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Anti-inflammatory properties of convolutamydine A and two structural analogues
Life Sciences 116 (2014) 16–24
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Anti-inflammatory properties of convolutamydine A and two structural analogues Patricia D. Fernandes a,⁎, Renata S. Zardo a,1, Gabriella S.M. Figueiredo a,1, Bárbara V. Silva b, Angelo C. Pinto b a Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas, Laboratório de Farmacologia da Dor e da Inflamação, Av. Carlos Chagas Filho, 373. Prédio do CCS, bloco J, sala 10, 21941-902 Rio de Janeiro, Brazil b Universidade Federal do Rio de Janeiro, Instituto de Química, Rio de Janeiro, Brazil
a r t i c l e
i n f o
Article history: Received 18 June 2014 Accepted 29 August 2014 Available online 6 September 2014 Keywords: Convolutamydine A Isatin Inflammation Anti-inflammatory drugs
a b s t r a c t Aims: Convolutamydine A is an oxindole alkaloid that can be isolated from a marine bryozoan. Due to the variety of biological effects, two analogues were synthesized and their anti-inflammatory properties were evaluated. Main methods: The anti-inflammatory effects of convolutamydine A and its analogues (ISA003 and ISA147) were investigated in a formalin-induced licking behaviour model, where mice received an intraplantar injection of formalin and their licking behaviour was evaluated for 30 min. Additionally, inflammatory parameters were evaluated in a subcutaneous air pouch (SAP) model of carrageenan-induced inflammation. Exudates were collected for leukocyte counts; measurement of protein, prostaglandin E2 (PGE2) and cytokines by ELISA; and analysis of nitric oxide (NO) using a nitrate conversion protocol. Cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) from RAW 264.7 cells were quantified by immunoblotting. Key findings: Convolutamydine A and its two analogues inhibited the formalin-induced licking response at doses as low as 0.01 mg/kg. An inhibitory effect was also observed on leukocyte migration and the production of NO, PGE2 and cytokines (IL-6 and TNF-α). The reduction in inflammatory parameters did not appear to be correlated with a direct reduction in the number of cells in the SAP, because a reduction in NO and PGE2 production by cultured macrophages was observed in addition to the inhibition of iNOS and COX2 enzyme expression. Significance: These results indicate that convolutamydine A and its two analogues have significant antiinflammatory effects. These substances can be improved to generate lead compounds for the synthesis of new anti-inflammatory drugs. © 2014 Elsevier Inc. All rights reserved.
Introduction Inflammation is a stereotyped response of living organisms to possible harmful stimuli (Di Rosa, 1972), in vascular tissues. This physiologic event has a protective role aiming at the removal of the offending agent by activating a cascade of events and production of inflammatory mediators. The ultimate goal is the destruction of the offending agent and tissue restoration. The current pharmacological therapies for inflammation consist primarily of non-steroidal anti-inflammatory drugs (NSAIDs). This class has a long history of clinical use and major deficiencies, and many of the drug discovery efforts in the area of inflammation have focused on the incremental improvement of this class of compounds. However, adverse side-effects (i.e., ulcers and bleeding) have limited their use. Many new molecules have been synthesized with the aim of seeking those with reduced side effects (reviewed by Carter et al., 2014).
⁎ Corresponding author. Tel.: +55 21 39386442. E-mail address: [email protected] (P.D. Fernandes). 1 Tel.: +55 21 39386442.
http://dx.doi.org/10.1016/j.lfs.2014.08.019 0024-3205/© 2014 Elsevier Inc. All rights reserved.
Amathia convoluta is a marine bryozoan species from which the oxindole alkaloid named Convolutamydine A was isolated. Because convolutamydine is only isolated from this Bryozoan in small amounts and due to its promising biological activities, it has been synthesized by several research groups (Kamano et al., 1995; Garden et al., 1997; Luppi et al., 2006; Cravotto et al., 2006; Silva et al., 2008). Garden et al. (1997) were the first group to propose the synthesis of a racemic mixture of convolutamydine A from isatin, a small, versatile, and widely applicable pharmacological molecule (da Silva et al., 2001; Silva, 2013; Vine et al., 2009). Convolutamydine A promoted alterations in HL-60 cell line (Kamano et al., 1995). In addition to convolutamydine A, the literature includes reports of other 3-substituted-3-hydroxyindolin-2-ones with applications in medicinal chemistry, such as active potent growth hormone secretagogues (Tokunaga et al., 2001), potassium channel openers (Hewawasam et al., 2002), anticonvulsants (Popp et al., 1980) and antinociceptives (Figueiredo et al., 2013). In our continuous search for bioactive substances, the purpose of the present work was to investigate the anti-inflammatory effects of convolutamydine A and its analogues in animal models of inflammation.
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Materials and methods Animals Male BALB/C mice (20–25 g), from our own animal facility, were housed in a room with constant temperature (22 ± 2 °C) and 12-h light/dark cycle and fed standard rodent chow and water. Before (12 h) the assays animals received only water in order to avoid food interference with compound absorption. Research protocol was approved by the Ethical Committee for Animal Research (Biomedical Science Institute/UFRJ; approval number ICBDFBC-015). General Acetylsalicylic acid (ASA) and carrageenan were purchased from Sigma (St. Louis, MO, USA), and acetic acid was purchased from Merck, Inc. All drugs were dissolved in phosphate buffer saline (PBS) just before use. ASA (200 mg/kg, p.o.) and dexamethasone (5 mg/kg, i.p.) were used as reference drugs. These doses were based on previous studies (Pinheiro et al., 2013) and calculated as ED50 (dose that reduced 50% the effect). Vehicle was used as negative control group.
17
Table 1 Name and substituent of each compound. Compound
Substituent (R)
Convolutamydine A ISA147 ISA003
4,6-Br 5-Br H
Formalin-induced licking behaviour test The licking behaviour was examined immediately after formalin hind paw injection. The procedure was similar to the method described by Hunskaar and Hole (1987). Animals received 20 μl of formalin (2.5% v/v) into the dorsal surface of the left hind paw. Animals were individually observed and the time that they spent licking the formalin-injected paw recorded. The nociceptive response develops in two phases: the first 5 min after formalin injection (first phase, neurogenic pain response); the second 15–30 min after formalin injection (second phase, inflammatory pain response). Mice were divided in the following groups: vehicle, convolutamydine A, analogues (0.01, 0.1 or 1 mg/kg, p.o.), and acetylsalicylic acid (100 mg/kg, i.p.) treated groups 1 h before formalin injection.
Synthesis of convolutamydine A and its analogues Subcutaneous air pouch (SAP) Isatins were suspended in acetone, and drops of diethylamine were added at room temperature, leading to convolutamydine A, ISA003 or ISA147 (Fig. 1 and Table 1). The structures of these compounds were determined by 1H-NMR and 13C-NMR as described by Garden et al. (1997). Administration of convolutamydine A and analogues A stock solution (200 mg/ml in dimethylsulphoxide, DMSO) was prepared with Convolutamydine A, ISA003 and ISA147. The doses of 0.1, 1 and 10 mg/kg (in tween 80) were orally administered to each animal. Vehicle group was composed by animals treated with DMSO/Tween 80. The final concentration of DMSO/tween 80 had no effect per se. Acute toxicity To evaluate a possible toxic effect we adapted the method used by Lorke (1983). Briefly, animals were treated with 150 mg/kg of convolutamydine A or its analogues. During 5 consecutive days several parameters (i.e., convulsion, sedation, respiration, and food and water intake) were observed. After five days the animals were euthanized with an overdose of ketamine/xilazine (150 mg/10 mg/kg). The stomachs of the animals were removed and opened to observe any signs of hyperemia and the presence or absence of ulcer.
Animals were used as described by Raymundo et al. (2011). After subcutaneous injection of 10 ml of sterile air in the dorsal region in three alternate days, an injection of sterile carrageenan suspension (1%; 1 ml) was done in the SAP formed. Mice were divided in the following groups: vehicle, convolutamydine A, analogues (0.1, 1 or 10 mg/kg, p.o.), and dexamethasone (5 mg/kg, i.p.) treated groups 1 h before carrageenan injection. Another vehicle-treated group was used in mice that received PBS (phosphate buffer saline, 1 ml) in SAP. After 24 h all groups were sacrificed, SAP was washed with 1 ml of sterile PBS and exudates collected. The total cell count was determined in a CellPocH-100iV Diff (Sysmex) haematology analyser. The exudates were also centrifuged (5000 ×g, 10 min, 4 °C) and aliquots of the supernatants were stored at −20 °C until the assays. IL-6, TNF-α,PGE2 and protein measurements The cytokines (TNF-α and IL-6) and prostaglandin E2 (PGE2) levels were quantified using enzyme-linked immunosorbent assays (ELISAs) according to the protocol supplied by the manufacturer (B&D, USA). BCA method (BCA™ Protein Assay Kit, Pierce) was used to measure protein content in exudates supernatants. Cell culture RAW 264.7 mouse macrophage cell line (ATCC TIB-71) was maintained in RPMI 1640 medium (with 10% foetal bovine serum, 2 mM glutamine and 15 mM HEPES) at 37 °C and 5% CO2. At confluence stage cells were scraped off and seeded in 96- or 12-well culture plates (2 × 106 cells/ml). Cytotoxicity assays by MTT
Fig. 1. General structure of convolutamydine A and analogues.
MTT assay was carried out as described by Denizot and Lang (1986). RAW 264.7 cells were treated with or without convolutamydine A or its analogues (10, 30 or 100 μM). After 24 h the medium was changed by 100 μl of RPMI with 0.5 mg/ml MTT and further incubated for 1 h at 37 °C, at 5% CO2. The medium was withdrawn and 100 μl of DMSO was added to the cells to dissolve the formazan. The absorbance was analysed using a FlexStation microplate reader (Molecular Devices, USA) at an absorbance wavelength of 570 nm. The control groups
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(1-Naphthyl) ethylenediamine dihydrochloride, 10% H3PO4) (Green et al., 1982). FlexStation microplate reader (Molecular Devices, USA) was used to measure the absorbance at 540 nm. Nitrite concentration was measured by comparison with a standard curve of sodium nitrite.
were composed by cells incubated with medium and was considered as 100% viable cells. Percentage of viable cells was used to express the results. Nitrate and nitrite measurement
Nitric oxide-trapping capacity of convolutamydine A and its derivatives To evaluate the production of NO in cell culture, the nitrite accumulated in the cell culture supernatants was measured according to the Griess reaction (Green et al., 1982). LPS (1 μg/ml)-activated cells incubated with convolutamydine A or analogues (10, 30 or 100 μM) were further incubated for 24 h, after which cell culture supernatant was collected to measure the nitrite concentration. Nitrate accumulated in SAP exudates was measured according to the method described by Bartholomew (1984) and adapted by Raymundo et al. (2011). The nitrite concentrations obtained after nitrate conversion (cited above) or from cell culture supernatants were measured by mixing equal parts of sample and Griess Reagent (1% sulphanilamide, 0.1% N-
Detection of iNOS and COX-2 enzyme expression Immunoblots were carried out as described previously (Oliveira et al., 2006). Briefly, RAW 264.7 cells were activated with LPS (1 μg/ml)
Convolutamydine A
1st phase
2 nd phase
300
* *
35
200
*
*
100
0
0
1st phase
ISA003
2 nd phase
400
300
* 35
200
*
*
100
0
70
Licking (seconds)
Licking (seconds)
70
0
1st phase
2
nd
ISA147
phase
400
300
35
200
*
*
100
kg
kg 1
m
g/
g/
g/ m
1 0.
01 0.
m
hi cl e AS A
Ve
kg
kg
g/ m
1
m
m
1 0.
01 0.
g/
g/
cl e AS A
hi Ve
kg
0 kg
0
Licking (seconds)
Licking (seconds)
400
Licking (seconds)
Licking (seconds)
70
To test the ability of convolutamydine A and its analogues to trap nitric oxide, a cell-free system was used as described in Matheus et al. (2007). Convolutamydine A and its analogues (at 100 μM) were incubated with 1 mM SNAP (S-nitroso-N-acetylpenicillamine). Nitrite accumulated in the supernatant after 6 h of incubation was measured using the Griess reaction (Green et al., 1982).
Fig. 2. The effects of convolutamydine A, ISA003 and ISA147 on the formalin-induced licking response in mice. Convolutamydine A or analogues (0.01, 0.1 or 1 mg/kg), acetylsalicylic acid (ASA, 100 mg/kg) or vehicle were orally administered to mice. Values mean ± S.D. (n = 6–10). *P b 0.05 compared to vehicle-treated mice using ANOVA followed by Bonferroni's test.
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and further incubated for 6 h with convolutamydine A or its analogues (3, 10 or 30 μM). Cells were lysed with cold lysis buffer (10% NP40, 150 mM NaCl, 10 mM Tris HCl pH 7.6, 2 mM PMSF, and 5 μM leupeptin). After determination of the protein concentration in the suspensions by the BCA method (BCA™ Protein Assay Kit, Pierce), the suspensions were boiled in application buffer (DTT 100 mM, Bromophenol Blue 0.1%). Aliquots of 30 μg of protein were submitted to electrophoresis in 10% polyacrylamide gel. Proteins were electrophoretically transferred onto nitrocellulose membranes. Membranes were incubated with anti-iNOS or anti-COX-2 (Cell signalling, USA) primary antibodies and further with secondary antibodies (anti-mouse IgG antibody conjugated to horseradish peroxidase). Proteins were detected using enhanced chemiluminescence (ECL) reagents and quantified using a ChemiDoc system (BioRad, USA).
Statistical analysis All results are presented as the mean ± S.D. Data was analysed in a GraphPad Prism™ (version 5.00, GraphPad Software Inc., San Diego, CA, U.S.A.) using analyses of variance (ANOVA) followed by Bonferroni's tests. P b 0.05 was considered to be significant.
Leukocytes (x 106 cell/ml)
100
PBS in SAP
19
Results Acute toxicity We first established whether convolutamydine A and its analogues have any toxic effects per se. Even when orally treated with 150 mg/kg of each substance mice did not show any type of behavioural changes. We also did not observe any lesions or bleeding on the stomach (data not shown). Anti-inflammatory activity The pretreatment of mice with 0.01, 0.1 or 1 mg/kg of each compound demonstrated that convolutamydine A and ISA003 significantly reduced the licking response to formalin injection at all doses tested, whereas ISA147 did not affect the response (Fig. 2). The inhibitory effect observed for convolutamydine A and its analogues is indicative of a possible anti-inflammatory effect. Accordingly, we evaluated convolutamydine A, ISA003 and ISA147 in another model of inflammation, the subcutaneous air pouch (SAP) model. The injection of carrageenan (1%) into the SAP produced a marked increase in the exudate volume and leukocyte number in the pouch, accompanied by an increase in almost seven-fold above the level of the control
Carrageenan in SAP
#
Conv. A
ISA003
ISA147
50
* *
* * *
* *
*
* *
0
500
PBS in SAP
#
400
Protein (μg/ml)
Carrageenan in SAP
300
*
* * *
200
* *
*
100
D
ex
Ve
hi
cl
e am V et eh ha ic so le ne 0. 1 m g 1 /kg m 10 g/k m g g/ kg 0. 1 m g 1 /kg m 10 g/k m g g/ kg 0. 1 m g 1 /kg m 10 g/k m g g/ kg
0
Fig. 3. Effects of convolutamydine A (Conv. A), ISA003 and ISA147 in leukocyte migration and protein leakage into the subcutaneous air pouch (SAP). The animals were pretreated with different doses of each substance, dexamethasone (5 mg/kg, i.p.) or vehicle 1 h before carrageenan (1%) injection into the SAP. Values mean ± S.D. (n = 6–10). #P b 0.05 compared to vehicle-treated mice receiving PBS in SAP and *p b 0.05 compared to vehicle-treated mice receiving carrageenan in SAP using ANOVA followed by Bonferroni's test.
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NO and PGE2 in vivo could simply be the result of a reduction in the number of cells that migrated into the SAP, we decided to carry out in vitro assays. We first investigated a possible direct cytotoxic effect of convolutamydine A, ISA003 and ISA147 on RAW 264.7 cells. Neither the compounds alone, at concentrations up to 100 μM, nor the compounds in the presence of LPS (1 μg/ml) affected cell viability (data not shown). Therefore, concentrations between 1 and 30 μM were used for the subsequent experiments. The 100 μM concentration was not used due to a possible effect from the diluent used (DMSO). After the 24 h incubation period, resting macrophages produced 3.8 ± 1.7 μM NO and 2.2 ± 1 pg/ml PGE2. In LPS-activated cells, the production of NO was dramatically increased to 71.5 ± 6.6 μM, and PGE2 levels reached 66.2 ± 6.9 pg/ml. Incubation of the cells with 10 μM convolutamydine A inhibited LPS-induced production of NO. ISA003 only inhibited the LPS-induced production of NO at higher concentrations, and ISA147 reduced the production of NO similarly to convolutamydine A. On the other hand, all three substances reduced the amount of PGE2 produced by LPS-activated cells (Fig. 6). In order to investigate the possibility that the inhibitory effect of convolutamydine A, ISA003 and ISA147 on NO production could be due to an NO-scavenging effect of the compounds, we utilized a “cellfree” protocol, where an NO donor, SNAP, was incubated with increasing concentrations of each substance. None of the tested doses was
group (PBS injected into the SAP). The pre-treatment of mice with convolutamydine A or its analogues (at doses of 0.1, 1 or 10 mg/kg) significantly suppressed the number of leukocytes in the exudates. The amount of protein that extravased to the SAP also had a 5-fold increase in carrageenan group. All doses of convolutamydine A and it analogues reduced the carrageenan-induced protein leakage. However, only the higher doses of ISA003 and ISA147 demonstrated an inhibitory effect (Fig. 3). Because convolutamydine A and its analogues significantly reduced cell migration into the SAP, we decided to further analyse other parameters that are present in carrageenan-induced inflammation. In this regard, we measured the amount of some cytokines (i.e., TNF-α and IL-6) in the exudates. Carrageenan induced 2.6- and 0.7-fold increases in the amount of TNF-α and IL-6, respectively. All tested doses of convolutamydine A and ISA147 (0.1, 1 and 10 mg/kg) reduced cytokine levels, whereas ISA003 only showed an effect at the 1 or 10 mg/kg doses (Fig. 4). Fig. 5 shows that both convolutamydine A and its analogues significantly and dose-dependently reduced NO and PGE2 production by the leukocytes that migrated into the SAP after carrageenan injection. To further evaluate the effect of convolutamydine A and its analogues and to eliminate the possibility that the reduction in cytokines,
2.5
PBS in SAP
Carrageenan in SAP Conv. A
TNF-α (pg/ml)
2.0
ISA003
ISA147
#
1.5 1.0
*
*
* *
0.5
* *
*
* *
0.0
PBS in SAP
2.5
Carrageenan in SAP
2.0
Conv. A
IL-6 (pg/ml)
# 1.5
ISA003
*
1.0
* *
*
*
*
0.5
ISA147
*
*
*
g/ k m g 10 g/k m g g/ kg 0. 1 m g 1 /kg m 10 g/k m g g/ kg 0. 1 m g 1 /kg m 10 g/k m g g/ kg
m
1
1 0.
D
ex
Ve
hi
cl
e am V et eh ha ic so le ne
0.0
Fig. 4. Effect of convolutamydine A (Conv. A), ISA003 and ISA147 on carrageenan-induced TNF-α or IL-6 production in the subcutaneous air pouch (SAP). The animals were pretreated with different doses of each substance, dexamethasone (5 mg/kg, i.p.) or vehicle. The results are presented as the mean ± S.D. (n = 6–10). #P b 0.05 compared to vehicle-treated mice receiving PBS in SAP and *p b 0.05 compared to vehicle-treated mice receiving carrageenan in SAP using ANOVA followed by Bonferroni's test.
P.D. Fernandes et al. / Life Sciences 116 (2014) 16–24
PBS in SAP
150
Carrageenan in SAP Conv. A
# NO (μM)
21
ISA003
ISA147
100
50
* * *
* *
*
* * *
*
0
PBS in SAP
80
Carrageenan in SAP
PGE2 (pg/ml)
# Conv. A
60
40
*
*
ISA003
ISA147
* * *
* *
* *
20
am V et eh ha icl so e ne 0. 1 m g 1 /kg m 10 g/k m g g/ kg 0. 1 m g 1 /kg m 10 g/k m g g/ kg 0. 1 m g 1 /kg m 10 g/k m g g/ kg
D
ex
Ve
hi
cl
e
0
Fig. 5. The effect of convolutamydine A (Conv. A), ISA003 and ISA147 on levels of nitric oxide (NO) and prostaglandin E2 (PGE2) accumulated in the subcutaneous air pouch (SAP). The animals were pretreated with different doses of each substance, dexamethasone (5 mg/kg, i.p.) or vehicle. The results are presented as the mean ± S.D. (n = 6–10). #P b 0.05 compared to vehicle-treated mice receiving PBS in SAP and *p b 0.05 compared to vehicle-treated mice receiving carrageenan in SAP using ANOVA followed by Bonferroni's test.
able to scavenge the NO liberated from SNAP and to reduce nitrite levels (data not shown). To examine if the inhibitory effects of convolutamydine A, ISA003 and ISA147 occurred due to an inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) expression, the levels of each protein were determined by western blot analysis. As shown in Fig. 7, the expression of the iNOS and COX-2 proteins was almost undetectable in unstimulated cells. However, upon LPS treatment, the iNOS and COX-2 proteins were markedly increased. Convolutamydine A, ISA003 and ISA147 significantly reduced the induction of iNOS and COX-2 protein expression. Discussion The search for new substances that affect and reduce the inflammatory process is still an area of intense research and interest. The anti-inflammatory drugs that are currently available are only partially effective due to their several side effects that can limit their continuous use. In this study, we showed that convolutamydine A and its two analogues (ISA003 and ISA147) have anti-inflammatory properties, reducing leukocyte migration and the levels of cytokines and nitric oxide. Formalin-induced licking behaviour is a widely used model to evaluate analgesic drugs. The subcutaneous injection of formalin induces a
licking response characterized by a nociceptive response (resulted from a direct activation of nociceptors, the first phase), whereas the second phase is associated with endogenous locally produced inflammatory and algesic mediators (e.g., bradykinin, histamine, serotonin, and prostaglandins), which activate the paw nociceptors (Parada et al., 2001). In a previous paper, we demonstrated that convolutamydine A, ISA003 and ISA147 (at doses of 1, 10 and 30 mg/kg) significantly reduced the formalin response (Figueiredo et al., 2013). Our present results showed that, even at lower doses (i.e., 0.01 or 0.1 mg/kg), convolutamydine A, ISA003 or ISA147 still significantly inhibited the formalin-licking behaviour. This effect may be due to an activity of the tested compounds against the inflammatory mediators that are liberated in the paw. A direct action against the nociceptors located in the paw maybe an alternate explanation. Because all three substances demonstrated a significant effect in the second phase (the inflammatory phase), we decided to further evaluate their effects in another model of inflammation. In this regard, we examined a model of carrageenan-induced inflammation in the subcutaneous air pouch (SAP). The injection of carrageenan represents a common model to study acute inflammation. The application of carrageenan causes rapid edema, linked to an exacerbated increase in vascular permeability, plasma extravasation and the production of inflammatory mediators (Di Rosa, 1972).
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P.D. Fernandes et al. / Life Sciences 116 (2014) 16–24
100 Conv. A
80
NO (μM)
ISA003
ISA147
#
* *
60
* *
40
*
20 0 100 Conv. A
PGE2 (pg/mL)
80
ISA003
ISA147
#
60
* *
*
*
*
* *
*
40 20
μM
μM
30
μM
10
1
μM
μM
30
μM
10
1
μM
μM
30
μM
10
1
-
-
0
+ LPS Fig. 6. The effects of convolutamydine A, ISA003 and ISA147 in NO and prostaglandin E2 (PGE2) production by RAW 264.7 cells. Cells were treated with either compounds (1, 10 or 30 μM) before activation or not with LPS (1 μg/ml). After 24 h incubation, the levels of NO or PGE2 that accumulated in the supernatant were measured. Values are mean ± S.D. (n = 4). #P b 0.05 when comparing the LPS-activated cells to the non-activated cells; *P b 0.05 comparing the LPS-activated cells that were pre-incubated with compounds to the LPS-activated cells by ANOVA followed by Bonferroni's test.
In the first hours after carrageenan injection neutrophils are the main cell to be recruited and to reach the inflammatory site. Few hours later macrophages and lymphocytes also gain the local and play pivotal roles in the evolution of the inflammation, producing and liberating cytokines. Amongst these cytokines, TNF-α and IL-6 exert a diversity of biological effects that can influence and participate in the developing and maintenance of acute and/or chronic inflammatory disease (Ershler and Keller, 2000). The total number of leukocytes and the levels of IL-6 and TNF-α induced by carrageenan were inhibited by convolutamydine A and its analogues. These results were similar to the effect obtained by dexamethasone, a corticosteroid. Endothelial nitric oxide synthase (eNOS) is a constitutive enzyme responsible for the production of low amounts of NO that participate in the maintenance of the vascular tone and prevent the adhesion and aggregation of leukocytes and platelets to the vascular wall. Inducible nitric oxide synthase (iNOS) is expressed in different cells after an activation process and the large amount of NO produced plays multiple roles in the inflammatory response, vasodilation and regulation of leukocyte rolling, migration, cytokine production and proliferation (Gurik et al., 2003). It has been shown that some iNOS inhibitors demonstrated an important effect in several inflammatory models, such as the air pouch model (Paya et al., 1997). Prostaglandin E2 (PGE2) is another mediator whose production is increased in the inflammatory process. Although PGE2 is an important participant in physiological events, it is also significantly involved in inflammation. Moreover, there is also evidence that prostaglandins
and NO can interfere with each other's synthesis (Prestes-Carneiro et al., 2007). NO was shown to either increase (Perkins and Kniss, 1999; Marnett et al., 2000) or decrease (Swierkosz et al., 1995; Clancy et al., 2000) the synthesis of prostaglandin, and prostaglandins were reported to increase (Lin et al., 1999) or decrease (D'Acquisto et al., 1998; Harbrecht et al., 1997) NO synthesis; these results suggest that a balance between the levels of NO and PGs is somehow important for cell homeostasis and that during an inflammatory event, both mediators can be generated, altering the synthesis of the other. Our results indicate that convolutamydine A and the two analogues reduced both NO and PGE2 levels. These effects are most likely due to a direct effect on enzyme (iNOS and COX-2) expression and not to a direct NO-scavenger effect. Our results demonstrated that convolutamydine A and its analogues (ISA003 and ISA147) have significant anti-inflammatory effects both in vivo and in vitro. There are some differences between the molecules. Convolutamydine A has two bromide radicals in positions 4 and 6. ISA003 does not have any substituent, and ISA147 has one bromide in position 5. It seems that the presence of two bromide atoms confer a better anti-inflammatory effect because smaller doses of convolutamydine A (i.e., 0.01 and 0.1 mg/kg, depending on the model) have stronger effects. Furthermore, the presence of only one bromide (or its absence) seems to be unimportant because both analogues (ISA147 and ISA003, respectively) have similar effects. One possible explanation for the differences in the effects of convolutamydine A and its analogues is that the presence of two
P.D. Fernandes et al. / Life Sciences 116 (2014) 16–24
500
iNOS
400
Densitometry (arbitrary units)
23
Conv. A
#
300
ISA003
ISA147
*
*
*
*
*
200
*
100
0 400
COX-2
Densitometry (arbitrary units)
Conv. A
#
300
*
200
ISA003
*
*
ISA147
*
*
*
*
*
100
μM
μM
30
μM
10
1
μM
μM
30
μM
10
1
μM
μM
30
μM
10
1
-
-
0
+ LPS Fig. 7. The effects of convolutamydine A, ISA003 and ISA147 on iNOS or cyclooxygenase-2 (COX-2) enzyme expression in RAW 264.7 cells. RAW 264.7 cells were activated or not with LPS (1 μg/ml) and then incubated with the compounds (1, 10 or 30 μM). After 6 h of incubation, western blot analysis was performed to visualize iNOS and COX-2 levels. The results are presented as the mean ± S.D. (n = 4). Statistical significance was calculated by ANOVA followed by Bonferroni's test. #P b 0.05 when comparing LPS-activated with non-activated cells; *P b 0.05 when comparing the LPS-activated cells that were pre-incubated with compounds to the LPS-activated cells.
bromides may facilitate and improve the interaction between the convolutamydine A molecule and its target, resulting in a lower effective dose.
to PDF, BV, ACP) and Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES, fellowship to RSZ). References
Conclusions To the best of our knowledge, this is the first study to demonstrate an anti-inflammatory effect of convolutamydine A and two analogues (ISA003 and ISA 147) both in vivo and in vitro. These results may be important because they raise the possibility of identifying new drugs for the treatment of inflammatory processes. Conflict of interest statement The authors declare no conflicts of interest.
Acknowledgements We would like to thank Mr Alan Minho for his technical assistance and Prof. Sonia Jancar for some suggestions. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq # 473106/2012-4 and INCT-INOFAR grant support and fellowship to PDF, ACP), Fundação Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ, grant support E-26/110.891/2013
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Green LC, Wagner DA, Glogowski J, Skipper PL, Wisnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 1982;126:131–8. Gurik TJ, Korbut R, Adamek-Gurik T. Nitric oxide and superoxide in inflammation and immune regulation. J Physiol Pharmacol 2003;54:469–87. Harbrecht BG, Kim YM, Wirant EA, Simmons RL, Billiar TR. Timing of prostaglandin exposure is critical for the inhibition of LPS- or IFN-gamma-induced macrophage NO synthesis by PGE2. J Leukoc Biol 1997;61:712–20. Hewawasam P, Erway M, Moon SL, Knipe J, Weiner H, Boissard CG, et al. Synthesis and structure–activity relationships of 3-aryloxindoles: a new class of calciumdependent, large conductance potassium (Maxi-K) channel openers with neuroprotective properties. J Med Chem 2002;45:1487–99. Hunskaar S, Hole K. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 1987;30:103–14. Kamano Y, Zhang H-P, Ichihara Y, Kizu H, Komiyama K, Pettit GR. Convolutamydine A, a novel bioactive hydroxyl oxindole alkaloid from marine bryozoan Amathia convoluta. Tetrahedron Lett 1995;36:2783–4. Lin WW, Chen BC, Hsu YW, Lee CM, Shyue SK. Modulation of inducible nitric oxide synthase induction by prostaglandinE2 in macrophages: distinct susceptibility in murine J774 and RAW 264.7 macrophages. Prostaglandins Other Lipid Mediat 1999;58: 87–101. Lorke DA. New approach to practical acute toxicity testing. Arch Toxicol 1983;54:275–87. Luppi G, Monari M, Corrêa RJ, Violante FA, Pinto AC, Kaptein B, et al. The first total synthesis of (R)-convolutamydine A. Tetrahedron 2006;62:12017–24. Marnett LJ, Wright TL, Crews BC, Tannenbaum SR, Morrow JD. Regulation of prostaglandin biosynthesis by nitric oxide is revealed by targeted deletion of inducible nitricoxide synthase. J Biol Chem 2000;275:13427–30. Matheus ME, Violante FA, Garden SJ, Pinto AC, Fernandes PD. Isatins inhibit cyclooxygenase-2 and inducible nitric oxide synthase in a mouse macrophage cell line. Eur J Pharmacol 2007;556:200–6. Oliveira SI, Fernandes PD, Mendes JGPA, Jancar S. Phagocytosis of apoptotic and necrotic thymocytes is inhibited by PAF-receptor antagonists and affects LPS-induced COX-2 expression in murine macrophages. Prostaglandins Lipid Mediat 2006;80:62–73.
Parada CA, Tambeli CH, Cunha FQ, Ferreira SH. The major role of peripheral release of histamine and 5-hydroxytryptamine in formalin-induced nociception. Neuroscience 2001;102:937–44. Paya M, Pastor PG, Coloma J, Alcaraz MJ. Nitric oxide synthase and cyclo-oxygenase pathways in the inflammatory response induced by zymosan in the rat air pouch. Brit J Pharmacol 1997;120:1445–52. Perkins DJ, Kniss DA. Blockade of nitric oxide formation down-regulatescyclooxygenase-2 and decreases PGE2biosynthesis in macrophages. J Leukoc Biol 1999;65:792–9. Pinheiro MMG, Fernandes SB, Fingolo CE, Boylan F, Fernandes PD. Anti-inflammatory activity of ethanol extract and fractions from Couroupita guianensis Aublet leaves. J Ethnopharmacol 2013;146:324–30. Popp FD, Parson R, Donigan BE. Synthesis of potential anticonvulsants, condensations of isatins with acetone and related ketones. J Pharmacol Sci 1980;69:1235–9. Prestes-Carneiro LE, Shio MT, Fernandes PD, Jancar S. Cross-regulation of iNOS and COX-2 by its products in murine macrophages under stress conditions. Cell Physiol Biochem 2007;20:283–92. Raymundo LJRP, Guilhon CC, Alviano DS, Matheus ME, Antoniolli AR, Cavalcanti SCH, et al. Characterization of the anti-inflammatory and antinociceptive activities of the Hyptis pectinata (L.) Poit essential oil. J Ethnopharmacol 2011;134:725–32. Silva BV. Isatin, a versatile molecule: studies in Brazil. J Braz Chem Soc 2013;24: 707–20. Silva BV, Torres JC, Garden SJ, Violante FA, Rezende MJC, et al. Quim Nova. 2008;31: 924–9. Swierkosz TA, Mitchell JA, Warner TD, Botting RM, Vane JR. Co-induction of nitric oxide synthase and cyclooxygenase: interactions between nitric oxide and prostanoids. Br J Pharmacol 1995;114:1335–42. Tokunaga T, Hume WE, Umezome T, Okazaki K, Ueki Y, Kumagai K, et al. Oxindole derivatives as orally active potent growth hormone secretagogues. J Med Chem 2001;44: 4641–9. Vine KL, Matesic L, Locke JM, Ranson M, Skropeta D. Cytotoxic and anticancer activities of isatin and its derivatives: a comprehensive review from 2000–2008. Anti Cancer Agents Med 2009;9:397–414.
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Anti-inflammatory properties of convolutamydine A and two structural analogues Patricia D. Fernandes a,⁎, Renata S. Zardo a,1, Gabriella S.M. Figueiredo a,1, Bárbara V. Silva b, Angelo C. Pinto b a Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas, Laboratório de Farmacologia da Dor e da Inflamação, Av. Carlos Chagas Filho, 373. Prédio do CCS, bloco J, sala 10, 21941-902 Rio de Janeiro, Brazil b Universidade Federal do Rio de Janeiro, Instituto de Química, Rio de Janeiro, Brazil
a r t i c l e
i n f o
Article history: Received 18 June 2014 Accepted 29 August 2014 Available online 6 September 2014 Keywords: Convolutamydine A Isatin Inflammation Anti-inflammatory drugs
a b s t r a c t Aims: Convolutamydine A is an oxindole alkaloid that can be isolated from a marine bryozoan. Due to the variety of biological effects, two analogues were synthesized and their anti-inflammatory properties were evaluated. Main methods: The anti-inflammatory effects of convolutamydine A and its analogues (ISA003 and ISA147) were investigated in a formalin-induced licking behaviour model, where mice received an intraplantar injection of formalin and their licking behaviour was evaluated for 30 min. Additionally, inflammatory parameters were evaluated in a subcutaneous air pouch (SAP) model of carrageenan-induced inflammation. Exudates were collected for leukocyte counts; measurement of protein, prostaglandin E2 (PGE2) and cytokines by ELISA; and analysis of nitric oxide (NO) using a nitrate conversion protocol. Cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) from RAW 264.7 cells were quantified by immunoblotting. Key findings: Convolutamydine A and its two analogues inhibited the formalin-induced licking response at doses as low as 0.01 mg/kg. An inhibitory effect was also observed on leukocyte migration and the production of NO, PGE2 and cytokines (IL-6 and TNF-α). The reduction in inflammatory parameters did not appear to be correlated with a direct reduction in the number of cells in the SAP, because a reduction in NO and PGE2 production by cultured macrophages was observed in addition to the inhibition of iNOS and COX2 enzyme expression. Significance: These results indicate that convolutamydine A and its two analogues have significant antiinflammatory effects. These substances can be improved to generate lead compounds for the synthesis of new anti-inflammatory drugs. © 2014 Elsevier Inc. All rights reserved.
Introduction Inflammation is a stereotyped response of living organisms to possible harmful stimuli (Di Rosa, 1972), in vascular tissues. This physiologic event has a protective role aiming at the removal of the offending agent by activating a cascade of events and production of inflammatory mediators. The ultimate goal is the destruction of the offending agent and tissue restoration. The current pharmacological therapies for inflammation consist primarily of non-steroidal anti-inflammatory drugs (NSAIDs). This class has a long history of clinical use and major deficiencies, and many of the drug discovery efforts in the area of inflammation have focused on the incremental improvement of this class of compounds. However, adverse side-effects (i.e., ulcers and bleeding) have limited their use. Many new molecules have been synthesized with the aim of seeking those with reduced side effects (reviewed by Carter et al., 2014).
⁎ Corresponding author. Tel.: +55 21 39386442. E-mail address: [email protected] (P.D. Fernandes). 1 Tel.: +55 21 39386442.
http://dx.doi.org/10.1016/j.lfs.2014.08.019 0024-3205/© 2014 Elsevier Inc. All rights reserved.
Amathia convoluta is a marine bryozoan species from which the oxindole alkaloid named Convolutamydine A was isolated. Because convolutamydine is only isolated from this Bryozoan in small amounts and due to its promising biological activities, it has been synthesized by several research groups (Kamano et al., 1995; Garden et al., 1997; Luppi et al., 2006; Cravotto et al., 2006; Silva et al., 2008). Garden et al. (1997) were the first group to propose the synthesis of a racemic mixture of convolutamydine A from isatin, a small, versatile, and widely applicable pharmacological molecule (da Silva et al., 2001; Silva, 2013; Vine et al., 2009). Convolutamydine A promoted alterations in HL-60 cell line (Kamano et al., 1995). In addition to convolutamydine A, the literature includes reports of other 3-substituted-3-hydroxyindolin-2-ones with applications in medicinal chemistry, such as active potent growth hormone secretagogues (Tokunaga et al., 2001), potassium channel openers (Hewawasam et al., 2002), anticonvulsants (Popp et al., 1980) and antinociceptives (Figueiredo et al., 2013). In our continuous search for bioactive substances, the purpose of the present work was to investigate the anti-inflammatory effects of convolutamydine A and its analogues in animal models of inflammation.
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Materials and methods Animals Male BALB/C mice (20–25 g), from our own animal facility, were housed in a room with constant temperature (22 ± 2 °C) and 12-h light/dark cycle and fed standard rodent chow and water. Before (12 h) the assays animals received only water in order to avoid food interference with compound absorption. Research protocol was approved by the Ethical Committee for Animal Research (Biomedical Science Institute/UFRJ; approval number ICBDFBC-015). General Acetylsalicylic acid (ASA) and carrageenan were purchased from Sigma (St. Louis, MO, USA), and acetic acid was purchased from Merck, Inc. All drugs were dissolved in phosphate buffer saline (PBS) just before use. ASA (200 mg/kg, p.o.) and dexamethasone (5 mg/kg, i.p.) were used as reference drugs. These doses were based on previous studies (Pinheiro et al., 2013) and calculated as ED50 (dose that reduced 50% the effect). Vehicle was used as negative control group.
17
Table 1 Name and substituent of each compound. Compound
Substituent (R)
Convolutamydine A ISA147 ISA003
4,6-Br 5-Br H
Formalin-induced licking behaviour test The licking behaviour was examined immediately after formalin hind paw injection. The procedure was similar to the method described by Hunskaar and Hole (1987). Animals received 20 μl of formalin (2.5% v/v) into the dorsal surface of the left hind paw. Animals were individually observed and the time that they spent licking the formalin-injected paw recorded. The nociceptive response develops in two phases: the first 5 min after formalin injection (first phase, neurogenic pain response); the second 15–30 min after formalin injection (second phase, inflammatory pain response). Mice were divided in the following groups: vehicle, convolutamydine A, analogues (0.01, 0.1 or 1 mg/kg, p.o.), and acetylsalicylic acid (100 mg/kg, i.p.) treated groups 1 h before formalin injection.
Synthesis of convolutamydine A and its analogues Subcutaneous air pouch (SAP) Isatins were suspended in acetone, and drops of diethylamine were added at room temperature, leading to convolutamydine A, ISA003 or ISA147 (Fig. 1 and Table 1). The structures of these compounds were determined by 1H-NMR and 13C-NMR as described by Garden et al. (1997). Administration of convolutamydine A and analogues A stock solution (200 mg/ml in dimethylsulphoxide, DMSO) was prepared with Convolutamydine A, ISA003 and ISA147. The doses of 0.1, 1 and 10 mg/kg (in tween 80) were orally administered to each animal. Vehicle group was composed by animals treated with DMSO/Tween 80. The final concentration of DMSO/tween 80 had no effect per se. Acute toxicity To evaluate a possible toxic effect we adapted the method used by Lorke (1983). Briefly, animals were treated with 150 mg/kg of convolutamydine A or its analogues. During 5 consecutive days several parameters (i.e., convulsion, sedation, respiration, and food and water intake) were observed. After five days the animals were euthanized with an overdose of ketamine/xilazine (150 mg/10 mg/kg). The stomachs of the animals were removed and opened to observe any signs of hyperemia and the presence or absence of ulcer.
Animals were used as described by Raymundo et al. (2011). After subcutaneous injection of 10 ml of sterile air in the dorsal region in three alternate days, an injection of sterile carrageenan suspension (1%; 1 ml) was done in the SAP formed. Mice were divided in the following groups: vehicle, convolutamydine A, analogues (0.1, 1 or 10 mg/kg, p.o.), and dexamethasone (5 mg/kg, i.p.) treated groups 1 h before carrageenan injection. Another vehicle-treated group was used in mice that received PBS (phosphate buffer saline, 1 ml) in SAP. After 24 h all groups were sacrificed, SAP was washed with 1 ml of sterile PBS and exudates collected. The total cell count was determined in a CellPocH-100iV Diff (Sysmex) haematology analyser. The exudates were also centrifuged (5000 ×g, 10 min, 4 °C) and aliquots of the supernatants were stored at −20 °C until the assays. IL-6, TNF-α,PGE2 and protein measurements The cytokines (TNF-α and IL-6) and prostaglandin E2 (PGE2) levels were quantified using enzyme-linked immunosorbent assays (ELISAs) according to the protocol supplied by the manufacturer (B&D, USA). BCA method (BCA™ Protein Assay Kit, Pierce) was used to measure protein content in exudates supernatants. Cell culture RAW 264.7 mouse macrophage cell line (ATCC TIB-71) was maintained in RPMI 1640 medium (with 10% foetal bovine serum, 2 mM glutamine and 15 mM HEPES) at 37 °C and 5% CO2. At confluence stage cells were scraped off and seeded in 96- or 12-well culture plates (2 × 106 cells/ml). Cytotoxicity assays by MTT
Fig. 1. General structure of convolutamydine A and analogues.
MTT assay was carried out as described by Denizot and Lang (1986). RAW 264.7 cells were treated with or without convolutamydine A or its analogues (10, 30 or 100 μM). After 24 h the medium was changed by 100 μl of RPMI with 0.5 mg/ml MTT and further incubated for 1 h at 37 °C, at 5% CO2. The medium was withdrawn and 100 μl of DMSO was added to the cells to dissolve the formazan. The absorbance was analysed using a FlexStation microplate reader (Molecular Devices, USA) at an absorbance wavelength of 570 nm. The control groups
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(1-Naphthyl) ethylenediamine dihydrochloride, 10% H3PO4) (Green et al., 1982). FlexStation microplate reader (Molecular Devices, USA) was used to measure the absorbance at 540 nm. Nitrite concentration was measured by comparison with a standard curve of sodium nitrite.
were composed by cells incubated with medium and was considered as 100% viable cells. Percentage of viable cells was used to express the results. Nitrate and nitrite measurement
Nitric oxide-trapping capacity of convolutamydine A and its derivatives To evaluate the production of NO in cell culture, the nitrite accumulated in the cell culture supernatants was measured according to the Griess reaction (Green et al., 1982). LPS (1 μg/ml)-activated cells incubated with convolutamydine A or analogues (10, 30 or 100 μM) were further incubated for 24 h, after which cell culture supernatant was collected to measure the nitrite concentration. Nitrate accumulated in SAP exudates was measured according to the method described by Bartholomew (1984) and adapted by Raymundo et al. (2011). The nitrite concentrations obtained after nitrate conversion (cited above) or from cell culture supernatants were measured by mixing equal parts of sample and Griess Reagent (1% sulphanilamide, 0.1% N-
Detection of iNOS and COX-2 enzyme expression Immunoblots were carried out as described previously (Oliveira et al., 2006). Briefly, RAW 264.7 cells were activated with LPS (1 μg/ml)
Convolutamydine A
1st phase
2 nd phase
300
* *
35
200
*
*
100
0
0
1st phase
ISA003
2 nd phase
400
300
* 35
200
*
*
100
0
70
Licking (seconds)
Licking (seconds)
70
0
1st phase
2
nd
ISA147
phase
400
300
35
200
*
*
100
kg
kg 1
m
g/
g/
g/ m
1 0.
01 0.
m
hi cl e AS A
Ve
kg
kg
g/ m
1
m
m
1 0.
01 0.
g/
g/
cl e AS A
hi Ve
kg
0 kg
0
Licking (seconds)
Licking (seconds)
400
Licking (seconds)
Licking (seconds)
70
To test the ability of convolutamydine A and its analogues to trap nitric oxide, a cell-free system was used as described in Matheus et al. (2007). Convolutamydine A and its analogues (at 100 μM) were incubated with 1 mM SNAP (S-nitroso-N-acetylpenicillamine). Nitrite accumulated in the supernatant after 6 h of incubation was measured using the Griess reaction (Green et al., 1982).
Fig. 2. The effects of convolutamydine A, ISA003 and ISA147 on the formalin-induced licking response in mice. Convolutamydine A or analogues (0.01, 0.1 or 1 mg/kg), acetylsalicylic acid (ASA, 100 mg/kg) or vehicle were orally administered to mice. Values mean ± S.D. (n = 6–10). *P b 0.05 compared to vehicle-treated mice using ANOVA followed by Bonferroni's test.
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and further incubated for 6 h with convolutamydine A or its analogues (3, 10 or 30 μM). Cells were lysed with cold lysis buffer (10% NP40, 150 mM NaCl, 10 mM Tris HCl pH 7.6, 2 mM PMSF, and 5 μM leupeptin). After determination of the protein concentration in the suspensions by the BCA method (BCA™ Protein Assay Kit, Pierce), the suspensions were boiled in application buffer (DTT 100 mM, Bromophenol Blue 0.1%). Aliquots of 30 μg of protein were submitted to electrophoresis in 10% polyacrylamide gel. Proteins were electrophoretically transferred onto nitrocellulose membranes. Membranes were incubated with anti-iNOS or anti-COX-2 (Cell signalling, USA) primary antibodies and further with secondary antibodies (anti-mouse IgG antibody conjugated to horseradish peroxidase). Proteins were detected using enhanced chemiluminescence (ECL) reagents and quantified using a ChemiDoc system (BioRad, USA).
Statistical analysis All results are presented as the mean ± S.D. Data was analysed in a GraphPad Prism™ (version 5.00, GraphPad Software Inc., San Diego, CA, U.S.A.) using analyses of variance (ANOVA) followed by Bonferroni's tests. P b 0.05 was considered to be significant.
Leukocytes (x 106 cell/ml)
100
PBS in SAP
19
Results Acute toxicity We first established whether convolutamydine A and its analogues have any toxic effects per se. Even when orally treated with 150 mg/kg of each substance mice did not show any type of behavioural changes. We also did not observe any lesions or bleeding on the stomach (data not shown). Anti-inflammatory activity The pretreatment of mice with 0.01, 0.1 or 1 mg/kg of each compound demonstrated that convolutamydine A and ISA003 significantly reduced the licking response to formalin injection at all doses tested, whereas ISA147 did not affect the response (Fig. 2). The inhibitory effect observed for convolutamydine A and its analogues is indicative of a possible anti-inflammatory effect. Accordingly, we evaluated convolutamydine A, ISA003 and ISA147 in another model of inflammation, the subcutaneous air pouch (SAP) model. The injection of carrageenan (1%) into the SAP produced a marked increase in the exudate volume and leukocyte number in the pouch, accompanied by an increase in almost seven-fold above the level of the control
Carrageenan in SAP
#
Conv. A
ISA003
ISA147
50
* *
* * *
* *
*
* *
0
500
PBS in SAP
#
400
Protein (μg/ml)
Carrageenan in SAP
300
*
* * *
200
* *
*
100
D
ex
Ve
hi
cl
e am V et eh ha ic so le ne 0. 1 m g 1 /kg m 10 g/k m g g/ kg 0. 1 m g 1 /kg m 10 g/k m g g/ kg 0. 1 m g 1 /kg m 10 g/k m g g/ kg
0
Fig. 3. Effects of convolutamydine A (Conv. A), ISA003 and ISA147 in leukocyte migration and protein leakage into the subcutaneous air pouch (SAP). The animals were pretreated with different doses of each substance, dexamethasone (5 mg/kg, i.p.) or vehicle 1 h before carrageenan (1%) injection into the SAP. Values mean ± S.D. (n = 6–10). #P b 0.05 compared to vehicle-treated mice receiving PBS in SAP and *p b 0.05 compared to vehicle-treated mice receiving carrageenan in SAP using ANOVA followed by Bonferroni's test.
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NO and PGE2 in vivo could simply be the result of a reduction in the number of cells that migrated into the SAP, we decided to carry out in vitro assays. We first investigated a possible direct cytotoxic effect of convolutamydine A, ISA003 and ISA147 on RAW 264.7 cells. Neither the compounds alone, at concentrations up to 100 μM, nor the compounds in the presence of LPS (1 μg/ml) affected cell viability (data not shown). Therefore, concentrations between 1 and 30 μM were used for the subsequent experiments. The 100 μM concentration was not used due to a possible effect from the diluent used (DMSO). After the 24 h incubation period, resting macrophages produced 3.8 ± 1.7 μM NO and 2.2 ± 1 pg/ml PGE2. In LPS-activated cells, the production of NO was dramatically increased to 71.5 ± 6.6 μM, and PGE2 levels reached 66.2 ± 6.9 pg/ml. Incubation of the cells with 10 μM convolutamydine A inhibited LPS-induced production of NO. ISA003 only inhibited the LPS-induced production of NO at higher concentrations, and ISA147 reduced the production of NO similarly to convolutamydine A. On the other hand, all three substances reduced the amount of PGE2 produced by LPS-activated cells (Fig. 6). In order to investigate the possibility that the inhibitory effect of convolutamydine A, ISA003 and ISA147 on NO production could be due to an NO-scavenging effect of the compounds, we utilized a “cellfree” protocol, where an NO donor, SNAP, was incubated with increasing concentrations of each substance. None of the tested doses was
group (PBS injected into the SAP). The pre-treatment of mice with convolutamydine A or its analogues (at doses of 0.1, 1 or 10 mg/kg) significantly suppressed the number of leukocytes in the exudates. The amount of protein that extravased to the SAP also had a 5-fold increase in carrageenan group. All doses of convolutamydine A and it analogues reduced the carrageenan-induced protein leakage. However, only the higher doses of ISA003 and ISA147 demonstrated an inhibitory effect (Fig. 3). Because convolutamydine A and its analogues significantly reduced cell migration into the SAP, we decided to further analyse other parameters that are present in carrageenan-induced inflammation. In this regard, we measured the amount of some cytokines (i.e., TNF-α and IL-6) in the exudates. Carrageenan induced 2.6- and 0.7-fold increases in the amount of TNF-α and IL-6, respectively. All tested doses of convolutamydine A and ISA147 (0.1, 1 and 10 mg/kg) reduced cytokine levels, whereas ISA003 only showed an effect at the 1 or 10 mg/kg doses (Fig. 4). Fig. 5 shows that both convolutamydine A and its analogues significantly and dose-dependently reduced NO and PGE2 production by the leukocytes that migrated into the SAP after carrageenan injection. To further evaluate the effect of convolutamydine A and its analogues and to eliminate the possibility that the reduction in cytokines,
2.5
PBS in SAP
Carrageenan in SAP Conv. A
TNF-α (pg/ml)
2.0
ISA003
ISA147
#
1.5 1.0
*
*
* *
0.5
* *
*
* *
0.0
PBS in SAP
2.5
Carrageenan in SAP
2.0
Conv. A
IL-6 (pg/ml)
# 1.5
ISA003
*
1.0
* *
*
*
*
0.5
ISA147
*
*
*
g/ k m g 10 g/k m g g/ kg 0. 1 m g 1 /kg m 10 g/k m g g/ kg 0. 1 m g 1 /kg m 10 g/k m g g/ kg
m
1
1 0.
D
ex
Ve
hi
cl
e am V et eh ha ic so le ne
0.0
Fig. 4. Effect of convolutamydine A (Conv. A), ISA003 and ISA147 on carrageenan-induced TNF-α or IL-6 production in the subcutaneous air pouch (SAP). The animals were pretreated with different doses of each substance, dexamethasone (5 mg/kg, i.p.) or vehicle. The results are presented as the mean ± S.D. (n = 6–10). #P b 0.05 compared to vehicle-treated mice receiving PBS in SAP and *p b 0.05 compared to vehicle-treated mice receiving carrageenan in SAP using ANOVA followed by Bonferroni's test.
P.D. Fernandes et al. / Life Sciences 116 (2014) 16–24
PBS in SAP
150
Carrageenan in SAP Conv. A
# NO (μM)
21
ISA003
ISA147
100
50
* * *
* *
*
* * *
*
0
PBS in SAP
80
Carrageenan in SAP
PGE2 (pg/ml)
# Conv. A
60
40
*
*
ISA003
ISA147
* * *
* *
* *
20
am V et eh ha icl so e ne 0. 1 m g 1 /kg m 10 g/k m g g/ kg 0. 1 m g 1 /kg m 10 g/k m g g/ kg 0. 1 m g 1 /kg m 10 g/k m g g/ kg
D
ex
Ve
hi
cl
e
0
Fig. 5. The effect of convolutamydine A (Conv. A), ISA003 and ISA147 on levels of nitric oxide (NO) and prostaglandin E2 (PGE2) accumulated in the subcutaneous air pouch (SAP). The animals were pretreated with different doses of each substance, dexamethasone (5 mg/kg, i.p.) or vehicle. The results are presented as the mean ± S.D. (n = 6–10). #P b 0.05 compared to vehicle-treated mice receiving PBS in SAP and *p b 0.05 compared to vehicle-treated mice receiving carrageenan in SAP using ANOVA followed by Bonferroni's test.
able to scavenge the NO liberated from SNAP and to reduce nitrite levels (data not shown). To examine if the inhibitory effects of convolutamydine A, ISA003 and ISA147 occurred due to an inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) expression, the levels of each protein were determined by western blot analysis. As shown in Fig. 7, the expression of the iNOS and COX-2 proteins was almost undetectable in unstimulated cells. However, upon LPS treatment, the iNOS and COX-2 proteins were markedly increased. Convolutamydine A, ISA003 and ISA147 significantly reduced the induction of iNOS and COX-2 protein expression. Discussion The search for new substances that affect and reduce the inflammatory process is still an area of intense research and interest. The anti-inflammatory drugs that are currently available are only partially effective due to their several side effects that can limit their continuous use. In this study, we showed that convolutamydine A and its two analogues (ISA003 and ISA147) have anti-inflammatory properties, reducing leukocyte migration and the levels of cytokines and nitric oxide. Formalin-induced licking behaviour is a widely used model to evaluate analgesic drugs. The subcutaneous injection of formalin induces a
licking response characterized by a nociceptive response (resulted from a direct activation of nociceptors, the first phase), whereas the second phase is associated with endogenous locally produced inflammatory and algesic mediators (e.g., bradykinin, histamine, serotonin, and prostaglandins), which activate the paw nociceptors (Parada et al., 2001). In a previous paper, we demonstrated that convolutamydine A, ISA003 and ISA147 (at doses of 1, 10 and 30 mg/kg) significantly reduced the formalin response (Figueiredo et al., 2013). Our present results showed that, even at lower doses (i.e., 0.01 or 0.1 mg/kg), convolutamydine A, ISA003 or ISA147 still significantly inhibited the formalin-licking behaviour. This effect may be due to an activity of the tested compounds against the inflammatory mediators that are liberated in the paw. A direct action against the nociceptors located in the paw maybe an alternate explanation. Because all three substances demonstrated a significant effect in the second phase (the inflammatory phase), we decided to further evaluate their effects in another model of inflammation. In this regard, we examined a model of carrageenan-induced inflammation in the subcutaneous air pouch (SAP). The injection of carrageenan represents a common model to study acute inflammation. The application of carrageenan causes rapid edema, linked to an exacerbated increase in vascular permeability, plasma extravasation and the production of inflammatory mediators (Di Rosa, 1972).
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100 Conv. A
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*
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*
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+ LPS Fig. 6. The effects of convolutamydine A, ISA003 and ISA147 in NO and prostaglandin E2 (PGE2) production by RAW 264.7 cells. Cells were treated with either compounds (1, 10 or 30 μM) before activation or not with LPS (1 μg/ml). After 24 h incubation, the levels of NO or PGE2 that accumulated in the supernatant were measured. Values are mean ± S.D. (n = 4). #P b 0.05 when comparing the LPS-activated cells to the non-activated cells; *P b 0.05 comparing the LPS-activated cells that were pre-incubated with compounds to the LPS-activated cells by ANOVA followed by Bonferroni's test.
In the first hours after carrageenan injection neutrophils are the main cell to be recruited and to reach the inflammatory site. Few hours later macrophages and lymphocytes also gain the local and play pivotal roles in the evolution of the inflammation, producing and liberating cytokines. Amongst these cytokines, TNF-α and IL-6 exert a diversity of biological effects that can influence and participate in the developing and maintenance of acute and/or chronic inflammatory disease (Ershler and Keller, 2000). The total number of leukocytes and the levels of IL-6 and TNF-α induced by carrageenan were inhibited by convolutamydine A and its analogues. These results were similar to the effect obtained by dexamethasone, a corticosteroid. Endothelial nitric oxide synthase (eNOS) is a constitutive enzyme responsible for the production of low amounts of NO that participate in the maintenance of the vascular tone and prevent the adhesion and aggregation of leukocytes and platelets to the vascular wall. Inducible nitric oxide synthase (iNOS) is expressed in different cells after an activation process and the large amount of NO produced plays multiple roles in the inflammatory response, vasodilation and regulation of leukocyte rolling, migration, cytokine production and proliferation (Gurik et al., 2003). It has been shown that some iNOS inhibitors demonstrated an important effect in several inflammatory models, such as the air pouch model (Paya et al., 1997). Prostaglandin E2 (PGE2) is another mediator whose production is increased in the inflammatory process. Although PGE2 is an important participant in physiological events, it is also significantly involved in inflammation. Moreover, there is also evidence that prostaglandins
and NO can interfere with each other's synthesis (Prestes-Carneiro et al., 2007). NO was shown to either increase (Perkins and Kniss, 1999; Marnett et al., 2000) or decrease (Swierkosz et al., 1995; Clancy et al., 2000) the synthesis of prostaglandin, and prostaglandins were reported to increase (Lin et al., 1999) or decrease (D'Acquisto et al., 1998; Harbrecht et al., 1997) NO synthesis; these results suggest that a balance between the levels of NO and PGs is somehow important for cell homeostasis and that during an inflammatory event, both mediators can be generated, altering the synthesis of the other. Our results indicate that convolutamydine A and the two analogues reduced both NO and PGE2 levels. These effects are most likely due to a direct effect on enzyme (iNOS and COX-2) expression and not to a direct NO-scavenger effect. Our results demonstrated that convolutamydine A and its analogues (ISA003 and ISA147) have significant anti-inflammatory effects both in vivo and in vitro. There are some differences between the molecules. Convolutamydine A has two bromide radicals in positions 4 and 6. ISA003 does not have any substituent, and ISA147 has one bromide in position 5. It seems that the presence of two bromide atoms confer a better anti-inflammatory effect because smaller doses of convolutamydine A (i.e., 0.01 and 0.1 mg/kg, depending on the model) have stronger effects. Furthermore, the presence of only one bromide (or its absence) seems to be unimportant because both analogues (ISA147 and ISA003, respectively) have similar effects. One possible explanation for the differences in the effects of convolutamydine A and its analogues is that the presence of two
P.D. Fernandes et al. / Life Sciences 116 (2014) 16–24
500
iNOS
400
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+ LPS Fig. 7. The effects of convolutamydine A, ISA003 and ISA147 on iNOS or cyclooxygenase-2 (COX-2) enzyme expression in RAW 264.7 cells. RAW 264.7 cells were activated or not with LPS (1 μg/ml) and then incubated with the compounds (1, 10 or 30 μM). After 6 h of incubation, western blot analysis was performed to visualize iNOS and COX-2 levels. The results are presented as the mean ± S.D. (n = 4). Statistical significance was calculated by ANOVA followed by Bonferroni's test. #P b 0.05 when comparing LPS-activated with non-activated cells; *P b 0.05 when comparing the LPS-activated cells that were pre-incubated with compounds to the LPS-activated cells.
bromides may facilitate and improve the interaction between the convolutamydine A molecule and its target, resulting in a lower effective dose.
to PDF, BV, ACP) and Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES, fellowship to RSZ). References
Conclusions To the best of our knowledge, this is the first study to demonstrate an anti-inflammatory effect of convolutamydine A and two analogues (ISA003 and ISA 147) both in vivo and in vitro. These results may be important because they raise the possibility of identifying new drugs for the treatment of inflammatory processes. Conflict of interest statement The authors declare no conflicts of interest.
Acknowledgements We would like to thank Mr Alan Minho for his technical assistance and Prof. Sonia Jancar for some suggestions. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq # 473106/2012-4 and INCT-INOFAR grant support and fellowship to PDF, ACP), Fundação Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ, grant support E-26/110.891/2013
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