Note: Descriptions are shown in the official language in which they were submitted.
W095/13805 ~2 ~ 7 ~ 7 ~ 7 PCIIUS94113239
-
USE OF NITRIC OXIDE SYNI~IASE I NH I I~
IN 'rElE lL~l~ l OF AUIY)INMUNE DISEASES
- 8Acr~jK~llJNL) OF THE INVENl~ON
MRL-lpr/lpr mice have been studied as a model for
human autoimmune diseases. This strain of mice develops a
spontaneous autoimmune disease characterized by
S lymrh~ ,yatl~, autoantibody production and inf lammatory
manifestations including nephritis, vasculitis, and
arthritis (Hang, L., et al., J. Exp. Ned. 155:1690-1701
(1982~ onh~rg~ R.~., et al., Clin. Exp. Rheumatol.
7:S35-S40 (1989) ) . Immune function ~hnr~ l ities also
include ~nh~nced constitutive macrophage class II antigen
expression, elevated levels of IFN-~y, TNF, IL-1, and IL-6
in isolated kidney, lymph node, and spleen cells, and an
~-nh~nrP~I state of macrophage "activation". These disease
manifestations are a result of both a single gene mutation
(lpr) of the Fas apoptosis gene on mouse ~1IL~ ~ 19 and
background genes from the NRL strain. Although the ~RL
genes contributing to disease manifestations have not been
identified, two loci contributing to renal disease have
been mapped to regions of mouse Chromosomes 7 and 12.
NO (nitric oxide), a multifunctional molecule
produced by diverse cell types, results from the
conversion of L-arginine to L-citrulline and NO by the
action of the enzyme nitric oxide synthase (NOS). NO has
been noted to promote relaxation of smooth muscle, serve
as a n~:uLoLL~lnsmitter, cause stasis and/or lysis of
microbes and tumor cells, and modulate function and
differentiation of hematopoietic cells.
NO also has potent proinflammatory actions. NO may
increase vascular permeability in inf lamed tissues .
Pain is also an important aspect of inflammation. Several
groups have now d~ ~L~ted that NO plays a role in the
Wo 95/13805 PCrtlJSs4/13239 ~
~:~7~74~ -2-
mediation of pain in in~lammation. Intradermal injection
of solutions containing N0 into humans caused a
dose-related O~ ~ ULL~rlCe of pain in the site tHolthusen,
H., and Arndt, J.o., Neuroscience Letters 165:71-74
5 (1994). Thus, pain, a hallmark symptom of inflammation,
can be induced by N0.
N0 has also been shown to cause increased production
of TNF and IL-1 by cells, and to increase the potential of
cells to produce 11YdLUY~ peroxide. Also, rabbit and
10 human chondrocytes have been shown to produce N0 and to
express inducible nitric oxide synthase (iNos) in response
to various cytokines and bacterial products. N0 is an
important mediator in immune complex vasculitis in rats
(Mulligan, M.S., et al., Brit. J. Pharmacol. 107:1159-1162
(1992) ~ . Some researchers have noted a role for N0 in
inflammatory bowel disease in guinea pigs (Miller, M.J.,
et al., J. Pharmacol . Exp. Ther. 264:11-16 (1993) ), and in
induced nephritis in mice or rats (Farrario, R., et al.,
J. Am Soc. Nephrol. 4:1847-1854 (1994)). Research in non-
20 human models of inflammatory tli~ c~c has also established
that N0 participates in experimentally-induced uveitis
(Parks, D.J., et al., Arch. ophthalmol. 112:544-546
(1994) ) .
Studies of humans with arthritis ~ L,~-te further
25 that N0 plays an i ~ Ldl.~ role in human arthritis.
Farrell et al . showed that patients with inf lammatory
arthritis (rheumatoid arthritis and osteoarthritis) had
elevated levels of the N0 catabolite nitrite in their
synovial fluid and serum (Farrell, A.J., et al., Ann.
30 Rheum. Dis. 51:1219-1222 (1992) ) . Patients with
rheumatoid arthritis had increased synovial f luid and
serum levels of niLLu~y.usine (Kaur, H., and Halliwell,
B., Febs Letters 350:9-12 (1994)).
WO 951~3805 ~ ~ ~ 6 7 4 7 PCTIUS941~3239
Cell-derived NO reacts with ,uyerc~ide (2'-) to form
peroxynitrite (ONOO-), which in turn may spontaneously
produce hydroxyl radical (HO ), a molecule with high
, potential for cell and tissue injury and destruction.
Other investigators have shown in a rat model of
inflammation that peroxynitrite is pro-inflammatory
(R~rhm;l~witz, D., et al., Gastroenterology 105:1681-1688
(1993) ) .
NiLL~LyLosine is formed by the action of
peroxynitrite on tyrosine, and it is felt to be a ctable
"footprint" or "track" of the presence of NO (Beckman,
J.S. et al., Methods Enzymol. 233:229-240 (1994) ) .
Nitrated proteins have been found associated with
macrophages and inflammation (by using an
anti-niL~ oLyLosine antibody) in atheromatous plaques in
human vessels (Beckman, J.S., et al., Biol. Chem. ~oppe-
Seyler 375:81-88 (1994) ), providing further evidence that
nitrotyrosine is formed in vivo in humans.
SUMMARY OF THE INVENTION
The invention relates to a method of treating or
preventing autoimmune d; C~ c-~s, such as rheumatoid
arthritis, insulin tl_~ t ~ het~c mellitus, systemic
lupus erythematosus and glomerulonephritis, comprising
administering, preferably enterally, to a patient in need
thereof an effective amount of a nitric oxide synthase
inhibitor or a nitric oxide scavenger.
13RTPIF DESCRTPTION OF T~F: DRAWING
The Figure is a bar graph showing the scores of
pathological characteristics in MRL-lpr/lpr mice either
treated with NG-monomethyl-L-arginine (NMMA) or left
untreated .
WO95/13805 Pcr/us94ll323s ~
217~
--4--
~n DES~ 'K I 1' ' lON OF T~E INVENTION
The level of nitric oxide has now been linked to the
manifestation of autoimmune ~;ceAcec~ particularly chronic
diseases, such as rheumatoid arthritis, insulin dependent
5 diabetes mellitus, systemic lupus erythematosus and
glomer-~1 on~Prhritis. The inhibitors of nitric oxide
synthase, known to synthesize nitric oxide in vivo, or
nitric oxide scavengers can be useful in the prevention or
treatment of autoimmune diCPACPC (C.jlkP~on et al., Arth.
10 Rheum. 36 (Suppl.):S219, 1993 (September)).
Inhibitors of nitric oxide synthase which can be used
in this invention are those known in the art and include
substrate analogs, such as aminogllAn;rlinp~ Na-amino-L-
arginine, N'J-methyl-L-arginine, Na-nitro-L-arginine, NG-
15 nitro-L-arginine methyl ester, and Na-iminoethyl-L-
ornithine, flavoprotein binders, such as diphenylene
iodonium, iodonium diphenyl and di-2-thienyl iodonium,
CAl-- ' 1 in binders, such as cA1~inpllrin~
trifluoroperazine, N- (4-aminobutyl) -5-chloro-2-
20 naphthalenesulfonamide and N- (6-aminohexyl) -1-
naphthA1PnPc-l1fonamide, heme binders, such as carbon
- ~P, depleters and analogs of tetrahydrobiopterin,
such as 2,4-diamino-6-lly~lLo,~y~yLimidine~ and induction
inhibitors, such as corticosteroids, TGF-~C-l, -2, 3,
25 interleukin-4, interleukin-10 and macrophage deactivation
factor (Nathan, The FASEB Journal, Vol. 6, Sept. 1992, pp.
3051-3064). Preferred are the substrate analogs of nitric
oxide synthase, Na-amino-L-arginine, N~-methyl-L-arginine,
Na-nitro-L-arginine, Na-nitro-L-arginine methyl ester, and
30 Na-iminoethyl-L-ornithine. Particularly preferred are NG-
amino-L-arginine, Na-methyl-L-arginine, N~-nitro-L-
arginine, and aminog~l~nidine. Most preferred is Na-
methyl-L-arginine.
7 ~ ~
WO 95/13805 ~ PCrlUS94113139
--5--
phAr~-~ce~ltically acceptable salts may also be
administered. Examples of suitable salts include acid
salts, such as I~YdL~Y~n chloride, llyd~o~ bromide,
hydrogen iodide, sulfate and acetate salts, as well as
basic salts, such as amine, ammonium, alkali metal and
AlkAl inP earth metal salts.
S~;~Vt:IIY~L:~ of nitric oxide are compounds which will
react with nitric oxide in vivo, such as hemoglobin (Wang
et al., Lffe Sciences 49:55-60 (7991)) and cr~h~lAm;nc
(Rajanayagam, C.G., et al., Brit. :r. Pharmacol. 108:3-5
(1993); Zatarain, J., et al., Clin~ Res. 41:783A (1993)).
The compounds described above are known in the art
and are commercially available.
The ~ '~ of the claimed invention can be
administered alone or in a suitable pharmaceutical
composition. Modes of administration are those known in
the art, such as enteral, parenteral or topical
application. Enteral is preferred and oral administration
is particularly preferred.
Suitable pharmaceutical carriers can be employed and
include, but are not limited to water, salt solutions,
alcohols, polyethylene glycols, gelatin, call-ollydl~tes
such as lactose, amylose or starch, magnesium stearate,
talc, silicic acid, viscous paraffin, fatty acid esters,
llydr ~,cy ` ylcellulose, polyvinyl pyrrolidone, etc. The
pharmaceutical preparations can be sterilized and if
desired, mixed with i:~llYi 1 i~ry agents, e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsif iers,
salts for influencing osmotic pressure, buffers,
flavorants, coloring, and/or aromatic substances and the
like which do not deleteriously react with the active
compounds .
~or parenteral application, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
WO 95/13805 ~ PCTIUS94/13239--
7 l1 7
--6--
i n~ l i n~ suppositories . Ampouleg are convenient unit
dosages. Oral applications are preferably administered in
the forms of c~rsl~lpc~ tablets and/or liquid formulations.
Unit form dosages are preferred. Topical applications can
5 be administered in the form of a liquid, gel or a cream.
It will be appreciated that the actual amounts of the
active ~ in a specif ic case will vary according to
the specific Cu~ uuu~-d being utilized, the particular
composition f ormulated, the mode of administration and the
10 age, weight and condition of the patient, for example.
Dosages for a particular patient can be det~rm;npd by one
of ordinary skill in the art using conventional
considerations, (e.g. by means of an appropriate,
conventional pharmacological protocol).
The invention is further specifically illustrated by
the following exemplification.
~FMpLIFIcATIoN
Nitrite/nitrate excretion
Mice were housed in metabolic cages (3 per cage) and
fed deionized-distilled sterile water and a defined
arginine and nitrate-free diet. Urine was collected into
isopropanol to inhibit bacterial growth. Urinary
nitrite/nitrate concentration was det~rmin~
~ue11_Lu~hotometrically as described before (Granger, D.L.
et al., J. Im~nunol. 146:1294 (1991)). Determinations were
done in duplicate or triplicate. Urinary protein was
measured by the Bradford assay. Total nitrate excretion
was then calculated based on the concentration and the
urine volume.
In animals cQn~'lm~n~ a nitrate-free diet, urinary
excretion of nitrite/nitrate accurately reflects the
endogenous production of NO. To determine the production
of NO in mice of different strains, we analyzed urine
~Wo95/13805 7 6~ 4~ PCrllJS94113239
collected daily under basal conditions. MR~-lpr/lpr mice
excrete more nitrite/nitrate than do C3H mice, when
analyzed over a 10 day period at age 3 months. Likewise,
when analyzed over time beginning at age 6 weeks, MRL-
5 lpr/lpr mice excrete higher levels of nitrite/nitrate than
do B6 mice as the mice age. Higher nitratelnitrite
excretion begin6 at approximately age 10 to 12 weeks
paralleling that of proteinuria. Oral administration of
50 mM NMMA in water to the MRL-lpr/lpr mice reduced the
high level nitrite/nitrate excretion. This signifies that
the nitrite/nitrate is a product of NOS, since NMMA is a
specif ic inhibitor of the enzyme . Levels of
nitrite/nitrate excretion in 5 month old mice of strains
MRL-+/+ (0.8 ,~mol per mouse per day) and B6-lpr/lpr (1.2
,umol per mouse per day) (3 mice in each group) were normal
These results indicate that neither the lpr gene per se
nor the MRL genetic background is adequate for the
expression of ~nhAn~ d nitrite excretion, and that both
the lpr gene and genetic f actors in the MRL ba._}.yL vulld are
npc~cs;~ry~
Nitric oxide l~rQduction in vitro and nitric oxide sYnthase
content
Spleen, liver, kidneys, lymph nodes, and peritoneal
cells were collected and quickly frozen in a dry
ice-ethanol slurry in a buffer-protease inhibitor cocktail
containing 100 ~M phenylmethylsulfonyl fluoride, 5 ,ug/ml
aprotinin, 1 ~g/ml ~:I-y ~ ~tin, and 5 ~Lg/ml pepstatin A.
The tissue cells were then disrupted with a pestle in
repeated freeze-thaw cycles. Cytosol was collected after
centrifugation, and assayed for protein and NOS activity
using a modification of procedures known previously
(Bredt, D.S. and Snyder, S.H., Proc. Natl. Acad. sci. USA
86:9030 (1989); Sherman, P.A., et al., Biochemistry
32:11600 (1993) ) . The assay buffer contained 50 mM HEPES
Wo 95113805 ! PCTIUS94/13239 ~
2~
(pH 7.S), 200 ,uM NADPH, l mM dithiothreitol, l0 ~M FAD,
100 ,uM tetrahydrobiopterin, and l0 ~M L-arginine. We used
L-arginine labeled with tritium in the gll~ni-l;nn position
(product number NEC453, New England Nuclear, Wilmington,
5 DE). Thirty microliters of sample were used in a total
reaction mixture of 50 microliters. Samples were done in
duplicate or triplicate.
Peritoneal macrophages from the normal BALB mice had
no F~nhAnl ~ of nitrite/nitrate production when treated
10 with endotoxin or IFN-~y alone, but the combination
Pnh :~nce~ the production greatly. In contra6t, peritoneal
macrophages from MRL-lpr/lpr mice had PnhAnc~-d responses
to treatment with endotoxin and murine IFN-~y alone, as
well as with -i nPd endotoxin IFN- r treatment. To
15 determine if there wa6 PnhAnred tissue iNos mR~A
expression, RNA was extracted from organs from BALB and
MRL-lpr/lpr mice, and then PYAmi n~rl by Northern analysis
for iNos mRNA expression. The iNoS mRNA (approximately
4.7 kilobases in size~ was noted in tissue from kidney and
20 spleen from MRL-lpr/lpr mice, but not in those tissues
from BALB mice. Various tissues and cells from
MRL-lpr/lpr and BALB mice were extracted and analyzed for
their abilities to convert ~4C-L-arginine ( labeled in the
gl1An;rlinn position) to I~C-L-citrulline. Peritoneal
25 macrophages and spleen extracts from MRL-lpr/lpr mice
displayed more NOS activity than did those from BALB mice,
while the activity in kidney extracts was not different.
By immunofluorescence analysis using a rabbit
anti-mouse iNoS antibody, we noted no NOS antigen
30 expression in spleen, liver, and kidney from BALB mice,
and none in liver or kidney from MRL-lpr/lpr mice.
However, spleens f rom MRL-lpr/lpr mice displayed large
numbers of cells containing the NOS antigen. Comparable
f indings were noted when we used a monospecif ic guinea pig
~ WO 95113805 ~ 1 7 6 ~ 4 7 PCr/US941~3239
_g_
anti-rat inducible NOS antibody. When ti6sue extracts
were analyzed for iNos antigen by; , ~cipitation and
lQtting techniques using a rabbit anti-mouse iNos
antibody, we did not detect antigen in extracts from
5 organs of BALB mice, although extracts from spleen and
kidney tissues from MRL-lpr/lpr mice had readily
~letect Ihle antigen .
NMMA treatment
Groups of 8 week old MRL-lpr/lpr mice were given
either sterile, distilled deionized water or water
containing 50 mM NMMA for ad libitum c...,ul~Lion. NMMA
was from CalBiochem (San Diego, CA) and from Dr. Owen
Griffith (Milwaukee, WI). Both groups of mice were
maintained on the defined nitrate free diet described
15 above. At weekly intervals, the mice were placed in
metabolic cages and 24 hour urine collections were
obtained. Urinary nitrite/nitrate was measured as
described above, and urinary protein was det~rm; nPd using
the Bradford assay (BioRad, Hercules, CA). After l0 weeks
20 of treatment, the mice were bled and sacrificed with
removal of the kidneys and knee joints.
Serum anti-DNA activity was det~ m; n-~cl by ELISA as
previously described . The kidneys were ; mh/~ d in
paraffin, sectioned and stained with hematoxylin and
25 eosin. Knee joints were decalcified in folic acid,
~mh~ ecl into paraffin, sectioned, and stained. Slides
were then read by a pathologist "blinded" as to the group
of origin . The amount of kidney and knee j oint disease
present in each specimen was quantitated as noted bef ore .
30 Briefly, glomeruli were graded for hypercellularity (0-4),
hyperlobularity ( 0-4 ), crescents ( 0-4 ), and necrosis
(0-4). A score was then derived by adding the grading of
these features of glomerular disease. Kidneys from normal
BALB mice usually have scores from 0-l. Vasculitis was
WO 95/13805 PCrlUS9~113239 ~
7 4~
--10--
noted when present in medium size vessels in the kidney
sections. The synovial score was derived by adding the
grading of synovial prolif eration ( 0-3 ) and subsynovial
inflammation (0-3). Knee joints from normal BALB mice
5 usually have scores from 0-0 . 5 .
Groups of MRJJ-lpr/lpr mice received either double
distilled water (n=10) or water containing 50 mM NMMA
(n=9 ) ad li~itum beginning at 8 weeks of age. Both groups
of mice received the def ined nitrate f ree diet . Mice were
10 treated for a total of 10 weeks. Mice in both groups
appeared clinically normal. However, two mice in the NMMA
group died during week 3 of treatment leaving 7 mice in
the NMMA group for analysis. Extensive autopsies
( i nr~ i ng careful histological examinations and culturing
15 of serum, urine, and organs) on these two mice and on a
comparable mouse that had received NNMA 4 weeks revealed
no evidence of microbial infection or other evident cause
of death. Administration of NMMA in the drinking water of
MRB-lpr/lpr mice effectively blocked nitrite/nitrate
20 excretion (and by inference nitric oxide production).
Also, mice receiving NMMA excreted significantly less
protein than did control mice; this difference became
apparent at week f ive of treatment .
Pathologic examination of the kidneys and knee joints
25 of mice from the two groups o~ mice revealed significantly
less disease in the NMMA-treated group. Renal disease as
mea6ured by the renal score, and arthritis as measured by
the synovial score were both signif icantly less in the
NMMA group as compared to the control group. The observed
30 differences for ~oint disease and renal disease are
statistically different (p<0.05 and p<0.02, respec'cively,
using the Mann-Whitney U test), while that for anti-DNA
antibody level is not. There was minimal to no glomerular
proliferation in mice treated with NMMA, while all but one
35 of the control mice had signif icant glomerular
~ WO 9511380S PCTIUS94113239
2~7~7~7
--11--
proliferation and hyperlobulation. The chronic
interstitial lymphocytic infiltrate seen in the kidneys of
-' all lpr congenic mice (including C3H-lpr/lpr mice that do
not develop glomerulonephritis) was present to comparable
degrees in both control and NMMA treated mice. Medium
vessel vasculitis appears sporadically in the kidneys of
untreated MRL-lpr/lpr mice with an overall incidence of
30%. Mild to moderate (1-2+) medium vessel vasculitis was
present in 3/10 kidneys from mice in the control group.
Mild vasculitis was seen in 1/7 kidneys from the NMMA
treated group. There was not a statistical difference in
vasculitis between the two groups, but the small numbers
of mice with vasculitis makes it dif f icult to draw f irm
conclusions regarding the effects of NMMA on this aspect
of ;nfl: -tion. Synovial proliferation was significantly
decreased in the mice treated with NMr~ hile only 3 of
7 mice in the NMMA-treated group had abnormal knee joints
with mild to moderate synovial proliferation and synovial
inflammation, all 10 mice in the control water group had
some degree of synovial proliferation and inflammation.
Levels of serum anti-double stranded DNA measured at age
18 weeks in the two groups were essentially equivalent
(see the Figure).
Formation of nitroso-hemocrlobi n
N0-Hb forms through an interaction of N0 with iron in
the heme group of hemoglobin (Huot, A.E., et al., Biochem.
Biopl2ys. Res. Commun. 182:151-157, (1992); Cantilena,
L.R.J., et al ., J. Lab. Clin. Med. 120:902-907 (1992) ) .
Whole blood from MRL-lpr/lpr mice at different ages was
analyzed for the presence of nitroso-hemoglobin (NO-Hb).
The blood samples were anticoagulated and ~ A~nin~(l by
electron paramagnetic resonance (EPR) at 77l~ using a
Bruker ESP300 spectrometer (Chamulitrat, ~. et al., Molec.
Pharmacol . 46:391-397 (1994) ) . Table 1 shows the mean +
WO95/13805 21~ 67 ~7 PCr/US94/13239 ~
--12--
SEM EPR units ~ ..u.llbe~ of animals ~YAm;nDcl). An
agc d~~ nt increase was observed in the amount of NO-Hb
in the blood of the ~:1;P.DA~:D~ mice. The levels of NO-Hb i~
were higher in MRL-lpr/lpr mice compared to s~ - a~c:
5 control mice without disease . The dif f erences were
statistically si~n; f; cAnt (p<0 . 05 at all ages analyzed) .
The presence of NO-Hb is another important sign that NO is
being over-eYpressed in these mice with autoimmune
nephritis and arthritis.
Table l
Mouse Age 12 weeks Age 16 weeks Age 18 to 20 weeks
MRL-lpr~lpr 1420 ~ 130 (n=16) 2045 i 226 (n=10) 4092 i 711 (n=711)
BALB/c (control) 970 i 221 (n=5) 1005 ~ 160 (n=160) 811 ~ 108 (n=8
The above studies .1 Lr ted an NO-mediated
15 modification of a protein (hemoglobin) in these mice. To
determine if ~;~eA~l tissue is modified by NO in
MRL-lpr/lpr mice, kidneys from normal BALB/c mice (20
weeks old) and MRL-lpr/lpr mice (20 weeks old) were
DYAm; nD~l by electron paramagnetic resonance (EPR) . For
20 these tissues, the spectrum of the control mouse was
subtracted from that of the MRL-lpr/lpr mouse, and the
resultant curve showed an easily detectable NO-non-heme
iron tyrosyl signal at g=2 . 04, as well as the typical
spectra f or NO-heme (presumably due to blood trapped
25 within the MRL-lpr/lpr kidney; see Chamulitrat, W., et
al ., IYolec. Ph~rmacol . 46:391-397 (1994) ) . It is
important to note that control kidney did not have these
NO-protein signals, signifying that the nitrosylated
_
W0 95113805 ~ ; 7 ~ 7 PCrtUS94/13239
--13--
non-heme protein might be causally related to the observed
severe renal pathology in these mice.
, ~
PrgtP;n nitration jn k;rlnpys from MRT-l~r/lDr ~ p
As noted above, N0 may react with ~u~eluxide and form
5 the highly reactive, tissue destructive molecule
peroxynitrite. It has been shown previously that cells
from MRL-lpr/lpr mice can vv~L~Lvduce reactive oxygen
species such as hydrogen peroxide, superoxide (Dang-Vu,
A.P. et al., .J. Immunol . 138:1757-1761 (1987) ) and nitric
10 oxide (Weinberg, J.B., et al., J. Exp. Med. 179:651-660
(1994) ) . A study was done to look for evidence that MRL-
lpr/lpr mice also uv~L~Loduce the destructive molecule
peroxynitrite (Beckman, J.S., et al., l~ethods Enzymol.
233: 229-240 ( 1994 ) ) .
Since peroxynitrite causes nitration of tyrosine
residues in proteins, a mono-specific, polyclonal
anti-tyrosine antibody was used to detect evidence of the
presence of peroxynitrite in ~l;cp~d kidneys of
MRL-lpr/lpr mice. Immunoblot analysis was performed on
20 protein extracts from kidneys of 20 week old normal
(BALB/c) and MRL-lpr/lpr mice.
Kidney tissue was homogenized with a glas6 pestle.
Proteins in soluble extracts (100 ,~lg per lane) were
separated by SDS-polyacrylamide gel electrophoresis and
25 transferred to nitrocPl lulocP (0.45 ,um, Novex) . Unbound
sites were blocked by incubation with 1% non-fat dry milk
in TBS (20 mM Tris, 500 mM NaCl, pH 7.5) for 60 min at
2 5 C . Membranes were incubated overnight at 2 5 C with a
polyclonal anti-niLLvLyLosine antibody (0.25 ~g/ml) in
30 milk/TBS. Immunoreactivity was visualized by incubation
with goat anti-rabbit IgG-HRP conjugate (Bio-Rad) (1:2000)
dilution in 1% milk/TBS, followed by enhanced
chemil~ i n~c-~n~e (ECL) detection (Amersham) .
Wo 95113805 PcrrUss4r1323s ~
7~7
--14--
While the immunoblot from kidneys from four
individual control mice showed only one protein band
reacting with the antibody, the immunoblot from kidneys
from MRL-lpr/lpr mice showed this same band plus four
5 other pL, i nPnt bands. This indicates that in
vivo-generated NO in MRL-lpr/lpr mice is converted to
peroxynitrite, and that this peroxynitrite subsequently
nitrates tyrosine residues in proteins in the kidneys.
Cat~ P activitY in kidnevs of MRL-lpr/lPr mice
The catalase content in kidneys from Z0 week old
normal (BALB/c) and MRL-lpr/lpr mice was analyzed.
Peroxynitrite or N0 can destroy catalase activity.
Catalase was measured by the di c:ArpP Irance of hydrogen
peroxide noted by absorbance at 240 nm (Beers, R.F., and
15 Sizer, R.W., J. Biol. Chem. 195:133 (1952) ) . Values shown
in Table 2 are the mean + SEM of replicate samples
expressed in units/mg protein. Table 2 shows that kidneys
from the control mice had high levels of catalase while
levels of catalase from MRL-lpr/lpr mice were very low.
2 0 Table 2
BALB/c mouse MRl,lpr/lpr mouse
Mousi~ 1 77 i 6 Mousc 1 12 il
Mouse 2 75 i 2 Mous~ 2 20
Mouse 3 67 i 5
25 Mousc 4 68 i 6
In a different type of analysis to determine
catalase, we studied protein extracts from kidneys by
~Iwo 9S/1380S PCrlUS94113239
2~7~7~7
--15--
PAGE, with subsequent vi~1~Al;7~A~tion of catala6e activity
by soaking the gels in phosphate buffer containing
- horseradish peroxidase and IIYdLOY~ peroxidase with
~;Am;nr h~n7idine.
Soluble extracts (25 ,ug/lane) were separated by
electrophoresis on a 6% native polyacrylamide gel. Bands
of catalase activity were v; !=IIA 1; 7~d by soaking the gels
for 45 min at 25C in 50 mM sodium phosphate, pH 7.0,
containing 0. l mM EDTA and 50 ,ug/ml horseradish
peroxidase. H202 was added to a final concentration of 5 . O
mM and the gel was incubated an additional 15 min. After
a brief rinse in water, stain development was initiated by
addition of 0.5 mg/ml rl;Am;nclb~n~idine-HCl in 50 mM sodium
phosphate, O. l mM EDTA, pH 7 . O.
Results showed clearly that kidneys from 4 different
BALB/c control mice contained large amounts of catalase,
while those from two NRL-lpr/lpr mice had markedly
~1; m; n; ~:h~ level5 of catala5e . However, if the mice had
been treated with oral NG-- Lhyl-L-arginine in vivo
(See NMMA ~t!at ~-)t above), the catalase level in kidneys
from three different MRL-lpr/lpr mice was normal
(comparable to that of the control mice). This signifies
that catalase activity (which may be inhibited by the
actions of NO or peroxynitrite) is markedly low in
NRL-lpr/lpr mice, and that the NO- (or peroxynitrite-)
mediated decrease in catalase is blocked by in vivo
administration of NG ~ y l-L-arginine .
n investiqationS
To determine if humans with arthritis have iNos
protein expressed in their synovial tissues, these tissues
were studied by immunof luorescence techniques using mouse
monoclonal anti-iNOS antibody (purchased from Transduction
Laboratories, Inc. ) . of six synovial samples removed from
WO 95/13805 PCTNS94/13239
~17~7~7
--16--
patients with advanced arthritis at the time of ; oint
rep' ~Ir L 6urgery, iNos antigen was detected in the
tissues of two of three rheumatoid arthritis patients and ,~
in one of three osteoarthritis patients. These studies
5 d~ LLc.te that patients have synovia that contain iNos,
and that it may be uv~le~Less~d in synovia of patients
with rheumatoid arthritis.
EOUIVAT .T~'NTS
Those skilled in the art will rf~ n; ~e, or be able
10 to ascertain using no more than routine experimentation,
many equivalents to the specific P~nhsr~ nts of the
invention described specif ically herein . Such equivalents
are intended to be t~n~ ,-cc~d in the scope of the
following claims.
