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The Anticollagenolytic Potential of Lymecycline in the Long-Term Treatment of Reactive Arthritis.

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195
THE ANTICOLLAGENOLYTIC POTENTIAL OF
LYMECYCLINE IN THE LONG-TERM TREATMENT OF
REACTIVE ARTHRITIS
ANNELI LAUHIO, TIM0 SORSA, OTSO LINDY, KIMMO SUOMALAINEN, HERKKO SAARI,
LORNE M.GOLUB, and YRJO T. KONTTINEN
Objective. We sought to determine the antiinflammatory properties of lymecycline in the long-term
treatment of reactive arthritis (ReA).
Methods. Quantitative assay of collagenase activity by densitometry after sodium dodecyl sulfate
polyacrylamide gel electrophoresis.
Results. Therapeutic levels of lymecycline do not
directly inhibit the activity of human neutrophil interstitial collagenase, but can prevent the oxidative activation of latent human neutrophil collagenase.
From the Department of Medical Chemistry, the Department of Bacteriology, the Department of Immunology, and the
Department of Periodontology, University of Helsinki, and the IVth
Department of Helsinki University General Hospital, Helsinki,
Finland, the Department of Oral Biology and Pathology, State
University of New York at Stony Brook, and the Institute of
Molecular Immunology, Hospital for Joint Diseases, New York
University Medical School, New York, New York.
Supported by the Academy of Finland, the Paivikki and
Sakari Sohlberg’s Foundation, the Oscar bflund’s Foundation, the
Finnish Dental Society, and NIDR grant R-37 DE-03987.
Anneli Lauhio, MD: Department of Medical Chemistry,
Department of Bacteriology, and Department of Immunology, UNversity of Helsinki; T h o Sorsa, DDS, PhD: Department of Periodontology and Department of Medical Chemistry, University of
Helsinki; Otso Lindy, MD: Department of Medical Chemistry,
University of Helsinki; Kimmo Suomalainen, DDS: Department of
Medical Chemistry and Department of Periodontology, University
of Helsinki; Herkko Saari, MD: IVth Department of Helsinki
University Central Hospital; Lome M. Golub, DMD, MSc: Department of Oral Biology and Pathology, State University of New York
at Stony Brook; Y j o T. Konttinen, MD, PhD: Institute of Molecular Immunology, Hospital for Joint Diseases, New York University
Medical School.
Address reprint requests to Timo Sorsa, DDS, PhD: Department of Periodontology, University of Helsinki, Mannerheimintie 172, SF-00300 Helsinki, Finland.
Submitted for publication April 26, 1991; accepted in revised form September 24, 1991.
Arthritis and Rheumatism, Vol. 35, No. 2 (February 1992)
Conclusion. This non-antimicrobial, anticollagenolytic property of lymecycline may contribute to its
therapeutic efficacy in the treatment of patients with ReA.
Reactive arthritis (ReA) is a condition characterized by a nonpurulent inflammation of a joint following infection occurring in the urogenital tract,
gastrointestinal tract, or pharyngeal area. Infectious
agents that have been reported in association with
ReA include Chlamydia, Yersiniu, Campylobacter,
Salmonella, Shigella, and possibly, Streptococcus or
gonococcus (1,2). In a recent placebo-controlled
study, a 3-month regimen of lymecycline (a form of
tetracycline) therapy was found to significantly reduce
the duration of Chlamydia trachomatis-induced arthritis, thus suggesting that the course of this form of
ReA can be modified by long-term antimicrobial therapy (2). In addition, the number of patients with tissue
destruction, as determined by radiologic examination,
was statistically significantly lower among ReA patients
treated with lymecycline than among ReA patients
treated with placebo (2). Since the radiographic examinations were not performed according to a strict protocol, it could not be directly concluded that lymecycline
therapy decreased tissue destruction, however (2).
Collagen degradation, which characterizes inflammatory diseases including the arthritides, has been
attributed in part to increased activity of interstitial
collagenases (3,4). This activity evidently results from
increased synthesis and activation of latent procollagenases, the latter of which is possibly mediated by
activation of proteinases and/or reactive oxygen species (3-6). A series of studies have demonstrated that
tetracyclines can inhibit mammalian interstitial collagenases by a mechanism independent of the antimicrobial efficacy of the drugs. The first such observation
LAUHIO ET AL
was a reduction in excess collagenase activity by
tetracycline therapy in germ-free rats, and this was
followed by demonstration of direct inhibition of neutrophil interstitial collagenase activity by different
concentrations and different types of these drugs
(minocycline, doxycycline, and chemically modified
non-antimicrobial tetracyclines [CMTs]) (7). An additional non-antimicrobial property of tetracyclines that
may be relevant to inflammation-mediated tissue destruction is their ability to suppress neutrophil function (8) and to scavenge reactive oxygen species (9). In
the studies described here, we examined the ability of
lymecycline to express these potential antiinflammatory properties.
o Lymecycline IC50 :310 FM
MATERIALS AND METHODS
Purified human neutrophil procollagenase that had
been optimally activated by 1 mM APMA (5,6) was incubated with native type I collagen (at 22°C for 20 hours) in the
presence of 0-1,OOO f l lymecycline. In separate experiments, neutrophil collagenase was activated with 5-10 @4
sodium hypochlorite (4,6) in the presence and absence of
2-15 p.M lymecycline. Collagenase activity was quantitated
by densitometric scannings of the sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), revealing
the characteristic collagen-degradation products resulting
from the collagenase action (5,6). Briefly, the degradation
products (aA fragments) were separated from intact native
soluble type I collagen a-chains on 8% SDS-PAGE gels, and
quantitated by densitometric scannings using an Ultroscan
Laser Densitometer model 2202 (LKB, Stockholm, Sweden). The values representing aA-chains were multiplied by
four-thirds, and their proportions compared with the total
collagen in the samples were used as measures of collagenase activity (5,6).
Figure 1. Inhibition of human neutrophil interstitial collagenase by
lymecycline. The concentration of lymecycline required to inhibit
50% of the enzyme activity (IC50) is indicated across the top.
due to its antimicrobial action (2). A possible explanation for the different behavior of Chlamydia-triggered
ReA could be the tendency of Chlamydia to induce a
chronic infection (10,ll). However, as we suggested in
the previous study, because tetracyclines have been
shown to inhibit collagenase activity (7) and suppress
neutrophil functions (8), further studies were needed
before the possible roles of these non-antimicrobial
effects in this important and complex issue could be
resolved. In the present in vitro studies, we have
shown that therapeutically attainable levels (5-10 pM
RESULTS
The concentration of lymecycline necessary to
inhibit 50% of the collagenase activity was found to be
310 pM (Figure 1). Furthermore, 2-15 pM lymecycline, a concentration which does not inhibit collagenase activity (Figure l), was found to efficiently inhibit
the oxidative activation of neutrophil collagenase by
5-10 pM NaOCl (Figure 2).
DISCUSSION
In a previous study, we found that long-term
treatment with lymecycline modifies the course of
Chlamydia-triggered ReA, but because a similar effect
was not found in enteric infection-associated ReA, it
was argued that the major effect of the drug was likely
Figure 2. The effect of lymecycline (LY) on the oxidative activation
of latent human neutrophil collagenase (LHNC) by NaOCI. LHNC
(5 pg of enzyme protein) was treated with buffer (lane l), 25 f l
APMA (lane 2), buffer (lane 3), 5 pM NaOCl (lane 4). 10 f l NaOCl
(lane 5 ) , 10 pM NaOCl + 2 pl4 LY (lane 6), 10 pM NaOCl + 5 ~ h f
LY (lane 7), 10 pM NaOCl + 10 phf LY (lane 8). or 10 f l NaOCl
+ 15 f l LY (lane 9). Indicated concentrations of NaOCl and
lymecycline were preincubated for I hour at 22°C; subsequently,
samples were incubated with native type I collagen for 20 hours at
22°C and analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. a = collagen a-chains; aA = characteristic 3/4cleavage products resulting from the action of collagenase.
I
LYMECYCLINE AS ANTIINFLAMMATORY AFTER LONG-TERM THERAPY
[7]) of lymecycline (tetracycline-L-methylenelysine,
which breaks down in vivo to lysine and tetracycline,
which serves as active metabolite [2]) cannot directly
inhibit the activity of human neutrophil interstitial
collagenase. These results are consistent with those in
earlier reports describing tetracycline as a less potent
inhibitor of mammalian interstitial collagenases than
doxycycline, minocycline, and CMT (7). However,
our present findings show that therapeutically attainable concentrations of lymecycline can prevent the
oxidative activation of latent human interstitial collagenases by hypochlorous acid (4,6).
In previous studies, tetracyclines have been
shown to scavenge reactive oxygen species (9). The
ability of lymecycline to scavenge the oxidative activation of latent human interstitial (pro)collagenases, as
found in the present study, may be the mode of action
in its therapeutic efficacy in the long-term treatment of
ReA. Further prospective, controlled studies of lymecycline, other tetracyclines, and/or non-antimicrobial
tetracycline-derivatives in the long-term treatment of
ReA are needed to examine the role of these effects in
vivo. Hypochlorous acid is a major myeloperoxidasederived reactive oxygen species generated by activated human neutrophils (4), and to determine the
possible clinical significance of this reactive oxygen
species, measurement of luminol-enhanced chemiluminescence could be used in the prospective, controlled trials.
Since a,-proteinase inhibitor, an endogenous
inhibitor of serine proteinases (4,12,13) and substrate
for interstitial collagenases (12,13), can be oxidatively
inactivated by NaOCl (4) as well as proteolytically
inactivated/degraded by interstitial collagenases (12,
13), it might be possible that therapeutic levels of
lymecycline/doxycycline can prevent general proteolytic events (in addition to collagenolysis) by maintaining the a,-proteinase inhibitory action (14).
Greenwald et a1 (15) recently administered
CMT to rats with adjuvant arthritis. They found that
treatment with CMT alone significantly reduced the
excess collagenase and gelatinase activities in the
periarticular tissues of those rats and decreased bone
resorption in the inflamed joints (as demonstrated by
histomorphometric analysis of microradiographs of
dissected joints). Radiographs of the tarsal joints obtained prior to killing (scored using double-blind techniques) did not reveal a significant reduction in joint
destruction with CMT alone; however, combined therapy with CMT plus flurbiprofen resulted in a dramatic
and significant reduction in bone erosion.
197
In a recent study of patients with rheumatoid
arthritis, Breedveld et a1 (16) demonstrated that 4
months of treatment with minocycline resulted in a
significant improvement in all disease parameters. The
patients were also taking a nonsteroidal antiinflammatory drug (NSAID) during the study period. In our
previous study, in which we found that tissue destruction (determined by radiologic examination) was statistically significantly lower among ReA patients
treated with long-term lymecycline therapy than
among those treated with placebo, both of the treatment groups had continued to take their NSAID
therapy (2). The results of our previous study (2) are
consistent with those of Breedveld et al (16). The
rationale for these clinical studies is based on the
demonstration that orally administered tetracyclines
can reduce the level of collagenase activity in extracts
of inflamed synovial and gingival tissues from patients
with rheumatoid arthritis and with periodontitis, respectively (7). Overall, the antiinflammatory potential of
lymecycline may contribute to its therapeutic efficacy in
the long-term management of the ReA patient (2).
ACKNOWLEDGMENT
The lymecycline preparation used for these studies
was generously provided by Farmitalia Carlo Erba, Helsinki, Finland.
REFERENCES
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Ingman T, Konttinen YT, Lee HM, Simon S, Hau J,
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IS. Greenwald R, Moak S, Golub L, Ramamurthy N, Zeng
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