U.S. patent application number 10/842474 was filed with the patent office on 2005-06-23 for treating server acute respiratory syndrome.
This patent application is currently assigned to HEMISPHERx BIOPHARMA. Invention is credited to Carter, William A., Strayer, David.
Application Number | 20050137154 10/842474 |
Document ID | / |
Family ID | 34107548 |
Filed Date | 2005-06-23 |
United States Patent
Application |
20050137154 |
Kind Code |
A1 |
Carter, William A. ; et
al. |
June 23, 2005 |
Treating server acute respiratory syndrome
Abstract
Severe acute respiratory syndrome is treated with a natural
human alpha interferon, a dsRNA or both natural human alpha
interferon and a dsRNA.
Inventors: |
Carter, William A.; (Spring
City, PA) ; Strayer, David; (Bryn Mawr, PA) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
1100 N GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
22201-4714
US
|
Assignee: |
HEMISPHERx BIOPHARMA
Philadelphia
PA
|
Family ID: |
34107548 |
Appl. No.: |
10/842474 |
Filed: |
May 11, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60470893 |
May 16, 2003 |
|
|
|
60517882 |
Nov 7, 2003 |
|
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Current U.S.
Class: |
514/44A ;
424/85.7 |
Current CPC
Class: |
A61K 31/7088 20130101;
A61K 2300/00 20130101; A61K 38/212 20130101; A61K 38/212 20130101;
A61K 31/7088 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/044 ;
424/085.7 |
International
Class: |
A61K 048/00; A61K
038/21 |
Claims
What is claimed is:
1. A method of treating severe acute respiratory syndrome
comprising administering to an infected subject natural human alpha
interferon.
2. A method of treating severe acute respiratory syndrome
comprising administering to an infected subject a dsRNA.
3. A method of treating severe acute respiratory syndrome
comprising the coordinated administration to an infected subject of
(1) a natural human alpha interferon and (2) dsRNA.
4. The method of claim 2 or 3 wherein the dsRNA is
rI.sub.n.multidot.r(C.s- ub.12U).sub.n., Poly A.multidot.Poly U or
rI.sub.n.multidot.r(C.sub.29,G).- sub.n, in which r is ribo.
5. The method of claim 1 wherein the interferon is administered
orally IV, IM or SQ.
6. A method of mitigating the effects of or conferring resistance
to severe acute respiratory syndrome comprising, prior to exposure,
to the SARS-CoV or shortly after exposure to the SARS-CoV, but
prior to the development of symptoms, administering to a subject
natural human .alpha.-interferon.
7. The method of claim 6 wherein the interferon is administered
orally IV, IM or SQ.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. provisional
patent application Ser. No. 60/470,893 filed May 16, 2003 and Ser.
No. 60/517,882 filed Nov. 7, 2003.
[0002] Procedures are provided for combating the effects of
coronavirus-induced conditions by the administration of an
.alpha.-interferon composed of a mixture of naturally occurring
.alpha.-interferons or a synthetic, specifically configured,
double-stranded ribonucleic acid (dsRNA) or both an
.alpha.-interferon and a dsRNA.
BACKGROUND
[0003] Severe Acute Respiratory Syndrome (SARS) is a new disease
that is rapidly spreading within China and other countries around
the world. Although, a combination of ribavirin, a synthetic,
non-interferon-inducing, broad spectrum antiviral nucleoside, and
corticosteroids is commonly used as therapy, especially in China,
laboratory testing by the National Institutes of Health (NIH) found
ribavirin to have no effect on this coronavirus. This lack of
efficacy suggests the need for an effective therapeutic
regimen.
DESCRIPTION OF THE INVENTION
[0004] Described is use of an .alpha.-interferon, preferably a
natural, multi-species .alpha.-interferon in the treatment of the
symptoms associated with SARS in patients including human patients
infected with the SARS virus, also referred to as the
SARS-associated coronavirus (SARS-CoV). Alternatively, a dsRNA may
be used in the treatment of the symptoms associated with
SARS-associated coronavirus in patents including human patients
infected with the SARS-associated coronavirus. Also described is
the coordinated use of both (1) an .alpha.interferon, preferably a
natural, multi-species .alpha.-interferon and conjointly therewith
(2) a dsRNA in the treatment of the symptoms associated with
SARS-associated coronavirus in patients including human patients
infected the SARS-associated coronavirus. Procedures for attaining
a favorable therapeutic and clinical result and compositions for
accomplishing the same are described. Preferably the dsRNA is
administered with the .alpha.-interferon and preferably the dsRNA
is rI.sub.n.multidot.r(C.sub.- 12U).sub.n., Poly A.multidot.Poly U
or rI.sub.n.multidot.r(C.sub.29,G).sub- .n, in which r is ribo.
[0005] In the context of the present invention, what is meant by
"coordinated" use is, independently, either (i) co-administration,
i.e. substantially simultaneous or sequential administration of the
.alpha.-interferon and of the dsRNA, or (ii) the administration of
a composition comprising the .alpha.-interferon and the dsRNA in
combination and in a mixture, in addition to optional
pharmaceutically acceptable excipients and/or vehicles.
[0006] For internal administration the .alpha.-interferon may, for
example, be formulated in conventional manner for oral or rectal
administration. Formulations for oral administration 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.
[0007] The .alpha.-interferon component of the therapeutic
procedures is preferably Alferon N Injection.RTM. the only approved
natural, multi-species, .alpha.-interferon available in the United
States. It is the first natural source, multi-species interferon
and is a consistent mixture of at least seven species of
.alpha.-interferon. In contrast, the other available
.alpha.-interferons are single molecular species of
.alpha.-interferon made in bacteria using DNA recombinant
technology. These single molecular species of .alpha.-interferon
also lack an important structural carbohydrate component because
this glycosylation step is not performed during the bacterial
process.
[0008] Unlike species of .alpha.-interferon produced by recombinant
techniques, Alferon N Injection.RTM. is produced by human white
blood cells which are able to glycosylate the multiple
.alpha.-interferon species. Reverse Phase HPLC studies show that
Alferon N Injection.RTM. is a consistent mixture of at least seven
species of alpha interferon (.alpha.2, .alpha.4, .alpha.7,
.alpha.8, .alpha.10, .alpha.16, .alpha.17). This natural-source
interferon has unique anti-viral properties distinguishing it from
genetically engineered interferons. The high purity of Alferon N
Injection.RTM. and its advantage as a natural mixture of seven
interferon species, some of which, like species 8b, have greater
antiviral activities than other species, for example, species 2b,
which is the only component of Intron A. The superior antiviral
activities for example in the treatment of chronic hepatitis C
virus (HCV) and (HIV) and tolerability of Alferon N Injection.RTM.
compared to other available recombinant interferons, such as Intron
A and Roferon A, have been reported.
[0009] It is reported Alferon N Injection.RTM. has activity against
a natural coronavirus infection in pigs. Transmissible
gastroenteritis (TGE) coronavirus causes an acute gastroenteritis
in swine. The diarrhea and dehydration caused by this viral
infection result in a high mortality rate in neonates with severity
inversely related to the age of the animal. In fact, in piglets
less than 14 days of age the morality/morbidity rate typically
approaches 100%. Piglets, ages 1-12 days treated with 1.0, 10.0, or
20.0 IU of Alferon N Injection.RTM. were found to have an increased
survival compared to the control group indicating benefit of this
natural mixture of .alpha.-interferons in combating this particular
coronavirus.
[0010] The invention includes methods of enhancing therapy against
coronaviruses by administering to patients interferons,
particularly natural human alpha interferon and together or
conjointly a synthetic, specifically configured, double-stranded
ribonucleic acid (dsRNA). The dsRNA of choice is Ampligen.RTM., a
synthetic, specifically configured, double-stranded ribonucleic
acid (dsRNA) which retains the immunostimulatory and antiviral
properties of other double-stranded RNA molecules (dsRNA) but
exhibits greatly reduced toxicity. Like other dsRNA, Ampligen.RTM.
can elicit the induction of interferon and other cytokines.
Ampligen.RTM. has the ability to stimulate a variety of
dsRNA-dependent intracellular antiviral defense mechanisms
including the 2',5'-oligoadenylate synthetase/RNase L and protein
kinase enzyme pathways.
[0011] The mismatched dsRNA may be of the general formula
rI.sub.n.multidot.r(C.sub.12U).sub.n. In this and the other
formulae that follow r=ribo. Other mismatched dsRNAs for use in the
present invention are based on copolynucleotides selected from poly
(C.sub.m,U) and poly (C.sub.mG) in which m is an integer having a
value of from 4 to 29 and are mismatched analogs of complexes of
polyriboinosinic and polyribocytidilic acids, formed by modifying
rI.sub.n.multidot.rC.sub.n to incorporate unpaired bases (uracil or
guanine) along the polyribocytidylate (rC.sub.m) strand.
Alternatively, the dsRNA may be derived from r(I).multidot.r(C)
dsRNA by modifying the ribosyl backbone of polyriboinosinic acid
(rI.sub.n), e.g., by including 2'-O-methyl ribosyl residues. The
mismatched may be complexed with an RNA-stabilizing polymer such as
lysine cellulose. Of these mismatched analogs of
rI.multidot.rC.sub.n, the preferred ones are of the general formula
rI.sub.n.multidot.r(C.sub.11-14,U).sub.n. or
rI.sub.n.multidot.r(C.sub.29- ,G).sub.n, and are described by
Carter and Ts'o in U.S. Pat. Nos. 4,130,641 and 4,024,222 the
disclosures of which are hereby incorporated by reference. The
dsRNA's described therein generally are suitable for use according
to the present invention.
[0012] Other examples of mismatched dsRNA for use in the invention
include:
[0013] r(I).multidot.r(C.sub.4, U)
[0014] r(I).multidot.r(C.sub.7, U)
[0015] r(I).multidot.r(C.sub.13, U)
[0016] r(I).multidot.r(C.sub.22, U)
[0017] r(I).multidot.r(C.sub.20, G) and
[0018] r(I).multidot.r(C.sub.p.multidot.23,G.sub.>p).
[0019] Alternatively the dsRNA may be the matched form, thus
polyadenylic acid complexed with polyuridylic acid (poly
A.multidot.poly U) may also be used.
[0020] When administered 24 hours prior to viral challenge,
ampligen has been demonstrated in viral cytopathic inhibition
assays and neutral red assays to inhibit human coronavirus strain
OC-43, thus suggesting protective activity of ampligen against
human chromavirus prior to an encounter with this virus.
[0021] .alpha.-interferon and/or the dsRNA may be administered for
therapy by any suitable route including oral, rectal, nasal,
topical (including transdermal, buccal and sublingual), vaginal and
parenteral (including subcutaneous, intramuscular, intravenous
intradermal, and intravitreal). It will be appreciated that the
preferred route will vary with the condition and age of the
recipient, the nature of the infection and the chosen active
ingredient.
[0022] As indicated above, severe acute respiratory syndrome (SARS)
is caused by a newly identified member of the coronavirus family.
Ampligen.RTM., a double-stranded RNA (dsRNA), is reported to
exhibit antiviral activity against the coronavirus, Mouse Hepatitis
Virus Type-3 (MHV-3) see Hepatology 3:837, 1983. MHV-3 is a
coronavirus which causes both a fulminant and a chronic form of
hepatitis depending on the mouse strain studied. Ampligen.RTM.
treatment had a positive effect against the MHV-3 coronavirus in
both mouse models. In an acute infection model, Balb/cJ mice
exposed to MHV-3 and then treated twice with Ampligen.RTM. survived
up to four times longer than untreated mice. Since no treatment was
given beyond 24 hours post-exposure, it is likely that additional
Ampligen.RTM. treatments would have had an even greater impact on
survival. In the chronic hepatitis model, C3H mice treated after
exposure to the MHV-3 coronavirus cleared the virus quickly and did
not develop chronic hepatitis. Thus, Ampligen.RTM. has shown
activity against the coronavirus, MHV-3, in two different mouse
models, increasing survival in the acute infection model and
completely abrogating the infection in the chronic model.
[0023] In a further study it has been determined Alferon.RTM.
inhibits SARS-CoV at a high specific activity in Vero 76 cells
(African green monkey) in culture. Alferon.RTM. is a highly
purified natural .alpha.-interferon obtained from human leucocytes
and consists of seven different .alpha.-interferon amino acid
sequences (.alpha.2, .alpha.4, .alpha.7, .alpha.8, .alpha.10,
.alpha.16, and .alpha.17). Inhibition was quantitated by visual
cytopathic effect (CPE), inhibition of the cellular uptake of the
vital dye, neutral red (NR), and by virus yield reduction. NR assay
was conducted immediately following visual assay. Fifty-percent
effective doses (EC5O) were calculated for both CPE and NR assays
by regression analysis. Quantitative values for viral yield
reduction assays were expressed as 90% effective concentrations
(EC9O), representing the drug concentration required to reduce
SARS-CoV titers by one log.sub.10 and were calculated by regression
analysis. Vein cells infected at a MOI of 0.001 visually exhibited
100% CPE over a 3-5 day incubation period without treatment.
Alferon.RTM. inhibited SARS-CoV at an ECSO=5,696+/-1,703 (SEM)
IU/ml (visual) and 10,740+/-5,161 (SEM) IU/ml (NR). Viral load
reduction by one log.sub.10 was 78,000+/-22,000 (SEM) IU/ml.
[0024] The beneficial effects of Alferon.RTM. are also reported by
Tan et al, Emerging Infectious Diseases--www.cdc.gov/eid--Vol. 10
No. 4, April 2004, in which several commercially available,
clinically approved compounds from several antiviral pharmacologic
classes screened to determine the presence of in vitro
anti-SARS-CoV activity. Of the 18 antiviral compounds tested
Alferon.RTM. was found to be the most active FDA-approved
interferon when tested against the SARS coronavirus. This contrasts
with the recombinant .alpha.-interferons, Roferon and Intron A,
which were not found to be active against the SARS coronavirus when
tested at much higher concentrations.
[0025] It is also reported the activity of interferon can
potentially be amplified by the addition of a double-stranded RNA
drug, Ampligen.RTM.. While interferon up-regulates certain
intracellular antiviral pathways, dsRNAs, like Ampligen.RTM., are
required to fully activate these important antiviral pathways. When
interferons are combine with Ampligen.RTM. synergistic antiviral
and antitumor effects are seen. Moreover, Ampligen.RTM. has already
shown strong antiviral activity in two separate animal models of
the coronavirus (MHV-3). Although uncertainty now exists regarding
the characteristics of this coronavirus, in the event it is
determined the SARS-associated coronavirus elaborates IFN
neutralizing products, Ampligen.RTM. has potential to override
these inhibitors and achieve an antiviral effect.
[0026] In addition, as a further attribute of the dsRNA arm of the
disclosed therapeutic combination therapy, synergistic antiviral
and antitumor effects have been demonstrated using Ampligen.RTM.
treatment in combination with all three types of interferon
(.alpha., .beta. and .gamma.). These synergistic effects have been
seen against HIV and a variety of different histologic tumor types.
Four human tumor cell lines were studied for their response to
antiproliferative effects of Ampligen.RTM. in combination with
various interferons. Results indicate that (1) Ampligen.RTM. worked
synergistically with all interferons in all cell lines studied; (2)
growth inhibition of cells resistant to interferons can be
potentiated by low doses of Ampligen.RTM.; (3) the
antiproliferative effect of interferons can be potentiated by
Ampligen.RTM. in Ampligen.RTM.-resistant cells; and (4)
Ampligen.RTM. works by a mechanism(s) other than, or in addition
to, the induction of interferon. See Montefiori, AIDS Res. and
Human Retroviruses 5:193-203, 1989 and Hubbell, Int. J. Cancer
37:359-365, 1986.
[0027] The recommended dosage of the components will depend on the
clinical status of the patient and the experience of the clinician
in treating similar infection. As a general guideline dosage of
Alferon N Injection.RTM. utilized for systemic infections is 5 to
10 million units (sq) thrice weekly. The Ampligen.RTM. dose
schedule is 400 mg by IV infusion twice weekly, although these
amounts and/or dosage frequency may be varied by the clinician in
response to the patient's condition. The components may be
administered at the same time, for instance as mixture of the
.alpha.-interferon and dsRNA, indepenently as the
.alpha.-interferon then the dsRNA or the .alpha.-interferon and the
dsRNA may be administered in a time-spaced manner.
EXAMPLE
[0028] Effects of Alferon N.RTM., an alfa-n3 human interferon, on
the replication of SARSCoV in vitro.
[0029] Vero 76 cells (African green monkey kidney) were obtained
from American Type Culture Collection (Manassas, Va.). The growth
medium was Eagle's minimum essential medium with non-essential
amino acids (MEM), 5% FBS and 0.1% NaHCO.sub.3. The test medium was
MEM supplemented with 2% FBS, 0.18% NaHCO.sub.3 and 50 .mu.g
gentamicin/ml. The SARS coronavirus, strain 200300592 (Urbani), was
obtained from James Corner (Centers for Disease Control, Atlanta,
Ga.). Human leukocyte derived interferon alfa-n3 (03-6600) was
kindly provided by Hemispherx Biopharma, Inc. (New Brunswick, N.J.)
as a stock solution of 5.times.10.sup.6 units/ml.
[0030] Using cytopathic effect (CPE) reduction assays read visually
and verified spectrophotometrically by neutral red (NR) uptake
assay of the same plate (Barnard et al., 2001), an interferon
alfa-n3 was evaluated for anti-SARSCoV activity in Vera 76 cells.
Virus at a multiplicity of infection of 0.001 was added to 96 well
plates seeded with near confluent monolayers of cells in which drug
had been serially diluted, using 10-fold or 1/2 log dilution
series. Addition of virus was within five minutes after exposure of
cells to drug.
[0031] The cells were incubated at 37.degree. C. until the
untreated virus controls displayed destruction of the monolayers
(100% CPE, 3-5 days). The plates were then scored for cytotoxicity
and viral CPE by microscopic examination, usually followed
immediately by neutral red staining and processing for
spectrophotometric reading. EC50 values (the concentration of
compound needed to inhibit the cytopathic effect to 50% of the
control value) and IC50 values (the concentration at which uptake
of neutral red or cytotoxic effects was reduced by 50% compared to
control cells) was calculated by regression analysis. Values were
expressed as mean.+-.the standard error of the mean. For the visual
assay, n=10 and for the NR assay, n=5. A selective index
(IC50/EC5O) for each compound was then calculated. Compounds found
active by these assays were then further evaluated for inhibitory
activity in two separate virus yield reduction assays (Barnard et
al., 2001). EC9O values were derived by regression analysis from
those assays and represent the concentration at which virus yields
were reduced by 1 log.sub.10. The EC9O values were averaged and the
average was expressed as the mean the .+-.standard error of the
mean.
[0032] It was found Alferon N.RTM. inhibited SARSCoV, with an
EC5O=5,696.+-.1703 IU by visual CPE inhibition assay and an EC5O
10,740.+-.5,161 IU/ml by NR assay. However, viral cytopathic
effects (CPE) were readily apparent at all dilutions tested,
although at higher compound doses, CPE was greatly reduced compared
to the virus replication controls (data not shown). This phenomenon
was verified by virus yield reduction assay in which each dilution
of drug was sampled and quantified for the presence of surviving
virus or newly produced virus. At each dilution of compound,
infectious virus was detected, with lower amounts of virus being
detected at the higher concentrations of compound (data not shown).
The concentration at which virus load was reduced by 1 log 10
(EC9O) 78 000.+-.22,000 IU/ml.
[0033] From these studies it was determined Alferon N.RTM. worked
well in reducing virus cytopathic effect, with an EC5O of
5,696.+-.1703 IU/ml.
[0034] Cinatl J, Morgenstern B, Bauer G, Chandra P, Rabenau H,
Doerr H W. Treatment of SARS with human interferons. Lancet 2003;
362: 293-4.
[0035] Barnard D L, Stowell V D, Seley K L, Hegde V R, Das S R,
Rajappan V P, et al. Inhibition of measles virus replication by
5'-nor carbocyclic adenosine analogues. Antiviral Chem Chemother
2001; 12: 241-250.
* * * * *