Mediators of Inflammation, 9, 223–228 (2000)
`
`Effect of ricinoleic acid in acute and
`subchronic experimental models of
`inflammation
`
`Celme Vieira1*, Stefano Evangelista2,CA,
`Rocco Cirillo1, Annalisa Lippi1, Carlo Alberto Maggi1
`and Stefano Manzini2
`
`Departments of 1Pharmacology and 2Preclinical
`Development, Menarini Ricerche spa, Pomezia
`(Roma) and Firenze, Italy
`
`CACorresponding Author
`Tel: +39 055 5680519
`Fax: +39 055 5680510
`E-mail: sevangelista@menarini-ricerche.i t
`
`* C.V. was a visiting scientist from Faculdade de Medicine
`de Riber`ao Preto – U.S.P. (Brazil).
`
`Research Paper
`
`OBSERVATIO N AL studies indicate that topical application
`of ricinoleic acid (RA), the main component of castor
`oil, exerts remarkable analgesic and anti-inflamma-
`tory effects. Pharmacological characterization has
`shown similarities between the effects of RA and those
`of capsaicin, suggesting a potential interaction of this
`drug on sensory neuropeptide-mediated neurogenic
`inflammation. The aim of this study was to assess RA
`anti-inflammatory activities in comparison with cap-
`saicin in several models of acute and subchronic
`inflammation. The acute inflammation was induced by
`intradermal injection of carrageenan in the mouse or
`by histamine in the guinea-pig eyelid. In either
`experiment, the extent of the oedema thickness was
`measured. Subchronic oedema was induced by com-
`plete Freund’s adjuvant injection in the ventral right
`paw of mice. Tissue substance P (SP) was measured in
`the carrageenan experiments by radioimmunoassay
`(RIA). It was found that the acute topical application of
`RA (0.9 mg/mouse) or capsaicin (0.09 mg/mouse) sig-
`nificantly increased the mouse paw oedema induced
`by carrageenan, while an 8-day repeated topical
`treatment with the same doses of both compounds
`resulted in a marked inhibition of carrageenan-
`induced paw oedema matched by a reduction in SP
`tissue levels. Similar effects were found against hista-
`mine-induced eyelid oedema in guinea-pigs after acute
`or repeated application of RA or capsaicin. RA and
`capsaicin given for 1Ð3 weeks reduced the established
`oedema induced by Freund’s adjuvant, a subchronic
`model of inflammation, particularly if given by the
`intradermal route. Either in mouse paw or in guinea-
`pig eyelid, capsaicin but not RA by itself produced a
`slight hyperemia and activation of a behavioural
`response (e.g. scratching of the eyelids). On the basis
`of the present results, RA may be seen as a new
`capsaicin-like, non-pungent anti-inflammatory agent
`suitable for peripheral application.
`
`Key words: Capsaicin, Carrageenan, Castor oil, Inflamma-
`tion, Ricinoleic acid, Substance P (SP)
`
`Introduction
`
`Ricinoleic acid (RA; [R-(Z)]–12-hydroxy–9-octadece-
`noic acid) is the main component of castor oil,
`accounting for about 90% of the total. Preliminary
`experiments in our laboratories indicated that RA,
`known for its laxative properties,1 could have a pro-
`or anti-inflammatory action following acute or repeti-
`tive local application, respectively. These activities
`closely resemble those previously described for cap-
`saicin or capsaicinoid analogues called vanilloids
`(olvanil, resiniferatoxin, scutigeral).2 – 4 On the other
`hand, part of the RA chemical structure has been used
`as a basis for the development of novel capsaicin-like
`compounds.5 The cloning of the vanilloid receptor/
`ion channel, termed VR1 is very recent6 and it has
`
`been shown that VR1 is activated by capsaicin and a
`number of other capsaicin-like drugs (vanilloid recep-
`tor agonists). Vanilloids specifically act through the
`activation and then the desensitization of a subset of
`primary afferent nerves involved in the genesis of
`neurogenic inflammation.7 Notably their acute pro-
`inflammatory effect is due to the stimulation of the
`release of sensory neuropeptides from peripheral
`axons of these nerves. In a subsequent phase there is a
`desensitization of these fibres and, if the dose and the
`duration of exposure to the agonists are appropriate,
`a depletion of the neuropeptide content of these
`nerves occurs.8 Desensitization and/or neuropeptide
`depletion determines an anti-inflammatory effect
`blocking the induction of neurogenic inflammation
`by endogenous or exogenous inflammatory stimuli.7
`
`ISSN 0962-9351 print/ISSN 1466-1861 online/00/050223-06 © 2000 Taylor & Francis Ltd
`DOI: 10.1080/09629350020025737
`
`223
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` EXHIBIT 1019
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`

`
`C. Vieira et al.
`
`Noteworthy is the fact that we found that RA is
`devoid of the pungent properties typical of capsaicin
`and that it does not exert any hyperalgesic effect
`towards heat and chemical nociceptive stimuli (Vieira
`et al., submitted – ref. 9). These similarities and the
`differences (lack of pungent properties) with capsai-
`cin render RA a promising analgesic and anti-inflam-
`matory agent via its local application. Long-term
`topical capsaicin application in rats has been shown
`to produce a reversible impairment of the primary
`afferent fibres in the skin.10
`In view of the above, we have assessed the effect of
`acute or repetitive RA topical treatment in several
`models of both acute and subchronic inflammation.
`
`Materials and methods
`
`Animals
`
`Male albino Dunkin–Hartley guinea-pigs (250–350 g)
`from Charles River (Calco, Italy) and male Swiss mice
`(20–25 g) from Harlan Nossan (Correzzana, Milan,
`Italy) were used. The animals were fed with a standard
`diet and water ad libitum. The experiments were
`performed in respect of the Principles of Laboratory
`Animal Care (NIH publication no. 85–23, revised
`1985) and in accordance with the Italian Health
`Ministry guidelines for the care and use of experi-
`mental animals.
`
`Oedema induction in the paw of mice
`
`Paw oedema was induced by intradermal (i.d.)
`injection in the right paw of 300 mg/0.04 ml of
`carrageenan. The thickness of the paw was measured
`in mm using a micro-calibrator. In acute experiments,
`the paw thickness was measured before and 1, 2, 3, 4,
`5 and 6 h after the carrageenan injection. In chronic
`experiments, the progression of oedema was assessed
`on the first day and the eighth day each hour during
`the 6-h period following the first or second carragee-
`nan injection, respectively. Single or repeated (8 days)
`topical applications of vehicle (peanut oil), RA
`(0.9 mg/mouse) or capsaicin (0.09 mg/mouse), at
`doses chosen in preliminary experiments, were
`applied on the ventral surface of the right paw. The
`first and the last application were made 30 min before
`the injection of carrageenan.
`In other experiments, the repeated topical applica-
`tion (8 days) of vehicle (peanut oil), RA (0.9 mg/
`mouse) or capsaicin (0.09 mg/mouse) or the repeated
`(4 days) i.d. administration of RA potassium salt
`(0.03 mg/mouse) or capsaicin (0.003 mg/mouse) on
`the ventral surface of the right paw was tested in the
`presence of complete Freund’s adjuvant (30 ml),
`injected in the hind-paw on the first day. The paw
`volume was monitored once a week for 3 weeks as
`described above.
`
`224
`
`Mediators of Inflammation · Vol 9 · 2000
`
`Eyelid oedema induction in guinea-pigs
`
`Eyelid oedema was induced by i.d. injection of
`10 mg/0.1 ml of histamine in the right superior eyelid
`of animals under a short anaesthesia by diethyl ether.
`The thickness of the eyelid was measured in mm using
`ophthalmic micro-callipers (Dixey, UK). In acute
`experiments, the eyelid thickness was measured
`before and 1, 2, 3, 4, 5 and 6 h after the histamine
`injection or maximal applicable doses of RA (100 mg/
`guinea-pig) and capsaicin (10 mg/guinea-pig).
`In subchronic experiments, the animals were
`treated topically for 8 or 22 days with vehicle (peanut
`oil), RA (0.9 mg/guinea-pig) or capsaicin (0.09 mg/
`guinea-pig), and the progression of the oedema was
`assessed on the first day during 6 h following the first
`histamine injection and then the eighth and the 22nd
`day each hour for a 6-h period after the second
`injection of histamine.
`
`Radioimmunoassay (RIA)
`
`For the tissue peptide measurements, the paws were
`rapidly removed and weighed. The samples were
`extracted with 3 ml of 2 N acetic acid at 95°C for
`15 min. They were then homogenized and centrifuged
`at 10 000 g for 30 min at 4°C, the supernatants were
`freeze-dried and stored at –20°C until their content of
`substance P-like immunoreactivity (SP-LI) was deter-
`mined by RIA. Lyophilized samples were reconstituted
`in 10 ml of 50 mM phosphate buffer (pH 7.4),
`containing 0.3% bovine serum albumin and 10 mM
`ethylene diamine tetraacetic acid (EDTA). The RIA
`assay for SP-LI was based on scintillation proximity
`assay technology, as previously described.11 The
`incubation mixture was composed of 100 ml of the
`reconstituted sample (diluted 1:30), 100 ml of
`125I-labelled tracer (about 6000 cpm), 100 ml of diluted
`antiserum (1:100 000 for SP) and 50 ml of scintillation
`proximity assay protein A reagent. After an overnight
`incubation at room temperature under gentle agi-
`tation, the samples were counted in a b-scintillation
`counter (2200CA, Canberra-Packard, USA).
`
`Chemicals
`
`RA (99% pure; Sigma) was diluted with peanut oil
`(Sigma); its soluble potassium salt (MEN 11938 or
`potassium [R-(Z)]–12-hydroxy–9-octadecenoate from
`Chemistry Department, Menarini Ricerche spa.,
`Pomezia, Italy) was used for i.d. treatment dissolved in
`saline. Capsaicin (Serva) was dissolved in 10% etha-
`nol, 10% tween 80 and 80% saline solution and
`applied locally in peanut oil. Histamine (Sigma) was
`dissolved in saline, carrageenan (type II, Sigma) in
`sterile phosphate buffer solution (PBS; Sigma). Com-
`plete Freund’s adjuvant was purchased from Sigma.
`[125I]- Bolton Hunter SP and scintillation proximity
`
`

`
`assay reagents were obtained from Amersham. The
`rabbit anti-SP serum (RAS 7451, cross-reactivity < 5%
`with SP 7–11, < 0.01% with neurokinin A (NKA) was
`from Peninsula Laboratories.
`
`Statistical analysis
`
`The data are expressed as means ± standard error of the
`mean (SEM). The statistical significance between
`groups was assessed using one-way analysis of variance
`followed by Bonferroni’s test. The Mann–Whitney U
`test was used for the “in vitro” experiments.
`
`Results
`
`Effects of acute and subchronic topical
`treatment with RA and capsaicin on
`carrageenan-induced paw oedema in mice
`
`As shown in Fig. 1, paw oedema induced by
`carrageenan was markedly increased by the topical
`application of RA (0.9 mg/mouse) or capsaicin
`(0.09 mg/mouse). The enhancement by RA or capsai-
`cin of the carrageenan-induced oedema reached its
`maximum between the second and fourth hours and
`then decreased over time, but still remained sig-
`nificant at the sixth hour as compared with the
`vehicle-treated paws (Fig. 1). No enhancement of
`carrageenan-induced oedema was detected with the
`vehicle (n = 5) or with a solution at the same pH of
`100 mg of RA (n = 5). Topical administration of
`vehicle, RA up to 10 mg/10 ml or capsaicin up to
`1 mg/10 ml did not produce per se any oedema in the
`paws of the mice.
`The treatment with RA (0.9 mg/mouse) for 8 days
`did not result per se in any oedematous effect or
`hyperemic reaction. Conversely, during the first days
`
`FIG. 1. Effects of vehicle, ricinoleic acid (RA, 0.9 mg/mouse)
`or capsaicin (Cps, 0.09 mg/mouse) on carrageenan-induced
`paw oedema expressed in mm. Ricinoleic acid, capsaicin and
`the vehicle were applied topically 30 min before carrageenan
`injection. Data are mean ± standard error (SE). n = 5–8 for
`each group. **p < 0.01; ***p < 0.001 versus vehicle group.
`
`Anti-inflammatory activity of ricinoleic acid
`
`FIG. 2. Carrageenan-induced oedema of mouse paw. Effects
`of 8-day treatment with ricinoleic acid (RA, 0.9 mg/mouse) or
`capsaicin (Cps, 0.09 mg/mouse). Data are mean ± standard
`error (SE). n = 5–8 for each group. **p < 0.01; ***p < 0.001
`versus vehicle group.
`
`of treatment, capsaicin (0.09 mg/mouse) produced a
`slight hyperemic reaction that disappeared within
`1–2 h from treatment. On the eighth day, the carragee-
`nan-induced oedema was virtually abolished in RA or
`capsaicin pre-treated animals (Fig. 2). Thus, the mice
`were unable to produce any oedema in response to
`carrageenan.
`
`Effect of topical and i.d. treatment with RA or
`capsaicin on Freund’s adjuvant-induced paw
`oedema in mice
`
`The intraplantar injection of Freund’s adjuvant in one
`of the hind-paws leads to localized, unilateral inflam-
`mation. As shown in Table 1, the paw oedema induced
`by Freund’s adjuvant peaked at the first 2 weeks after
`the induction and then declined. Topical treatment
`with RA for 8 days was effective in reducing the
`oedema formation at 1 week observation time. On the
`second and third weeks after the oedema induction,
`RA did not affect the increase in thickness of the paws
`as compared with the vehicle-treated group. Capsai-
`cin in the applied dose did not induce significant
`inhibition in the oedema evoked by Freund’s adjuvant
`(Table 1).
`Conversely, the i.d. administration of the potassium
`salt of RA (MEN 11938) was able to exert a marked
`anti-inflammatory effect that lasted for 3 weeks (Table
`1). The values of reduction in oedema formation were
`68, 67 and 85% of control at 1, 2 and 3 weeks after
`Freund’s adjuvant injection. Similarly, i.d. capsaicin
`significantly affected oedema formation, the sig-
`nificance of the effect lasting only for 2 weeks; the
`oedema was reduced by 50, 57 and 51% at 1, 2 and 3
`weeks after its induction (Table 1). The i.d. injection
`of capsaicin (0.003 mg/mouse), but not RA (0.03 mg/
`mouse), produced nociceptive reactions during the
`first 2–3 days of treatment.
`
`Mediators of Inflammation · Vol 9 · 2000
`
`225
`
`

`
`C. Vieira et al.
`
`Table 1. Paw oedema induced by Freund’s adjuvant in mice. Animals were treated by local application of ricinoleic acid (RA;
`900 mg/mouse) or capsaicin (90 mg/mouse) for 8 days or intradermally administered for 4 days with MEN 11938 (30 mg/mouse),
`the potassium salt of RA, or capsaicin (3 mg/mouse). The paw oedema was measured once a week after the injection of Freund’s
`adjuvant
`
`Local application for 8 days
`Vehicle
`RA (900 mg/mouse)
`Capsaicin (90 mg/mouse)
`Intradermal treatment for 4 days
`Vehicle
`MEN 11938 (30 mg/mouse)
`Capsaicin (3 mg/mouse)
`
`First week
`
`Second week
`
`Third week
`
`1.39 ± 0.10
`0.82 ± 0.10**
`0.87 ± 0.13
`
`1.19 ± 0.09
`0.38 ± 0.10**
`0.59 ± 0.09**
`
`0.79 ± 0.05
`0.61 ± 0.13
`0.70 ± 0.08
`
`1.20 ± 0.12
`0.40 ± 0.13**
`0.51 ± 0.08**
`
`0.60 ± 0.10
`0.40 ± 0.07
`0.48 ± 0.09
`
`0.63 ± 0.10
`0.09 ± 0.13*
`0.31 ± 0.15
`
`Data are mean ± standard error (SE). n = 8–10 for each group. *p < 0.05; **p < 0.01 versus respective vehicle group.
`
`Effect of topical application of RA or capsaicin
`on guinea-pig eyelid
`
`The topical administration of capsaicin resulted in a
`local hyperemic response and activation of behav-
`ioural defensive responses including scratching of the
`eyelid. Neither hyperemia nor aversive behaviour was
`observed following RA topical treatment. A slight
`eyelid oedema with the peak at 2 h following applica-
`tion with high doses of RA (100 mg/guinea-pig, 0.23 ±
`0.01 mm) or capsaicin (10 mg/guinea-pig, 0.35 ±
`0.005 mm) alone was observed.
`
`Effects of acute and subchronic topical
`treatment with RA and capsaicin on eyelid
`oedema induced by histamine in guinea-pig
`
`The injection of histamine in the right eyelid rapidly
`induced an oedema that reached the maximum
`thickness at the first hour (Fig. 3). During i.d. injection,
`
`the animals showed aversive response behaviour, but
`no scratching response on the eyelid was usually
`observed. As shown in Fig. 3, the oedema was
`increased by the topical application of RA (0.9 mg/
`guinea-pig) and capsaicin (0.09 mg/guinea-pig).
`The topical application of RA (0.9 mg/guinea-pig)
`for 8 days did not result per se in any oedematous
`effect or hyperemic reaction. Instead, capsaicin
`(0.09 mg/guinea-pig) produced hyperemic and pain-
`ful reactions that disappeared 1–2 h after the treat-
`ment. The intensity of these responses gradually
`decreased starting from 2–3 days and was almost
`abolished at the end of the treatment. As shown in Fig.
`4, the eyelid oedema induced by histamine was
`markedly reduced after 8 days of topical treatment
`with RA (0.9 mg/guinea-pig) or capsaicin (0.09 mg/
`guinea-pig). A similar profile of oedema reduction was
`also found after 3 weeks of topical treatment with RA
`(0.9 mg/guinea-pig) or capsaicin (0.09 mg/guinea-
`pig), the values of eyelid oedema at the peak (first
`hour) being: 1.37 ± 0.06, 0.70 ± 0.05 and 0.85 ±
`
`FIG. 3. Histamine-induced oedema of guinea-pig eyelid.
`Effect of vehicle, ricinoleic acid (RA, 0.9 mg/guinea-pig) and
`capsaicin (Cps, 0.09 mg/guinea-pig). Ricinoleic acid, capsai-
`cin and the vehicle were administered topically 30 min
`before histamine injection. Data are mean ± standard error
`(SE). n = 5–8 for each group. *p < 0.05; **p < 0.01;
`***p < 0.001 versus vehicle group.
`
`226
`
`Mediators of Inflammation · Vol 9 · 2000
`
`FIG. 4. Histamine-induced oedema of guinea-pig eyelid.
`Effect of 8-day treatment with ricinoleic acid (RA, 0.9 mg/
`guinea-pig) or capsaicin (Cps, 0.09 mg/guinea-pig). Data are
`mean ± standard error (SE). n = 5–8 for each group. *p < 0.05;
`**p < 0.01; ***p < 0.001 versus vehicle group.
`
`

`
`for vehicle-, RA- and capsaicin-treated
`0.05 mm
`groups, respectively.
`
`Effect of repeated treatment of RA and
`capsaicin on mouse paw SP levels
`
`The levels of SP were not different in carrageenan-
`inflamed and non-inflamed paws (1.30 ± 0.28 and
`1.09 ± 0.34 pmol/g tissue). The repeated (8 days) local
`administration of RA, at doses able to produce marked
`anti-inflammatory effects (see above), reduced the SP
`content by 58% in the inflamed paw of mice (0.54 ±
`0.12 pmol/g tissue; p < 0.05 as compared with the
`inflamed paw treated with vehicle). Repeated topical
`capsaicin administration did not significantly reduce
`SP paw levels (0.73 ± 0.14 pmol/g tissue).
`
`Discussion
`
`The main result of this study is that RA topically
`administered for 8 days exerts a marked anti-inflam-
`matory effect in several models of inflammation. The
`anti-inflammatory effect was comparable with that
`obtained with a 10-fold lower dose of capsaicin.
`RA, when administered acutely, produced an addi-
`tive inflammatory effect on carrageenan- or histamine-
`induced oedema in mice or guinea-pigs, respectively,
`but at a variance with capsaicin, acute topical RA
`administration did not produce any nociceptive or
`hyperemic reaction. Capsaicin is known to induce
`neurogenic inflammation (e.g. vasodilatation, protein
`extravasation and oedema) through an acute release of
`vasoactive peptides, such as SP, neurokinin A and
`calcitonin gene-related peptide (CGRP) from sensory
`nerve terminals.12–14 It is noteworthy that capsaicin-
`induced oedema or plasma protein extravasation is
`almost completely absent in genetically tachykinin-
`deficient mice.15 A release of tachykinins from sensory
`terminal nerves leading to an activation of their
`receptors present on the skin surface may explain the
`acute additive inflammatory effect of RA in our
`experimentally induced oedemas.
`Although carrageenan injection leads to the genera-
`tion of a range of inflammatory mediators, the
`noticeable reduction of paw oedema thickness
`induced by the NK1 receptor antagonist CP 96.345 in
`rats16 and the almost complete disappearance of paw
`oedema in mice induced by the other NK1 receptor
`antagonist CP 122,721 (Vieira et al., unpublished
`observations) support the involvement of SP in this
`inflammatory oedema. Furthermore, in NK1 receptor
`knockout mice the carrageenan oedema was sig-
`nificantly reduced.17
`As concerns the anti-inflammatory effect of RA
`resulting from an 8-day treatment, a desensitization
`action on peripheral nerve endings, already described
`for capsaicin, may explain, at least in part, such a
`phenomenon. In keeping with the above, the noci-
`
`Anti-inflammatory activity of ricinoleic acid
`
`ceptive reactions observed during the first days only
`after capsaicin application or i.d. injection disap-
`peared on the second or third day of treatment. It is
`known that repeated exposure to capsaicin results in
`a desensitization of chemosensitive sensory affer-
`ents,18 depletion of sensory peptides such as SP19,20
`and a loss of local tissue reactions upon chemical
`irritation. 20 Desensitization of a flare reaction induced
`by a prolonged local exposure to capsaicin, i.d.
`bradykinin and histamine has been described in
`human skin.21 The anti-inflammatory effect of RA and
`capsaicin found in our experimental conditions was
`more marked towards carrageenan than histamine;
`different mechanisms of action of these inflammatory
`agents can be responsible for these differences in
`desensitization. Histamine has been described to
`excite small diameter afferent neurones and evoke the
`release of neuropeptides from vasoactive local nerve
`endings.22 It is interesting to note that paw oedema
`induced by histamine was abolished in capsaicin-
`denervated rats.23 Our findings in the guinea-pig
`eyelid show that the histamine-induced oedema is
`also dependent on other mediators.
`On the other hand, carrageenan paw oedema was
`blunted by the local application of RA or capsaicin and
`it was matched by a significant reduction of SP tissue
`levels at least after the local application of RA.
`In contrast to what happens in acute inflammation,
`in subchronic inflammation induced by Freund’s
`adjuvant, RA was barely more effective than capsaicin
`in reducing oedema formation. Freund’s adjuvant is
`able to produce a relatively stable inflammation that
`was decreased partially by local application of RA and
`was markedly affected by i.d. injection of both RA and
`capsaicin. The more effective counteraction of the
`oedema induced by i.d. versus topical administration
`of both compounds is probably due to a stronger
`desensitization induced by this route. On the other
`hand, Freund’s adjuvant-induced chronically inflamed
`tissue in the rat has been reported to be associated
`with changes in SP content in nerves supplying the
`inflamed paws24 and systemic capsaicin administra-
`tion reduced the oedema formation concomitantly
`with the reduction of SP in nerve tissues.25
`The relevance of the data presented should be
`associated with the observation that RA does not
`possess the pungent and painful effect of capsaicin
`but maintains its anti-inflammatory activities. Topical
`capsaicin has shown therapeutic potential in the
`treatment of cutaneous disorders such as post-
`herpetic neuralgia, painful diabetic neuropathy, pruri-
`tus, psoriasis, post-mastectomy pain syndrome, vulvar
`vestibulitis,26 – 29 but its utility appears to be limited
`primarily by its irritant properties. RA has the
`potential to be a new capsaicin-like substance
`endowed with anti-inflammatory effects on several
`models of inflammation without the pungent charac-
`teristics of capsaicin.
`
`Mediators of Inflammation · Vol 9 · 2000
`
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`
`

`
`C. Vieira et al.
`
`ACKNOWLEDGEMENTS. We would like to thank Dr R. Terracciano for the
`development and synthesis of MEN 11938, Mrs L. Mylander for revision of
`the English text and Ms C. Azzurrini for her secretarial assistance and Dr G.
`B. Oliveira from the Faculty of Medical Sciences of Ribeirao Preto, USP, Brazil
`and CNPq Brazil.
`
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`Received 6 August 2000;
`accepted 8 September 2000
`
`228
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`Mediators of Inflammation · Vol 9 · 2000