Bioclwmiall Pluumtlco/ory. Vol. 44. No.6. pp. 1165-1170, 1992.
`Printed in Great Britain.
`
`0006-2952/92 ss.oo + 0.00
`© 1992. Peraamon Press Ltd
`
`INHIBITION OF ENZYMATIC ACTIVITY OF
`PHOSPHOLIPASES A 2 BY MINOCYCLINE AND
`DOXYCYCLINE
`WALDEMAR PRUZANSKr,•t ROBERT A. GREENWALD,* IAN P. STREET,§
`FRANCE LALIBERTE,§ EVA STEFANSKI* and PETER VADAS*
`•Inflammation Research Group, The Wellesley Hospital, University of Toronto, Toronto, Ontario,
`Canada; ;Long Island Jewish Medical Center, New Hyde Park, NY, U.S.A.; and
`§Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, P.O., Canada
`
`(Received 24 February 1992; accepted 27 May 1992)
`
`Abllract-Extracellular phospholipase& A1 play an important role in articular and extra-articular
`inflammatory processes. Secretory non-pancreatic phospholipase A 2 (PLA2) has been implicated in the
`pathogenesis of articular inflammation in rheumatoid arthritis, whereas pancreatic PLA2 contributes to
`the tissue damage associated with acute pancreatitis. Since in experimental models lipophilic tetracyclines
`such as minoc:ycline and doxycycline are antiinflammatory, we examined their effects on PLA2 activity
`using two assay systems in vitro. We found that minocycline and to a lesser degree doxycycline were
`markedly inhibitory to both pancreatic and non-pancreatic PLA2• Using [I4C)oleic acid labeled
`Escherichia coli membrane phospholipids as substrate, the 1c50 values for minocycline and doxycycline
`were 3.6 x 10-s M (18 ,.,g/mL) and 0.98 x w-• M (47/lg/mL), respectively. In a scooting mode assay
`using the synthetic phospholipid 1-palmitoyl-2-(10-pyrenedecanoyl)-3-L-phosphatidylmethanol as
`substrate, IC50 values for minoc:ycline were 5/lM (2.47 /lg/mL) for non-pancreatic PLA 2 and 8/lM
`(3.95/lg/mL) for pancreatic PLA2• Addition of excess calcium up to 50 mM did not reverse the
`inhibitory activity of tetracyclines. We conclude that lipophilic tetracyclines inhibit PLA 2, probably by
`interaction with the substrate, and may be a useful adjunct in the therapy of inflammatory conditions
`in which PLA2 is implicated pathogenetically.
`
`synovial fluid (SF) [16, 17]. Serum PLA2 activity
`correlates with disease activity in adult rheumatoid
`[20) and juvenile chronic [21] arthritis. Intra-articular
`injections of a native purified PLA2 [22] or
`(23]
`of recombinant human
`induce
`rh-PLA2
`experimental synovitis closely resembling the pro-
`liferative rheumatoid process.
`Since PLA2 is proinflammatory, and present in
`the synovial milieu, it was of interest to investigate
`whether tetracycline derivatives modulate the activity
`of this enzyme. Our studies have shown that
`minocycline and, to a lesser degree. doxycycline
`substantially inhibit the PLArsubstrate interaction.
`
`METHODS
`
`Several semi-synthetic tetracycline antibiotics, such
`as minocycline and doxycycline, have been found to
`be powerful
`inhibitors of the neutral matrix
`metalloproteinases collagenase and gelatinase (1-9].
`Inhibitory activity has been demonstrated both in
`vitro and in vivo in tissues as diverse as inflamed
`gingiva, rheumatoidsynovium, cornea, osteoarthritic
`cartilage, and cancer cells (see Ref. 10 for review).
`Minocycline has also been found to inhibit the
`synthesis of prostaglandin E2 (PGE2) and 6-keto
`PGF1cr by gingival fibroblasts [11). It has also been
`reported that minocycline suppresses some clinical
`manifestations of adjuvant and/or collagen-induced
`arthritis [12-14]. However, extensive studies with
`minocycline and doxycycline in rats with adjuvant
`arthritis have failed to confirm a significant effect on
`joint score and paw swelling while at the same time
`demonstrating clearly the ability of these compounds
`(after in vivo administration) to inhibit collagenase
`and gelatinase [15].
`Several years ago a new enzyme, low molecular
`weight, secretory phospholipase A 2 (PLA2II), was
`discovered [16, 17). This PLA2 was found to belong
`to the non-pancreatic group II phospholipases [18]
`on the basis of its novel amino acid sequence [19).
`Originally, this PLA2 was detected in rheumatoid
`
`t Corresponding author; Dr. W. Pruzanski, The
`Wellesley Hospital, Room 104, Jones Building, Toronto,
`Ontario, M4Y 113, Canada. Tel. (416) 926-7785; FAX
`(416) 966-5046.
`II Abbreviations: PLA2, phospholipase A2; SF, synovial
`fluid; rh, recombinant human; and TET, tetracyclines.
`
`Minocycline and doxycycline were obtained
`from the Sigma Chemical Co., St. Louis, MO.
`Recombinant human group II PLA2 (rh-PLA2), Lot
`No. 2789, was a gift of Dr. J. Browning, Biogen,
`Cambridge, MA. Human group II extracellular
`PLA2 (SF-PLA2) was purified from rheumatoid
`synovial fluid as described [24). Cell-free human
`synovial fluids were obtained from rheumatoid,
`osteoarthritic, and psoriatic inflamed joints. Naja
`naja PLA2 was purchased from Sigma, and repurified
`as described [25). Crotalus adamanteus PLA2 (Crot.
`adamanteus), Crotalus atrox PLA2 (Crot. atrox) and
`porcine pancreatic I
`Dr. Reddy's Laboratories, Ltd., et al.
`Sigma. Purified hum
`v.
`of Dr. B. Stemby,
`Galderma Laboratories, Inc.
`'
`Homogenates of
`IPR2015-__
`Exhibit 1023
`1165
`
`

`
`Biochtmical Plumrulco/ogy. Vol. 44, No. 6, pp. 1165-1170, 1992.
`Printed in Great Britain.
`
`0006-2952/92 $5.00 + 0.00
`© 1992. Pergarnon Press Ltd
`
`INHIBITION OF ENZYMATIC ACTIVITY OF
`PHOSPHOLIPASES A 2 BY MINOCYCLINE AND
`DOXYCYCLINE
`WALDEMAR PRUZANSKI,*t ROBERT A. GREENWALD,t IAN P. STREET,§
`FRANCE LALIBERTE,§ EVA STEFANSKI* and PETER VADAS*
`•rnftammation Research Group, The Wellesley Hospital, University of Toronto, Toronto, Ontario,
`Canada; *Long Island Jewish Medical Center, New Hyde Park, NY, U.S.A.; and
`§Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, P.Q., Canada
`
`(Received 24 February 1992; accepted 27 May 1992)
`
`Abstract-Extracellular phospholipases A 2 play an important role in articular and extra-articular
`inftammatory processes. Secretory non-pancreatic phospholipase A 2 (PLA2) has been implicated in the
`pathogenesis of articular inftammation in rheurnatoid arthritis, whereas pancreatic PLA2 contributes to
`the tissue darnage associated with acute pancreatitis. Since in experimental models lipophilic tetracyclines
`such as minocycline and doxycycline are antiinftammatory, we examined their effects on PLA 2 activity
`using two assay systems in vitro. We found that minocycline and to a lesser degree doxycycline were
`markedly inhibitory to both pancreatic and non-pancreatic PLA2• Using [1 4C)oleic acid labeled
`Escherichia coli membrane phospholipids as substrate, the ICso values for minocycline and doxycycline
`were 3.6 x w-s M (181J8/mL) and 0.98 x w-• M (47 !Jg/mL), respectively. In a scooting mode assay
`the synthetic phospholipid 1-palmitoyl-2-(10-pyrenedecanoyi)-3-L-phosphatidylmethanol as
`using
`substrate, IC50 values for minocycline were 5 !JM (2.47 ~Jg/mL) for non-pancreatic PLA2 and 8 1-1M
`(3.95 !Jg/mL) for pancreatic PLA2• Addition of excess calcium up to 50 mM did not reverse the
`inhibitory activity of tetracyclines. We conclude that lipophilic tetracyclines inhibit PLA 2, probably by
`interaction with the substrate, and may be a useful adjunct in the therapy of inftammatory conditions
`in which PLA2 is implicated pathogenetically.
`
`Several semi-synthetic tetracycline antibiotics, such
`as minocycline and doxycycline, have been found to
`be powerful
`inhibitors of
`the neutral matrix
`metalloproteinases collagenase and gelatinase [1-9).
`Inhibitory activity has been demonstrated both in
`vitro and in vivo in tissues as diverse as inftamed
`gingiva, rheumatoid synovium, cornea, osteoarthritic
`cartilage, and cancer cells (see Ref. 10 for review).
`Minocycline has also been found to inhibit the
`synthesis of prostaglandin E 2 (PGE 2) and 6-keto
`PGF1a by gingival fibroblasts [11). It has also been
`reported that minocycline suppresses some clinical
`manifestations of adjuvant and/or collagen-induced
`arthritis [12-14). However, extensive studies with
`minocycline and doxycycline in rats with adjuvant
`arthritis have failed to confirm a significant effect on
`joint score and paw swelling while at the same time
`demonstrating clearly the ability of these compounds
`(after in vivo administration) to inhibit collagenase
`and gelatinase [15].
`Several years ago a new enzyme, low molecular
`weight, secretory phospholipase A 2 (PLA2II), was
`discovered [16, 17). This PLA 2 was found to belong
`to the non-pancreatic group II phospholipases [18)
`on the basis of its novel amino acid sequence [19).
`Originally, this PLA2 was detected in rheumatoid
`
`t Corresponding author: Dr. W. Pruzanski, The
`Wellesley Hospital, Room 104, Jones Building, Toronto,
`Ontario, M4Y 113, Canada. Tel. (416) 926-7785; FAX
`(416) 966-5046.
`II Abbreviations: PLA 2, phospholipase A 2; SF, synovial
`fluid; rh, recombinant human; and TET, tetracyclines.
`
`synovial fluid (SF) (16, 17). Serum PLA 2 actiVIty
`correlates with disease activity in adult rheumatoid
`(20] and juvenile chronic (21) arthritis. Intra-articular
`injections of a native purified PLA 2 (22) or
`recombinant human
`(23)
`induce
`rh-PLA 2
`of
`experimental synovitis closely resembling the pro-
`liferative rheumatoid process.
`Since PLA2 is proinflammatory, and present in
`the synovial milieu, it was of interest to investigate
`whether tetracyclinederivatives modulate the activity
`of this enzyme. Our studies have shown that
`minocycline and, to a lesser degree, doxycycline
`substantially inhibit the PLA;rsubstrate interaction.
`
`METHODS
`
`Minocycline and doxycycline were obtained
`from the Sigma Chemical Co., St. Louis, MO.
`Recombinant human group II PLA2 (rh-PLA2), Lot
`No. 2789, was a gift of Dr. J. Browning, Biogen,
`Cambridge, MA. Human group II extracellular
`PLA2 (SF-PLA2) was purified from rheumatoid
`synovial fluid as described (24). Cell-free human
`synovial ftuids were obtained from rheumatoid,
`osteoarthritic, and psoriatic inflamed joints. Naja
`naja PLA2 was purchased from Sigma, and repurified
`as described (25). Crotalus adamanteus PLA 2 (Crot.
`adamanteus), Crotalus atrox PLA 2 (Crot. atrox) and
`porcine pancreatic PLA 2 also were obtained from
`Sigma. Purified human pancreatic PLA2 was a gift
`of Dr. B. Sternby, University of Lund, Sweden.
`Homogenates of deep and superficial human
`
`1165
`
`Exh. 1023
`
`

`
`1166
`
`100
`
`W. PRUZANSKI et al.
`
`90
`80
`c
`~ 70
`:c
`:E
`60
`.s
`..
`c
`so
`~ ..
`40
`
`0..
`
`30
`20
`10
`
`10
`
`25
`
`~g/ml
`
`so
`
`100
`
`Fig. 1. Inhibition of PLA2 purified from rheumatoid
`synovial fluid (SF-PLA 2) by minocycline and doxycycline.
`The tetracyclines were preincubated for 30 min with PLA 2•
`Values are means ± SD of triplicate experiments. Control
`value: 134 ± 7 U/mL.
`
`100
`90
`80
`c
`~ 70
`:c
`:c .5
`60
`50
`c
`40
`30
`20
`10
`0
`
`GI
`~
`GI
`Q.
`
`20
`
`25
`
`10
`15
`pg/ml
`Fig. 2. Inhibition of SF-PLA2 and of recombinant human
`(rh) PLA2 by minocycline. Values are means ± SD of
`triplicate experiments. Respective control values were
`137 ± 18 and 97 ± 2 U/mL.
`
`osteoarthritic cartilage were prepared as described
`[26].
`PLA 2 enzymatic activity. PLA2 activity was
`assayed using [14C]oleic acid-labeled Escherichia coli
`membrane phospholipid as substrate [24]. Reaction
`mixtures contained 10 mg bovine serum albumin,
`2 mM CaCl2, 8 x 108 radiolabeled E. coli and 0.1 M
`Tris-HCl buffer, pH7.5, in a volume of 1.5mL.
`Reaction mixtures were incubated at 37° for 30 min
`and the reaction was terminated by filtration through
`a 0.45 ,um Millipore filter. Assays were performed
`in duplicate in substrate excess. Enzyme activities
`were corrected for non-enzymatic hydrolysis [24].
`PLA 2 scooting mode assay. A scooting mode assay
`(27, 28] has been developed for the PLA2 from
`synovial fluid. The activities of the rh SF-PLA2 and
`purified PLA2 from porcine pancreas were measured
`using the synthetic phospholipid 1-palmitoyl-2-(10-
`pyrenedecanoyl)-3-L-phosphatidylmethanol (10-Py-
`PM). Vesicles containing different mole fractions of
`minocycline were prepared by mixing chloroform
`solutions of the phospholipid and the inhibitor,
`followed by removal of the solvent under a stream
`of nitrogen. The phospholipid-inhibitor mixtures
`were
`then resuspended
`in 1 mM ethylenegly-
`(EG TA)
`colbis( aminoethylether )tetra-acetate
`(pH 9.0) and sonicated in a bath sonicator. Reactions
`contained 10 ,uM 10-Py-PM, 2.5 mM CaCl2, 100 mM
`KCl, and 50 mM Tris-HCl ( pH 9.0). Reactions were
`incubated at 3r for 4 min and quenched, and the
`products were analyzed by HPLC (Street et al.,
`unpublished Observations).
`
`RESULTS
`Using E. coli membrane phospholipids, mino-
`cycline in concentrations of 1 x 10- M to 1 x 10-4 M
`(5-50 ,ug/mL) was
`found
`to
`inhibit substrate
`hydrolysis by SF-PLA2 in a concentration-dependent
`manner. The IC50 was 3.6 x 10-s M (18 ,ug/mL).
`Doxycycline
`tested
`in
`concentrations
`from
`0.1 x 10-4 M to 2.0 x 10-4 M (5-100 ,ug/mL) was
`also inhibitory with an 1c50 of 0.98 X 10-4 M (47 ,ug/
`mL)
`(Fig. 1). Comparison of SF-PLA2 and
`
`Table 1. Inhibitors of group I and group II phospholipases
`Az
`
`Inhibitor
`
`Gelb inhibitor
`Dennis inhibitor
`(PE)t
`Dennis inhibitor
`(PC)§
`Minocycline
`
`ICso (,uM)
`rh SF-PLA2*
`Pancreatic PLA2t
`65
`
`2
`
`0.048
`
`0.69
`5
`
`0.005
`
`0.14
`8
`
`• Recombinant human (synovial type) PLA2 •
`t Purified PLA2 from porcine pancreas.
`t PE = ethanolamine head group.
`§ PC = choline head group.
`
`recombinant human PLA2 showed that minocycline
`was more inhibitory to rh-PLA 2 (Fig. 2).
`Minocycline also inhibited the rh-PLA2 and the
`porcine pancreatic PLA2 when 10-Py-PM was used
`as the substratein the assay. The IC50 values obtained
`for the rh-PLA2 and the porcine pancreatic PLA2
`were 5 ,uM (2.47 ,ug/mL) and 8 ,uM (3.95 ,ug/mL),
`respectively. For comparison a number of substrate
`analogue-inhibitors were also tested for their ability
`to inhibit PLA2 in the 10-Py-PM assay (Table 1).
`The phosphonate substrate analogue, described by
`Jain et al. [29] and called Gelb inhibitor, and the
`amidophospholipids, described by Yu et al. [30] and
`called Dennis inhibitors, were found to be effective
`inhibitors of both the rh-PLA2 and the porcine
`pancreatic PLA2• The impact of calcium con-
`centration on the inhibitory activity of minocycline
`and doxycycline was tested at concentrations from
`7 to 50 mM. Addition of excess calcium did not
`reverse the inhibitory activity of the tetracyclines
`(data not shown).
`Preincubation of 40 ,uM minocycline (20 ,ug/mL)
`and 104 ,uM doxycycline (50 ,ug/mL) with group I
`
`Exh. 1023
`
`

`
`Tetracyclines and phospholipase A 2
`
`1167
`
`Table 2. Inhibition of group I and group II phospholipase A 2 activity by
`minocycline and doxycycline
`Minocycline, 40 "M
`(20pg/mL)
`
`Doxycycline, 104 "M
`(50pg/mL)
`
`Phospholipase A2
`
`Control
`rh-PLA 2t
`Control
`SF-PLA2§
`Control
`Human pancreatic
`Control
`Porcine pancreatic
`
`Activity (U/mL)
`
`91 ± 2•
`14 ± 1 (85)*
`137 ± 18
`47 ± 4 (66)
`106 ± 2
`5 ± 3 (95)
`223 ± 4
`100 ± 3 (55)
`
`134 ± 1
`30 ± 3 (78)
`68 ± 8
`36 ± 3 (53)
`106 ± 2
`95 ± 9 (10)
`223 ± 4
`194 ± 13 (13)
`
`• Mean ± SD of 3 experiments in duplicate.
`t Recombinant human (synovial type) PLA2.
`t Values in parentheses indicate mean percent of inhibition.
`§ Native purified synovial ftuid PLA2.
`
`and group II phospholipases A 2 (Table 2) showed
`that minocycline was consistently more inhibitory
`than doxycycline. Minocycline inhibited PLA2 in
`rheumatoid, osteoarthritic and psoriatic synovial
`fluids (62-68% inhibition) and in the homogenate
`of superficial and deep human osteoarthritic articular
`cartilage
`(62-71%
`inhibition). The
`respective
`inhibition by doxycycline was 26-57 and 33-38%.
`Naja naja (group I) PLA2 activity was strongly
`inhibited by minocycline
`(72%) but not by
`doxycycline (12% ). Phospholipases A2 from the
`venoms of Crot. atrox and Crot. adamanteus (Group
`II enzymes) were inhibited by minocycline (22
`and 43%, respectively) but not by doxycycline.
`Preincubation of minocycline (40 JlM, 20 llg/mL)
`and doxycycline (104JlM, 50 Jlg/mL) for 30 min with
`the substrate showed inhibition essentially identical
`to
`that obtained when
`the tetracyclines were
`preincubated with the enzymes (data not shown).
`
`DISCUSSION
`
`Minocycline and doxycycline are highly Iipid
`soluble tetracyclines (TET) that easily penetrate
`body tissues and fluids including synovial fluid [31).
`Both these TET, as weil as others which have been
`modified so as to remove their antibiotic activity,
`were found to exert marked inhibitory activity on
`matrix metalloproteinases, such as collagenases,
`gelatinase and macrophage elastase (see Ref. 10 for
`review).
`Synovia! ftuids and tissues from patients with
`rheumatoid arthritis treated with minocycline have
`markedly lower collagenase activity as compared
`to pretreatment values [5]. Doxycycline inhibits
`type XI collagen by
`digestion of exogenous
`homogenates of human articular cartilage [8).
`These studies [1-10) prompted trials to attenuate
`experimental arthritis by TET. lt was suggested that
`minocycline suppressed collagen II and adjuvant
`arthritis in the rat [12-14). However, in another
`
`study, oral TET given as a single agent to rats had
`no substantial antiinftammatory activity as assessed
`by joint swelling and paw diameter but it inhibited
`collagenase activity. A combination of the non-
`steroidal agent fturbiprofen and TET potently
`inhibits inftammation and normalized radiographic
`jointdarnage [15). Thus, simultaneaus administration
`of cyclooxygenase inhibitors and lipid-soluble TET
`seems to have a more pronounced beneficial effect
`in experimental arthritis than when each agent is
`given separately.
`In several small series of patients with rheumatoid
`or reactive arthritis, minocycline and doxycycline
`were shown to have some beneficial effect on arthritis
`[32-34). Tetracyclinehydrochloride given to patients
`with rheumatoid arthritis showed no significant
`benefit [35). However, these were all open trials and
`were statistically inconclusive.
`The impact of tetracyclines on the arachidonic
`acid cascade has not been studied extensively.
`Preliminary study has shown that tetracycline and
`minocycline inhibit the activity of partially purified
`PLA2 from venom of Crot. adamanteus [36). lt was
`also reported that minocycline added to the cultures
`of gingival fibroblasts inhibits the synthesis of PGE2
`and 6-keto PGF1"' [11).
`The above Observation combined with the fact
`that TET are inhibitory to several metalloproteinases
`prompted us to investigate the impact of TET on a
`panel of structurally distinct phospholipases A2•
`Since in several models, low molecular weight
`found
`to have pro-
`extracellular PLA2 was
`inftammatory activity [22, 23, 37, 38), inhibition of
`this enzyme by TET would serve as an additional
`rationale to use TET in arthritis alone or in
`combination with antiinflammatory agents. Our
`study has shown that minocycline and doxycycline
`markedly
`inhibit proinftammatory synovial-type
`phospholipase A2 in both E. coli assay and in the
`scooting mode assay. Minocycline was a morepotent
`inhibitor than doxycycline. Both TET showed
`
`Exh. 1023
`
`

`
`W. PRUZANSKI et al.
`1168
`in vesicles containing only a 0.048 ,uM concentration
`selectivity towards certain PLA2s. Minocycline was
`of the inhibitor.
`markedly inhibitory to both group I and II PLA2 of
`human origin. In the snakevenom PLA2sminocycline
`Minocycline codispersed with 10-Py-PM was
`was slightly more inhibitory to group I PLA2 (Naja
`inhibitory to both the rh SF-PLA2 and the porcine
`naja) than to group II (Crot. atrox and Crot.
`pancreatic PLA2• However, inhibition of these
`adamenteus). Doxycycline substantially inhibited
`enzymes was observed only when minocycline was
`human group II PLA2s, but had no inhibitory activity
`present in the assay at concentrations approaching
`against pancreatic PLA2 and snake venom PLA2s.
`that of the substrate. Minocycline is one of the most
`It is not clear why minocycline was found to be more
`lipophilic tetracyclines and will partition essentially
`completely into the Iipid phase. Thus, at the
`inhibitory than doxycycline whereas the opposite is
`true in the studies of collagenase inhibition [5, 10].
`concentration of minocycline required for 50%
`Perhaps some differences in lipophilic properties
`inhibition of the rh SF-PLA2 (5 ,uM) the inhibitor
`may account for the above phenomenon. An increase
`constituted 30 mol% of the substrate vesicle. At this
`of calcium concentration in the reaction mixtures
`very high interfacial concentration, minocycline may
`did not reverse the inhibitory activity of TET,
`cause structural changes to the substrate vesicle and
`thereby suggesting that chelation of calcium by TET
`it is likely that most of the inhibition is due to these
`is not the mechanism of PLA2 inhibition.
`effects and dilution of the substrate within the
`Many group I PLA2s have a high affinity for
`vesicle. However, as this study only involved the
`measurement of initial rates, the possibility that
`vesicles containing anionic phospholipids, and
`hydrolyse these substrates without leaving the lipid-
`minocycline does bind at the active site cannot be
`excluded completely.
`water interface [27]. U nder these conditions ( scooting
`mode hydrolysis), it is possible to differentiate
`Irrespective of the mechanism of the inhibitory
`between inhibitors which bind at the catalytic site of
`activity of TET, they may potentially be useful as
`the enzyme and those compounds which exert their
`adjuncts in the treatment of inflammatory arthritides.
`inhibitory effect by decreasing the affinity of the
`TET may also be used in acute pancreatitis.
`enzyme for the lipid-water interface [28].
`Pancreatic PLA2 converts Iecithin to lysolecithin
`which may cause marked inflammatory reaction [44].
`Kinetic studies with PLA2 are complicated by the
`fact that binding of the enzyme to the lipid-
`In acute pancreatitis the release of !arge amounts of
`water interface must precede catalytic turnover.
`pancreatic PLA2 into the pancreatic and adjacent
`Compounds which affect the organization of the
`tissues has been weil documented [45]. Furthermore,
`substrate interface, and consequently the amount of
`the circulating Ievels of PLA2 in theserum of patients
`absorbed enzyme, can alter apparent turnover rates
`with pancreatitis may increase up to 800-fold from
`the normal Ievei [46]. Since TET markedly inhibit
`by up to several orders of magnitude. By monitoring
`the kinetics of interfacial catalysis in the scooting
`pancreatic PLA2-substrate interaction, their use in
`conditions in which this enzyme is excessively
`mode [29] it is possible to differentiate between
`compounds which inhibit PLA2 by desorption of the
`activated and released from the pancreas may be
`clinically beneficial.
`enzyme from the substrate interface (nonspecific
`inhibitors) and compounds which inhibit PLA2
`directly by binding at the active site. In the scooting
`mode the PLA2 has a very high affinity for the
`substrate interface and remains tightly bound to the
`substrate vesicle during and after hydrolysis of all of
`the available substrate [39]. The effect of nonspecific
`inhibitors is minimized and it is possible to detect
`desorption of the enzyme from
`the interface.
`Examples of compounds which inhibit the action of
`interface structure
`include
`PLA2 by affecting
`lipocortin [40], alkanols [41], cationic amphiphiles
`[42] and aristolochic acid [43]. None of these
`compounds has any effect on PLA2 activity in the
`scooting mode [29].
`The group II rh SF-PLA2 has a high affinity for
`the anionic interface of 10-Py-PM vesicles and
`remains tightly bound to the vesicle surface for
`many catalytic cycles (Street et al., unpublished
`Observations). Therefore, it was of interest to
`examine minocycline and a number of known
`substrate analogue inhibitors of PLA2 in the 10-Py-
`PM assay system. The results presented in Table 1
`show that the phosphonate inhibitor (Gelb inhibitor)
`and particularly the amidophospholipids described
`by Yu et al. [30] were potent inhibitors of both the
`rh SF-PLA2 and the porcine pancreatic PLA2• In
`the case of
`the amidophospholipid analogue
`possessing the ethanolamine head group (Dennis-
`PE), 50% inhibition ofthe rh SF-PLA2 was observed
`
`1. Golub LM, Lee HM, Lehrer G, Ramamurthy N
`and McNamara TF, Minocycline reduces gingival
`collagenolytic activity during diabetes: Preliminary
`observations and a proposed new mechanism of action.
`J Periodont Res 18: 516-524, 1983.
`2. Golub LM, Wolff M, Lee HM, McNamara TF,
`Ramamurthy NS, Zambon J and Ciancio S, Further
`evidence that tetracyclines inhibit collagenase activity
`in human crevicular fluid and from other mammalian
`sources. J Periodont Res 20: 12-23, 1985.
`3. Gomes BC, Golub LM and Ramamurthy NS,
`Tetracyclines inhibit parathyroid hormone induced
`bone resorption in organ culture. Experientia 40: 1273-
`1275, 1985.
`4. Rifkin RB, Golub LM, Sanavi R, Vernillo AT,
`Kleckner AP, McNamara TF, Auszmann JM and
`Ramamurthy NS, Effects of tetracyclines on rat
`Osteoblast collagenase activity and bone resorption in
`vitro.
`In: The Biological Mechanisms of Tooth
`Movement and Craniofacial Adaptation (Ed. Dav-
`idovitch Z), pp. 85-90. EBSCO Media, Birmingham
`AL, 1992.
`5. Greenwald RA, Golub LM, Lavietes B, Ramamurthy
`NS, Gruber B, Laskin RS and McNamara TF,
`
`Acknowledgements-This work was supported by grants-
`in-aid from the Medical Research Council of Canada and
`The Arthritis Society.
`
`REFERENCES
`
`Exh. 1023
`
`

`
`Tetracyclines and phospholipase A 2
`
`1169
`
`Tetracyclines inhibit human synovial collagenase in
`vivo andin vitro. J Rheumato/14: 28--32, 1987.
`6. Golub LM, McNamara TF, D'Angelo G, Greenwald
`RA and Ramamurthy NS, A nonantibacterial chemi-
`cally modified tetracycline inhibits mammalian col-
`lagenase activity. J Dent Res 66: 1310-1314, 1987.
`7. Burns FR, Stack MS, Gray RD and Paterson CA,
`Inhibition of purified collagenase from alkali-burned
`rabbit corneas. Invest Ophthalmol Vis Sei 30: 1569-
`1575, 1989.
`8. Yu LP Jr, Smith GN, Hasty KA and Brandt KD,
`Doxycycline inhibits Type XI collagenolytic activity of
`extracts from human osteoarthritic cartilage and of
`gelatinase. J Rheumato/18: 1450-1452, 1991.
`9. ZuckerS, Lysik RM, Ramamurthy NS, Golub LM,
`Wieman JM and Wilkie DP, Diversity of melanoma
`membrane proteinases: Inhibition of collagenolytic and
`cytolytic activity by minocycline. J Natl Cancer Inst 75:
`517-525, 1985.
`10. Golub LM, Ramamurthy NS, McNamara TF, Green-
`wald RA and Rifkin BR, Tetracyclines
`inhibit
`connective tissue breakdown: New therapeutic impli-
`cations for an old family of drugs. Crit Rev Oral Med
`Pathol 2: 297-322, 1991.
`11. E!Attar TMA, Lin HS and Schulz R, Effect of
`minocycline on prostaglandin formation in gingival
`fibroblasts. J Periodont Res 23: 285-286, 1988.
`12. Breedveld FC, O'Brien JM, Brinckerhoff CE and
`Trentham DE, Suppression of collagen and adjuvant
`arthritis by a tetracycline. Arthritis Rheum 31 (Suppl):
`S88, 1988.
`13. Sewell KL, Fury E, Dynesius-Trentham R, Tomyl
`K and Trentham DE, Evidence that minocycline
`suppresses collagen and adjuvant arthritis by altering
`the expression of T cell derived, collagen-binding
`proteins. Arthritis Rheum 32 (Suppl): S134, 1989.
`14. Sewell KL, Furrie SE and Trentham DE, The
`therapeutic effect of minocycline in experimental
`arthritis. Mechanism of action. Arthritis Rheum 33
`(Suppl): S106, 1991.
`15. Greenwald RA, Moak SA, Ramamurthy NSD and
`Golub LM, Tetracyclines suppress metalloproteinase
`activity in adjuvant arthritis and, in combination with
`flurbiprofen, ameliorate bone damage. J Rheumatal
`19: 927-938, 1992.
`16. Vadas P and Pruzanski W, The roJe of extracellular
`phospholipase A 2 in inflammation. Adv lnflammation
`Res 7: 51-62, 1983.
`17. Vadas P and Pruzanski W, RoJe of secretory
`phospholipase A 2 in the pathology of disease. Lab
`Invest 55: 391-399, 1986.
`18. Heinrikson RL, Kreuger ET and Keim PS, Aminoacid
`sequence of phospholipase A 2-a from the venom
`of Crotalus adamanteus. A new classification of
`phospholipases A 2 based upon structural determinants.
`J Bio/ Chem 252: 4913-4921, 1977.
`19. Seilhamer JJ, Pruzanski W, Vadas P, Plant S, Miller
`JA, Kloss J and Johnson LK, Cloning and recombinant
`expression of phospholipase A 2 present in rheumatoid
`arthritic synovial fluid. J Bio/ Chem 264: 5335-5338,
`1989.
`20. Pruzanski W, Vadas P, Stefanski E and Urowitz M,
`Phospholipase A 2 activity in sera and synovial fluids in
`rheumatoid arthritis and osteoarthritis. J Rheumatal
`12: 211-216, 1986.
`21. Silverman E, Pruzanski W, Laxer R, Albin-Cook K,
`Stepanovic E and Vadas P, Correlation ofphospholipase
`A 2 Ievel and disease activity in juvenile rheumatoid
`arthritis (JRA). Arthritis Rheum 30 (Suppl): S127,
`1987.
`22. Vadas P, Pruzanski W, Kim J and Fornasier V, The
`proinflammatory effect of intra-articular injection of
`
`soluble human and venom phospholipase A 2• Am J
`Patho/134: 807-811, 1989.
`23. Bomalaski JS, Lawton P and Browning JL, Human
`extracellular recombinant phospholipase A 2 induces an
`inflammatory response in rabbit joints. J lmmuno/146:
`3904-3910, 1991.
`24. Stefanski E, Pruzanski W, Sternby B and Vadas P,
`Purification of a soluble phospholipase A 2 from synovial
`fluid in rheumatoid arthritis. J Bioehern (Tokyo) 100:
`1297-1303, 1986.
`25. Deerns RA and Dennis EA, Phospholipase A 2 from
`cobra venom (Naja naja naja). Methods Enzymol 71:
`703-710, 1981.
`26. Pruzanski W, Bogoch E, Stefanski E, Wloch M and
`Vadas P, Enzymatic activity and distribution of
`phospholipase A 2 in human cartilage. Life Sei 48: 2457-
`2462, 1991.
`27. Berg OG, Yu B-Z, Rogers J and Jain MK, Interfacial
`catalysis by phospholipase A 2: Determination of the
`interfacial kinetic rate constants. Biochemistry 30:
`7283-7297, 1991.
`28. Jain MK and Gelb MH, Phospholipase A 2-catalyzed
`hydrolysis of vesicles: Uses of interfacial catalysis in
`the scooting mode. Methods Enzymol 197: 112-125,
`1991.
`29. Jain MK, Yuan W and Gelb MH, Competitive
`inhibition of phospholipase A 2 in vesicles. Biochemistry
`28: 4135-4139, 1989.
`30. Yu L, Deerns RA, Hajdu J and Dennis EA, The
`interaction of phospholipase A2 with phospholipid
`analogues and inhibitors. J Bio/ Chem 265: 2657-2664,
`1990.
`tetracycline and
`31. Ehrlich GE, Concentration of
`minocycline in joint effusions following oral adminis-
`tration. Pennsyl Med 75: 47-49, 1972.
`32. Breedveld FC, Dijkmans BAC and Mattie H,
`Minocycline treatment for rheumatoid arthritis: An
`open dose finding study. J Rheumato/17: 43-46, 1990.
`33. Pott H-G, Wittenborg A and Junge-Hülsing G, Long-
`term antibiotic treatment in reactive arthritis. Lancet
`1: 245-246, 1988.
`34. Panayi GS and Clark B, Minocycline in the treatment
`of patients with Reiter's syndrome. Cl in Exp Rheumatal
`7: 100-101. 1989.
`35. Skinner M, Cathcart ES, Mills JA and Pinals RS,
`Tetracycline in the treatment of rheumatoid arthritis.
`Arthritis Rheum 14: 727-732, 1971.
`36. Sugatani J, Saito K and Honjo I, In vitro actions of
`some antibiolies on phospholipases. J Antibiot (Tokyo)
`32: 734-739, 1979.
`37. Pruzanski W, Vadas P and Fornasier V, lnflammatory
`effect of
`intradermal administration of soluble
`phospholipase A 2 in rabbits. J Invest Dermatal 87:
`380-383, 1986.
`38. Edelson JD, Vadas P, Villar J, Mullen JBM
`and Pruzanski W, Acute Jung injury induced by
`phospholipase A 2• Structural and functional changes.
`Am Rev Respir Dis 143: 1102-1109, 1991.
`39. Jain MK, Rogers J, Johagirdar DV, Marecek JF and
`Ramirez F, Kinetics of
`interfacial catalysis of
`phospholipase A 2 in intravesical scooting mode and
`heterofusion of anionic and zwitterionic vesicles.
`Biochim Biophys Acta 860: 435-447, 1986.
`40. Flower RJ, Wood JN and ParenteL, Macrocortin and
`the mechanism of action of the glucocorticoids. Adv
`Inflammation Res 7: 61-70, 1984.
`41. Jain MK and Berg OG, The kinetics of interfacial
`catalysis by phospholipase A 2 and regulation of
`interfacial activation: Hopping versus scooting. Biochim
`Biophys Acta 1002: 127-156, 1989.
`42. Goldhammer AR, Jain MK and Cordes EH,
`Phospholipases. 111. Effects of ionic surfactants on the
`
`Exh. 1023
`
`

`
`1170
`
`phospholipase-catalyzed hydrolysis of unsonicated egg
`Iecithin Iiposomes. J Membr Bio/ 23: 293-304, 1975.
`43. Vishwanath BS, Kini RM and Gowda TV, Charac·
`terization of three edema-inducing phospholipase A 2
`enzymes from habu (Trimeresurusflavoviridis) venom
`and their interaction with the alkaloid aristolochic acid.
`Toxicon 25: 501-515, 1987.
`44. Nevalainen TJ, The rote of phospholipase A in acute
`pancreatitis. Scand J Gastroenteroll