12U).
(a) Protection by Poly(I):Poly(C.sub.12U) is by Selective
Activation of TLR3
[0078] TLR3 Activation is Linked to Expression of
IFN-.alpha./.beta., 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-.alpha./.beta., IL-6 or IL-12 when stimulated
with poly(I):poly(C).
[0079] Poly(I):Poly(C.sub.12U) Induces Host Defense Gene Modulation
through Highly Selective Activation of TLR3. 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-.alpha./.beta., IL-6, and IL-12. The
mice were also subsequently challenged by exposure to Punta Toro
virus (PTV). Protection from the viral challenge was exquisitely
sensitive to treatment with poly(I):poly(C.sub.12U). Viral
protection conferred by poly(I):poly(C.sub.12U) was completely
abolished for the case of TLR3-deficient mice. When contrasted to
the partial but significant effectiveness of poly(I):poly(C) in
TLR3.sup.-/- mice, it is clear that the structural substitutions of
uridine in the cytidine strand of poly(I):poly(C.sub.12U) are
responsible for the highly specific, TLR3-dependent pathway.
Furthermore, measurements of IFN-.alpha./.beta. and IL-6 directly
link PTV protection or lack thereof to the modulation of these
cytokines.
[0080] This selective targeting of the TLR3 signaling pathway
represents a significant advantage for therapeutic applications of
poly(I):poly(C.sub.12U) 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).
(b) Binding of dsRNA to TLR3 Requires Helical Conformation of
dsRNA
[0081] 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.
[0082] Using mutational analysis, Bell (2005, 2006) modified
putative TLR3 binding sites in the positively-charged patches and
observed formation of a dsRNA/TLR3 complex by size-exclusion
chromatography. Despite the presence of numerous positively-charged
residues, only two amino acids N541 and H539 were required for
binding. The amido group of H539 could interact with dsRNA by
hydrogen binding. Proximity of the second positively-charged
residue N541 was also important, albeit the role of this amino acid
was not as clear. Mutation to negatively-charged aspirate prevented
binding by dsRNA, however conversion to a neutral alanine residue
had no effect on binding by dsRNA.
[0083] Binding to TLR3 Requires Helical Conformation of dsRNA.
Following the structural determination of most likely dsRNA binding
surfaces on TLR3, Choe (2005) further proposed that the helical
symmetry of dsRNA structure is necessary for the creation of the
symmetric dimer form of activated, membrane-associated TLR3. In the
ternary complex at the membrane surface, two symmetrically opposed
TLR3 molecules are linked to either side of the helix of the common
dsRNA.
[0084] As discussed above, using mutational analysis, Bell (2005,
2006) defined two highly conserved residues (N541 and H539) that
are necessary for binding of dsRNA to TLR3. Moreover, the
constraining requirement for ligand binding to both of these
residues of TLR3 is satisfied only by the minor groove architecture
of the (helical) conformation of dsRNA. When dsRNA phosphate binds
in proximity to the charge sensitive H539, then the amide side
(H541) becomes aligned with hydrogen bonding site of a 2' dsRNA
hydroxyl only when helical dimensions are utilized.
(c) Helical Conformation of dsRNA and Alteration Thereto
Accompanying Ligand Interactions are Precisely Characterized by
Circular Dichroism
[0085] 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.
[0086] dsRNA Characterization. Gray (1995) showed that CD, applied
in the mixing curve protocol, complemented ultraviolet absorption
measurements to determine the stoichiometry of duplex RNA
(A-G:C-T(U)). In this approach, the optical property is analyzed as
a function of the added ratios of individual strands. The
magnitudes of CD difference plots were maximal for 50:50 mixtures.
Further, isodichroic behavior correlated with the formation of
higher ordered or intra strand structures.
[0087] Ligand Interactions. Ghazaryan (2006) studied the ligand
interaction of dsRNA with a family of positively charged pyridinium
porphyrins. From CD measurements they found that minor
modifications of porphyrin structure led to profound differences in
mode of their attachment to the double helical structure. Whereas
TEtOHPyP4 associated by intercalation, TMetAlPyP4 attached by
forming an external, self-stacking assembly.
[0088] Using circular dichroism, Brown (2002) showed that ADAR1, a
human dsRNA, (chimeric) converted from the A to Z.alpha. form upon
binding to adenosine deaminase. Corroboration was provided by
crystallization of the complex and Raman spectroscopy. Sorrentino
(2003) studied the powerful enzymatic degradation of dsRNA by human
pancreateic ribonuclease (HP-RNase). Circular dichroism of the
RNA/enzyme complex revealed that multi-site attachment of the dsRNA
to HP-RNase was responsible for the destabilization of the RNA
helix.
[0089] Stability of dsRNA. Studying the rRNA component of the 70S
ribosomal complex, Sumita (2005) showed that pseudouridine
substitutions stabilized the dsRNA helix based upon structural
information provided by circular dichroism (CD). Specifically,
pseudouridine substitutions created duplex regions with closing
base pairs and water-mediated hydrogen bonds. Stabilization by
Mg.sup.++ was also characterized by CD in this study. Investigating
the stability of RNA-DNA hybrids with variants in base composition,
Lesnik (1995) showed that more stable hybrids retain ellipticity at
210 nm, a wavelength characteristic of the single component RNA
band (A-form hybrid). In contrast, less stable hybrids showed
lowered 210 nm ellipticity, values which were intermediate between
the RNA and DNA components.
[0090] A double-stranded RNA composition may be analyzed by high
performance liquid chromatography (HPLC) as shown in FIGS. 1A, 1B
and 1C. Analysis of a representative lot of AMPLIGEN.RTM.
(rintatolimod) poly(I):poly(C.sub.12U) 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.sub.12U) 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.
[0091] For the lyophilized (freeze-dried) preparation (FIG. 1B),
aggregates maybe present and elute at 13 and 15 min. A small
fraction of monomers inosine, cytosine and uridine, elute at 1 min.
The overall purity of poly I:polyC.sub.12U (Ampligen.RTM.)
determined by HPLC is represented by the sum of the 7.4, 8.7 and 10
minute peaks and is 96-99%. "New dsRNA" comprises 1-4% and is
different from Ampligen by its size and physico-chemical properties
as discussed herein.
[0092] The identity of each peak is determined by analysis with a
photodiode array (PDA) detector as shown in FIGS. 2A, 2B and 2C. At
each selected retention time, a UV absorption scan of wavelengths
from 200 nm to 360 nm was obtained. Duplex poly(I):poly(C.sub.12U)
and individual poly(I) and poly(C.sub.12U) 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.sub.12U), respectively
(FIG. 2A). Peak absorption centered at about 265 nm indicates the
presence of the poly(C.sub.12U) strand (FIG. 2B). 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.
[0093] FIG. 21 shows the relative size of AMPLIGEN vs new rugged
dsRNA (peak 5 minutes)
[0094] Common name: poly(I):poly(C.sub.12U) predominant species
[0095] Chemical name: poly(inosinic acid):poly((cytidylic
acid).sub.12(uridylic acid))
[0096] CAS registry number: 3864-92-5
[0097] Other names: YY057
[0098] Shown in FIG. 22 are a partial view of
poly(I):poly(C.sub.12U) partially hybridized strands and the
interaction of bases of individual poly(I) and the poly(C.sub.12U)
strands. Single inosine bases bind to cytosine bases, but not to
the uridine base. In this structure, 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.
[0099] Molecular formula:
(13C.sub.10H.sub.11N.sub.4O.sub.7P).sub.n:((12C.sub.9H.sub.12N.sub.3O.sub-
.7P)(C.sub.9H.sub.11N.sub.2O.sub.8P)).sub.n
[0100] Molecular size: about 1,200,000 daltons
The number of repeat units (n) corresponding to the size of
poly(I):poly(C.sub.12U) of approximately 1.2 Mda is 2000 base pairs
or 166 full helical turns.
TABLE-US-00002 TABLE 2 Molecular Weight (MW) of Components. MW
Repeat Unit Repeat Unit MW Inosine 5' mono- 330 13 4527 phosphate
Cytidine 3' mono- 305 12 3880 phosphate Uridylic acid 306 1 324
Overall Average: 318 N/A Sum: 8730
[0101] Common name: poly(I):poly(C.sub.12U) variant minor species
(286,000 daltons)
[0102] Chemical name: poly(inosinic acid):poly((cytidylic
acid).sub.12(uridylic acid))
[0103] Shown in FIG. 23 are a partial view of
poly(I):poly(C.sub.12U) partially hybridized strands and the
interaction of bases of individual poly(I) and the poly(C.sub.12U)
strands. Single inosine bases bind to cytosine bases, but not to
the uridine base. In this structure, 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.
[0104] Molecular formula:
(13C.sub.10H.sub.11N.sub.4O.sub.7P).sub.n:((12C.sub.9H.sub.12N.sub.3O.sub-
.7P)(C.sub.9H.sub.11N.sub.2O.sub.8P)).sub.n
[0105] Molecular size: about 286,000 daltons
[0106] 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.
[0107] Circular dichroism (CD) 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.sub.12U) 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.sub.12U). 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.RTM. (rintatolimod)
poly(I):poly(C.sub.12U), 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.sub.12U) is dependent on the complementary base
pairing of double-stranded structures and the complex chirality of
the resultant helical structure.
[0108] It has been demonstrated by UV and CD spectroscopy that the
biological activity of dsRNA is dependent on these specific spatial
and steric configurations. Since perturbation of helical structure
results in loss of the chiral centers characteristic of the
secondary structure, the analysis and monitoring of secondary
structure by CD provides a method to characterize the
physico-chemical properties of poly(I):poly(C.sub.12U) that are
associated with its bioactivity.
[0109] 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.sub.278/CD.sub.245, is
unique to poly(I):poly(C.sub.12U). In a second CD analysis,
ellipticity is measured during heating. As poly(I):poly(C.sub.12U)
is heated and thermally denatured, the individual poly(I) and
poly(C.sub.12U) strands unwind due to the breakdown of hydrogen
bonding between complementary base pairs. When the temperature
derivative of ellipticity is plotted, 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.
[0110] The wavelength scan detects two peaks: a first peak at 245
nm corresponding to the doubled stranded helix of the
poly(I):poly(C.sub.12U) and a second peak at 278 nm corresponding
to the stacking of the nucleic acid's base pairs. As shown
previously in FIGS. 4 and 5, 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.
[0111] The following table summarizes CD wavelength scans obtained
by isolation of fractions of the three reversed phase HPLC peaks
previously discussed in FIGS. 1B and 1C. 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.sub.12U):
7.0 (poly C.sub.12U) 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.sub.12U.
[0112] This result underscores the greater stability of rugged
dsRNA during the reversed phase isolation, in which all
polynucleotides experience binding and elution. Whereas
poly(I):poly(C.sub.12U) is separated into the component 7 and 10
minute polynucleotide strands, the 5 minute, rugged dsRNA, retains
the double stranded conformation.
TABLE-US-00003 PREP HPLC: Peak Analysis by Circular Dichroism
Circular Dichroism Peak 278/245 nm response requirement Lot (min)
(0.69-0.79) 0303SD 5 min 0.78 7 min 12.3 10 min 1.74 0301SD 5 min
0.79 7 min 2.49 10 min N/A
Only 5 minute exhibits double stranded base stacking character:
[0113] *Significant 245 nm response reflecting double strand helix
(245 nm)
[0114] *Acceptable 245/278 Ratio reflecting base pairs=278 nm
(chiral centers in backbone).
[0115] Precision. AMPLIGEN.RTM. (rintatolimod)
poly(I):poly(C.sub.12U), lot 9807CD, at a concentration of 2.5
mg/mL was repeatedly assayed to investigate the precision of the CD
assay. The percent relative standard deviations (% RSD) for the
melting temperature (T.sub.M), for the width at half-height for the
first derivative of the melting curve and for the ratio of
measurements of the CD peaks at 278 nm and 245 nm were calculated
as 0.76%, 9.09%, and 1.41%, respectively. This demonstrated that CD
assay of AMPLIGEN.RTM. (rintatolimod) poly(I):poly(C.sub.12U) acts
in a precise manner during thermal analysis for the determination
of T.sub.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.
[0116] Specificity. This CD method for characterizing
poly(I):poly(C.sub.12U) 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.RTM.
(rintatolimod) poly(I):poly(C.sub.12U). The specificity of this
method, in regards to analysis of single versus double-stranded
nucleic acids, was demonstrated by comparing scanning profiles and
melting temperature curves. See FIG. 7. The scans of
double-stranded molecules such as poly(I):poly(C.sub.12U),
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.sub.12U). See FIGS. 8-17. Furthermore, each of
the CD scans was unique for the molecular species being
assayed.
[0117] The specificity of the assay was also investigated to
assess, unequivocally, the ability to detect compounds of closely
related structure.
(a) Double-stranded ribonucleic acids of different nucleotide base
composition, such as poly(I):poly(C.sub.12U), poly(I):poly(C), and
poly(A):poly(U). (FIGS. 10, 12 and 14). (b) AMPLIGEN.RTM.
(rintatolimod) poly(I):poly(C.sub.12U) that meets the polymer size
specification. (c) Double-stranded ribonucleic acid formulated from
poly(I) and poly(C.sub.xU.sub.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).
[0118] The specificity of assays for dsRNA that differed in their
nucleotide base composition was evidenced by comparison of CD scans
and melting curves of similar, but different, double-stranded
molecules, such as poly(I):poly(C.sub.12U), poly(I):poly(C), and
poly(A):poly(U). CD scanning profiles appear to be similar, as seen
with the scans of AMPLIGEN.RTM. poly(I):poly(C.sub.12U) and
poly(I):poly(C). But calculations of the ratios obtained at 278 nm
and 245 nm, and subsequent t-test statistical analysis for equal
means showed that the CD scan of AMPLIGEN.RTM. (rintatolimod)
differs significantly from similar dsRNA having different
nucleotide base compositions. Specificity for the dsRNA of
different nucleic acid base composition was also demonstrated by
their thermal melting curves. Thermal melt curves for dsRNA
differed significantly from each other. Statistical analysis
(t-test for equal means) of data from the plots of the first
derivative of the melting curves confirmed that the results
obtained for their respective T.sub.M and width at half-height are
significantly different. Therefore, specificity of the CD method
differentiates AMPLIGEN.RTM. (rintatolimod) from other dsRNA
molecules by parameters of both the scan and the thermal melt
profiles.
[0119] The CD method is specific for detection of
poly(I):poly(C.sub.12U) formulated from polymers not meeting the
aforementioned specifications for size. When one or both polymers
of the poly(I):poly(C.sub.12U) molecule is outside the 4-8 S size
specification, the results from the CD analysis of these molecules
do not meet specifications for AMPLIGEN.RTM. (rintatolimod) in
regards to T.sub.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.
Relative to the data obtained from the thermal melt analyses of
AMPLIGEN.RTM. (rintatolimod) formulations, the
CD.sub.278/CD.sub.245 ratio determinations were less specific. CD
scans alone did not differentiate between poly(I):poly(C.sub.12U)
and non-poly(I):poly(C.sub.12U) formulations that did not meet
manufacturing and/or release specifications for polymer size.
[0120] As discussed above, the specificity of CD analysis is
sensitive to the size of the single-stranded polymer strands. In
addition, when the size difference between the complementary
single-stranded polymer components, poly(I) and poly(C.sub.12U), is
2.4 S or greater, the CD thermal melt analyses will differentiate
poly(I):poly(C.sub.12U) from similar molecules not meeting the
specification for the complementary polymer size differential.
[0121] CD analysis can distinguish between poly(I):poly(C.sub.12U)
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.sub.12U) 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.sub.xU.sub.y)
polymer. The ratio of cytidine to uridine in the
poly(C.sub.xU.sub.y) polymer affects the melting temperature
(T.sub.M) as well as the width at half height of the first
derivative of the melting curve. When the ratio of cytidine to
uridine is less than 11:1, there is less double strandedness or
base pairing (between polyinosinic acid and polycytidylic acid
complementary strands of the duplex RNA helix) than that for
AMPLIGEN.RTM. (rintatolimod). This results in lower observed
T.sub.m's and larger widths at half-height for the first derivative
of the thermal melt curves relative to those observed for
poly(I):poly(C.sub.12U). Increasing the cytidine to uridine ratio
of the poly(C.sub.xU.sub.y) strand increases the base pairing
between the complementary strands of the helix and, therefore,
increases the observed T.sub.M and decreases the observed width at
half-height of the first derivative of the thermal curve. The
CD.sub.278/CD.sub.245 ratio determinations were demonstrated to be
less sensitive to differences in the C:U ratio in AMPLIGEN.RTM.
(rintatolimod) formulations.
[0122] Both the size of the complementary polymer strands and the
C:U ratio of the poly(C.sub.12U) strand contribute to double
strandedness of a poly(I):poly(C.sub.12U) molecule. The double
strandedness, in turn, contributes to the efficacy of the drug
product as discussed in the introduction. Therefore, CD method is
an important analytical tool for characterization of
poly(I):poly(C.sub.12U). Although CD scans and determinations of
the CD.sub.278/CD.sub.245 ratio are less specific than the thermal
melt analysis determinations of T.sub.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.sub.12U).
Bioactivity and Stability of Rugged dsRNA
[0123] Bioactivity of dsRNA and poly(I):poly(C.sub.12U) were
measured, and then compared utilizing a ligand-binding assay.
Stability was measured using the product release test, reverse
phase HPLC assay.
[0124] A summary of the results is presented below, followed by
more detailed discussion. The combination of enhanced bioactivity
and much greater stability under the thermal stress of 40.degree.
C. illustrate the "ruggedness" of this novel variant dsRNA (i.e.,
rugged dsRNA) and suggest that it will be more bioavailable than
most of the dsRNA molecules in a formulation of Ampligen.RTM.
(rintatolimod). [0125] 1. Bioactivity of rugged dsRNA shows
two-fold greater binding affinity as compared to unselected dsRNA
Rugged dsRNA binding sites become unsaturated at a ratio of 0.50:1
(TLR3: rugged dsRNA) or higher. But binding sites for Ampligen.RTM.
(rintatolimod) poly(I):poly(C.sub.12U) become unsaturated at a
ratio of 0.20:1 (TLR3: unselected dsRNA) or higher. [0126] 2.
Stability of rugged dsRNA is four-fold greater than unselected
dsRNA Ampligen.RTM. (rintatolimod) poly(I):poly(C.sub.12U) is
stable (i.e., S.sub.w,20>10.0) for less than 90 days when
subjected to hydrolysis under thermal stress of 40.degree. C. By
contrast, rugged dsRNA is stable for greater than 360 days under
the same conditions. [0127] 3. Stability and bioactivity data show
that rugged dsRNA is more bioavailable than unselected dsRNA From
these stability and bioactivity considerations, rugged dsRNA is
more bioavailable for the relevant signaling receptor that conveys
the therapeutic benefit. The rugged dsRNA has the additional
benefit of maintaining long-term stability at ambient temperatures,
which has important clinical implications for treating populations
in regions of the world without adequate refrigeration
capabilities.
Bioactivity Background
[0128] Toll-like receptors (TLR) are signaling molecules
recognizing pathogen-associated molecular patterns (PAMP) and
activating innate immune defense mechanisms. 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.RTM. (rintatolimod) is comprised of dsRNA molecules that
act through TLR3 binding and downstream signaling events. While
poly(I):poly(C) signaling has alternate routes, the
poly(I):poly(C.sub.12U) pathway acts exclusively through TLR3
binding as AMPLIGEN.RTM. (rintatolimod) treatment protects
TLR3.sup.++ but not TLR3.sup.-/- mice from Punta Toro virus
infection. TLR3.sup.-/- cells do not produce IFN upon
poly(I):poly(C.sub.12U) treatment while IFN is induced by
poly(I):poly(C) in TLR3 knockout cells.
[0129] The 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.
[0130] The effect of length and structure of dsRNA on TLR3 binding
and IFN induction is known. Inosine.sub.30 (I.sub.30):poly(C) or
poly(I):Cytosine.sub.30 (C.sub.30) induced interferon (IFN), but
shorter dsRNA stretches do not induce IFN. Compared to them,
however, IFN induction by poly(I):poly(C) was always superior.
I.sub.20:C.sub.20, I.sub.30:C.sub.30, and I.sub.40:C.sub.40 were
ineffective IFN inducers. Therefore, characterizing AMPLIGEN.RTM.
(rintatolimod) by its TLR3 binding capacity is a biomarker to
predict its biological activity.
Bioactivity Method
[0131] A range of ratios of TLR3-ECD to unselected Ampligen.RTM.
(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.RTM.
(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.
[0132] The following method is an adaptation of the experimental
procedures used to characterize TLR3 ligand binding at a molecular
level. Since TLR3-ECD (1.12.times.10.sup.2 Kda) and
poly(I):poly(C.sub.12U) (0.2-2.times.10.sup.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 .mu.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).
[0133] The binding of TLR3-ECD to poly(I):poly(C) or
poly(I):poly(C.sub.12U) 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.
[0134] FIG. 18 shows the resulting chromatograms obtained from the
reacted mixture of TLR3-ECD/poly(I):poly(C.sub.12U) compared to
component injections of TLR3-ECD and poly(I):poly(C.sub.12U) alone,
respectively.
[0135] Characterization of Peaks. Identification and quantitation
of TLR3-ECD in size-exclusion chromatography fractions is possible
in an ELISA format. The commercially-available 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.
[0136] AMPLIGEN.RTM. (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.RTM. (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.
[0137] Bioactivity Results. Results in Table 3 show the percentage
of free TLR3-ECD that remains in a series of reactions using
different ratios of TLR3-ECD to dsRNA. These studies were conducted
with either unselected AMPLIGEN.RTM. (rintatolimod) as well as
rugged dsRNA.
[0138] Binding of TLR3-ECD to rugged dsRNA is more effective than
binding of TLR3-ECD to unselected AMPLIGEN.RTM. (rintatolimod). An
approximately 2-fold greater ratio of TLR3-ECD is required to
"unsaturate" rugged dsRNA (-0.50:1) as compared to AMPLIGEN.RTM.
(rintatolimod) (0.25:1). Also, 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.
TABLE-US-00004 TABLE 3 Bioactivity Measurements of Unselected vs.
Rugged dsRNA. Unselected AMPLIGEN .RTM. (rintatolimod), Molar Lot #
0701HE Rugged dsRNA Ratio of dsRNA/TLR3 dsRNA/TLR3 TLR3 to Complex
Area Free TLR3 Complex Area Free TLR3 dsRNA % Area % % Area %
0.20:1 99.0 0.978 99.4 0.577 0.25:1 78.4 21.6 99.1 0.880 0.33:1
20.9 79.1 92.9 7.086 0.50:1 58.9 41.1 60.3 39.723 0.67:1 15.4 84.6
11.3 88.660
[0139] The TLR3 binding of rugged dsRNA is 2-fold better than
receptor binding of unselected AMPLIGEN.RTM. (rintatolimod). Free
TLR3 (area>10%) appears at a TLR3:dsRNA ratio of 0.25:1 for
unselected AMPLIGEN.RTM. (rintatolimod) as compared to a 0.50:1 for
rugged dsRNA
[0140] Stability of Rugged dsRNA. Stability of
poly(I):poly(C.sub.12U) was measured at an accelerated temperature
condition of 40.degree. C. as compared to the long-term storage
temperature of from 2.degree. C. to 8.degree. C. As shown in FIG.
19, the size of poly(I):poly(C.sub.12U) decays at this temperature
as measured by analytical ultracentrifugation (S.sub.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(.sub.20,w), whereas the
size of poly(I):poly(C.sub.12U) at more than 180 days indicates a
loss of bioactivity at about 8.0 S(.sub.20,w).
[0141] FIG. 20 shows the results of a second stability indicating
parameter, the reversed phase HPLC assay, previously described,
that separates poly(I):poly(C.sub.12U) into its individual strands.
It is clearly evident that hydrolysis begins with the poly(I)
strand followed by the poly(C.sub.12U) strand. HPLC results show
that loss of size does not begin until commencement of the
hydrolysis of the second strand poly(C.sub.12U); 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.sub.12U)
strands.
[0142] Importantly, 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.sub.12U) strands. This conclusively shows
that rugged dsRNA is not only "rugged" but can form spontaneously
from smaller strands of degraded poly(I):poly(C.sub.12U).
[0143] Patents, patent applications, books, and other publications
cited herein are incorporated by reference in their entirety.
[0144] In stating a numerical range, it should be understood that
all values within the range are also described (e.g., one to ten
also includes every integer value between one and ten as well as
all intermediate ranges such as two to ten, one to five, and three
to eight). The term "about" may refer to the statistical
uncertainty associated with a measurement or the variability in a
numerical quantity which a person skilled in the art would
understand does not affect operation of the invention or its
patentability.
[0145] All modifications and substitutions that come within the
meaning of the claims and the range of their legal equivalents are
to be embraced within their scope. A claim which recites
"comprising" allows the inclusion of other elements to be within
the scope of the claim; the invention is also described by such
claims reciting the transitional phrases "consisting essentially
of" (i.e., allowing the inclusion of other elements to be within
the scope of the claim if they do not materially affect operation
of the invention) or "consisting of" (i.e., allowing only the
elements listed in the claim other than impurities or
inconsequential activities which are ordinarily associated with the
invention) instead of the "comprising" term. Any of these three
transitions can be used to claim the invention.
[0146] It should be understood that an element described in this
specification should not be construed as a limitation of the
claimed invention unless it is explicitly recited in the claims.
Thus, the granted claims are the basis for determining the scope of
legal protection instead of a limitation from the specification
which is read into the claims. In contradistinction, the prior art
is explicitly excluded from the invention to the extent of specific
embodiments that would anticipate the claimed invention or destroy
novelty.
[0147] Moreover, no particular relationship between or among
limitations of a claim is intended unless such relationship is
explicitly recited in the claim (e.g., the arrangement of
components in a product claim or order of steps in a method claim
is not a limitation of the claim unless explicitly stated to be
so). All possible combinations and permutations of individual
elements disclosed herein are considered to be aspects of the
invention. Similarly, generalizations of the invention's
description are considered to be part of the invention.
[0148] From the foregoing, it would be apparent to a person of
skill in this art that the invention can be embodied in other
specific forms without departing from its spirit or essential
characteristics. The described embodiments should be considered
only as illustrative, not restrictive, because the scope of the
legal protection provided for the invention will be indicated by
the appended claims rather than by this specification.
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