Eur opean Rev iew for Med ical and Pharmacol ogical Sci ences 2013; 17: 2449-2456 Blood pressure reducing effects of piromelatine and melatonin in spontaneously hypertensive rats

L. HUANG 1,2 , C. ZHANG 1, Y. HOU 1, M. LAUDON 4, M. SHE 3, S. YANG 3, L. DING 3, H. WANG 2, Z. WANG 5, P. HE 1, W. YIN 1,3

1Institute of Cardiovascular Research, Key Laboratory for Arteriosclerosis of Hunan Province, University of South China, Hengyang, Hunan, People's Republic of China, China 2Department of Operative Surgery, University of South China, Hengyang, Hunan, People's Republic of China, China 3Department of Biochemistry and Molecular Biology, School of Life Sciences and Technology, University of South China, Hengyang, Hunan, People's Republic of China, China 4Drug discovery, Neurim Pharmaceuticals Ltd, Tel-Aviv, Israel 5Department of Laboratory Animal Science, University of South China, Hengyang, Hunan, People's Republic of China, China

Abstract. – BACKGROUND : Recently, wide - Key Words: spread interest has grown regarding melatonin Piromelatine, Melatonin, Hypertension, Sponta - treatment of hypertension including its cardiopro - neously hypertensive rats, Metabolic diseases. tective effects. Studies in rodents indicate that melatonin plays a role in the pathogenesis of hy - pertension in rats with metabolic syndrome. Piromelatine, a melatonin agonist, serotonin Introduction 5-HT-1A and 5-HT-1D agonist and serotonin 5- HT2B antagonist is a multimodal agent with sleep promoting, anti-diabetic, analgesic, anti- The pineal hormone melatonin is secreted with neurodegenerative, anxiolytic and antidepres - a marked circadian rhythm. The endogenous sant potential, currently in development for the rhythm of secretion is generated by the suprachi - treatment of insomnia. asmatic nuclei, entrained to the light/dark cycle AIM: In this report we assessed the effects of and conveys information concerning the daily cy - piromelatine and melatonin treatment on blood cle to body physiology functions such as sleep, pressure in spontaneously hypertensive rats 1 (SHR) and normotensive Wistar-Kyoto (WKY) rats. immune system and glucose regulation . Recent MATERIALS AND METHODS: Five groups of data indicate that impaired melatonin production 12-wk-old rats (10/group) were treated for 5 weeks is involved in several cardiovascular pathologies with a vehicle, piromelatine (5, 15 and 50 mg/kg including hypertension and ischemic heart dis - BW) and melatonin (10 mg/kg BW) and an age- ease 2. In addition, some reported that melatonin matched WKY control group. Systolic blood pres - can reduce increased blood pressure both in hy - sure (tail-cuff method) was measured weekly at 3 9:00 a.m. and at 9:00 p.m. The rats body weight, pertensive humans and animals . The blood pres - plasma glucose, insulin, triglyceride, adiponectin, sure lowering effect of melatonin was demon - total cholesterol, HDL and LDL/VLDL cholesterol strated in healthy women taking contraceptive were also measured . pills, postmenopausal women with hormone re - RESULTS: Our results showed that both pirome - placement therapy, healthy men and hypertension latine and melatonin reduced SHR blood pressure 4 significantly both during the morning and the patients . evening. Piromelatine, but not melatonin, also re - Diminished melatonin production at night is duced SHR body weight gain and both significant - consistently reported in hypertensive patients ly decreased plasma glucose and insulin levels with nondipping blood pressure and in patients and increased adiponectin levels. with coronary heart disease. Pinealectomy, which CONCLUSIONS: Piromelatine, similar to mela - was associated with decreased melatonin produc - tonin, has an antihypertensive effect and also at - tenuates body weight, improves metabolic pro - tion, caused vasoconstriction and temporary hy - files and might be useful in the treatment of hy - pertension in adult rats while continuous light pertension and the metabolic syndrome. (24 hours/day), which prevented the nocturnal

Corresponding Author: Weidong Yin, MD; e-mail: [email protected] 2449 L. Huang, C. Zhang, Y. Hou, M. Laudon, M. She, S. Yang, L. Ding, H. Wang, Z. Wang, et al rise of melatonin plasma levels, also resulted in Materials and Methods suppression of circadian heart rate and blood pressure variability 1. Melatonin has been shown Materials to reduce blood pressure, oxidative load and to Piromelatine (C17H16N2O4) and melatonin increase nitric oxide bioavailability in sponta - were provided by Neurim Pharmaceuticals Ltd. neously hypertensive rats (SHR). Blood pressure (Tel-Aviv, Israel), both were dissolved in 1% di - decreased after 6 weeks of melatonin treatment methylsufoxide (DMSO)/water solution. (10 m/kg) of SHR, which was associated with a reduction of interstitial renal tissue inflammation, Animals and Treatment decreased oxidative stress and attenuation of ex - 12-wks-old male spontaneously hypertensive pression of nuclear factor kappa B in the kidney 5. rats (SHR) were randomly divided into 5 groups In the same model of hypertension, melatonin, (n=10 in each group): control SHR group, in addition to lowering mean arterial pressure piromelatine (5 mg/kg/day) treated SHR group, and causing a heart rate reduction, restored plas - piromelatine (15 mg/kg/day) treated SHR group, ma norepinephrine concentration and the propor - piromelatine (50 mg/kg/day) treated SHR group tion of β1/ β2 receptors in the heart, enhanced and melatonin (10 mg/kg/day) treated SHR maximal relaxation of mesenteric arteries and group. In addition age-matched Wistar-Kyoto improved baroreflex responses, which relate to rats (WKY) were divided into 5 groups (n=10 in its antioxidant effects. Acute administration of each group) with the same treatment as the SHR melatonin lowered blood pressure and reduced to serve as control. Both drugs were given with norepinephrine blood levels in SHR 6-8 . intraperitoneal injection in 18:00 every day. All In vitro , melatonin attenuated constriction of animals were housed at a temperature of 23±1°C aortic ring in SHR were demonstrated to inhibit in individual cages and freely fed a regular pellet phospholipase-C cascade independently on mela - diet ad libitum. All rats were subjected to alter - tonin receptor or α1- adrenoceptor blockade and nate 12 h periods of dark and light (lights on 6:00 prevent excess oxidative load related with the an - a.m.-6:00 p.m.). tioxidant enzyme superoxide dismutase 9. Melatonin secretion decreased in fructose fed Blood Pressure Measurements rats, an animal model of the metabolic syndrome, Systolic blood pressure (SBP) was determined that developed hypertension and the administra - weekly in conscious restrained rats by tail-cuff tion of melatonin blunted this blood pressure plethysmography at 9:00 a.m. and at 9:00 p.m.. rise 10 . Before the beginning of the experiments, rats The plasma half-life of melatonin is only 35 to were conditioned three to four times to the proce - 50 minutes. Attempts have been made to remedy dure and when blood pressure was determined, this problem via a controlled-release formulation the reported values represented the mean of three of melatonin and by developing various indolic to four separate determinations. After five weeks and non-indolic melatonergic agonists. of treatment, blood pressure was determined by Piromelatine (former name is Neu-P11) is a direct puncture of the carotid. Body weights melatonin agonist, serotonin 5-HT-1A and 5-HT- (BW) and food consumption were measured 1D agonist and serotonin 5-HT2B antagonist. once a week. Piromelatine is a multimodal agent with sleep promoting, anti-diabetic, analgesic, anti-neurode - Biochemical Parameters generative, anxiolytic and antidepressant poten - Plasma was separated by centrifugation (12,000 tial, currently in development for the treatment of rpm, 4 min) and stored at –80 °C. Glucose was de - insomnia. termined by a chemical colorimetric method Recent studies have shown that piromelatine (BioAssay Systems, Hayward, CA, USA). Plasma promoted sleep 11 , exerts antidepressant and anxi - insulin and adiponectin were determined by olytic activities in rodent models 12 and inhibited ELISA (Biocompare, San Francisco, CA, USA. weight gain and improved insulin sensitivity in Plasma triglycerides (TG), total Cholesterol (TC), high-fat/high-sucrose-fed rats 13 . In the present high-density lipoprotein (HDL) and low-density study we investigated the potential effects of lipoprotein/very low-density lipoprotein piromelatine and melatonin on blood pressure (LDL/VLDL) cholesterol were determined by and metabolic profiles of SHR with established commercially available enzymatic methods hypertension. (BioAssay Systems, Hayward, CA, USA).

2450 ( Bo chan body / g m e h t i F ( i s cr untr mm ± tion and 5 . 5 mm mg/ tre (15 e r p e e w a.m tr - n i n o t ues. the cal S (p. pre wa h t Blo St and com 18 t c e p s to 9:00 tre a toni n Y K W mo wa data B p D eat l g li c easd n a 3.5 a o Body Af Al at m. s s < ly f i n u g ss te dy . s s 6 week . tm od tis s kg e od 1 g k 10 k 5 pa m me men 12 me o Hg .3 i Hg 4 si A ged O n at a ure i l t 0.5). r u ivl e n u blo gni . si f n e r wei m er did c i . t s me m en t f o e e w g ti ri m. - e n ed quant e r 6 t SH t ra W ive n m gnifi l ± p e nt re , l i p ni m , c t n a P t at so . ca e oc fi s by t od r 8 f ressu a 1 p o y wei at in g/ H re s asur e vel at resu r t n i ght icane d e t a e SH 4.2 f . s on e m o r ei an ve not e t w wit h R a n m , wi o H ica ± s k tu WKY < l e kg g me sp ha d f n a al 9: d y a ght t Howe 18.3% p f e cant pa d i g a d o lo B n a Ana - 8 1 i , 5.7 rna d o ght R n-t 0. th we tat re age of t ec ntly , of l mm Hg, 00 a e S f o 17 c e f p t n e m d me nt re r co 0 me 14 d vel ff groups ( ON A VA e n i t a l ison ss 5 R H re ti l no iv e re 1 ek e e w 17 5 r mm < . SHR o b p 5 ect ( of , s t a bl vel n of an reduci 5 a ) . ) b e h t 0 la u ly . t at 15 mg/ kg ra .4 nt) op , ver tr s .m higher r 5 .7 re < ood 0 tonin h t eac ma ed 1 mg /kg, si s re p dif .4 ts ol and h t o B dat y, o s a e r c e d - k 1.9 SHR , ) 5 0 . d Hg to ± ( the e 5 d e t o n Re . ± 0.5 d o o F f Te l b a d u t s in d o o r i p ± g i s WKY in compar mel d l o rke p SHR 5 and fer encs hing p mg/kg Fod of t a r untreated 5.8 pres r .1 h ± r u s n and 5.1 sult body eatment, 3 18 < < thir d caus igher pir t s e t ac ar c fi i n g dly r 0%, in 4.5 e n i t a l e m o r i p y. and atonin 0 5 at m ats o r i p was 0 9: 0.05) mm - e e co s 50 s a ef o c ome mm 18 d e w I was .0 122.8 e ur 9:00 co Tr rats . ) 00 s e f o ed n i t a l rda pirom ws a mmHg, fe ef ee m Con dur ed 5 wei a n xpres 1 e (a. a e WKY r n i t a l e m hypertension mg /k mpar f a a e h T ) y l t n cons .8 u s fec t espct Hg cts betwen g k / g k lat in at a 2 1 Hg). p si nc y l r (Tabl as and t m. SHR r e t -o ing t the e m t .m. p.m. o ght s i t p m e reat e gnifi ± ± d e s u 9:00 sump -w ignificant e of ld and elati s untr co a g on ider y s i e 4.2 e d 6. me s a ed five so with systoli d n the e e i p t n mela o r p of iv ment . e pirome pi p m ( 2 e c i l o t s ats ( can n o f c 1 n p In iro th ne o r 143.5 < p. k as e h t I). ely, eated as s a e r m ed rom e o t pared d n a 72. n a m WK WKY h t i w l e m mean l -o s < wi ti m. e e w 0.05) e m ur tu m tly li were me latin e addition, s a w m to T (161.8 d on mean a 0.05) 3 S stat i c t c e s d th had e Hg, dy. he e n during d e by nin s k e o t a (145.7 reduc Y BP al e m l me l b al e m Hg r blood la lo a ± la ± t a S SHR atu 5 wit l nor sys val do o o ti wer rat s a rat s y RH nu - tine 12 ni n sti i eh t 5. 4.4 i n re 05 dn t no 10 en ne z of o n in re a t) ± ± h 9 9 e ------f t and

Table I. Effect of 5 weeks administration of piromelatine and melatonin on systolic blood pressure and body weight (10 per group). mela

Time of Treatment SHR+ SHR+ SHR+ SHR+ WKY+ WKY+ WKY+ to WKY+ SBP period SHR 5 mg/kg 15 mg/kg 50 mg/kg 10 mg/kg 5 mg/kg 15 mg/kg 50 mg/kg 10nin mg/kg measurement (week) (mmHg) piromelatine piromelatine piromelatine melatonin WKY piromelatine piromelatine piromelatine melatonin

(mmHg) (mmHg) (mmHg) (mmHg) (mmHg) (mmHg) (mmHg) (mmHg) (min mHg) sp

9 am 1 169.2 ± 4.6 164.2 ± 4.8 165.5 ± 3.6 164.1 ± 4.5 164.0 ± 4.4 124.9 ± 3.5 o a 125.1 ± 4.5a 122.2 ± 3.4a 125.1 ± 3.3a 128.3 ± 5.1a nta 9 am 2 172.4 ± 4.1 166.2 ± 5.2 163.6 ± 4.4 166.3 ± 4.9 1b58.142±3.37.7± 3.7a 127.2 ± 4.8a 124.1 ± 4.8a 127.3 ± 2.9a 127.2 ± 3.8a 9 am 3 177.1 ± 7.4 169.8 ± 4.9 159.b2 ± 3.17 62.6 ± 4.6b 157.8 ± 3.5b 123.2 ± 3.5a 123.9 ± 4.1a 122.8 ± 3.9a 122.9 ± 3.6a 123.4 ± 3.5a neo 9 am 4 178.9 ± 5.5 168.1b± 5.1 161.9 ± 5.6b 160.9 ± 4.4b 155.3 ± 4.1b 122.8 ± 2.4a 122.7 ± 3.5a 125.1 ± 3.8a 125.3 ± 4.2 126.8 ± 2a .9 usly 9 am 5 181.8 ± 6.2 172.3b± 5.9 161.8 ± 5.5b 156.3 ± 4.2b 151.9 ± 4.5b 126.8 ± 5.7a 126.5 ± 4.1a 124.1 ± 4.6a 122.1 ± 4.2a 124.3 ± 3.9a 9 pm 1 158.7 ± 5.7 158.2 ± 4.4 158.4 ± 3.9 155.2 ± 4.1 155.1 ± 3.8 a 1171.244±.23±.9 2.9a 119.5 ± 3.5a 120.5 ± 2.9a 121.5 ± 3.5a hyper 9 pm 2 162.5 ± 6.3 157.8 ± 5.1 151.b5 ± 5.2 152 ± 3.7 b 152.5 ± 4.7b 121.5 ± 3.2a 120.8 ± 3.3a 122.6 ± 2.7a 123.1 ± 3.4a 120.4 ± 3.1a 9 pm 3 168.1 ± 6.8 158.3 ± 6.2 144.b6 ± 4.18 44.5 ± 5.2b 145.6 ± 4.1b 123.5 ± 2.4a 122.9 ± 2.8a 124.2 ± 3.4a 124.1 ± 2.5a 118.9 ± 2.4a

b b b b a a a a a t 9 pm 4 170.9 ± 7.1 156.5 ± 5.2 146.2 ± 3.2 142.8 ± 3.5 145.9 ± 3.5 120.8 ± 3.4 121.4 ± 4.4 118.8 ± 2.9 120.5 ± 3.4 120.2 ± 3.8 ensive 9 pm 5 175.4 ± 5.1 160.8 ± 5.2 145.b7 ± 3.15 40.7 ± 5.8b 143.5 ± 4.4b 122.8 ± 4.2a 120.9 ± 3.9a 115.6 ± 4.1a 118.7 ± 3.8a 119.2 ± 4.1a 245

Data are means ± SD, Significant differences ( p < 0.05): acompared to corresponding SHR, bcompared to untreated SHR. Systolic blood pressure (SBP) was determined in con - rats

1 scious restrained rats by tail-cuff plethysmography (weeks 1-4) and by direct puncture of the carotid (week 5). L. Huang, C. Zhang, Y. Hou, M. Laudon, M. She, S. Yang, L. Ding, H. Wang, Z. Wang, et al

Plasma Glucose and Insulin weeks treatment with melatonin the triglycerides As shown in Figure 2a, plasma glucose concen - level had been significantly suppressed compared tration was significantly higher in the SHR com - with SHR (0.66±0.06 mmol/L vs. 0.75±0.11 pared with WKY rats ( p < 0.05; WKY vs. corre - mmol/L ), but piromelatine had no effect ( p > sponding SHR). After 5 weeks of treatments with 5 0.05, vs. SHR). There were no significant differ - mg/kg, 15 mg/kg and 50 mg/kg piromelatine , plas - ences in plasma levels of TC, LDL-C and HDL- ma glucose was decreased significantly by 27.3%, C between SHR and WKY despite of treatment 34.5% and 61.5%, respectively, in the treated SHR with piromelatine or melatonin (data not shown). (p < 0.05) compared to untreated SHR. The plasma level of insulin in SHR was significantly higher Effects of Treatments on than that in WKY rats (Figure 2b). The treatment Plasma Adiponectin with 5 mg/kg, 15 mg/kg and 50 mg/kg of pirome - As shown in Figure 4, the plasma level of latine or 10 mg/kg melatonin resulted in a signifi - adiponectin in SHR was significantly lower than cant decrease in plasma level of insulin in SHR in WKY ( p < 0.05). Treatment with either 15 (34.5%, 56.9%, 67.2% and 51.5%, respectively, p mg/kg and 50 mg/kg piromelatine or 10 mg/kg < 0.05) compared to untreated SHR. melatonin could markedly increase the plasma In the WKY rats, 15 mg/kg and 50 mg/kg of level of adiponectin in SHR ( p < 0.05) and the piromelatine significantly decreased the plasma plasma level of adiponectin of SHR administrated level of glucose while 5 mg/kg and 50 mg/kg of with 15 mg/kg and 50 mg/kg piromelatine even piromelatine and 10 mg/kg of melatonin signifi - reached a level comparable to the WKY rats. cantly decreased insulin plasma levels compared to untreated WKY rats (Figure 1b). Discussion Effects of Treatments on Rat’s Lipid Metabolism Hypertension, obesity, insulin resistance and Figure 3 shows the concentrations of triglyc - their associated vascular complications are reaching erides. The level of triglycerides in SHR was sig - epidemic proportion worldwide 15 . Here, we demon - nificantly higher than that in WKY ( p < 0.05; strate that piromelatine like melatonin markedly re - WKY vs. corresponding SHR , Figure 3). After 5 duced hypertension, decreased weight gain and im -

A Control Control B 5 mg/kg piromelatine 5 mg/kg piromelatine 15 mg/kg piromelatine 15 mg/kg piromelatine 50 mg/kg piromelatine 50 mg/kg piromelatine 10 mg/kg melatonin 10 mg/kg melatonin ) ) g g ( ( n n i i a a g g t t h h g g i i e e w w y y d d o o B B

Treatment period (weeks) Treatment period (weeks)

Figure 1. The effect of piromelatine on body weight of SHR and WKY rats (A) SHR body weight. B, WKY body weight. Data are expressed as means ± SD (n=10), * p < 0.05 treated vs. untreated rats of the same genotype.

2452 Blood pressure reducing effects of piromelatine and melatonin in spontaneously hypertensive rats ) ) L L / / l l o o m m m m ( ( e n s i l o u c s u l n i g a a m m s s a a l l P P

l e e e n l e e e n o n in n i o n n n i r ti t i n r i i ti n t a t o t t t o n la l la t n la la la t o e e e la o e e e la C m m m e C m m m e o o o o o o m r ir r m r r r i i g i i i g p p p k p p p k g g / g / k g g g g g /k / /k /k /k /k g g g m g g g m 0 0 m m m 1 m m m 1 5 5 0 5 5 0 A 1 5 1 5 B

Figure 2. Plasma glucose and insulin concentrations by piromelatine and melatonin treatments. A, Plasma glucose. B, Plasma insulin. Data are expressed as means ± SD (n=10), * p < 0.05 treated vs. untreated rats of the same genotype, +p < 0.05 WKY vs. corresponding SHR. proved glucose and insulin homeostasis in the SHR increases soon after the onset of darkness, peaks in rat model of hypertension. The endogenous produc - the middle of the night (between 2 and 4 a.m.) and tion of melatonin is regulated by a circadian gradually falls during the second half of the night. rhythm, and timing of melatonin administration In this study, all drugs were injected at 6 p.m., the may be important 16 . In humans, melatonin secretion onset of the dark period of the rats. ) L ) / l l o m / m g µ m ( ( e n i d t i c r e e c n y o l p g i i r d t a a a m m s s a a l l P P

l e e e n o n n i l e e e n r i in i n o n n i t t t t o r i in i n n la a la t t t t t o o e l e a n la a la t C e l o e l e a m m e C e l m m m e ro o ro m m i ir i ro o ro m p p g i ir i p /k p p g g g p /k k g k g g g / /k / k g k g g g m / /k / g g g m m m 0 g m 1 m m 0 5 5 0 m 1 1 5 5 5 0 1 5 Figure 3. The effect of piromelatine and melatonin on Figure 4. The effect of piromelatine and melatonin on plasma triglyceride concentrations. Data are expressed as means ± adiponectin plasma adiponectin. Data are expressed as means ± SD (n=10), * p < 0.05 treated vs. untreated rats of the same SD (n=10), * p < 0.05 treated vs. untreated rats of the same genotype, + p < 0.05 WKY vs. corresponding SHR. genotype, + p < 0.05 WKY vs. corresponding SHR.

2453 L. Huang, C. Zhang, Y. Hou, M. Laudon, M. She, S. Yang, L. Ding, H. Wang, Z. Wang, et al

The major new finding of our study was that a creases daily secretion of insulin stimulated by 5-wks treatment with both piromelatine and mela - glucose intake and insulin secretion by isolated tonin were able to significantly reduce both morn - pancreatic islets 23 ,24 . Melatonin was effective in ing and evening blood pressure in SHR. Melatonin treatment of metabolic syndrome induced by has been shown to reduce systolic blood pressure high caloric diet or by antipsychotic drugs. Mela - in Spraque-Dawley rats fed with high fructose 17 . tonin decreased plasma glucose (13%), leptin The hypotensive effect of piromelatine might be (28%) and triglyceride (28%) levels but had no mediated via melatonin receptors. The involve - effect on plasma insulin level in Sprague Dawley ment of melatonin receptors in regulation of blood rats with diet-induced obesity 21 . Furthermore, in pressure was further supported by the finding that pinealectomized high-fat diet rats, body weight the hypotensive effect of microinjection of mela - gain and feed efficiency were increased 4-wks af - tonin into specific brain structures was almost ter surgery. Adipose tissue weight, insulinemia, completely prevented by luzindole, an antagonist and glycemia had a tendency to increase. Treat - of the melatonin MT1/MT2 receptors 18 . ment with melatonin prevented in part these In our previous work piromelatine effectively changes. These data demonstrate that melatonin decreased body weight gain in obese rats estab - may act as a regulator of body weight in a model lished using high-fat/high-sucrose-fed for 5 of obesity and may prevent some of the side ef - months 13 . In addition, daily administration of fects on glucose homeostasis such as decreased melatonin (0.2 microg/mL) via drinking water insulin sensitivity 21 . decreased weight in 10 months-old but not in 3- Insulin resistance (IR) plays a key role in the months-old rats 19 . It is notable that in ovariec - pathogenesis of the insulin resistance syndrome 25 . tomized (Ovx) rats melatonin reduced food in - IR caused by hyperglycemia and dyslipidemia take and partially prevented the increase of body seems to be linked to many features of the meta - weight. Furthermore, melatonin affected body bolic diseases. The role of central nervous system weight in a model closer to that observed in in regulating plasma glucose has been demonstrat - Western populations, i.e. Sprague Dawley rats ed in supra-chiasmatic nuclei (SCN)-lesion stud - fed with a high-fat diet and prevented diet-in - ies. Without a functioning SCN, cortisol and glu - duced decrease of insulin sensitivity 20 . Daily cose do not rise before the beginning of the active melatonin administration to middle-aged male period (morning arousal) 26 ,27 . Additionally, damag - rats was also shown to decrease body weight, in - ing the SCN also eliminates the circadian physiol - traabdominal adiposity, and plasma insulin and ogy of the pineal gland and loss of the pineal leptin concentrations while increasing physical melatonin alters glucose homeostasis in pinealec - activity, body temperature, and basal plasma cor - tomized rats 28 . ticosterone levels 21 . These results suggest that the We also observed that piromelatine as well as decrease in endogenous melatonin secretion may melatonin improved insulin intolerance in SHR alter energy regulation in middle age, resulting in as verified by a significant reduction of glucose detrimental metabolic consequences. and insulin levels with no change in total choles - Melatonin has no effect in rats on normal body terol, LDL-C and HDL-C levels. These results weight but it alters body fat mass which is elevated were coinciding with the findings of our previous during aging or in genetically obese rats 20 . report on the effects of piromelatine on body In this study piromelatine, but not melatonin, weight gain in obese rats 13 . decreased body weight gain in SHR with no in - Accumulating evidence indicate that plasma fluence on food intake. We hypothesized that adiponectin is lower (hypoadiponectinemia) in higher body temperature and/or higher locomotor patients with diabetes, hypertension, and dyslipi - activity in piromelatine treated rats might result demia than BMI-matched controls, indicating in a decreased body weight. This hypothesis is that hypoadiponectinemia is associated with an consistent with the results of our previous inves - increased prevalence of coronary risk factors 29 . tigation on the effects of piromelatine on body Using adiponectin to prevent cardiovascular dis - weight gain in obese rats 13 . ease might be a therapeutic strategy. In the cur - In models with altered body weight, such as rent work , piromelatine significantly increased middle-aged, obese rats and high-fat/high- the plasma adiponectin levels. So we can specu - sucrose-fed rats melatonin may improve insulin late that piromelatine may be a candidate drug to sensitivity 13, 22 . It has been documented that prevent and treat cardiovascular diseases associ - pinealectomy causes glucose intolerance and de - ated with the metabolic syndrome.

2454 Blood pressure reducing effects of piromelatine and melatonin in spontaneously hypertensive rats

To our knowledge, this is the first evidence of and improves hypertension in spontaneously hy - a positive effect of piromelatine on overweight pertensive rats. Am J Physiol Renal Physiol 2003; and glucose levels in SHR, supporting the propo - 284: 447-454. sition that piromelatine like melatonin may ame - 6) GIROUARD H, C HULAK C, L EJOSSEC M, L AMONTAGNE D, DE CHAMPLAIN J. Chronic antioxidant treatment im - liorate hypertension, overweight and insulin re - proves sympathetic functions and beta-adrenergic sistance in humans. pathway in the spontaneously hypertensive rats. J Since these benefits occurred in young-adult Hypertens 2003; 21:179-188. rats, before aging induced metabolic and vascular 7) GIROUARD H, C HULAK C, L EJOSSEC M, L AMONTAGNE D, complications, piromelatine might help to pre - DE CHAMPLAIN J. Vasorelaxant effects of the chronic vent cardiovascular disease associated with hy - treatment with melatonin on mesenteric artery and aorta of spontaneously hypertensive rats. J pertension and insulin resistance. Hypertens 2001; 19: 1369-1377. 8) GIROUARD H, D ENAULT C, C HULAK C, DE CHAMPLAIN J. Treatment by N-acetylcysteine and melatonin in - Conclusions crease cardiac baroreflex and improves antioxi - dant reserve. Am J Hypertens 2004; 17: 947-954. The results of our study suggest that piromela - 9) K-L AFLAMME A, W U L, F OUCART S, DE CHAMPLAIN J. Im - tine , a novel melatonin agonist, possess the ef - paired basal sympathetic tone and alpha1-adren - fects of melatonin in attenuating the development ergic responsiveness in association with the hy - potensive effect of melatonin in spontaneously of hypertension in adult SHR. We have demon - hypertensive rats. Am J Hypertens 1998; 11: 219- strated that piromelatine treatment may not only 229. reduce body weight and blood pressure but also 10) LEIBOWITZ A, P ELEG E, S HARABI Y, S HABTAI Z, S HAMISS A, improve glucose homeostasis and may help to GROSSMAN E. The role of melatonin in the patho - prevent vascular complications in hypertensive/ genesis of hypertension in rats with metabolic insulin-resistant patients. syndrome. Am J Hypertens 2008; 21: 348-351. 11) LAUDON M, U RADE Y, H UANG Z. Piromelatine. A novel melatonin agonist: effects on sleep and EEG power ––––––––––––––––– –– Acknowledgements spectra in rats. Sleep 2008; 31(Suppl): Abst 34. 12) TIAN SW, L AUDON M, H AN L, J UN G, F U-L IAN H, Y U- This work was supported by grants from the National Natur - FENG Y, H AI -F ENG D. Antidepressant and anxiolytic al Sciences Foundation of China (81100106,81200590) and effects of the novel melatonin agonist Neu-P11 in the Scientific Research Fund of Hunan Provincial Education rodent models. Acta Pharmacol Sinica 2010; 31: Department (11C1093 and 11C1095). 775-783. 13) SHE M, D ENG X, G UO Z, L AUDON M, H U Z, L IAO D, ––––––––––––– –– ––– –– HU X, L UO Y, S HEN Q, S U Z, Y IN W. Neu-P11, a nov - Conflict of Interest el melatonin agonist, inhibits weight gain and im - Moshe Laudon is an employee of Neurim Pharmaceuticals proves insulin sensitivity in high-fat/high-sucrose- Ltd. All other Authors declare that they have no conflicts of fed rats. Pharmacol Res 2009; 59: 248-253. interest. 14) AITMAN TJ, P RAVENEC M, K URTZ TW, S COTT J. The spontaneously hypertensive rat: a model to dis - sect cellular defects in glucose and fatty acid me - tabolism. Pathol Biol (Paris) 1998; 46: 693-694. References 15) NDUHIRABANDI F, DU TOIT EF, L OCHNER A. Melatonin and the metabolic syndrome: a tool for effective 1) BRZEZINSKI A. Melatonin in humans. N Engl J Med therapy in obesity-associated abnormalities? Acta 1997; 336: 186-195. Physiol (Oxf) 2012. 2) PAULIS L, Š IMKO F. Blood pressure modulation and 16) CAJOCHEN C, K RÄUCHI K, W IRZ -J USTICE A. Role of cardiovascular protection by melatonin: potential melatonin in the regulation of human circadian mechanisms behind. Physiol Res 2007; 56: 671- rhythms and sleep. J Neuroendocrinol 2003; 15: 684. 432-437. 3) SIMKO F, P AULIS L. Melatonin as a potential antihyper - 17) LEIBOWITZ A, P ELEG E, S HARABI Y, S HABTAI Z, S HAMISS A, tensive treatment. J Pineal Res 2007; 42: 319-322. GROSSMAN E. The role of melatonin in the patho - 4) ARANGINO S, C AGNACCI A, A NGIOLUCCI M, V ACCA AM, genesis of hypertension in rats with metabolic LONGU G, V OLPE A, M ELIS GB . Effects of melatonin syndrome. Am J Hypertens 2008; 21: 348-351 . on vascular reactivity, catecholamine levels, and 18) NAKAHARA K, K AWANO T, S HIOTA K, M URAKAMI N. Ef - blood pressure in healthy men. Am J Cardiol fects of microinjection of melatonin into various 1999; 83: 1417-1419. brain regions of Japanese quail on locomotor ac - 5) NAVA M, Q UIROZ Y, V AZIRI N, R ODRIGUEZ -I TURBE B. tivity and body temperature. Neurosci Lett 2003; Melatonin reduces renal interstitial inflammation 345: 117-120.

2455 L. Huang, C. Zhang, Y. Hou, M. Laudon, M. She, S. Yang, L. Ding, H. Wang, Z. Wang, et al

19) RASMUSSEN DD, M ITTON DR, L ARSEN SA, Y ELLON SM. Ag - 24) PICINATO MC, H ABER EP, C ARPINELLI AR, C IPOLLA -N E- ing-dependent changes in the effect of daily mela - TO J. Daily rhythm of glucose-induced insulin se - tonin supplementation on rat metabolic and behav - cretion by isolated islets from intact and ioral responses. J Pineal Res 2001; 31: 89-94. pinealectomized rat. J Pineal Res 2002; 33: 20) PRUNET -M ARCASSUS B, D ESBAZEILLE M, B ROS A, L OUCHE 172-177. K, D ELAGRANGE P, R ENARD P, C ASTEILLA L, P ÉNICAUD L. 25) EINHORN D, R EAVEN GM, C OBIN RH, F ORD E, G ANDA Melatonin reduces body weight gain in Sprague OP, H ANDELSMAN Y, H ELLMAN R, J ELLINGER PS, K ENDALL Dawley rats with diet-ind-uced obesity. En - D, K RAUSS RM, N EUFELD ND,P ETAK SM, R ODBARD HW, docrinology 2003; 144: 5347-5352. SEIBEL JA, S MITH DA, W ILSON PW . American College of Endocrinology position statement on the insulin 21) WOLDEN -H ANSON T, M ITTON DR, M CCANTS RL, Y ELLON resistance syndrome. Endocr Pract 2003; 9: 237- SM, W ILKINSON CW, M ATSUMOTO AM, R ASMUSSEN DD . Daily melatonin administration to middle-aged 252. male rats supper-ses body weight, intraabdominal 26) LA FLEUR SE, K ALSBEEK A, W ORTEL J, F EKKES ML, B UIJS adiposity, and plasma leptin and insulin indepe- RM. A daily rhythm in glucose tolerance: a role for ndent of food intake and total body fat. En - the suprachiasmatic nucleus. Diabetes 2001; 50: docrinology 2000; 141: 487-497. 1237-1243. 22) RASMUSSEN DD, B OLDT BM, W ILKINSON CW, Y EL - 27) REITER RJ, T AN DX, K ORKMAZ A. The circadian mela - LON SM, M ATSUMOTO AM . Daily melatonin ad - tonin rhythm and its modulation: possible impact ministration at middle age suppresses male on hypertension. J Hypertens 2009; 27(Suppl 6): rat visceral fat, plasma leptin, and plasma in - 17-20. sulin to youthful levels. Endocrinology 1999; 28) LA FLEUR SE, K ALSBEEK A, W ORTEL J, VAN DER VLIET J, 140: 1009-1012. BUIJS RM. Role for the pineal and melatonin in glu - 23) LIMA FB, M ACHADO UF, B ARTOL I, S ERAPHIM PM, S UMI - cose homeostasis: pinealectomy increases night- DA DH, M ORAES SM, H ELL NS, O KAMOTO MM, S AAD time glucose concentrations. J Neuroendocrinol MJ, C ARVALHO CR, C IPOLLA -N ETO J. Pinealectomy 2001; 13: 1025-1032. causes glucose intolerance and decreases adi - 29) OKAMOTO Y. Adiponectin provides cardiovascular pose cell responsiveness to insulin in rats. Am J protection in metabolic syndrome. Cardiol Res Physiol 1998; 275: 934-941. Pract 2011; 2011: 313179.

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