вход по аккаунту


Tenidap in contrast to several available nonsteroidal antiinflammatory drugs potently inhibits the release of activated neutrophil collagenase.

код для вставкиСкачать
21 1
Neutrophils contain a collagenase that is stored in
a latent form within the specific granule. With cellular
activation, the latent enzyme is activated in association
with the production of a variety of oxidants, including
hypochlorous acid. We evaluated 4 nonsteroidal antiinflammatory drugs (NSAIDs) currently on the market
and the new antiinflammatory/antirheumatic drug
tenidap for their effects on the release of activated
collagenase. In contrast to the 4 NSAIDs, tenidap profoundly inhibited the release of activated collagenase.
This inhibition was predominantly due to interference
with activation of the latent enzyme, rather than interference with enzyme release. The inhibition of collagenase activation was associated with a profound reduction in myeloperoxidase activity and in hypochlorous
acid production. These observations demonstrate that
tenidap has properties that set it apart from conventional NSAIDs and suggest that it may be a particularly
From the Division of Clinical Immunology and Rheumatology, Birmingham Veterans Administration Medical Center and The
University of Alabama at Birmingham, and the Central Research
Division, Pfizer, Inc., Groton, Connecticut.
Supported by a Veterans Administration Career Development Award and by the Central Research Division, Pfizer, Inc. Dr.
Blackburn’s work was supported by a Veterans Administration
Merit Review Award and by NIH grant AR-20614.
Warren D. Blackburn, Jr., MD: Birmingham VA Medical
Center and The University of Alabama at Birmingham; Leland D.
Loose, PhD: Central Research Divison, Pfizer, Inc.; Louis W.
Heck, MD: Birmingham VA Medical Center and The University of
Alabama at Birmingham; W. Winn Chatham, MD: The University of
Alabama at Birmingham.
Address reprint requests to Warren D. Blackburn, Jr., MD,
Research Service (151-F), Birmingham Veteran’s Administration
Hospital, 700 19th Street South, Birmingham, AL 35233.
Submitted for publication March 13, 1990; accepted in
revised form September 13, 1990.
Arthritis and Rheumatism, Vol. 34, No. 2 (February 1991)
useful agent in the treatment of inflammatory rheumatic
Neutrophils contain several proteases that
likely play an important role in the irreversible tissue
injury characteristic of a number of chronic inflammatory disorders (1-3). One of these proteases is a
collagenase that is stored within the neutrophilspecific granule in a latent form (43). During cell
stimulation, this protease can be activated by a unique
mechanism, which entails interaction between the
oxygen radical-generating system and the azurophilic
neutrophil granule protein, myeloperoxidase ( 6 ) . Superoxide radicals, products of the membraneassociated NADPH (nicotinamide-adenine dinucleotide phosphate) oxidase system, undergo dismutation
to hydrogen peroxide, a myeloperoxidase substrate.
Myeloperoxidase converts hydrogen peroxide to hypochlorous acid, which may directly activate latent
neutrophil collagenase (6). Once activated, collagenase is capable of specific proteolysis of types I, 11,
and I11 collagen (3,which may contribute to the
irreversible tissue injury seen in disorders such as
rheumatoid arthritis (RA).
Previous work has suggested that certain antirheumatic compounds may influence the activity of
neutrophil collagenase. For example, gold compounds
can interfere with activated collagenase, but only in
the presence of organomercurial compounds (7,8).
Penicillamine can scavenge hypochlorous acid, and
may thus interfere with collagenase activation (9).
Little is known about the effect of available antirheumatic compounds on the release and cell-associated
activation of neutrophil collagenase. The present
study was undertaken after it was observed that
tenidap, the novel combined lipoxygenasekyclooxygenase inhibitor (lo), is capable of potently inhibiting
activation of neutrophil collagenase.
Materials. Cytochrome C, bovine serum albumin
(BSA), bovine superoxide dismutase, taurine, DTNB [5,5’dithio-bis(2-nitrobenzoic acid)], naproxen, ibuprofen, piroxicam, and indomethacin were obtained from Sigma (St.
Louis, MO). Hanks’ balanced salt solution (HBSS) was
obtained from Gibco (Grand Island, NY). Tenidap (CP66,248) was obtained from Pfizer Central Research (Groton,
CT) .
Neutrophil preparation. Whole human blood from
normal volunteers was obtained by venipuncture into heparinized syringes. Most of the red blood cells were removed
by dextran sedimentation, and the neutrophils were separated by density centrifugation over Ficoll-Hypaque. The
neutrophil-rich fraction was washed, and residual red blood
cells were removed by hypotonic lysis (11). In the assays
described below, cell viability was assured by determining
their ability to exclude trypan blue dye. In each assay, cell
viability routinely exceeded 95%.
Drug preparation. Because of their limited solubility
at neutral pH, naproxen, ibuprofen, piroxicam, and indomethacin were initially dissolved in 0.1M NaOH, and then
further diluted in water prior to adding to the cell suspensions. The addition of the diluted compounds to the cell
suspension did not significantly alter the pH of the suspensions. Suspensions of control cells contained the same
amounts of the diluted NaOH buffer. Tenidap was diluted
with water prior to its addition to the cell suspensions.
Monomeric IgG. Monomeric IgG was prepared from
Cohn fraction I1 by DEAE cellulose chromatography, as
previously described (12).
Superoxide production. Neutrophils, at a concentration of 1.25 x lo6 cells/ml, were incubated in the presence of
cytochrome C in wells coated with either BSA or with 5 pg
of human IgG. Cells were incubated for 20 minutes at 37”C,
aspirated, and immediately placed on ice. The cells were
removed by centrifugation, and the superoxide dismutaseinhibitable reduction of cytochrome C was determined spectrophotometrically (13). For these experiments, test compounds were added back to supernatants at equivalent final
concentrations to eliminate variability due to absorption by
the compound tested.
Hypochlorous acid production. Neutrophils (1 x lo7/
ml) were suspended in HBSS containing 15 mM taurine. The
neutrophil suspension was then incubated in wells either
coated with BSA or precoated with human IgG and then
blocked with BSA. After 20 minutes, the cell suspensions
were aspirated, placed on ice, and 5-thio-2-nitrobenzoic acid
(450 nM) was added. Cells were removed by centrifugation,
and their light absorption at 412 nm was determined (14). In
each experiment, test compounds were added back to the
supernatants to eliminate any alterations in the light absorption secondary to the test compounds.
Myeloperoxidase activity. Neutrophils (1.25 X 106/ml)
were incubated for 60 minutes at 37°C in either BSA or
IgG-coated wells that had been blocked with BSA. After
incubation, the wells were aspirated, and the cells were
removed by centrifugation. Myeloperoxidase was also extracted from whole neutrophils with 1M NaCl and separated
from cell fragments by centrifugation. The supernatant was
dialyzed against HBSS. Myeloperoxidase activity was determined by adding supernatant to 300 pl of 0.2M sodium
acetate buffer, pH 4.5, containing 17 mg of 2,2’-azino-di-(3ethylbenzthiazoline) sulfonic acid and to 500 pl of 0.003%
hydrogen peroxide. The change in absorbance at 412 nm was
then determined spectrophotometrically, as described by
Shindler et a1 (15).
Collagenase activity. Neutrophil suspensions (5 x lo6
cells/ml, 125 pl/well) were added to the IgG-coated and
BSA-blocked wells, and the mixtures were incubated for 45
minutes at 37°C. As controls, similar incubations were
performed with polymorphonuclear cells (PMN) in BSAblocked wells containing no IgG. Following incubation, the
cell suspensions were centrifuged at 750g for 5 minutes at
4°C. The supernatants were removed and treated with diisopropyl fluorophosphate (DFP; final concentration 10-3M) to
inactivate serine proteases; they were then evaluated for
collagenase activity.
Collagenase activity in the DFP-treated supernatants
was determined by incubating, in triplicate, 200-pl aliquots
of supernatant with 3H-labeled reconstituted type I collagen
fibrils in 7-mm flat-bottom tissue culture wells (Linbro, no.
76-032-05; Flow Laboratories, McLean, VA), as described
by Johnson-Wint (16). The reconstituted fibrils in each well
contained 75 pg of a mixture of 3H-labeled and unlabeled
collagen, with an activity of 7,000 counts per minute.
To determine the total radioactivity potentially released from the fibrils in each experiment, the reconstituted
fibrils were also incubated with a mixture of clostridial
collagenase (250 mg/ml of HBSS). To maximize the sensitivity and specificity of the assay, incubations were performed for 18 hours in triplicate at 37°C (17). At the end of
the incubation period, the supernatants were aspirated from
each well, and the radioactivity was determined by counting
in a liquid scintillation counter. More than 99% of the
radioactivity applied to each well was recovered from the
wells incubated with bacterial collagenase. The average cpm
released by fibrils incubated with supernatants obtained
from cells incubated in BSA-coated wells was subtracted
from the cpm measured in each supernatant. The resulting
triplicate values for each PMN supernatant were averaged
and were then divided by the average cpm released by the
bacterial collagenase to determine the percentage of fibril
lysis produced by each PMN supernatant. The total amount
of collagen that was released during the 18-hour incubation
period was then calculated and divided by the incubation
time to yield values for the collagenase activity (nanograms
of collagen degraded/minute) in each supernatant. Studies
that have been performed with serial dilutions of collagenase
that contain supernatants have demonstrated that the assay
is linear over the range of values reported here (data not
In parallel experiments, the release of total collagenase into the PMN supernatants was determined by activat-
ing latent collagenase in the supernatants with 1.0 mM
mersalyl (1 8) prior to the addition of the supernatants to the
radiolabeled collagen fibrils. To avoid underestimation of the
total amount of collagenase released due to inhibition of
protease activity by oxidative metabolites generated during
PMN activation, the supernatants used for these determinations were derived from PMN that were activated in the
presence of 1.O mM sodium azide. Incubations and calculations of collagenase activity in the mersalyl-treated supernatants were performed as described above.
A series of cyclooxygenase inhibitors (ibuprofen, naproxen, indomethacin, and piroxicam) was
assessed for their ability to inhibit the release of
activated neutrophil collagenase (Table 1). The addition of ibuprofen or naproxen did not inhibit the
release of activated enzyme. Piroxicam and indomethacin had modest inhibitory effects on the release of
activated collagenase, but only at supratherapeutic
concentrations. Interestingly, as demonstrated in Figure 1 , the novel combined cyclooxygenase and lipoxygenase inhibitor tenidap dramatically inhibited the
release of activated collagenase, even at concentrations found in clinical studies (data supplied by manufacturer, Pfizer Central Research). In the presence of
tenidap (30 pglml), only 11.6% of the released collagenase was activated, compared with a 33% activation
rate in the absence of tenidap.
The inhibition of activated collagenase release
noted with tenidap prompted us to perform further
experiments to delineate the mechanism of inhibition.
Inhibition by tenidap could be explained by at least 3
different potential mechanisms: 1) tenidap could interfere with the function of activated collagenase, 2)
tenidap could interfere with the release of collagenase,
or 3) tenidap could interfere with the activation of the
latent enzyme. To address the first possibility, activated collagenase was generated by stimulating neutrophils with the phorbol ester phorbol myristate acetate (PMA) in the absence of tenidap. Cells were
removed by centrifugation, and the supernatants were
assayed for collagenase activity. In parallel experiments, tenidap was added to the same supernatants,
and the collagenase activity was again determined. As
shown in Figure 2, the addition of tenidap to the
already activated enzyme did not alter its ability to
cleave type I collagen.
To determine whether tenidap interfered with
the release of collagenase, the collagenase activity in
supernatants obtained from cells activated in the pres-
Table I. Inhibition of activated neutrophil collagenase release by
antirheumatic agents*
Peak therapeutic % inhibition at
drug concentration peak concen(PW
69 23
>200 >42
>400 >92
* Neutrophils were incubated with the peak therapeutic concentration of the indicated compound for 30 minutes at 37"C, and then
placed in wells containing surface-bound IgG. Cell-free supernatants
were assayed for collagenase activity (see Materials and Methods
for details). To determine the 50% inhibitory concentration (IC,,),
dose-response studies were performed, and curve fitting was done
assuming a log-linear relationship between the response and the
drug concentration, with optimum fit obtained by minimizing the
sum of the squares error.
ence or absence of tenidap was compared after activation of secreted latent enzyme with the organomercurial compound mersalyl. As demonstrated in Figure
3, the addition of tenidap to the cells only slightly
inhibited (by 22%) the total amount of collagenase
released extracellularly.
These observations suggested that the mechanism of inhibition noted with tenidap was most likely
through interference with activation of the latent enzyme. Since latent collagenase may be activated by
neutrophil-produced hypochlorous acid, experiments
were designed to measure the effect of tenidap on the
- - .-.
- -V
Figure 1. Effects of various concentrations of tenidap on the release of activated collagenase by surface-bound I&. Purified human
neutrophils were incubated with the indicated concentration of
tenidap for I5 minutes at 25"C, and then for 45 minutes in IgGcoated wells, as described in Materials and Methods. Cell-free
supernatants were assayed for collagenase activity. Control (untreated) supernatants released 1 I .8 ng of collagenase per minute.
Values are the mean 2 SEM of 6 observations.
2 14
z 0+
5 40
Figure 3. Effects of tenidap on total neutrophil collagenase release.
Figure 2. Effects of tenidap on activated neutrophil collagenase.
Cell-free supernatants were generated, as described in Materials and
Methods, in the absence (control) or presence of tenidap, before or
after activation of neutrophils. Bars show the mean and SEM of 6
observations. HNC = human neutrophil collagenase.
generation of hypochlorous acid. As indicated in Figure 4, the addition of tenidap inhibited the production
of hypochlorous acid by neutrophils. The addition of
tenidap to hypochlorous acid, however, did not alter
the level of hypochlorous acid measured by our assay
(data not shown), which suggests that simple scavenging of this product could not account for these observations.
Alterations in hypochlorous acid generation
could be due to an effect either on the generating
enzyme myeloperoxidase or on its substrate. To test
the former possibility, myeloperoxidase-containing supernatants were prepared from both activated neutrophils and whole cell extracts, as described in Materials
and Methods. As demonstrated in Figure 5 , tenidap, in
a dose-related manner, inhibited the activity of myeloperoxidase extracted from normal human neutrophils. Supernatants from cells stimulated by surfacebound IgG were also assayed for myeloperoxidase
activity in the presence and absence of tenidap, and
inhibition of myeloperoxidase activity comparable
with that observed in the experiments with the neutrophi1 extracts was seen (data not shown).
Hydrogen peroxide, the myeloperoxidase substrate that is converted to hypochlorous acid, is
formed by the dismutation of superoxide radicals. We
next determined if tenidap altered the production of
superoxide. The addition of tenidap almost completely
inhibited the production of superoxide radicals by
neutrophils stimulated by surface-bound IgG (Figure
Collagenase activity was assessed in supernatants generated by
surface-bound IgG-activated cells in the absence (control; mersalyltreated) or presence of tenidap, before or after activation of neutrophils, and before or after activation of latent collagenase with
mersalyl. Bars show the mean and SEM of 6 observations.
6). In dose-response studies, tenidap concentrations as
low as 5 pgiml continued to inhibit superoxide production. It is noteworthy that when neutrophils were
stimulated by PMA, superoxide production was not
only not inhibited, it was augmented. This finding
suggests that the effect of tenidap on surface-bound
IgG-mediated superoxide production was not due to
scavenging of superoxide radicals.
Since tenidap did not inhibit PMA-stimulated
superoxide production, it was important to determine
if the effects of tenidap on collagenase activation were
unique to neutrophils that had been stimulated by
surface-bound IgG. As shown in Table 2, PMA-
Figure 4. Effects of tenidap on neutrophil hypochlorous acid pro-
duction. Neutrophils were incubated with the indicated concentrations of tenidap for 15 minutes at 37°C prior to incubation in
IgG-coated wells. Hypochlorous acid production was measured as
described in Materials and Methods. Bars show the mean and SEM
of at least 4 observations.
Figure 5. Effects of tenidap on myeloperoxidase activity. Myeloperoxidase was extracted from whole human neutrophils as
described in Materials and Methods. Various concentrations of
tenidap were added to the extract prior to its addition to the reaction
mixture. Myeloperoxidase activity was expressed as the change in
optical density (O.D.) at 412 nm. Bars show the mean and SEM of
at least 4 observations.
stimulated collagenase activation and hypochlorous
acid production were indeed inhibited by tenidap.
In chronic inflammatory disorders such as rheumatoid arthritis, a hallmark of tissue injury is the
irreversible degradation of connective tissue molecules such as collagen. There are several cellular
sources of collagenase in the rheumatoid joint. These
include mononuclear phagocytes, fibroblasts, and neutrophils. Neutrophils are the predominant cellular constituent of synovial fluid and are frequently found at
the cartilage-pannus interface in patients with RA.
The physiologic neutrophil activator which most potently causes the release of activated collagenase is
surface-bound IgG (17). Collagenase that is released
and activated in this manner may play an important
pathologic role, since it is released contiguous to
degradable connective tissue molecules. Therapeutic
agents capable of inhibiting the activation of neutrophi1 collagenase may therefore have a role in altering
the course of tissue injury.
The observations presented in this study demonstrate that the novel combined cyclooxygenase and
5-lipoxygenase inhibitor tenidap is, in contrast with
currently available cyclooxygenase inhibitors, capable
of potently inhibiting the activation of neutrophil collagenase. This is due to interference with the production of 2 neutrophil products, hypochlorous acid and
Figure 6 . Effects of tenidap on neutrophil superoxide production.
Neutrophils were incubated with the indicated concentrations of
tenidap for 15 minutes at 37°C prior to incubation in IgG-coated
wells. Superoxide production was measured spectrophotometrically, as described in Materials and Methods. Bars show the mean
and SEM of at least 6 observations.
superoxide radicals. Hypochlorous acid, in addition to
directly activating collagenase, has other deleterious
effects on cells and molecules in the inflammatory
environment. It is toxic to cellular membranes (19,20),
it can decrease the viscosity of solutions of hyaluronic
acid (a major component of proteoglycans) (21,22),
and it can form N-chloroamines, which are capable of
inactivating antiproteases (23).
The effects of tenidap on superoxide production
are of interest. When neutrophils are activated by
surface-bound IgG, superoxide production is inhibited. In contrast, superoxide production resulting from
stimulation by the phorbol ester PMA is slightly enhanced. These observations suggest that the effect of
tenidap on surface-bound IgG-stimulated neutrophil
superoxide production is not due to scavenging super-
Table 2. Effect of tenidap on PMA-mediated neutrophil collagenase
activation and hypochlorous acid production*
10 pgiml
20 p g h l
30 p g h l
(% of control)
acid production
(% of control)
(% of control)
12 2 4
37 2 21
16 i 2
12.5 2 2.9
16.4 2 2.6
7.6 2 2.2
114 k 2.0
120 2 1.5
129 2 2.0
* Neutrophils were preincubated with the indicated concentration of
tenidap, transferred to bovine serum albumin-coated wells to which
phorbol myristate acetate (PMA; 500 nglml) had been added, and
incubation was continued. Cells were removed, and supernatants
were assayed as described in Materials and Methods. Values are the
mean ? SEM of at least 4 observations.
oxide radicals or to interference with the superoxidegenerating capacity of the neutrophil. Rather, these
observations suggest that tenidap m a y interfere with
an early step, prior t o activation of t h e C-kinase, in the
signal transduction pathway in the cell.
Tenidap is presently undergoing phase I1 clinical studies for the treatment of RA. It appears to b e
well tolerated (24,25), a n d clinical response to the
agent is prompt (24,25). The results of this study
suggest that tenidap has unique therapeutic properties
which set it apart from conventional nonsteroidal
antiinflammatory drugs a n d which may be of relevance
in preventing t h e tissue injury commonly seen in
disorders such as rheumatoid arthritis.
I. Weissmann G: Lysosomes and rheumatoid joint inflammation. Arthritis Rheum (suppl) 20:S193-S204, 1977
2. Janoff A: At least three human neutrophil lysosomal
proteases are capable of degrading joint connective
tissue. Ann NY Acad Sci 256:402-408, 1975
3. Starkey PM, Barrett AJ, Burleigh MC: The degradation
of articular cartilage collagen by neutrophil proteases.
Biochim Biophys Acta 483:386397, 1977
4. Hasty KA, Hibbs MS, Kang AH, Mainardi CL: Secreted forms of human neutrophil collagenase. J Biol
Chem 2615645-5650, 1986
5 . Hasty KA, Jeffrey JJ, Hibbs MS, Welgus HG, The
coliagen substrate specificity of human neutrophil collagenase. J Biol Chem 262:10048-10052, 1987
6. Weiss SJ, Peppin G, Ortiz X, Ragsdale C, Test ST:
Oxidative autoactivation of latent collagenase by human
neutrophils. Science 227:747-749, 1985
7. Mallya SK,Van Wart HE: Mechanism of inhibition of
human neutrophil collagenase by gold(1) chrysotherapeutic compounds. J Biol Chem 264:1594-1601, 1989
8. Mallya SK, Van Wart HE: Inhibition of human neutrophi1 collagenase by gold(1) salts used in chrysotherapy.
Biochem Biophys Res Comm 144:101-108, 1987
9. Cuperus RA, Muijsers AO, Wever R: Antiarthritic drugs
containing thiol groups savenge hypochlorite and inhibit
its formation by myeloperoxidase from human leukocytes: a therapeutic mechanism of these drugs in rheumatoid arthritis. Arthritis Rheum 28:1228-1233, 1985
10. Moilanen E, Alanko J , Asmawi MZ, Vapaatalo H:
CP-66,248,a new anti-inflammatory agent, is a potent
inhibitor of leukotriene B4 and prostanoid syntheses in
human polymorphonuclear leucocytes in vitro. Eicosanoids 1:35-39, 1988
11. Blackburn WD Jr, Heck LW, Koopman WJ, Gresham
HD: A low molecular weight, heat-labile factor enhances neutrophil Fc receptor-mediated lysosomal en-
zyme release and phagocytosis. Arthritis Rheum 30:
1006-1014, 1987
12. Blackburn WD, Koopman WJ, Schrohenloher RE,
Heck LW: Induction of neutrophil enzyme release by
rheumatoid factors: evidence for differences based on
molecular characteristics. Clin Immunol Immunopathol
40:347-355, 1986
13. Blackburn WD, Heck LW: Neutrophil activation by
surface bound IgG: pertussis toxin insensitive activation. Biochem Biophys Res Comm 152:136-142, 1988
14. Weiss SJ, Klein R, Slivka A, Wei M: Chlorination of
taurine by human neutrophils: evidence for hypochlorous acid generation. J Clin Invest 70598-607, 1982
15. Shindler JS, Childs RE, Bardsley WG: Peroxidase from
human cervical mucus: the isolation and characterisation. Eur J Biochem 65:325-331, 1976
16. Johnson-Wint B: A quantitative collagen film collagenase assay for large numbers of samples. Anal Biochem
104:175-1 8 I , 1980
17. Chatham WW, Heck LW, Blackburn WD: Liganddependent release of active neutrophil collagenase. Arthritis Rheum 33:228-234, 1990
18. Harris ED, Vater CA: Methodology of collagenase
research: substrate purification, enzyme activation and
purification, Collagenase in Normal and Pathological
Connective Tissues. Edited by DE Woolley, JM Evanson, Chichester, John Wiley & Sons, 1980
19. Sepe SM, Clark RA: Oxidant membrane injury by the
neutrophil myeloperoxidase system. I. Characterization
of a liposome model and injury by myeloperoxidase,
hydrogen peroxide and halides. J Immunol 134:18881895, 1985
20. Sepe SM,Clark RA: Oxidant membrane injury by the
neutrophil myeloperoxidase system. 11. Injury by stimulated neutrophils and protection by lipid-soluble antioxidants. J Immunol 134:1896-1901, 1985
21. Greenwald RA, Moy WW: Effect of oxygen-derived free
radicals on hyaluronic acid. Arthritis Rheum 23:455463, 1980
22. Baker MS, Green SP, Lowther DA: Changes in the
viscosity of hyaluronic acid after exposure to a myeloperoxidase-derived oxidant. Arthritis Rheum 32:461467, 1989
23. Weiss SJ, Lampert MB, Test ST: Long-lived oxidants
generated by human neutrophils: characterization and
bioactivity. Science 222:625-627, 1983
24. Katz P, Borger AP, Loose LD: Evaluation of CP-66,248
[5-chloro-2,3-dihydro-2-oxo-3(2-thienylcarbonyl)-indole1-carboxamide] in rheumatoid arthritis (abstract). Arthritis Rheum 31 (suppl 4): S52, 1988
25. Davis JS, Loose L, Borger AP: Clinical efficacy of
CP-66,248 [5-chloro-2,3-dihydro-2-oxo-3-(2-thienylcarbony1)-indole-I-carboxamide]in osteoarthritis (abstract). Arthritis Rheum 31 (suppl 4):S72, 1988
Без категории
Размер файла
621 Кб
contrast, potently, severa, neutrophils, collagenase, drug, inhibits, release, tenidap, nonsteroidal, antiinflammatory, available, activated
Пожаловаться на содержимое документа