Canadian Journal of Physiology and Pharmacology
Analgesia Additive Interaction between Tadalafil and Morphine in Experimental Animal Model
Journal: Canadian Journal of Physiology and Pharmacology
Manuscript ID cjpp-2019-0674.R2
Manuscript Type: Article
Date Submitted by the 24-Apr-2020 Author:
Complete List of Authors: Mehanna, Mohammed; Beirut Arab University, Pharmaceutical technology Domiati, Souraya ; Department of Pharmacology and Therapeutics, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon, Nakkash Chmaisse,Draft Hania ; Department of Pharmacology and Therapeutics, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon, El Mallah, Ahmed ; Department of Pharmacology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue:
Keyword: Morphine, Antinociception, Isobolographic analysis, Nitric oxide, Tadalafil
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1 Analgesia Additive Interaction between Tadalafil and Morphine in Experimental
2 Animal Model
3 Mohammed Mehanna1*, PhD, Souraya Domiati2, Pharm D, MSc, PhD; Hania Nakkash Chmaisse3, PhD;
4 Ahmed El Mallah4, PhD;
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6 1Department of Pharmaceutical technology, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon,
8 2Department of Pharmacology and Therapeutics, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon,
10 3Department of Pharmacology and Therapeutics, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon, 11 [email protected] 12 4Department of Pharmacology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt,
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20 *correspondent email: [email protected]; Beirut Arab University, Beirut Campus, Tarik El Jadida; Phone
21 number 96101300110 ext. 2867; P.O. Box 11-5020 Riad El Solh 11072809- Beirut, Lebanon
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24 Abstract:
25 Since both morphine and tadalafil have been proven to exert some of their analgesic activity
26 through modulation of the NO-cGMP pathway, the aim of the current study is to evaluate the
27 pharmacologic interaction between tadalafil and morphine in order to decrease the dose of
28 morphine and subsequently its side effects. The assessment was carried out through isobolographic
29 analysis relative to ED50s of both morphine and tadalafil obtained by tail-flick test on BALB/c
30 mice. Morphine and tadalafil ED50s calculated from the dose-response curves were 8303 µg/kg
31 and 2080 µ/kg, respectively. The experimental ED50 values of morphine and tadalafil in their
32 mixture were 4800 µ/kg and 1210 µ/kg, respectively. Those results showed an additive interaction 33 between morphine and tadalafil presentedDraft by a total fraction value for the mixture of 1160 µ/kg. 34 This outcome can be interpreted by the fact that both drugs share common pathways namely, NO-
35 cGMP and opioid receptors. As a conclusion, morphine and tadalafil combination showed an
36 additive effect against acute pain, which is mediated through the central nervous system, thus
37 providing a rationale for combining them in order to decrease morphine dose and thus minimizing
38 its side effect.
39
40 Keywords: Antinociception, analgesia, Drug-interaction, Isobolographic analysis, Morphine,
41 Nitric oxide, Phosphodiesterase 5 inhibitor, Tadalafil.
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46 1. Introduction
47 Nitric oxide (NO) is produced in different cell types through the action of specific neuronal and
48 non-neuronal nitric oxide synthases on L-arginine, as a substrate. NO plays a major role in diverse
49 physiologic functions relevant to white blood cells, blood vessels, and the central nervous system
50 via targeting the cytosolic enzyme soluble guanylyl cyclase which converts guanosine -5’
51 triphosphate (GTP) into the second messenger cyclic guanosine monophosphate (cGMP). The
52 latter has many cellular targets among which cGMP-dependent protein kinases and cyclic
53 nucleotide-gated ion channels control up/down nociceptors expressions. cGMP is further degraded
54 by the specific phosphodiesterases 5, 6 and 9 to GMP which terminates the signal transduction 55 (Florentino, Galdino et al. 2015, Florentino,Draft Silva et al. 2017). In fact, the complex role of NO- 56 cGMP system in the modulation of nociception has been proven in both acute and chronic pain on
57 central and peripheral levels (Ambriz-Tututi, Velazquez-Zamora et al. 2005, Gediz, Nacitarhan et
58 al. 2015). NO formation is induced and terminated within few seconds after reacting with its
59 intracellular targets (Sharma, Al-Omran et al. 2007). Therefore, the cornerstone of pain
60 management is NO biosynthesis modulation and cGMP formation. Accordingly, different
61 phosphodiesterase inhibitors have been investigated for their analgesic effect. Sildenafil, tadalafil,
62 vardenafil, and other phosphodiesterase inhibitors, used in the treatment of erectile dysfunction,
63 have been proven to exert an antinociceptive effect in different pain models (Ambriz-Tututi,
64 Velazquez-Zamora et al. 2005, Rocha, Silva et al. 2011, Pasternak 2014). In fact, according to
65 Rocha, Silva et al.(2011), tadalafil antinociceptive effect was due to inhibition of neutrophil influx
66 and tumor necrosis factor alpha release in an inflammatory pain model in rat. NO was proven by
67 Otari and Upasani (2015) to play an essential role in in tadalafil analgesic effect in carrageenan
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68 pain model. Furthermore, Mehanna et al (2018) demonstrated that tadalafil analgesic effect
69 involved several receptors and mediators at the central and peripheral pain pathways.
70 Morphine, a well-defined analgesic, exerts its action mainly on mu as well as delta and kappa
71 opioid receptors (Pasternak 2014). In 1997, Granados-Soto et al. proved that morphine peripheral
72 analgesia is associated with the activation of L-arginine-NO-cGMP pathway (Granados-Soto,
73 Rufino et al. 1997). Moreover, several studies have shown that morphine induces nitric oxide
74 synthase formation thus leading to the elevation of nitric oxide concentration at the supraspinal
75 level, which in turn contributes to opioid-suppression of perception of pain signals in the higher
76 centers (Granados-Soto, Rufino et al. 1997, Basbaum, Bautista et al. 2009, Ghelardini, Di Cesare
77 Mannelli et al. 2015). In fact, opioid analgesic effect was demonstrated by conditional gene
78 knockout approaches to involve supraspinal,Draft spinal, and peripheral actions (Sawynok & Liu 2014).
79 Although morphine is a potent analgesic agent, it should be used with caution as it can induce
80 several side effects ranging from nausea, vomiting, pruritus, sedation up to respiratory depression
81 and death, especially at high doses. Moreover, long term use of morphine can lead to tolerance and
82 physical addiction (Dumas and Pollack 2008, Hong, Flood et al. 2008). As a consequence, in order
83 to decrease the dose of morphine and accordingly its side effects, several studies have investigated
84 the beneficial pharmacologic interaction of morphine with different drugs such as sildenafil and
85 zaprinast, two phosphodiesterase 5 inhibitors. In fact, additive antinociception was proven between
86 sildenafil and morphine while synergistic antinociception was demonstrated between zaprinast and
87 morphine (Yoon, Choi et al. 2006, Yoon, Park et al. 2008, Chen, Ma et al. 2010).
88 In light of the aforementioned facts, the aim of the current study is to investigate and evaluate the
89 potential possible interaction between tadalafil and morphine with respect to their analgesic
90 effects.
91
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92 2. Material and methods
93 2.1 Animals
94 Animal handling and experimental procedures were performed in accordance with the regulations
95 and guidelines stipulated by the Institutional Animal Care and Use Guidelines (IACUG) at Beirut
96 Arab University, Lebanon (IRB number 2016A-0043-P-P-0165) as well as the Canadian Council
97 on Animal Care (CCAC). Male BALB/c mice (20-25g), housed in polyacrylic cages under
98 standard conditions, were used. Animals had free access to water and standard laboratory chow.
99 2.2 Chemicals and drugs
100 Tadalafil and morphine sulfate (15mg/ml) used in the experimental work were obtained from 101 Pfizer and Renaudin, respectively. TadalafilDraft and morphine were dissolved in polyethylene glycol 102 400 (Fluka analytica) and normal saline, correspondingly to be administered intraperitoneally.
103 2.3 Apparatus for pain assessment
104 A tail flick analgesic apparatus type 812, Ugo Basile®, Germany was used in addition to a
105 plexiglass restrainer.
106 2.4 Experimental procedure
107 Tail flick test was used to study the nociceptive effect of tadalafil, morphine and their combinations
108 in addition to determine their ED50 (Bannon and Malmberg 2007). A total of 105 mice were used.
109 To determine the most suitable time latency for the isobolographic analysis, two different doses of
110 tadalafil (1 and 1.5 mg/kg) and two doses of morphine (2.5 and 5mg/kg) were administered
111 intraperitoneally as previously determined in other studies (Mehanna et.al 2018; Fennessy and Lee
112 1972). Polyethylene glycol 400 and normal saline groups served as negative controls for tadalafil
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113 and morphine, respectively. Consequently, a total of 6 groups of mice of 5 each were used. Sample
114 size was calculated based on “resource equation” method (Charan & Kantharia 2013).
115 To determine tadalafil, morphine and their combinations ED50s, mice were divided into 15 groups,
116 five mice each, and received either morphine or tadalafil or their combinations according to the
117 experimental protocol. Mice were placed in a plexiglass restrainer allowing only their tails to be
118 exposed to the radiant heat at an intensity of 8 delivered by the apparatus 5 cm away from the tip
119 of the tail to induce thermal pain. Reaction time, the interval between exposing the tail to the
120 radiant heat and its withdrawal, was recorded. A cut-off time of 20 seconds was imposed as
121 protection against tissue damage. After recording the first latency until the brisk withdrawal of the
122 tail, which served as a baseline value, vehicle/drug solutions were injected intraperitoneally, then
123 the latency time was re-measured at differentDraft time intervals.
124 An isobolographic analysis was used to determine the nature of pharmacologic interaction between
125 intraperitoneal administration of morphine and tadalafil. As a first step, the ED50 value of each
126 drug was determined from the dose-response curve. Then, morphine and tadalafil were co-
127 administered at their ED50, 1/2, 1/4, 1/6 and 1/8. From the dose-response curves of the combined
128 treatment, ED50 values of morphine and tadalafil in the mixture were calculated and these dose
129 combinations were used to plot the isobologram. The theoretical additive dose combination and
130 the total fraction value were calculated to describe the magnitude of the interaction (Tallarida 2011,
131 Miranda, Noriega et al. 2014).
132 2.5 Calculations and statistical analysis.
133 ED50s of tadalafil and morphine were determined by plotting the logarithmic concentrations of
134 different doses versus the responses in the tail flick test, calculated as the percentage change from
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135 baseline. Values were expressed as mean ±standard error. Calculations were performed using
136 Graph Pad Prism 3 software. The fraction value was computed using the following equation:
퐸퐷50 표푓 푚표푟푝ℎ푖푛푒 푐표푚푏푖푛푒푑 푤푖푡ℎ 푡푎푑푎푙푎푓푖푙 퐸퐷50 표푓 푡푎푑푎푙푎푓푖푙 푐표푚푏푖푛푒푑 푤푖푡ℎ 푚표푟푝ℎ푖푛푒 137 Fraction value = 퐸퐷50 푓표푟 푚표푟푝ℎ푖푛푒 푔푖푣푒푛 푎푙표푛푒 + 퐸퐷50 푓표푟 푡푎푑푎푙푎푓푖푙 푔푖푣푒푛 푎푙표푛푒
138 Fraction values close to1 designate an additive effect, values greater than one indicate an
139 antagonistic interaction, while values less than one point at synergistic effect (Arguelles, Torres-
140 Lopez et al. 2002, Yoon and Choi 2003, Yoon, Choi et al. 2006, Tallarida 2011).
141 3. Results
142 Both morphine and tadalafil as well as their combination didn’t induce observed motor impairment
143 during experimentation. In fact, spontaneous activity expressed as vertical movements with both 144 forelimbs off the floor were recorded Draft by a video-camera during different periods between the 145 readings (Fleming et al, 2013).
146 All groups’ baseline latencies in the tail flick test were equal (table 1). In the control groups, the
147 latency time did not differ through the 24 hours period while both morphine and tadalafil
148 administration resulted in a significant dose-dependent increase in latency time as compared to the
149 control (figures 1 and 2). Moreover, as depicted from these figures, the morphine peak response
150 was between half and one hour and that of tadalafil was at approximately 1.5 to 2 hours. In
151 addition, tadalafil resulted in a steady response for up to 12 hours.
152 The dose-response curves of morphine and tadalafil resulted in ED50 values (95% confidence
153 intervals) of 8303 µ/kg (CI: 6817-1011.3) and 2080 µ/kg (CI: 10-4680), respectively (figure 3).
154 To estimate the ED50 of morphine-tadalafil combination, ED50 values and fractions (1/2, 1/4, 1/6
155 and 1/8) of ED50 of each drug were administered concurrently in an equal dose ratio. Percentage
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156 change of latency time from the baseline of the combination in the tail-flick test revealed an
157 increase in withdrawal latency in a dose-dependent manner (figure 4).
158 Isobolographic analysis revealed an additive interaction between tadalafil and morphine in the tail-
159 flick test (figure 5). The experimental ED50 values (95% confidence interval) of tadalafil and
160 morphine in their mixture, determined by the combination of different fractions of the ED50s, were
161 1210 µ/kg (CI: 140-10240) and 4800 µ/kg (CI: 570-40400), respectively. The total fraction value
162 for the mixture was 1.16 which is statistically not different from 1.
163 4. Discussion
164 The obtained results showed a significant central antinociceptive effect of morphine and tadalafil 165 in the tail-flick test, a validated methodDraft for assessing acute thermal central pain (Gardmark, 166 Hoglund et al. 1998, Bannon and Malmberg 2007). In fact, although it is known that morphine
167 decrease the pain behavior expressed by the tail withdrawal response which is susceptible to false
168 positive outcomes due to motor depression, no observed motor impairment was detected in
169 morphine group as compared to the control (Steven Negus, S. 2019). Morphine exerts its action
170 mainly through mu-opioid receptor activation (Chen, Ma et al. 2010) in addition to the nitric oxide-
171 cGMP pathway, as proven by several studies (Granados-Soto, Rufino et al. 1997, Arguelles,
172 Torres-Lopez et al. 2002, Ozdemir, Bagcivan et al. 2011). Similarly, tadalafil has been shown to
173 have an analgesic effect mediated via guanylyl cyclase which regulates the intracellular cGMP
174 concentration (Rocha, Silva et al. 2011). In fact, cGMP is involved in the modulation of
175 nociception transmission and is hydrolyzed by phosphodiesterase enzymes to GMP.
176 Phosphodiesterase inhibitors, such as sildenafil and vardenafil, have been shown to have a
177 potential analgesic effect in different pain models (Ambriz-Tututi, Velazquez-Zamora et al. 2005,
178 Gediz, Nacitarhan et al. 2015). More recently, our research group highlighted the antinociceptive
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179 effect of tadalafil and the involvement of opioid as well as NO-cGMP pathway in its effect
180 (Mehanna, Domiati et al. 2018). Thus, both tadalafil and morphine share a common pharmacologic
181 site of action which is the nitric oxide-cGMP pathway.
182 Although morphine is a potent analgesic used to treat severe pain, yet its use is accompanied by
183 several side effects ranging from nausea and vomiting to respiratory depression and tolerance
184 (Dumas and Pollack 2008, Hong, Flood et al. 2008). In fact, Mu-opioid receptor agonists overdose
185 epidemic raise a concern to develop safer ones. Accordingly, therapeutic strategies currently used
186 and investigated focuses on combining mu-opioid receptor agonist with non-opioid analgesic in
187 order to increase the therapeutic index and to decrease opioid dose that lead to milder side effects 188 and better compliance (Li, 2019). As a result,Draft several studies have been carried out to examine the 189 effect of different agents on morphine activity such as dexmedetomidine, lamotrigine, simvastatin,
190 and sildenafil (Arguelles, Torres-Lopez et al. 2002, Yoon, Park et al. 2008, Lin, Yeh et al. 2009,
191 Kabalak, Ekmekcioglu et al. 2013, Ghasemi, Moradi et al. 2015). In the current study, the
192 isobolographic analysis showed an additive interaction between morphine and tadalafil indicating
193 that this combination can augment the antinociceptive effect of both agents which is mediated
194 through the central nervous system. Such analogous action phenomenon was demonstrated by Ha
195 Yoon et.al who reported an additive interaction between morphine and sildenafil in rat formalin
196 test (Yoon, Park et al. 2008).
197 The synergistic effect between drugs occurs when an agent is able to alter the other agent kinetics
198 or act on complementary steps or pharmacological mechanisms. Meanwhile, if the drugs share the
199 same site or mechanism of action, a synergistic effect is less likely to be described (Yoon and Choi
200 2003, Yoon, Park et al. 2008). Therefore, the last case can be applied on morphine and tadalafil
201 combination since their analgesic action is mediated through common sites namely, NO-cGMP
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202 pathway and opioid receptors as demonstrated by Roche et al. and Mehanna et.al., respectively
203 (Rocha, Silva et al. 2011, Mehanna, Domiati et al. 2018).
204 The proven additive effect of tadalafil and morphine is similar to the results reported by Yoon
205 et.al. who displayed, through the isobolographic analysis, an additive interaction between
206 morphine and sildenafil (Yoon, Park et al. 2008). On the other hand, the combination of morphine
207 and zaprinast, another phosphodiesterase inhibitor, exhibited a synergistic effect (Yoon, Choi et
208 al. 2006). This discrepancy can be attributed to the enzymes inhibition selectivity since zaprinast
209 is a phosphodiesterase 5, 6 and 9 inhibitor while sildenafil and tadalafil are more selective
210 inhibitors of phosphodiesterase 5 (Merkel 1993, Francis and Corbin 2005, Coward and Carson 211 2008, Resta, Masi et al. 2016). Other factors,Draft that may also affect the results, include the utilized 212 pain model, animal species, and the route and dose of drugs administration.
213 As a conclusion, the obtained results indicate that tadalafil, morphine and their combination are
214 effective against acute pain mediated through the central nervous system. Moreover, an additive
215 effect is shown upon concomitant administration of tadalafil with morphine providing a rationale
216 for combining them in order to decrease morphine dose and thus minimizing its side effect.
217 Nevertheless, further studies are required. In fact, a range of fixed proportions of both drugs should
218 be studied in different pain models. Corresponding side effects also should be assessed (Li, 2019,
219 Stevenson, G.W et al., 2020)
220 Acknowledgement :-
221 Author contribution:
222 Authors' contributions: The idea of the research was suggested by Ahmed El Mallah. The study
223 design was elaborated by all authors. Souraya Domiati performed the experimental work and takes
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224 responsibility for the integrity of the data and the accuracy of the data analysis. She also wrote the
225 manuscript. Mohammad Mehanna was involved from the early steps of manuscript preparation.
226 All authors were involved in revising the manuscript critically for important intellectual content,
227 and all approved the final version to be published.
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318 Yoon, M. H. and J. I. Choi (2003). "Pharmacologic interaction between cannabinoid and either
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320 Yoon, M. H., J. I. Choi, S. J. Kim, C. M. Kim, H. B. Bae and S. T. Chung (2006). "Synergistic
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325 test." J Korean Med Sci 23(6): 1033-1038.
326
327 Table 1 Baseline latency time of different doses of morphine and tadalafil on the tail flick test
Agent (dose) Latency time (second) Control 2.44±0.16 Morphine (2.5mg/kg) 2.46±0.21 Morphine (5mg/kg) 2.26±0.16 Tadalafil (1mg/kg) 2.38±0.18 Tadalafil (1.5mg/kg) 2.25±0.10 328 n=5 329 Values are expressed as mean ±SEM 330 ANOVA test was used to compare the means (p=0.902) 331
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10
9
8
7
6
5 2.5mg/kg 4 5mg/kg control 3 Morphine latency time latency (second) Morphine 2
1
0 Draft 0 5 10 15 20 25 30 Time (hour) 336
337
338 Figure 1: Time course of intraperitoneal administration of morphine on the latency time in tail
339 flick test in mice
340 Each line represents the mean ± SEM of the latency time of a certain dose of morphine or the control which is
341 normal saline (n=5)
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10
8 1mg/kg 6 1.5mg/kg 4 control Tadalafil latency time (second) latency Tadalafil 2
0 0 5 10 15 20 25 Time (hour) 348
349 Figure 2: Time course of intraperitoneal administration of tadalafil on the latency time in tail 350 flick test in mice Draft 351 Each line represents the mean ± SEM of the latency time of a certain dose of tadalafil or the control which is
352 polyethylene glycol 400 (n=5)
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A 1200
1000
800
600
400 % Change from baseline
200
0 3 3.2 3.4 3.6 3.8 4 4.2 Log dose of morphine (µg/Kg)
B 90.00 80.00 70.00 Draft 60.00 50.00 40.00 30.00 % Change from baseline 20.00 10.00 0.00 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 Log dose of tadalafil (µg/Kg)
Figure 3: Dose response curve of morphine (A) and tadalafil (B) for percentage change in latency time from baseline in tail flick test after 1.5 hours of drug administration
Each point represents the response mean ±SEM of a certain dose (n=5). % change from baseline= (latency time – latency time at baseline)*100/latency time at baseline
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% Change from baseline 100
50
0 A B C D E Dose of combined morphine and tadalafil*
Figure 4: Percentage change from baseline of mice receiving the combination of morphine and
tadalafil in tail flick test
% change from baseline= (latency time – latencyDraft time at baseline)*100/latency time at baseline % change from baseline is expressed as mean ± SEM (n=5). *A: ED50 of morphine +ED50 of tadalafil *B: ½ ED50 of morphine + ½ ED50 of tadalafil *C: ¼ ED50 of morphine + ¼ ED50 of tadalafil *D: 1/6 ED50 of morphine + 1/6 ED 50 of tadalafil *C: 1/8 ED 50 of morphine +1/8 ED 50 of tadalafil
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2.5
2 µg /kg) 3 1.5 E 1 T 0.5 ED 50 Tadalafil (10 ED 50 Tadalafil 0 0 2 4 6 8 10 3 ED50 Morphine (10 µg/kg)
Figure 5: Isobologram for the interactionDraft between morphine and tadalafil in tail flick test.
Theoretical ED50 (T); Experimental ED50 (E)
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