Activities of 2-phthalimidethanol and 2-phthalimidethyl nitrate, phthalimide analogs devoid of the glutarimide moiety, in experimental models of inflammatory pain and edema

Article abstract of DOI:10.1016/j.pbb.2014.04.008

The reintroduction of thalidomide in the pharmacotherapy greatly stimulated the interest in the synthesis and pharmacological evaluation of phthalimide analogs with new and improved activities and also greater safety. In the present study, we evaluated the activities of two phthalimide analogs devoid of the glutarimide ring, namely 2-phthalimidethanol (PTD-OH) and 2-phthalimidethyl nitrate (PTD-NO), in experimental models of inflammatory pain and edema in male C57BL/6J mice. Intraplantar (i.pl.) injection of carrageenan (300 μg) induced mechanical allodynia and this response was inhibited by previous per os (p.o.) administration of PTD-OH and PTD-NO (750 mg/kg) and also by thalidomide (500 or 750 mg/kg). The edema induced by carrageenan was also inhibited by previous p.o. administration of PTD-OH (500 and 750 mg/kg) and PTD-NO (125, 250, 500 or 750 mg/kg), but not by thalidomide. Carrageenan increased tumor necrosis factor (TNF)-α and CXCL1 concentrations and also the number of neutrophils in the paw tissue. Previous p.o. administration of PTD-NO (500 mg/kg) reduced all the parameters, while PTD-OH (500 mg/kg) reduced only the accumulation of neutrophils. Thalidomide, on the other hand, was devoid of effect on these biochemical parameters. Plasma concentrations of nitrite were increased after p.o. administration of the phthalimide analog coupled to a NO donor, PTD-NO (500 mg/kg), but not after administration of PTD-OH or thalidomide. In conclusion, our results show that small molecules, structurally much simpler than thalidomide or many of its analogs under investigation, exhibit similar activities in experimental models of pain and inflammation. Finally, as there is evidence that the glutarimide moiety contributes to the teratogenic effect of many thalidomide analogs, our results indicate that phthalimide analogs devoid of this functional group could represent a new class of analgesic and anti-inflammatory candidates with potential greater safety.

Full text of DOI:10.1016/j.pbb.2014.04.008

PBB-71945; No of Pages 8
Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior
journal homepage: www.elsevier.com/locate/pharmbiochembeh
1
2
3
Activities of 2-phthalimidethanol and 2-phthalimidethyl nitrate,
phthalimide analogs devoid of the glutarimide moiety, in experimental
models of inflammatory pain and edema
4Q1 Adriana M. Godin a, Débora P. Araújo b, Raquel R. Menezes a, Ana Mercy S. Brito a, Ivo S.F. Melo a,
5
6
7
Giovanna M.E. Coura a, Darly G. Soares a, Leandro F.S. Bastos d, Flávio A. Amaral d, Lucas S. Ribeiro d, Daiane Boff d,
Julliana R.A. Santos c, Daniel A. Santos c, Mauro M. Teixeira d, Ângelo de Fátima b,
Renes R. Machado a, Márcio M. Coelho a,
⁎
OOF
a
8
9
10
11
Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, Pampulha CEP 31270-901, Belo Horizonte, MG, Brazil
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, Pampulha CEP 31270-901, Belo Horizonte, MG, Brazil
Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, Pampulha CEP 31270-901, Belo Horizonte, MG, Brazil
b
c
d
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, Pampulha CEP 31270-901, Belo Horizonte, MG, Brazil
1 2 a r t i c l e i n f o
a b s t r a c t
13
14
15
16
17
Article history:
The reintroduction of thalidomide in the pharmacotherapy greatly stimulated the interest in the synthesis and 25
pharmacological evaluation of phthalimide analogs with new and improved activities and also greater safety. 26
In the present study, we evaluated the activities of two phthalimide analogs devoid of the glutarimide ring, 27
namely 2-phthalimidethanol (PTD-OH) and 2-phthalimidethyl nitrate (PTD-NO), in experimental models of 28
inflammatory pain and edema in male C57BL/6J mice. Intraplantar (i.pl.) injection of carrageenan (300 μg) 29
induced mechanical allodynia and this response was inhibited by previous per os (p.o.) administration of PTD- 30
OH and PTD-NO (750 mg/kg) and also by thalidomide (500 or 750 mg/kg). The edema induced by carrageenan 31
was also inhibited by previous p.o. administration of PTD-OH (500 and 750 mg/kg) and PTD-NO (125, 250, 500 or 32
750 mg/kg), but not by thalidomide. Carrageenan increased tumor necrosis factor (TNF)-α and CXCL1 concentra- 33
tions and also the number of neutrophils in the paw tissue. Previous p.o. administration of PTD-NO (500 mg/kg) 34
reduced all the parameters, while PTD-OH (500 mg/kg) reduced only the accumulation of neutrophils. Thalido- 35
mide, on the other hand, was devoid of effect on these biochemical parameters. Plasma concentrations of nitrite 36
were increased after p.o. administration of the phthalimide analog coupled to a NO donor, PTD-NO (500 mg/kg), 37
but not after administration of PTD-OH or thalidomide. In conclusion, our results show that small molecules, 38
structurally much simpler than thalidomide or many of its analogs under investigation, exhibit similar activities 39
in experimental models of pain and inflammation. Finally, as there is evidence that the glutarimide moiety 40
contributes to the teratogenic effect of many thalidomide analogs, our results indicate that phthalimide analogs 41
devoid of this functional group could represent a new class of analgesic and anti-inflammatory candidates with 42
Received 31 January 2014
Received in revised form 8 April 2014
Accepted 18 April 2014
Available online xxxx
18
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24
Keywords:
Phthalimide analogs
2-Phthalimidethanol
2-Phthalimidethyl nitrate
Thalidomide
Inflammation
Pain
potential greater safety.
43
© 2014 Published by Elsevier Inc. 44
48
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49
1. Introduction
Matthews and McCoy, 2003) and allowed its reintroduction, initially 57
(1998) in the treatment of patients with erythema nodosum leprosum 58
and later on (2006) in the treatment of patients with multiple myeloma 59
50
51
52
53
54
55
56
The best known phthalimide analog is thalidomide (2-phthalimido
glutarimide; Fig. 1), originally developed and marketed as a hypnotic,
(for a review, see Matthews and McCoy, 2003).
60
UNCORRECTED PR
sedative and antiemetic drug in the late 1950s, but withdrawn in 1961
after the association of its use with teratogenic effects (Brynner and
Stephens, 2001). Extensive investigations during the next three decades
showed the immunomodulatory and anti-inflammatory activities of
thalidomide (Asher and Furnish, 2013; Brynner and Stephens, 2001;
The reintroduction of thalidomide greatly stimulated the interest in 61
the synthesis and pharmacological evaluation of phthalimide analogs 62
with new and improved activities and also greater safety. Usually, 63
phthalimide analogs have been developed aiming anti-inflammatory, 64
analgesic, immunomodulatory, anti-angiogenic and anti-tumor proper- 65
ties. Some of these analogs inhibit the production of the inflammatory 66
cytokine tumor necrosis factor (TNF)-α (Chaulet et al., 2011; de 67
Almeida et al., 2007; Mazzoccoli et al., 2012; Miyachi et al., 1997a, 68
1997b; Stewart et al., 2007, 2010; Teubert et al., 1998) and also the 69
⁎
Corresponding author. Tel.: +55 31 3409 6948.
E-mail addresses: mmcoelho@farmacia.ufmg.br, marciocoelhobh@yahoo.com.br
(M.M. Coelho).
http://dx.doi.org/10.1016/j.pbb.2014.04.008
0091-3057/© 2014 Published by Elsevier Inc.
Please cite this article as: Godin AM, et al, Activities of 2-phthalimidethanol and 2-phthalimidethyl nitrate, phthalimide analogs devoid of the
glutarimide moiety, in experime..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.04.008

2
A.M. Godin et al. / Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx
2.1.2. Syntheses of PTD-OH
111
Phthalimide (1.0 mmol) was added slowly to the ethanolamine 112
(1.25 mmol) at 100 °C and stirred for 4 h. The reaction mixture was 113
stirred at room temperature for 12 h (Scheme 1). The progress of the re- 114
action was monitored by TLC. Boiling water was added to the residue 115
and the obtained white solid was vacuum filtered, with yield 65%; Mp 116
126.6–128.4 °C. IR (ATR): 3474, 2954, 2944, 2888, 1768, 1694, 1430, 117
1396, 1378, 1058, 994, 726, 534. 1H NMR (200 MHz, CDCl3): 3.83 (s, 118
4H), 7.65–7.80 (m, 4H). 13C NMR (50 MHz, CDCl3): 40.6, 60.6, 123.2, 119
131.8, 134.0, 168.7. Anal. Calcd. for C10H9NO3. Calcd. (%): C, 62.82; H, 120
4.74; N, 7.33. Found (%): C, 62.99; H, 4.66; N, 7.13.
121
2.1.3. Syntheses of PTD-NO
122
This compound was obtained by mixing fuming nitric acid 123
(1.0 mmol) and the precursor compound (0.10 mmol) at −5 °C and 124
stirring for 2 h (Scheme 1). The reaction mixture was poured into a 125
mixture of water and ice. The pH was adjusted to 6 by adding CaCO3. 126
The obtained white solid was vacuum filtered, with yield 93%; Mp 127
87.1–89.5 °C. IR (ATR): 3062, 3030, 2978, 2886, 1774, 1716, 1608, 128
1422, 1402, 1288, 982, 870, 722, 530. 1H NMR (200 MHz, DMSO-d6): 129
3.95 (t, 2H), 4.71 (t, 2H), 7.86 (br s,4H). 13C NMR (50 MHz, DMSO-d6): 130
34.8, 70.8, 123.1, 131.4, 134.4, 167.5. Anal. Calcd. for C10H8N2O5. Calcd. 131
(%): C, 50.85; H, 3.41; N, 11.86. Found (%): C, 51.09; H, 3.32; N, 11.77. 132
Fig. 1. Chemical structures of (A) 2-phthalimidethanol (PTD-OH), (B) 2-phthalimidethyl
nitrate (PTD-NO) and (C) thalidomide (2-phthalimidoglutarimide).
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
activity of cyclooxygenases (Noguchi et al., 2002; Sano et al., 2004;
Suizu et al., 2003) and nitric oxide (NO) synthases (Noguchi et al.,
2004; Shimazawa et al., 2004). The antitumoral activity of some phthal-
imide analogs may be related to their inhibitory effects on angiogenesis
(Noguchi et al., 2005; Sano et al., 2006) and also on tubulin polymeriza-
tion (Iguchi et al., 2008; Li et al., 2006; Yanagawa et al., 2006).
Some investigators, while developing new phthalimide analogs,
have also adopted a strategy that has been widely used with steroidal
(SAIDs) and non-steroidal (NSAIDs) anti-inflammatory drugs, that is,
the coupling of NO donors. This strategy led to the development of
new compounds with improved efficacy and greater safety in preclinical
assays such as nitroacetylsalicylic acid (Fiorucci et al., 2000; Turnbull
et al., 2008), nitroparacetamol (Romero-Sandoval et al., 2007),
nitronaproxen (Young et al., 2005) and nitroprednisolone (Tallet et al.,
2002). Regarding the phthalimide analogs coupled to NO donors, the in-
vestigation is still preliminary but it has been shown that some of them
exhibit cytotoxic activity against various cancer cell lines (Wang et al.,
2.2. Animals
133
Eight-week-old male C57BL/6J mice were used. The animals had free 134
access to food and water and were maintained in a room with a 12 h 135
light-dark cycle for at least 3 days before the experiment to allow accli- 136
matization. The experiments were carried out at a room temperature of 137
27 °C. This temperature was used because the thermoneutral zone for 138
mice and rats ranges between 26 and 34 °C, a temperature range that 139
markedly differs from that of standard laboratory environments which 140
could be stressful and affect many aspects of physiology and behavior 141
of the rodents (Gaskill et al., 2009; Gordon, 1990). All experiments 142
were conducted according to the ethical guidelines for investigation of 143
experimental pain in conscious animals (Zimmermann, 1983) and by 144
the Ethics Committee on Animal Experimentation of the Federal Univer- 145
sity of Minas Gerais (CETEA — UFMG. Protocol 48/2011).
146
8Q7 2 2009) and also antinociceptive and anti-inflammatory activities (Dos
88
89
90
91
92
93
94
95
96
97
98
99
Santos et al., 2001).
2.3. Drugs
147
In the present study, we evaluated the activities of two phthalimide
analogs devoid of the glutarimide ring, 2-phthalimidethanol (PTD-OH)
and 2-phthalimidethyl nitrate (PTD-NO) (Fig. 1), in experimental
models of inflammatory pain and edema. These analogs represent
much simpler molecules when compared with many of the phthalimide
analogs synthesized and evaluated by other investigators (Chaulet et al.,
2011; de Almeida et al., 2007; Mazzoccoli et al., 2012; Miyachi et al.,
1997a, 1997b; Stewart et al., 2007, 2010; Teubert et al., 1998). In addi-
tion, our interest in the phthalimide analogs devoid of the glutarimide
moiety derives from the evidence provided by different studies that
this functional group may contribute to the teratogenic effect (Huang
Suspensions of PTD-OH, PTD-NO, thalidomide (FUNED, Brazil) and 148
phenobarbital (Aventis Pharma, Brazil) were prepared in 0.5% carboxy- 149
methylcellulose (CMC) sodium salt (Sigma, USA) suspension in saline 150
immediately before the experiments. Suspensions were administered 151
per os (p.o.) in a volume of 12 ml/kg. λ-Carrageenan (Sigma, USA) 152
suspension was prepared in saline.
153
2.4. Evaluation of mechanical allodynia
154
Tactile thresholds were measured by using an electronic von Frey 155
apparatus (electronic pressure-meter, Insight, Brazil), according to 156
method described by Cunha et al. (2004), with minor modifications. 157
The mice were kept individually in acrylic cages (10 × 10 cm with 158
18 cm-high walls) whose floor was a metal grid. The hand-held force 159
transducer, fitted with a polypropylene tip (0.5 mm2) filament, was 160
directed upwards from below the grid and gradually pressed on the 161
plantar surface of the right hind paw. The test consisted of evoking a 162
hind paw flexion reflex. The endpoint was characterized by the removal 163
of the paw usually followed by flinching movements. After the paw 164
withdrawal, the intensity of the pressure was automatically recorded. 165
The value for the response was obtained by averaging five measure- 166
ments. Mice were habituated to the experimental apparatus daily, ap- 167
proximately 60 min a day, for 4 days before the experiments. On the 168
experimental day, baseline paw withdrawal threshold of each animal 169
100 and McBride, 1997; Lepper et al., 2004).
101 2. Methods
102 2.1. Synthesis of phthalimide analogs
103 2.1.1. Chemicals and reagents
104
All chemicals were obtained from commercial suppliers and used
105 without further purification. Melting points were determined in a
106 Gehaka PF 1500 apparatus and are uncorrected. 1H nuclear magnetic
107 resonance (NMR) spectra and 13C NMR spectra were recorded on a
108 Bruker Avance DPX/200. Chemical shift values (δ) were given in parts
109 per million (ppm). Infrared (IR) spectra were recorded on a Spectro
110 One Perkin Elmer.
Please cite this article as: Godin AM, et al, Activities of 2-phthalimidethanol and 2-phthalimidethyl nitrate, phthalimide analogs devoid of the
glutarimide moiety, in experime..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.04.008

A.M. Godin et al. / Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx
3
Scheme 1. Synthesis of PTD-OH and PTD-NO.
170 was determined. After that, the animals were divided into the experi-
171 mental groups in such a way that the mean thresholds of the different
172 groups were similar. Carrageenan (300 μg, 20 μl) was injected via the
173 intraplantar (i.pl.) route 1 h after p.o. administration of PTD-OH, PTD-
174 NO, thalidomide (125, 250, 500 or 750 mg/kg) or vehicle (CMC 0.5%,
175 12 ml/kg). The paw withdrawal threshold of each animal was again
176 measured at 2, 4 and 6 h after carrageenan injection. In an additional
177 protocol, we investigated the effect induced by p.o. administration of
178 PTD-OH, PTD-NO, thalidomide (500 mg/kg) or vehicle (CMC 0.5%,
179 12 ml/kg) on the paw withdrawal threshold of naive animals that
180 were not injected with carrageenan. The results were expressed as the
181 absolute withdrawal threshold (in grams).
instructions supplied by the manufacturer (R&D Systems, Minneapolis, 221
USA). The results were expressed as pg/100 mg of tissue. 222
The number of neutrophils in the paw tissue was indirectly deter- 223
mined by quantifying mieloperoxydase (MPO) activity. The pellet 224
samples were frozen and thawed three times in liquid nitrogen. Upon 225
thawing, the samples were centrifuged (10,000 r.p.m.) for 15 min 226
at 4 °C and 25 μl of the supernatant were used for the MPO assay. 227
The enzymatic reaction was assessed by adding 25 μl of 1.6 mM 228
tetramethylbenzidine prepared in DMSO. The mixture was incubated 229
for 5 min at 37 °C. Then, 100 μl of 0.02% hydrogen peroxide were 230
added, followed by incubation for 5 min at 37 °C. After 10 min, the reac- 231
tion was stopped by adding 100 μl of 1 M sulphuric acid. The absorbance 232
OOF
was measured at 450 nm. Previously, a standard curve indicating the 233
182 2.5. Evaluation of paw edema
range of linear relationship between neutrophils and optical density 234
(O.D.; expressed in relative units) was prepared. Results were expressed 235
183
Paw edema was measured with a plethysmometer (Model 7140,
as number of neutrophils per 100 mg of tissue.
236
184 Ugo Basile, Italy). The basal volume of the right hind paw was measured
185 before any treatment. After determination of the basal volume, the ani-
186 mals were divided into the experimental groups in such a way that the
187 mean volumes of the different groups were similar. Carrageenan (300
188 μg, 20 μl) was injected via the i.pl. route 1 h after p.o. administration
189 of PTD-OH, PTD-NO, thalidomide (125, 250, 500 or 750 mg/kg) or vehi-
190 cle (CMC 0.5%, 12 ml/kg). The paw volume of each animal was again
191 measured at 2, 4 and 6 h after carrageenan injection. The results were
192 expressed as the paw volume change (μl) in relation to the basal values.
2.8. Evaluation of plasma concentrations of nitrite
237
PTD-OH, PTD-NO, thalidomide (500 mg/kg) or vehicle (CMC 0.5%, 238
12 ml/kg) were administered p.o. in animals fasted for 12 h. A naive 239
group of animals that was not submitted to any intervention was also 240
used to provide the blank samples. One hour later, each animal was anes- 241
thetized via the i.p. route with a mixture of ketamine (100 mg/kg) and 242
xylazine (10 mg/kg). After laparotomy, a blood sample was collected 243
from the abdominal artery of each animal in a heparinized tube and im- 244
mediately centrifuged at 14,000 rpm for 10 min at 4 °C. Then, the plasma 245
samples were transferred to plastic tubes and stored at −70 °C until 246
analysis. Plasma samples were analysed by using a procedure based on 247
the Griess reaction (Sun et al., 2003). Briefly, 60 μl of Griess reagent (1% 248
sulfanilamide and 0.1% napthylethylenediamine in 2.5% phosphoric 249
acid) was added to 60 μl of plasma samples. After 10 min, the absorbance 250
of the samples was measured at 540 nm. A standard curve of nitrite con- 251
centrations against absorbance, with concentrations ranging from 5 to 252
240 μM, was used to quantify the concentrations of nitrite in the samples. 253
193 2.6. Evaluation of motor activity
194
The motor activity of the animals was evaluated in a rota-rod appa-
195 ratus. The animals were trained on the apparatus for 3 days before the
196 experiment. On the experimental day, the animals were placed on a ro-
197 tating (14 rpm) and the time they spent on it was measured. The cut-off
198 time was 120 s. After determination of the baseline values, the animals
199 were treated with PTD-OH, PTD-NO, thalidomide (500 or 750 mg/kg),
200 phenobarbital (40 mg/kg; positive control) or vehicle (CMC 0.5%,
201 12 ml/kg) and 1 h later they were again tested in the apparatus.
The results were expressed in μM.
254
202 2.7. Evaluation of TNF-α and CXCL1 concentrations and the number of
203 neutrophils in the paw tissue
2.9. Statistical analysis
255
While registering the experimental parameters within each protocol, 256
the observer was not aware of the treatments. Data were presented as 257
204
Carrageenan (300 μg, 20 μl) was injected via the i.pl. route 1 h after
205 p.o. administration of PTD-OH, PTD-NO, thalidomide (500 mg/kg) or
206 vehicle (CMC 0.5%, 12 ml/kg). The animals were euthanized by cervical
207 dislocation 4 h later and the paw tissue was removed and frozen at
208 −70 °C. The methods used to quantify TNF-α and CXCL1 concentra-
mean
standard error of the mean. Two-way ANOVA followed by 258
Bonferroni post hoc test was used to analyze withdrawal thresholds 259
and paw volumes differences. One-way ANOVA followed by Newman– 260
Keuls post-hoc test was used to analyze biochemical parameters and 261
motor activity differences. A p b 0.05 was considered significant. A 262
GraphPad® 5.0 (San Diego, USA) software was used to perform statistical 263
UNCORRECTED PR
209 tions and the number of neutrophils were based on those described
210 by Guerrero et al. (2006) and Coelho et al. (2008). Upon thawing, the
211 tissue (0.1 g of tissue per 1.9 ml of phosphate buffered saline solution
212 containing 0.05% Tween-20, 0.1 mM phenylmethylsulphonyl fluoride,
213 0.1 mM benzamethonium chloride, 10 mM EDTA, 2 μg/ml aprotinin A
214 and 0.5% bovine serum albumin) was homogenized and centrifuged
215 (10,000 r.p.m.) for 15 min at 4 °C. The supernatant and the pellet (resus-
216 pended in 0.5% hexadecyltrimethyl ammonium bromide buffer pH 5.4)
217 were stored separately at −70 °C until further analysis of TNF-α and
218 CXCL1 concentrations and number of neutrophils. The supernatant
219 samples, diluted in phosphate buffered saline (1:3), were used to
220 determine TNF-α and CXCL1 concentrations by ELISA, following the
analysis and prepare the figures.
264
3. Results
265
3.1. Effects induced by the phthalimide analogs on the mechanical allodynia 266
I.pl. injection of carrageenan induced a marked and long lasting me- 267
chanical allodynia that was already observed 2 h after injection. Both 268
phthalimide analogs, PTD-OH (Fig. 2A) and PTD-NO (Fig. 2B), adminis- 269
tered 1 h before carrageenan at the dose of 750 mg/kg, inhibited the 270
Please cite this article as: Godin AM, et al, Activities of 2-phthalimidethanol and 2-phthalimidethyl nitrate, phthalimide analogs devoid of the
glutarimide moiety, in experime..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.04.008

4
A.M. Godin et al. / Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx
Table 1
t1:1
t1:2
t1:3
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
mechanical allodynia. The anti-allodynic effect was observed at 2 and 4 h
after injection of carrageenan. Thalidomide, used as a reference phthali-
mide analog, also exhibited an anti-allodynic effect (Fig. 2C). The highest
dose (750 mg/kg) inhibited the mechanical allodynia along the whole ex-
perimental period, while the effect induced by the dose of 500 mg/kg was
observed only at 4 h after carrageenan injection. The phthalimide analogs
and thalidomide (500 mg/kg) were devoid of activity in naive animals
that were not injected with carrageenan (Fig. 2D).
To rule out lack of motor coordination or muscle relaxation as possi-
ble confounding effects to the antinociceptive activity of the phthalimide
analogs, we evaluated the effect induced by the previous (1 h) adminis-
tration of the three compounds on the motor coordination of the mice.
PTD-OH (500 mg/kg), PTD-NO and thalidomide (500 or 750 mg/kg)
did not alter the time mice spent on the rotating rod. However, the
highest dose (750 mg/kg) of the PTD-OH and also phenobarbital
(40 mg/kg) exerted an inhibitory effect on the motor coordination
(Table 1). Thus, although PTD-OH, PTD-NO and thalidomide inhibited
mechanical allodynia induced by carrageenan, lack of motor coordina-
tion may account, at least partially, for the reduced display of experimen-
tal pain behavior by mice treated with PTD-OH at the dose of 750 mg/kg.
Effects induced by PTD-OH, PTD-NO, thalidomide (500 or 750 mg/kg, p.o. — 1 h) or phe-
nobarbital (40 mg/kg, p.o. — 1 h) on the time spent on the rotating rod.
Treatment
Baseline
Time spent on the rotating rod (s)
1 h
t1:4
t1:5
Vehicle
120
120
119
120
120
119
120
119
0
0
1
0
0
1
0
1
120
119
117
115
45
117
112
17
0
1
2
t1:6
Thalidomide 500
Thalidomide 750
PTD-OH 500
PTD-OH 750
PTD-NO 500
PTD-NO 750
Phenobarbital
t1:7
t1:8
3
t1:9
9a
2
t1:10
t1:11
t1:12
t1:13
8
3a
a
Significantly different from respective vehicle-treated group at the same time point
(p b 0.001), n = 8.
t1:14
inhibited the paw edema, an effect observed at 4 and 6 h after carra- 296
geenan injection (Fig. 3A). On the other hand, an anti-edema effect 297
was induced at all doses (125, 250, 500 or 750 mg/kg) of PTD-NO, 298
with the higher doses inducing a more enduring effect (Fig. 3B). 299
Thalidomide, used as a reference phthalimide analog, did not exhibit 300
an anti-edema activity in this experimental model (Fig. 3C).
301
291 3.2. Effects induced by the phthalimide analogs on the paw edema
292
I.pl. injection of carrageenan induced a marked and long lasting paw
3.3. Effects induced by the phthalimide analogs on the paw TNF-α and 302
CXCL1 concentrations and the number of neutrophils in the paw tissue 303
293 edema that was already evident 2 h after injection. Both phthalimide
294 analogs, administered 1 h before carrageenan, inhibited the paw
295 edema. Only the two highest doses of PTD-OH (500 and 750 mg/kg)
Four hours after i.pl. injection of carrageenan, there was an increase 304
in the paw concentrations of TNF-α and CXCL1 and also the number of 305
Fig. 2. Effects induced by (A) PTD-OH, (B) PTD-NO or (C) thalidomide (125, 250, 500 or 750 mg/kg, p.o. — 1 h) on the mechanical allodynia induced by i.pl. injection of carrageenan
(300 μg) or by (D) PTD-OH, PTD-NO or thalidomide (500 mg/kg, p.o.) on the paw withdrawal threshold of naive animals that were not injected with carrageenan. * and ** indicate sig-
nificantly different from vehicle (p b 0.05 and p b 0.01, respectively), n = 7–8.
Please cite this article as: Godin AM, et al, Activities of 2-phthalimidethanol and 2-phthalimidethyl nitrate, phthalimide analogs devoid of the
glutarimide moiety, in experime..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.04.008

A.M. Godin et al. / Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx
5
3.4. Effects induced by the phthalimide analogs on the plasma concentra- 312
tions of nitrite 313
Plasma samples from naive animals showed concentrations of nitrite 314
close to 6 μM. The plasma concentrations of nitrite, evaluated 1 h after 315
administration of vehicle (CMC 0.5%, 12 ml/kg), PTD-OH or thalidomide 316
(500 mg/kg) did not differ from that found in the samples from naive 317
animals (ranged between 9 and 14 μM). On the other hand, the plasma 318
concentration of nitrite was increased (close to 39 μM) after the 319
OOF
Fig. 3. Effects induced by (A) PTD-OH, (B) PTD-NO or (C) thalidomide (125, 250, 500 or
750 mg/kg, p.o. — 1 h) on the paw edema induced by i.pl. injection of carrageenan
UNCORRECTED PR
(300 μg). *, ** and ** indicate significantly different from vehicle (p b 0.05, p b 0.01 and
p b 0.001, respectively), n = 8.
306 neutrophils. PTD-NO, at the highest dose (500 mg/kg), reduced all the
307 responses, while at the lowest dose (250 mg/kg) reduced the concen-
308 trations of TNF-α (Fig. 4B) and CXCL1 (Fig. 4C). On the other hand,
309 PTD-OH (500 mg/kg) reduced only the number of neutrophils
310 (Fig. 4A), while thalidomide was devoid of activity in the models used
311 (Fig. 4).
Fig. 4. Effects induced by PTD-OH, PTD-NO or thalidomide (250 or 500 mg/kg, p.o. — 1 h)
on the number of neutrophils (A) and the concentrations of TNF-α (B) and CXCL1 (C) in
the paw of the mice 4 h after i.pl. injection of carrageenan (300 μg). V = vehicle. * and
*** indicate significantly different from vehicle (p b 0.05 and p b 0.001, respectively).
# indicates significantly different from control (p b 0.001), n = 8.
Please cite this article as: Godin AM, et al, Activities of 2-phthalimidethanol and 2-phthalimidethyl nitrate, phthalimide analogs devoid of the
glutarimide moiety, in experime..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.04.008

6
A.M. Godin et al. / Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx
320 administration of the phthalimide analog coupled to a NO donor,
321 PTD-NO (Fig. 5).
The mechanical allodynia and paw edema induced by carrageenan 364
were associated with increased paw concentrations of TNF-α and 365
CXCL-1 and also increased neutrophil infiltration, indirectly evaluated 366
by MPO activity. It has been widely demonstrated that carrageenan in- 367
duces a local inflammatory reaction involving the production of media- 368
tors such as NO (Handy and Moore, 1998; Nakamura et al., 1996), 369
eicosanoids (Seibert et al., 1994; Tonussi and Ferreira, 1994; Vinegar 370
et al., 1987), chemokines (Loram et al., 2007) and cytokines (Chen 371
et al., 1994; Cunha et al., 2000), among others. By using specific neutral- 372
izing antiserum to cytokines or deficient animals, some authors have 373
demonstrated the role of both TNF-α and CXCL-1 to the mechanical 374
allodynia and paw edema induced by carrageenan (Cunha et al., 1992; 375
Loram et al., 2007; Rittner et al., 2005; Rocha et al., 2006). In addition, 376
the role of local neutrophil infiltration and activation in these responses 377
has also been shown (Cunha et al., 2008; Salvemini et al., 1996) and it is 378
suggested that their contribution derives from their ability to produce 379
322 4. Discussion
323
Recently, driven mainly by the reintroduction of thalidomide in the
324 pharmacotherapy, there is a great and increasing interest in the synthe-
325 sis and pharmacological evaluation of phthalimide analogs with new
326 and improved activities and also greater safety. Many analogs of thalid-
327 omide, containing both the phthalimide and the glutarimide moieties,
328 have been synthesized and shown to exhibit a wide diversity of
329 activities, including anti-inflammatory and antinociceptive ones, in pre-
330 clinical pharmacological assays (Chaulet et al., 2011; de Almeida et al.,
331 2007; Li et al., 2006; Noguchi et al., 2002, 2004; Sano et al., 2006). In
332 the present study, we showed that two phthalimide analogs devoid of
333 the glutarimide moiety and thus structurally simpler in comparison to
334 thalidomide or its most investigated analogs, exhibit activities in exper-
335 imental models of inflammatory pain and edema, but do not change the
336 nociceptive threshold in naive animals.
chemokines, cytokines, NO and reactive oxygen species.
380
Aiming to provide insight into the mechanisms that could contribute 381
to the anti-inflammatory and antinociceptive activities of PTD-OH and 382
PTD-NO, we investigated the effects induced by both analogs and also 383
by thalidomide on the paw concentrations of TNF-α and CXCL-1 and 384
number of neutrophils after carrageenan injection. Both PTD-OH and 385
PTD-NO, but not thalidomide, partially reduced the accumulation of 386
neutrophils induced by carrageenan. Regarding the effects induced on 387
the paw concentrations of TNF-α and CXCL-1, only the phthalimide 388
analog coupled to a NO donor, PTD-NO, exhibited an inhibitory activity. 389
Although thalidomide has been regarded as a traditional TNF-α inhibi- 390
tor, its effects on the production of this cytokine are not as straightfor- 391
ward as it would be expected. There are studies showing both 392
inhibitory (Moreira et al., 1993; Sampaio et al., 1991) and stimulatory 393
(Corral et al., 1999; Haslett et al., 1998; Marriott et al., 2002) effects in- 394
duced by thalidomide, depending on the doses or concentrations used, 395
the stimulus used to trigger TNF-α production, whether it is an 396
in vitro or an in vivo assay, among other factors. Although we did not 397
use neutralizing antiserum or antibodies or deficient animals in the 398
present study, the increased concentrations of TNF-α and CXCL-1 in 399
the paw tissue of animals injected with carrageenan, summed up with 400
the literature data, allows us to suggest that these mediators may con- 401
tribute to the mechanical allodynia in our experimental model. It must 402
be emphasized that both phthalimide analogs, in spite of inhibiting 403
the mechanical allodynia induced by carrageenan, exerted differential 404
effects on the production of TNF-α and CXCL-1. It may be hypothesized 405
that the effects induced by the analogs on the production or action of 406
other mediators may contribute to their concordant effects on the 407
337
The two phthalimide analogs investigated in the present study were
338 PTD-OH and PTD-NO, both devoid of the glutarimide moiety. PTD-OH is
339 characterized by the coupling of a side chain with the alcohol functional
340 group (-OH) while in the PTD-NO there is the replacement of this
341 hydroxyl by an NO donor. The investigation of these two analogs in
342 the same experimental models allowed us to characterize and compare
343 their pharmacological profiles and to investigate whether the
344 glutarimide moiety is essential for the anti-inflammatory activity.
345
In the present study we used the experimental model of inflamma-
346 tion induced by carrageenan to investigate the activities of the two
347 phthalimide analogs and to compare them with thalidomide. Previous
348 treatment of the mice with both PTD-OH and PTD-NO inhibited both
349 the mechanical allodynia and the paw edema induced by carrageenan.
350 Thalidomide, on the other hand, inhibited only the mechanical
351 allodynia. Although Rocha et al. (2006) have shown that thalidomide in-
352 hibits the paw edema induced by carrageenan, such an effect was not
353 observed in the present study. The reasons for this difference are not
354 clear, but may be related to different doses of thalidomide and mice
355 strains used in the studies. Other investigators have shown varying
356 effects induced by thalidomide in preclinical assays depending on the
357 doses and concentration of the drug, stimuli used to induce the inflam-
358 matory response and also the dosing and time courses assessed (Corral
359 et al., 1999; Haslett et al., 1998; Marriott et al., 2002; Moreira et al.,
360 1993; Sampaio et al., 1991). However, noteworthy is the fact that the
361 two phthalimide analogs, structurally simpler when compared with
362 thalidomide, induced more marked effects than the most known
363 compound of the class.
mechanical allodynia induced by carrageenan.
408
The strategy of coupling NO donors to clinical or experimental drugs 409
has been highly promoted in the last years aiming to obtain new drugs 410
with greater safety and efficacy (Fiorucci, 2001; Iwanaga et al., 2004; 411
Keeble and Moore, 2002; Miller and Megson, 2007; Tallet et al., 2002). 412
This strategy has been more frequently applied to SAIDs, NSAIDs and 413
cardiovascular drugs. Recently, Wang et al. (2009) and Dos Santos 4Q134
et al. (2001) coupled NO donors to some phthalimide analogs and 415
showed that these compounds exhibit cytotoxic activity against various 416
cancer cell lines and also antinociceptive and anti-inflammatory 417
activities.
418
In the present study, we extend this investigation by evaluating the 419
activity of a phthalimide analog coupled to a NO donor, PTD-NO, and 420
comparing it with that of the parent compound, PTD-OH. As expected, 421
PTD-NO administration resulted in increased nitrite levels, an effect 422
that was not observed after administration of PTD-OH. Increased nitrite 423
levels have also been observed after administration of nitroparacetamol 424
(Futter et al., 2001), nitroacetylsalicylic acid (Carini et al., 2004) and 425
nitronaproxen (Rossoni et al., 2006). Our results show that PTD-OH 426
and PTD-NO, in spite of inhibiting the mechanical allodynia and paw 427
edema induced by carrageenan, may induce these effects by activating 428
different mechanisms. Clearly, the phthalimide analog coupled to the 429
Fig. 5. Effects induced by PTD-OH, PTD-NO or thalidomide (500 mg/kg, p.o. — 1 h) on
the plasma concentrations of nitrite. *** indicate significantly different from vehicle
(p b 0.001). n = 7.
Please cite this article as: Godin AM, et al, Activities of 2-phthalimidethanol and 2-phthalimidethyl nitrate, phthalimide analogs devoid of the
glutarimide moiety, in experime..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.04.008

A.M. Godin et al. / Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx
7
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430 NO donor, PTD-NO, exerted a wider effect on the biochemical parame-
431 ters when compared to PTD-OH, inhibiting both the production of
432 TNF-α and CXCL-1. This wider effect could be associated with the fact
433 that all doses of PTD-NO, but only the two highest doses of PTD-OH,
434 inhibited the paw edema induced by carrageenan. It has been shown
435 that NO may mediate the antinociceptive and anti-inflammatory effects
436 induced by several classes of drugs in different experimental models
437 (Alves et al., 2004; Brock and Tonussi, 2008; Fernandes et al., 2002;
438 Soares et al., 2000). Thus, it is proposed that NO release could contribute
439 to the wider effect induced by PTD-NO both on the production of in-
440 flammatory mediators and paw edema. Data from other studies provide
441 additional clues to understand why NO-donating compounds may
442 exhibit increased anti-inflammatory activity and also greater safety
443 when compared with the parent compounds. These include greater in-
444 hibitory effect on caspase activity, transduction via the NF-κB pathway
445 and Janus kinase and p38 pathways (Keeble and Moore, 2002; Rigas,
446 2007). However, it must be emphasized that the role of NO in the mod-
447 ulation of inflammation and pain is clearly dual, as there is evidence of
448 both inflammatory/nociceptive and anti-inflammatory/antinociceptive
449 roles, depending mainly on the site of production and tissue concentra-
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OOF
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452 tivities PTD-OH and PTD-NO contributes to further increase the interest
453 in the pharmacological evaluation of phthalimide analogs. Noteworthy,
454 the glutarimide moiety is not essential for the anti-inflammatory and
455 antinociceptive activities. In addition, our results show that compounds
456 structurally much simpler than thalidomide or many of its analogs
457 under investigation exhibit similar activities in experimental models
458 of pain and inflammation. Finally, as there is evidence that the
459 glutarimide moiety contributes to the teratogenic effect of many thalid-
460 omide analogs (Huang and McBride, 1997; Lepper et al., 2004), our re-
461 sults indicate that phthalimide analogs devoid of this functional group
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Lepper ER, Ng SS, Gütschow M, Weiss M, Hauschildt S, Hecker TK, et al. Comparative 546
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We thank Fundação de Amparo à Pesquisa do Estado de Minas
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466 Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e
467 Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de
468 Ensino Superior (CAPES) and Pró-Reitoria de Pesquisa/UFMG for finan-
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Please cite this article as: Godin AM, et al, Activities of 2-phthalimidethanol and 2-phthalimidethyl nitrate, phthalimide analogs devoid of the
glutarimide moiety, in experime..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.04.008