U.S. patent application number 16/304550 was filed with the patent office on 2019-08-22 for treatment of alphavirus-induced inflammation.
This patent application is currently assigned to GRIFFITH UNIVERSITY. The applicant listed for this patent is GRIFFITH UNIVERSITY. Invention is credited to Lara Josefina Herrero, Surendran Mahalingam.
Application Number | 20190255097 16/304550 |
Document ID | / |
Family ID | 60410929 |
Filed Date | 2019-08-22 |
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United States Patent
Application |
20190255097 |
Kind Code |
A1 |
Mahalingam; Surendran ; et
al. |
August 22, 2019 |
TREATMENT OF ALPHAVIRUS-INDUCED INFLAMMATION
Abstract
Disclosed is a method of treating alphavirus infections,
particularly in humans, in which pentosan polysulfate is
administered to an infected subject. Whilst not effecting the viral
load in a subject, the pentosan polysulfate acts to reduce
inflammation in tissues, such as the muscles, and in the joints of
a subject. In addition, cartilage damage in the joints may be
reduced. The reduction in inflammation and/or cartilage damage acts
to reduce the severe pain experienced by subjects suffering from
alphavirus infections, such as Ross River virus, chikungunya virus
and Barmah Forest virus.
Inventors: |
Mahalingam; Surendran;
(Arundel, AU) ; Herrero; Lara Josefina;
(Southport, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GRIFFITH UNIVERSITY |
Nathan, Queensland |
|
AU |
|
|
Assignee: |
GRIFFITH UNIVERSITY
Nathan, Queensland
AU
|
Family ID: |
60410929 |
Appl. No.: |
16/304550 |
Filed: |
May 26, 2016 |
PCT Filed: |
May 26, 2016 |
PCT NO: |
PCT/AU2016/050408 |
371 Date: |
November 26, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61P 31/14 20180101; A61P 29/00 20180101; A61K 31/737 20130101;
A61P 19/02 20180101; Y02A 50/382 20180101; A61K 9/0019
20130101 |
International
Class: |
A61K 31/737 20060101
A61K031/737; A61K 9/00 20060101 A61K009/00; A61P 31/14 20060101
A61P031/14; A61P 19/02 20060101 A61P019/02; A61P 29/00 20060101
A61P029/00 |
Claims
1. A method of treating a subject having an alphavirus infection
comprising administering parenterally by intra-articular or
intra-muscular injection an amount of pentosan polysulfate or a
salt thereof effective to reduce alphavirus induced inflammation
and/or alphavirus induced cartilage damage in the subject.
2. The method of claim 1 wherein the inflammation is in the joints
and/or muscles.
3. The method of claim 2 wherein the inflammation is in the
joints.
4. The method of claim 2 wherein the inflammation is in the
muscles.
5. The method of claim 2 wherein the inflammation is in the joints
and the muscles.
6. The method of claim 1 wherein the alphavirus is selected from
the group consisting of Ross River virus, chikungunya virus and
Barmah Forest virus.
7. The method of claim 6 wherein the virus is Ross River virus.
8. The method of claim 6 wherein the virus is chikungunya
virus.
9. The method of claim 6 wherein the virus is Barmah Forest
virus.
10. The method of claim 1 wherein administration is
intra-articular.
11. The method of claim 1 wherein administration is
intra-muscular.
12. The method of claim 1 wherein the pentosan polysulfate is the
sodium salt.
13. The method of claim 1 wherein administration is daily.
14. The method of claim 1 wherein administration is twice
daily.
15. The method of claim 13 wherein the amount administered is
within the range of from 0.5 to 5.0 mg/kg body weight of
subject.
16. The method of claim 15 wherein the amount is within the range
of from 1.0 to 5.0 mg/kg body weight of subject.
17. The method of claim 16 wherein the amount is within the range
of from 2.0 to 5.0 mg/kg body weight of subject.
18. The method claim 17 wherein the subject is a human.
19-20. (cancelled)
21. A method of treating a subject having an alphavirus infection
selected from the group consisting of Ross River virus, chikungunya
virus and Barmah Forest virus comprising administering daily
parenterally by intra-articular or intra-muscular injection an
amount of sodium pentosan polysulfate effective to reduce
alphavirus induced inflammation and/or alphavirus induced cartilage
damage in the subject.
Description
TECHNICAL FIELD
[0001] This invention relates to the treatment of animals,
especially humans, infected with alphaviruses and more particularly
to a treatment directed to the reduction of inflammation and/or
cartilage damage arising from the infection.
BACKGROUND
[0002] Arthropod-borne arthritogenic alphaviruses such as Ross
River virus (RRV) and chikungunya virus (CHIKV) cause large
epidemics of severe musculoskeletal disease. They have been
progressively expanding their global distribution, regularly
emerging in new regions of the world (1, 2). The hallmark of
alphavirus disease is crippling joint pain and arthritis, which
often has an extended duration leaving patients bed-ridden and
incapacitated. In 2014-2015, CHIKV further expanded its global
distribution by entering the Americas and is circulating in several
Caribbean islands. As of 23.sup.rd May 2016, the Pan American
Health Organization (PAHO) reported an estimated total of over 1 5
million cases since 2014, with 100,000 reported in 2016 so far;
additionally, the first report of local autochthonous CHIKV
transmission in mainland USA was reported in July 2014 (3, 4). Due
to the expanding range of alphaviral infections, understanding the
mechanisms by which alphaviruses cause debilitating arthritic
disease has become increasingly important, especially as there are
no specific treatments available (5).
[0003] The severe arthralgia/arthritis in the joints caused by
alphaviruses can be both acute and chronic. Ultrasonography of
CHIKV patients with joint pain reveals striking tenosynovitis, bone
erosion and synovial thickening (6). RRV antigen has been detected
by immunofluorescence in synovial monocytes and macrophages during
the early phase of illness (7), and in basal epidermal and eccrine
duct epithelia three days after the onset of RRV exanthem (8).
Using antigen staining and RT-PCR, RRV has also been detected in
synovial effusions more than one month after the onset of symptoms,
providing evidence of persistent infection in the inflamed synovium
(9).
[0004] The synovial space of joints is glycan-rich, containing high
levels of glycosaminoglycan (GAGs) frequently linked to protein
backbones that form proteoglycan structures. Chondrocytes are the
major cell type producing the matrix of articular cartilage that is
rich in proteoglycans (14). However, there have been no studies to
elucidate the impact of alphaviruses on cartilage and the
proteoglycan matrix of the joint.
[0005] In the work described herein, we show RRV infection results
in similar histopathology of the joint to that observed in
rheumatoid arthritis (RA). This includes pannus-like formation,
immune infiltration and cartilage damage. We further show that
treatment with pentosan polysulfate (PPS) ameliorates the severity
of both RRV and CHIKV clinical disease, overall reduction in both
immune infiltrates and soluble pro- inflammatory factors. We also
observed a change in the kinetics of the soluble factors involved
in macrophage activation. In RRV-infection treatment also reduced
the loss of articular cartilage and protected the level of
proteoglycans in the cartilage matrix, altering the expression of
cartilage components including aggrecan and collagen in. Overall we
show that PPS is a safe and effective treatment for both acute and
chronic RRV-infection.
SUMMARY
[0006] Accordingly the present invention consists in a method of
treating a subject having an alphavirus infection comprising
administering parenterally an amount of pentosan polysulfate or a
salt thereof effective to reduce alphavirus induced inflammation
and/or alphavirus induced cartilage damage in the subject.
[0007] In another aspect, the present invention consists in the use
of pentosan polysulfate or a salt thereof in the preparation of a
medicament for the treatment of alphavirus induced inflammation
and/or to reduce alphavirus induced cartilage damage in a subject
having an alphavirus infection.
[0008] In a further aspect, the present invention consists in a
composition comprising pentosan polysulfate and a pharmaceutically
acceptable carrier for use in treating alphavirus induced
inflammation and/or to reducing alphavirus induced cartilage damage
in a subject having an alphavirus infection.
[0009] Whilst the subject to be treated may be an animal,
preferably the subject is a human infected with an alphavirus.
BRIEF DESCRIPTION OF DRAWINGS
[0010] FIG. 1. RRV-infection results in damages to the cartilage in
joint tissues.
[0011] 20-day-old C57BL/6 mice were infected s.c. With 10.sup.4 pfu
RRV or mock-infected with diluent alone. Infection resulted in
extensive inflammation, pannus formation, articular cartilage
thinning, disruption of the proteoglycans and upregulation of
cartilage associated genes. (A, B) For histological analysis the
joints of RRV-infected mice were sacrificed at peak disease at 10
days p.i., perfused with 4% PFA, knee joint tissues removed,
paraffin-embedded and 5 .mu.m sections generated. Sections were
stained with (A) H&E or (B) Safranin O/fast green. Annotations;
(B) bone, (C) cartilage, (P) pannus. Images are representative of
at least 5 mice per group (magnification 100.times.). (C) The width
of cartilage from mock- and RRV-infected mice were measured in five
areas per mouse and averaged with each point representing one
mouse. Cartilage degradation from the medial femoral condyle (MFC)
and the medial tibia plateau (MTP) of RRV mice was assessed as
outlined in the methods, mock control mice show a score of 1. Data
represent mean .+-.SEM of 5 mice per group. (D) Similarly, at both
early time points (i) and at peak disease (ii) total RNA from ankle
joint tissues was isolated and analysed for mRNA expression by
qRT-PCR. Data was normalised to the housekeeping gene HPRT1 and
expressed as relative expression compared to mock-infected controls
(as represented by the dashed line). Each bar represents the mean
+/- the SEM for 5-6 mice per group, *p<0.05, ***p<0.001
one-way ANOVA Dunnett's post test.
[0012] FIG. 2. Pentosan polysulfate reduces the severity of acute
RRV-induced inflammatory disease.
[0013] PPS treatment reduced the level of disease signs, prevented
severe weight loss and reduced the level of inflammatory
infiltrates into the joint and muscle tissues protecting the muscle
tissue from extensive RRV-induced damage. 20-day-old C57BL/6 mice
were infected s.c. with 10.sup.4 pfu RRV or mock-infected with
diluent alone then either treated daily i.p. with PPS at 3 mg/kg in
100 .mu.L PBS or mock-treated with PBS alone. (A) Mice were scored
for the development of hind-limb dysfunction and displayed a
reduction in RRV-disease severity with PPS treatment. Mock-infected
mice were scored zero for the duration of the experiment
*p<0.05, **p<0.01 using a Mann-Whitney test. (B) Weight was
monitored at 24-hour intervals ***p<0.001 significantly reduced
weight loss of RRV-infected PPS-treated compared to RRV-infected
mock-treated -using two-way ANOVA with Bonferroni post test. (C)
For histological analysis, mice were sacrificed at 7 or 10 days
p.i., perfused with 4% PFA, quadriceps and knee joint tissues
removed, paraffin-embedded, 5 .mu.m sections generated. Sections
were stained with H&E. (D) Quadriceps muscles were removed from
RRV-infected `pentosan` and `PBS` treated mice at day 7 and 10
p.i.. cells were isolated, counted and stained for CD45, Grl,
CD11b, pan NK/NKT, CD3 and CD19 expression. Total leukocyte
(CD45.sup.hi), inflammatory monocyte (Gr1.sup.hiCD11b.sup.hi),
NK/NKT (CD45.sup.hi pan NK.sup.hi) and T cell (CD3.sup.hi)
populations were determined among total live (PI-negative)
infiltrated cells using various gating strategies, analysed by
Student t test. Each data point represents the mean +/- SEM of 5 to
10 mice and is representative of 3-4 independent experiments.
[0014] FIG. 3. PPS-treatment does not alter the kinetics of viral
replication.
[0015] Both infectious virus and viral RNA levels were measured
indicating PPS-treatment did not affect viral clearance. Mice were
infected s.c. with RRV or mock-infected with diluent alone then
either treated daily i.p. with PPS or mock-treated with PBS alone.
At days 1, 2, 3, 7 and 10 p.i. the serum, quadriceps and ankle
tissues were harvested, homogenised and the viral load determined
by (A) plaque assay on Vero cells for infectious virus or (B) by
qPCR for viral RNA with nsp3 specific primers using a standard
generated from serial dilutions of RRV T48 infectious plasmid. Each
data point represents a single mouse, line indicates the median
value. *p<0.05 using two-way ANOVA with Bonferroni post test for
plaque assay and unpaired Mann-Whitney for PCR.
[0016] FIG. 4. PPS-treatment alters the expression of soluble
factors in RRV-inflammatory disease.
[0017] 20-day-old C57BL/6 mice were infected s.c. with 10.sup.4 pfu
RRV or mock-infected with diluent alone then either treated daily
i.p. with PPS at 3 mg/kg in 100 .mu.L PBS or mock-treated with PBS
alone (mock-infected mock-treated=Mock control, mock-infected
PPS-treated=PPS alone, RRV-infected mock-treated=RRV alone,
RRV-infected PPS-treated=RRV+PPS). At days 1, 3 and 10 p.i. serum
was collected and analysed for level of soluble factors using Bio-
Plex Pro Mouse Cytokine 23-plex kits (Biorad). (A) PPS treatment
reduced the levels pro- inflammatory factors at day 10 p.i., (B)
increased levels of chemo-attractants factors at day 1 p.i. and (C)
altered the kinetics of M2 cytokine IL-10. Red line indicates the
level of mock control. Each data point represents the mean +/-
standard error of 5 to 6 mice. (A, B) *p<0.05, ** p<0.01,
***p<0.001 one-way ANOVA with Tukey's post test. (C) *p<0.05,
** p<0.01, ***p<0.001 using two-way ANOVA with Bonferroni
post test. Astrices directly on top of bars are compared to mock
control levels.
[0018] FIG. 5. Pentosan polysulfate treatment protects the joints
from RRV-induced cartilage damage.
[0019] 20-day-old C57BL/6 mice were infected s.c. with 10.sup.4 pfu
RRV or mock-infected with diluent alone then either treated daily
i.p. with PPS at 3 mg/kg in 100 .mu.L PBS or mock-treated with PBS
alone. For histological analysis, mice were sacrificed at peak
disease at 10 days p.i., perfused with 4% PFA, whole legs removed,
paraffin-embedded and 5 pm sections generated. Sections were
stained with (A) Masson's Trichrome or (B) Safranin 0/fast green
and showed an increase in collagen fibres, improvement in the
skeletal muscle tissue morphology and protection of the
proteoglycan matrix with treatment. Annotations; (B) bone, (C)
cartilage, (P) pannus, (M) muscle. Images are representative of at
least 5-8 mice per group. (C) The width of cartilage and epiphyseal
plate from mice were measured in five areas of per mouse and
averaged with each point representing one mouse. Cartilage
degradation from the medial femoral condyle (MFC) and the medial
tibia plateau (MTP) of at least five mice per group was assessed as
outlined in the methods with mock control mice showing a score of
1. Data represent mean .+-.SEM of 5 mice per group.
[0020] FIG. 6. Pentosan polysulfate treatment counteracts the
dysregulation of the cartilage matrix components caused by
RRV-infection.
[0021] PPS treatment significantly reduced the early expression of
aggrecan and collagen II and the expression of ADAMTS-5 and TIMP-3
at peak disease. 20-day-old C57BL/6 mice were infected s.c. with
10.sup.4 pfu RRV or mock-infected with diluent alone then either
treated daily i.p. with PPS at 3 mg/kg in 100 .mu.L PBS or
mock-treated with PBS alone (mock-infected mock-treated=Mock
control, mock-infected PPS-treated=PPS alone, RRV-infected mock-
treated=RRV alone, RRV-infected PPS-treated=RRV+PPS). At days 1, 3
and 10 p.i. joint tissues were removed, RNA extracted and real time
PCR performed to evaluate gene regulation of key mediators of the
proteoglycan matrix of joint cartilage. Results were normalised to
the housekeeping gene HPRT1 and are expressed as fold of change
compared to the mock control samples. Each data point represents
the mean +/- standard error of 5 to 6 mice and is representative of
two independent experiments. *p<0.05, ** p<0.01,
***p<0.001 using two-way ANOVA with Bonferroni post test.
[0022] FIG. 7. Pentosan-polysulfate reduces the severity of acute
CHIKV- inflammation without affecting the kinetics of viral
infection.
[0023] 25-day-old C57BL/6 mice were infected s.c. with CHIKV or
mock-infected with diluent alone then either treated daily i.p.
with PPS at 3 mg/kg in 100 .mu.L PBS or mock-treated with PBS
alone. (A) CHIKV-induced footpad swelling was assessed daily by
measuring the height and width of the perimetatarsal area of the
hind foot. PPS treatment resulted in a significant reduction in
swelling. (B) H&E stained histological analysis showed PPS
treatment decreased the level of inflammatory infiltrates in
CHIKV-infected mouse joints at peak swelling 3 days p.i.. Both
infectious virus and viral RNA levels were measured indicating
PPS-treatment did not affect viral clearance. At days 1 and 3 and 7
p.i. the serum, quadriceps and ankle tissues were harvested,
homogenised and the viral load determined by (C) plaque assay on
Vero cells for infectious virus or (D) by qPCR for viral RNA in
joint tissues with CHIKV E2 specific primers. Each data point
represents a single mouse, line indicates the median value. **
p<0.01 using two-way ANOVA with Bonferroni post test for foot
swelling and plaque assay and Mann Whitney for PCR.
[0024] FIG. 8. PPS-treatment alters soluble factors in
CHIKV-inflammatory disease.
[0025] 25-day-old C57BL/6 mice were infected with CHIKV or diluent
alone then either treated daily i.p. with PPS or mock-treated with
PBS alone. (A) Kinetics of IL-10 were altered with PPS-treatment.
Grey line indicates the level of mock control. Each data point
represents the mean +/- standard error of 5 to 6 mice. *p<0.05,
** p<0.01, ***p<0.001 using two-way ANOVA with Bonferroni
post test. Astrices directly on top of bars are compared to mock
control levels. (B) Levels pro-inflammatory factors were decreased
at peak swelling by day 3 p.i. with PPS treatment *p<0.05, **
p<0.01, ***p<0.001 one-way ANOVA with Tukey's post test.
DESCRIPTION OF EMBODIMENTS
[0026] The inflammation arising out of the alphavirus infection is
in the joints and/or muscles. Alternatively, the inflammation may
be confined to the joints or it may be confined to the muscles. In
some embodiments, the inflammation will be in both the joints and
the muscles.
[0027] Although the present invention is directed towards
alphavirus infections generally, in some embodiments, the
alphavirus infection will be selected from the group consisting of
Ross River virus, chikungunya virus and Barmah Forest virus.
[0028] In particular, the present invention in one embodiment is
directed towards Ross River virus.
[0029] In particular, the present invention in another embodiment
is directed towards chikungunya virus.
[0030] In particular, the present invention in another embodiment
is directed towards Barmah Forest virus.
[0031] Administration of the effective amount of pentosan
polysulfate to a subject infected with an alphavirus is
parenteral.
[0032] In some embodiments when a joint is suffering from
inflammation, administration is intra-articular.
[0033] In other embodiments when a muscle is suffering from
inflammation, administration is intra-muscular.
[0034] Generally, a daily dose of pentosan polysulfate will be
administered. Such dosing may be into one or more joints, one or
more muscles, or into both one or more joints and muscles.
Alternatively, a single dose may be administered intravenously to
treat inflammation in both joints and muscles.
[0035] It is, however, within the scope of the invention to
administer on two more occasions on a daily basis, depending on the
severity of the symptoms of the subject.
[0036] Of course it will be recognised that treatment to reduce
cartilage damage is undertaken by intra-articular or intravenous
administration.
[0037] Based on the animal studies disclosed herein, the amount to
be administered is within the range of from 0.1 to 5.0 mg/kg body
weight of subject.
[0038] In some embodiments, the amount to be administered is within
the range of from 1.0 to 5.0 mg/kg body weight of subject.
[0039] In some embodiments, the amount to be administered is within
the range of from 2.0 to 5.0 mg/kg body weight of subject.
[0040] Treatment of subjects is by administration to the subject of
pentosan polysulfate. Owing to its solubility and ready
availability, preferably the sodium salt of pentosan polysulfate is
used. However, other salts such as magnesium and calcium may also
be used.
[0041] Commercially, Bene-PharmaChem has supplied their PPS in 1 ml
glass ampoules containing 100mg PPS/ml. Because of the ready
availability of this sterile injectable product it is preferred to
be used in the present invention for treating humans.
[0042] The Bene-PharmaChem product comprises: sodium pentosan
polysulfate (PPS) 100 mg, sodium phosphate 2.2mg, sodium hydrogen
phosphate 6.8mg, adjusted to pH 6.5 with sodium hydroxide and water
for injection, USP, qs. 1mL.
[0043] It is, however, within the scope of this invention to use
alternative formulations. For example, standard textbooks in the
field of this invention, such as Remington's Practice of Pharmacy
teach such alternatives.
[0044] Likewise, for veterinary applications, a product such as
Cartrophen Vet.RTM. (Biopharm Australia) may be used.
[0045] In order to better understand the nature of this invention,
set out below is a description of a series of experiments carried
out to demonstrate the effectiveness of PPS in the treatment of
subjects infected with an alphavirus and suffering from joint
and/or muscle inflammation and cartilage damage.
[0046] Methods
[0047] Virus and Cells. Stocks of the wild-type T48 strain of RRV
were generated from the full- length T48 cDNA clone (kindly
provided by Dr Richard Kuhn, Purdue University) (20). Stocks of
CHIKV Mauritius strain were propagated in BHK-21 cells. All
titrations were performed by plaque assay on Vero cells as
described previously (21).
[0048] Mice. C57BL/6 wild type (WT) mice were obtained from the
Animal Resources Centre (Perth, Australia) and bred in- house. All
animal experiments were performed in accordance with the guidelines
set out by the Griffith University Animal Ethics Committee. C57BL/6
mice of twenty- to twenty-five day old, were inoculated
subcutaneously (s.c.) with 10.sup.4 pfu virus. Injections of RRV in
PBS to a volume of 50 .mu.L was in the thorax as described
previously (11), and CHIKV in PBS to a volume of 20 .mu.L in the
ventral side of the footpad as described previously (10).
Mock-infected mice were inoculated with PBS alone. Mice were
weighed and scored for disease signs every 24 h. RRV disease scores
were assessed based on animal strength and hind-leg paralysis as
outlined previously (22). Swelling of the footpad induced by CHIKV
was assessed by measuring the height and width of the
perimetatarsal area of the hind foot using Kincrome digital vernier
calipers.
[0049] Treatment with pentosan polysulfate (Cartrophen Vet.RTM.,
Biopharm Australia) or vehicle alone was given intraperitoneally
(i.p.) at 3 mg/kg in 100 .mu.L PBS (vehicle), daily for the
duration of the experiment, commencing from the day of virus
infection. In long-term experiments, PPS treatment was delivered
orally by adding to drinking water at a concentration of 100 mg/L
which is equivalent to a dose of 25 mg/kg/day based on the daily
water consumption of a C57BL/6 mouse as previously reported (23).
Using the human to animal conversion outlined by Regan-Shaw et al.,
(24) 25 mg/kg/day is the equivalent of the human dose of 2 mg/kg
which is the recommended dose for Elmiron.RTM..
[0050] Histology. Mice were sacrificed and perfused with 4%
paraformaldehyde (PFA). Tissues were collected and fixed in 4% PFA,
followed by paraffin embedding. Ankles and knee joints were
decalcified prior to embedding. Sagittal sections of five micron
thickness were prepared and stained with haematoxylin and eosin (H
& E), Masson's trichrome or Safranin O/Fast Green. Cartilage
thickness and damage was measured at 200.times. magnification from
the medial femoral condyle (MFC) and the medial tibial plateau
(MTP) by averaging five random points of measurement (separated by
at least 20 .mu.M of distance) per region per mouse and graphed as
the mean .+-.SEM of 5 mice per group. Epiphyseal thickness was
measured from central sagittal sections by averaging five random
points of measurement (separated by at least 20 .mu.M of distance)
per region per mouse and graphed as the mean .+-.SEM of 5 mice per
group system. Cartilage degradation from the medial femoral condyle
(MFC) and the medial tibial plateau (MTP) was assessed according to
a modified semi-quantitative scoring system of Glasson et at, (25)
where 1=normal cartilage; 2=alteration of the proteoglycan matrix
assessed by Safranin O stain; 3=alteration of the proteoglycan
matrix and loss of lamina splendens; 4=a score of 2-3 thinning
either the transitional or radial cartilage; 5=a score of 2-3 plus
thinning of both the transitional and radial layers.
[0051] Multiplex. The level of serum cytokines was determined using
multiplex bead arrays kits according to the manufacturer's
instructions (Bio-Plex Pro Mouse Cytokine 23-plex kits) (Biorad,
Hercules, CA). Data was acquired using a Luminex 200.TM. (Biorad)
and analysed using the Bio-plex Manager' 6.1 software (Biorad).
[0052] Real-Time PCR. Preparation of RNA was performed from cell
pellets using TRIzol (Life Technologies, Victoria, Australia)
according to the manufacturer's instructions. Quantification of
total RNA was measured by NanoDrop 1000 spectrophotometer (Thermo
Scientific, Victoria, Australia). Extracted total RNA (20 ng/.mu.L)
was reverse-transcribed using an oligo (dT) primer and reverse
transcriptase (Sigma Aldrich, Sydney, Australia) according to the
manufacturer's instructions.
[0053] Gene expression- SYBR.RTM. Green Real-time PCR was performed
using 10 ng of template cDNA on a CFX96 TouchTM Real-Time PCR
System in 96-well plates, using QuantiTect Primer Assay kits
(Qiagen, Hilden, Germany) for HPRT1, or purchased from primers from
Sigma-Aldrich with the sequences outlined in table 1.
TABLE-US-00001 TABLE 1 Primer sequences Gene Forward Reverse
TGF.beta. CAA CGC CAT CTA TGA AAG CCC TGT ATT CCG GAA AAC C TCT CC
Aggrecan GCC CAA GAA CAG TAC TGC TAG GTT GGT TGA AAT GGT CCC A
Collagen CAG AAC ATC ACC TAC TTC AAC ATC GTT GGA I CAC TGC AA ACC
CTG Collagen AGA ACA GCA TCG CCT CTT GCC CCA CTT ACC II ACC TG AGT
GT BMP-1/ AGC AGG CTG CAG TTC GAA TGT GTT CCG GGC mTLD TCA GAC AGC
ATA GTG CAT ADAMTS-4 CAC TGA CTT CCT GGA GGA AAA GTC GTC GGT CAA
TGG TTA T AGA TGG A ADAMTS-5 GAT GAT CAC GAA GAG TCA CAT GAA TGA
TGC CAC TAC GA CCA CAT MMP-3 TGG AGC TGA TGC ATA TGA AGC CAC CAA
CAT AGC CC CAG GA MMP-9 GGA ACT CAC ACG ACA GAA ACT CAC ACG CCA TCT
TCC A GAA GAA TTT TIMP-3 GGC ACT CTG GTC TAC TTT CAG AGG CTT CCG
ACT ATT AAG CA TGT GA RRV nsp3 CCG TGG CGG GTA TTA AAC ACT CCC GTC
GAC primer* TCA AT AAC AGA *RRV nsp3 Probe ATT AAG AGT GTA GCC ATC
C
[0054] Viral load quantification-Standard curve was generated using
serial dilutions of RRV T48 infectious plasmid DNA as described
previously (26). Quantification of viral load was performed using
SsoAdvanced Universal Probes Supermix (BioRad) in 12.5 .mu.L
reaction volume to detect nsP3 region RNA (table 1) (26).
[0055] All reactions were performed using BioRad CFX96 Touch.TM.
Real-Time PCR Detection System on 96-well plates. Cycler conditions
were as follows: (i) PCR initial activation step: 95.degree. C. for
15 min, 1 cycle and (ii) 3-step cycling: 94.degree. C. for 15 sec,
follow by 55.degree. C. for 30 sec and 72.degree. C. for 30 sec, 40
cycles. Dissociation curve was acquired using CFX Manager.TM.
software to determine specificity of amplified products. Standard
curve was plotted and copy numbers of amplified products were
interpolated from standard curve using Prism Graphpad software to
determine viral load. The fold change in mRNA expression relative
to mock- infected samples for each gene was calculated with the
.DELTA..DELTA.Ct method. Briefly, .DELTA..DELTA.Ct=.DELTA.Ct
(RRV-infected)-.DELTA.Ct (Mock-infected) with .DELTA.Ct=Ct (gene of
interest)-Ct (housekeeping gene--HPRT). The fold change for each
gene was calculated as 2.sup.-.DELTA..DELTA.Ct.
[0056] Detection of leukocyte infiltrates in quadriceps. Quadriceps
muscles were removed and processed as described previously (10).
Briefly, tissues were incubated with 3 mg/Ml collagenase IV and 1
mg/mL DNase I in 100 .mu.L RPMI 1640 at 37.degree. C. for 1.5 h
then resuspended in 5 mL RPMI and passed through a 40 .mu.m cell
strainer. Cells were washed, pelleted and treated with 1.times. RBC
lysis buffer for 5 min, and counted. To determine percentages and
numbers of specific leukocyte populations, cells were treated with
Fc Block (2.4G2; BD) for 5 min at 4.degree. C.and labelled with
fluorochrome-conjugated anti-mouse antibodies, including
anti-CD3-FITC (145-2C11, BD), anti-CD19-APC (MB19-1, eBioscience),
anti-CD11b-PE (M1/70, BD), anti-Grl-APC (RB6-8C5, eBioscience) and
anti-pan-NK/NKT antigen-PE (U5A2-13, BD) in various combinations in
the presence of biotinylated anti-CD45 (30-F11, eBioscience),
followed by treatment with streptavidin PE-Cy7 at 4.degree. C.for
30 min. Cells were resuspended in 500 .mu.L PBS containing 2% FCS
and 1 .mu.g/mL propidium iodine (PI), and analysed by the CyAn ADP
flow cytometer (Beckman Coutler) with Kaluza software.
[0057] Statistical analysis. Body mass, plaque assay, multiplex
(FIG. 4C and 8A), real-time PCR (FIG. 6) and joint swelling were
analysed using two-way ANOVA with Bonferroni post- test. Real-time
PCR (FIG. 1D), multiplex (FIGS. 4 B and 8B) and histology (FIG. 5C)
were analysed using a one-way ANOVA with a Dunnett's or Tukey's
post-test. Flow cytometry data and histology (FIG. 1C) were
analysed using unpaired Students t-test. All data was tested for
normality using the D'Agostino-Pearson normality test prior to
analysis with these parametric tests. Clinical scores and and
real-time PCR for viral load were analysed using the non-parametric
Mann-Whitney test. Statistics were performed with GraphPad Prism
5.0
[0058] Results
[0059] Ross River virus infection stimulates the production of
proteases ADAMTS-4, MMP-3 and MMP-9 and causes damage to the
articular cartilage in joints.
[0060] To determine if RRV infection affects the cartilage of
joints, we infected C57BL/6 mice with RRV and isolated joint tissue
for histological analysis. At peak disease, extensive joint
inflammation was observed along with pannus-like formation and
thinning of the articular cartilage in H&E stained sections
(FIG. 1A). Further analysis of joint sections, stained with
Safranin O, revealed considerable disruption of the proteoglycans
in the cartilage matrix, as seen by the difference in Safranin O
staining intensity (which is directly proportional to the
proteoglycan content) between infected and mock tissues (FIG. 1B)
(27). Quantification of cartilage thickness and damage was measured
from the MFC and MTP and showed RRV- infection results in an
average 20 .mu.m reduction in articular cartilage thickness and
cartilage damage characterised by alteration of the proteoglycan
matrix and loss of lamina splendens (FIG. 1C). Furthermore,
RRV-infection resulted in an early significant increase (24 hours
p.i.) of the enzymes; A disintegrin and metalloproteinase with
thrombospondin motifs (ADAMTS)-4 (p<0.001), matrix
metalloproteinase (MMP)-3 (p<0.05) and MMP-9 (p<0.01)
compared to mock infected controls. These are known to cause
cartilage damage by degrading aggrecan, collagen, proteogylcans and
the extracellular matrix (FIG. 1Di). At peak disease, stimulators
of cartilage growth, matrix-transforming growth factor (TGF)
.beta.1 (p<0.01) and bone morphogenetic protein (BMP)-1
(p<0.001) were also significantly increased in response to
RRV-infection compared to mock-infected controls (FIG. 1Dii). The
results suggest that RRV infection results in cartilage degradation
and thinning which is associated with arthritic disease
symptoms.
[0061] Pentosan polysulfate reduces the severity of RRV-induced
disease and inflammation
[0062] To assess the potential of PPS as a treatment strategy in
alphaviral disease, mice were infected with RRV or mock-infected
with PBS alone and then treated i.p. with either PPS at 3 mg/kg or
with vehicle daily. PPS treatment resulted in a 65% decrease
(p>0.05) in clinical disease score in RRV-infected mice (FIG.
2A) and a corresponding protection from disease-associated weight
loss (p>0.001) (FIG. 2B).
[0063] To better characterise the reduction in disease, we assessed
inflammation and tissue damage in RRV-infected mock and PPS-treated
mice. Tissues from RRV-infected mice were collected at the start
and end of peak disease (day 7 and 10 p.i.) for histological
analysis and flow cytometry. No inflammation was observed in the
quadriceps muscle or ankle joint of control mock-infected
mock-treated or mock-infected PPS-treated mice (FIG. 2C).
Consistent with previous studies, RRV-infected mock-treated mice
showed extensive inflammation and myositis in quadriceps muscle at
day 7 p.i. (FIG. 2C,D) and around the ankle joint (FIG. 2C) (11,
21, 22, 28). In contrast, RRV-infected PPS-treated mice showed
markedly reduced numbers of infiltrating cells in both the muscle
and the joint tissues (FIG. 2C).
[0064] In order to characterise the effect of PPS treatment on both
lymphoid and myeloid infiltrating cells, we analysed the cell
populations in the quadriceps muscles at days 7 and 10 p.i. by flow
cytometry. At day 7 p.i. PPS treatment uniformly reduced the
numbers of all CD45.sup.+ infiltrating leukocytes including
reductions (p<0.05) in the monocytes, T cell and NK cell
populations (FIG. 2D). By day 10 p.i. PPS treatment showed a
decrease in the T cell and NK cell populations (p<0.05) with
similar numbers of CD45.sup.+ cells and monocytes (FIG. 2D). At day
10 p.i. treatment also altered the NK cell to total cell ratio
resulting in a reduction in the overall percentage of NK cells in
the quadriceps muscle (data not shown).
[0065] Reduced disease in treated mice is not due to decreased
viral burden.
[0066] Viral titres in the serum of PPS-treated and mock-treated
mice were comparable at all days tested, indicative of equivalent
systemic replication (FIG. 3A). Similarly, RRV titres recovered
from quadriceps muscles were comparable (FIG. 3A). Interestingly,
RRV titres in the ankle tissues showed the most variation between
PPS-treated and mock-treated mice (FIG. 3A). By day 3 p.i. there
was a slight reduction in RRV titres for PPS-treated mice; by day 7
p.i titres were comparable; and then by day 10 p.i. RRV titres in
PPS-treated mice were elevated compared to mock-treated mice
(p<0.05). The results from day 10 p.i. suggest that
PPS-treatment may have an effect on viral clearance within the
joint. To assess viral clearance, qPCR to quantify viral RNA was
performed on the joint tissues (FIG. 3B). Although the trend
appears to suggest a lower level of specific RRV RNA in the joint
tissue of PPS-treated RRV-infected mice in the joint at ay 10 p.i.,
this was not statistically significant compared to mock-treated
mice, confirming that PPS-treatment does not affect viral
clearance.
[0067] Treatment with pentosan polysulfate increases the level of
anti-inflammatory IL-10 and decreases pro-inflammatory factors
associated with RRV disease.
[0068] A range of pro-inflammatory factors and chemoattractants
mediate or contribute to alphaviral disease (29). To elucidate
whether PPS-treatment affects the production of soluble immune
mediators during RRV infection, sera from PPS-treated and
mock-treated mice were analysed using multiplex and compared to
mock-infected, PPS-treated or mock-treated mice. As expected, RRV
infection resulted in an increase (p<0.05) of pro-inflammatory
factors (both cytokines and chemoattractants) at peak disease (FIG.
4). PPS-treatment also altered the levels of the M2
anti-inflammatory cytokine IL-10. IL-10 kinetics corresponded to
the kinetics of disease with serum levels increasing over time in
RRV-infected, mock-treated mice. PPS-treatment resulted in an early
surge of IL-10 in RRV-infected mice, being significantly elevated
at both days 1 and 3 p.i. (p<0.001 and 0.01 respectively) (FIG.
4A). Additionally, PPS treatment significantly reduced the serum
levels of IL-1 a, IL-2, IL-6, CCL-2 and MIP-1.alpha. at peak
disease (p<0.05) (FIG. 4B).
[0069] Pentosan polysulfate treatment protects the joints from
cartilage damage associated with RRV infection.
[0070] Recently we showed that RRV could infect osteoblasts and
infection results in systemic bone loss including the tibial
epiphysis and vertebrae (30-32). To determine the effect of PPS
treatment on RRV-induced muscle and joint damage, the tissues of
RRV-infected and mock- infected PPS-treated and mock-treated mice
were processed for histological analysis using Masson's trichrome
and Safranin O/Fast Green staining. Masson's trichrome staining of
the tibialis anterior showed PPS treatment protected the morphology
of striations within the skeletal muscle with sections of collagen
formation characteristic of muscle repair and fibrosis (33). PPS
treatment also prevented RRV-induced thinning of the epiphyseal
plate, protecting against cartilage loss (FIG. 5A, C). Safranin
O/Fast Green staining of the cartilage in the knee joint revealed
that PPS treatment protected the proteoglycan matrix of the
articular cartilage, preventing the loss of articular cartilage
observed in RRV-infected mock-treated mice as well as maintaining
chondrocyte morphology (FIG. 5B, C).
[0071] To further characterise the mechanism of PPS treatment we
analysed the genes involved in enzyme degradation of cartilage
(ADAMTS-4, ADAMTS-5, MMP-3 and MMP-9), stimulation of cartilage
protection and synthesis (TGF-.beta.1 and BMP-1) and cartilage
matrix proteins (aggrecan, collagen I and collagen II). As shown in
FIG. 1D, RRV-infection caused an increase in ADAMTS-4, which
remains elevated at day 3 p.i (p<0.01), but drops by the time of
peak disease (day 10 p.i), where there was a surge in ADMATS-5.
RRV- infection also resulted in a late rise (at peak disease) of
tissue inhibitor of metalloproteinases (TIMP)-3 (p<0.001); known
to inhibit both ADAMTS-4 and ADAMTS-5 (FIG. 6A, 6C). PPS-treatment
significantly reduced the levels of ADAMTS-5 and TIMP-3 at peak
disease (p<0.01), but not at the early stages of infection.
RRV-infection also resulted in an increase of cartilage components
aggrecan, collagen I and collagen II, that is largely reduced with
PPS-treatment (FIG. 6B). The genes associated with signalling
pathways for cartilage development (TGF-.beta.1 and BMP-1) and the
metalloproteinases were unaffected by PPS (FIG. 6).
[0072] Pentosan polysulfate treatment is a safe long-term treatment
strategy for chronic RRV disease.
[0073] To assess PPS-treatment for a long-term treatment in
patients with chronic symptoms, mice were RRV-infected and treated
orally with PPS or mock-treated in drinking water. Long-term PPS
treatment resulted in no adverse clinical signs in the mice for the
3-month duration of the experiment. RRV-infected mock-treated mice
showed extended disruption of the cartilage components with a
three-fold elevation of aggrecan (p<0.01) (Table 2). PPS-treated
mice showed less joint damage and significantly decreased the
expression of aggrecan back to base-line levels (p<0.001). The
levels of ADAMTS-4 expression were also reduced (p<0.01).
TABLE-US-00002 TABLE 2 Long-term PPS treatment decreases the
expression of aggrecan and ADAMTS-4 in RRV infection. Mock RRV RRV
+ PPS Gene Fold SEM n Fold SEM n Fold SEM n ADAMT 0.8345 0.203 6
1.2261 0.237 5 0.1689* 0.299 5 ADAMT 1.0854 0.320 6 1.5759 0.373 5
0.8305 0.179 5 MMP- 1.1335 0.349 6 1.6958 0.440 5 2.5028 1.221 5
MMP- 1.0066 0.083 6 1.0858 0.201 5 0.8604 0.247 5 9 3 2 7 Collagen
1.0128 0.117 3 0.8550 0.141 5 0.4014 0.074 5 Collagen 1.0357 0.179
3 0.9584 0.235 5 0.1719 0.055 5 II 3 8 6 BMP-1 1.0597 0.267 3
1.6204 0.295 5 1.4697 0.702 5 TIMP- 1.3321 0.772 6 1.4287 0.318 5
0.6328 0.136 5
[0074] Pentosan polysulfate is a potential treatment for
CHIKV-induced inflammation, reducing disease by altering the
cytokine response.
[0075] Given the expanding range of the alphavirus-CHIKV, together
with the current lack of therapeutic treatment options, we sought
to determine the broader application of PPS on alleviating
CHIKV-induced disease. PPS treatment decreased the level of joint
swelling of CHIKV-infected mice corresponding to a reduction in
inflammatory cells infiltrating into the joint (FIG. 7A, B).
Furthermore, as seen in RRV-infection, PPS-treatment did not affect
the kinetics of virus infection (FIG. 7C) and did not increase the
viral persistence in the joint tissues, with similar levels of
viral RNA detected three weeks p.i. (FIG. 7D). The reduced disease
also correlated to an early surge in anti-inflammatory IL-10 (FIG.
8A) and reduced the levels of soluble factors CCL-2, IL-6, IL-9 and
G-CSF at peak disease (day 3 p.i.) (FIG. 8B). These collective
results, whereby PPS reduces the disease severity of two critical
alphaviral diseases suggest that PPS may be a promising broad-range
treatment for alphavirus disease manifestations in general.
[0076] Discussion
[0077] The mechanisms by which alphaviruses trigger arthritis and
myositis are the focus of ongoing studies. Alphavirus-induced
disease has many similarities to rheumatoid arthritis (RA)
including common inflammatory pathways and the key involvement of
macrophages (11,12). The innate immune response is critical in the
pathogenesis of alphaviral disease, mediating cell recruitment,
viral clearance and inflammation (28, 29, 34). In particular,
monocytes and macrophages are the major cellular contributors to
disease progression and severity (29). In RA, monocytes play a
significant role in disease development and cartilage destruction
through the production of pro-inflammatory factors. (35). Despite
these clear similarities, the potential of alphavirus infection to
damage the articular cartilage in the joint tissues has not been
investigated. We now propose that, analogous to RA, RRV-infection
leads to an immune poly-arthritis that causes cartilage thinning
that contributes to clinical signs associated with alphaviral
disease.
[0078] It has long been recognised that the joint tissue is a
critical site of viral replication, and we recently identified
osteoblasts as a source of infectious virus, being susceptible to
RRV infection (31). We showed that RRV-infection results in bone
loss by disrupting the receptor activator of nuclear factor
.kappa..beta. ligand (RANKL) and osteoprotegerin (OPG) ratio (31).
We now describe thinning of articular cartilage in an RRV disease
mouse model correlating to a significant increase in
metalloproteinases including ADAMTS-4 and ADAMTS-5. These result in
histopathological findings similar to mild onset RA.
[0079] A recent study, using CCR2.sup.-/- mice infected with CHIKV,
showed that when normal monocyte trafficking is disrupted by this
receptor knockout the major inflammatory infiltrates became
neutrophil dominant (36). This replaces the usual macrophage
dominance of the cellular response observed in alphavirus
infections. Comparing the histopathological findings in the feet of
CHIKV-infected wild-type (WT) and CCR2.sup.-/- mice, Poo et al (36)
observed that the neutrophil shift resulted in cartilage damage. We
also observe cartilage thinning in immunocompetent C57BL/6 mice
(with functional macrophage trafficking) following RRV infection,
demonstrating that macrophages may also play a critical role in
RRV-induced inflammation including cartilage thinning.
[0080] The use of glycans as novel therapeutics has developed
momentum in recent years (37). The interaction of glycans with
growth factors, extracellular proteases, protease inhibitors,
cytokines/chemokines and adhesive proteins regulate various
physiopathologies and diseases including cancer, atherosclerosis
and thrombosis (38). In addition many pathogens, including viruses,
exploit host glycans to cause infection. The therapeutic potential
of this class of molecule to alleviate viral-induced arthritis and
inflammatory disease has not been studied and currently remains
unknown.
[0081] Pentosan polysulfate is a semisynthetic polysaccharide
derivative that chemically and structurally resembles other GAGs,
including heparin. In contrast to many other GAGs, PPS is
bioavailable in both its injectable and oral forms and produces
limited toxic side effects, even when administered in high doses
(39). In a clinical setting, PPS has been used as an
anti-thrombotic agent for several decades, due to its ability to
bind preferentially to the glycocalyx of circulating blood cells
(40). In more recent times, PPS has been identified as having
anti-inflammatory properties and is currently approved in the
United States for the management of patients with interstitial
cystitis, having an excellent long-term safety profile (15).
Furthermore, injectable forms of PPS are currently used to treat
osteoarthritis in veterinary medicine (19).
[0082] Given the promising results of PPS treatment of a range of
inflammatory conditions particularly arthritis, and the lack of
studies on PPS in treating virus-associated pathologies, we tested
the efficacy of PPS to treat alphavirus-induced arthritis. PPS
treatment significantly reduced the acute disease signs and the
muscle and joint inflammation of both RRV- and CHIKV-induced
disease. This corresponded to a reduction in serum levels of
pro-inflammatory factors at peak disease. In diabetic kidney
nephropathy, disease pathogenesis is dependent on the cellular
infiltration of macrophages and pro-inflammatory and
chemoattractant factors, similar to those associated with
alphavirus-arthritis. These include CCL-2, RANTES and CXCL1, TNF-a
(29, 41). Our data are consistent with the treatment observed in
diabetic nephropathy, in which PPS reduced the macrophage
infiltration and suppressed the induction of pro-inflammatory
factors (41).
[0083] At the onset of RRV-disease (day 6-7 p.i), there is a surge
in the levels of IL-10 (42). Of interest to the present study is
the recent observation demonstrating that a surge in IL-10 resulted
in a phenotype switch of monocytes/macrophages (43, 44). This
suggests that IL-10 is part of a repair signal that activates
specific cellular and molecular cascades to facilitate tissue
recovery (43). PPS-treatment altered the kinetics of RRV-induced
soluble pro- and anti-inflammatory factors, promoting a swing to
anti-inflammatory cytokines with an early induction of IL-10 that
enhances myogenesis (43). Furthermore IL-10 inhibits the synthesis
of pro-inflammatory soluble factors including, IL-1.alpha., IL-2,
IL-6, TNF-.alpha. and CCL-2 (45), previously associated with
increased severity of alphaviral disease. Overall the early PPS-
induced increase of IL-10 may act to reduce inflammation, but also
enhance tissue repair, thereby providing a key mechanism by which
PPS-treatment reduces the severity of alphaviral disease.
[0084] In addition to the reported action of PPS in reducing
pro-inflammatory factors, PPS inhibits both the alternative and
classical pathways of complement activation (46). For example, in
RRV-pathogenesis, complement activation is essential for the
development of severe RRV disease (34). Furthermore, this is
specific to RRV-activation of complement via the MBL pathway and is
independent of the classical and alternative pathways (28), and
therefore it is unlikely that the reduction in RRV disease observed
with PPS treatment is due to its effect on complement.
[0085] Treatment with PPS also resulted in protection of the i)
epiphysis, 2) articular cartilage and 3) proteoglycan matrix. The
anti-inflammatory effect of PPS is due in part to its ability to
inhibit IL-6 (47). In our results, serum levels of IL-6 were
significantly reduced in PPS treated RRV- infected mice at peak
disease. We have shown that the disruption of the RANKL and OPG
ratio during RRV-infection occurs in an IL-6 dependent manner such
that inhibition of IL-6 protects from RRV-induced bone loss(31).
Additionally, studies on CCL-2 have recently demonstrated that
inhibition of CCL-2 can both inhibit osteoclast differentiation and
protect against CHIKV induced bone loss (30, 48). Therefore, it is
likely that PPS protection against RRV-induced bone loss is due to
its ability to inhibit both IL-6 and CCL-2. PPS also stimulates
hyaluronan synthesis by synovial fibroblasts and proteoglycan
synthesis by chondrocytes (49). This may explain the observed
protection of both the articular cartilage and the proteoglycan
matrix in PPS-treated RRV-infected mice. PPS also promotes the
proliferation and chondrogenic differentiation of adult human bone
marrow mesenchymal stem cells (50), further explaining the PPS
protection of articular cartilage thinning that we see in
RRV-infection.
[0086] Although the molecular mechanism of PPS action remains
unclear, PPS can repress MMP, including ADAMTS expression and
inflammation, as well as NF-.kappa..beta. activation. It also
enhances proteoglycan synthesis, including the production of
aggrecan and hyaluronan (49) and has been shown to be efficacious
as both a treatment and a prophylactic (51). The results of this
study demonstrate that RRV-infection results in cartilage thinning,
increasing the levels of ADAMTS-4 and ADMATS-5 (aggrecanase 1 and 2
respectively), which in turn disrupts the proteoglycan matrix in
the cartilage, similar to that reported in osteoarthritis. It also
has been demonstrated recently that PPS blocks aggrecan breakdown
by both binding directly to ADAMTS molecules and inhibiting their
action, and by increasing the affinity between ADAMTS and its
inhibitor TIMP-3 (52). We therefore hypothesise that one of the
underlying mechanisms by which PPS treatment prevents cartilage
thinning in RRV-induced arthritis, occurs by blocking aggrecan
breakdown.
[0087] In humans, long-term clinical use of PPS is extremely well
tolerated and highly efficacious for periods greater than 12 months
(53). Alphaviruses can produce chronic musculoskeletal ailments
over a prolonged period of time. A long-term therapeutic strategy
is therefore required for effective treatment of alphavirus-induced
arthritis. We have shown that PPS not only alleviates the acute
signs of RRV-induced arthritis but also protects the cartilage over
the long-term without compromising host viral clearance. Given that
PPS promotes an anti- inflammatory immune state without promoting
viral persistence, it is an attractive drug-repurposing candidate
for the long-term treatment of RRV-associated inflammation and
disease.
[0088] To date, non-steroidal anti-inflammatory drugs are the
primary therapeutic means to alleviate the symptoms of
alphavirus-associated inflammatory disease. These drugs can cause a
variety of undesired side effects and may compromise immunity in
treated patients (54). Studies by our group in the past have
examined a number of drug candidates for the treatment of
alphavirus disease. Bindarit (a CCL-2 inhibitor), while effective
in reducing alphavirus induced arthritis and myositis, is currently
not a drug that is available for human use (30, 55). Enbrel, while
available for human use, was found to suppress the antiviral
response and enhance viral replication thereby worsen disease (56).
Similarly, methotrexate, a licensed drug for the treatment of RA,
increased the onset of RRV-induced musculoskeletal disease and the
influx of inflammatory cell infiltrates into the skeletal muscle
tissue (57). Here we show PPS treatment significantly reduced both
the acute clinical signs and the inflammation in the muscle
(myositis) and the joint (arthritis) in alphavirus disease.
Additionally PPS has positive and extensive long-term human safety
data, and is available as an approved drug by a number of
regulatory authorities globally. We therefore conclude that PPS is
a promising therapeutic candidate for alphaviral disease; and may
also be effective in other infectious inflammatory conditions.
[0089] To this end, the Australian Therapeutic Goods Administration
has provided its approval for the evaluation of PPS in four Ross
River virus infected subjects. Those subjects were treated with PPS
intramuscularly in accordance with the dosage regimen as described
herein.
[0090] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
above-described embodiments, without departing from the broad
general scope of the present disclosure. The present embodiments
are, therefore, to be considered in all respects as illustrative
and not restrictive.
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