Antihypertensive effects of fluoxetine and L-5-hydroxytryptophan in rats

Life Sciences, Vol. 25, pp . 1237-1242 Printed in the U.S .A .

Pergamon Press

ANTIHYPERTENSIVE EFFECTS OF FLUOXETINE AND L-5-HYDROXYTRYPTOPHAN IN RATS Ray W. Fuller, Donald R. Holland, Terence T . Yen, Kerry G. Bemis and Nancy B. Stamm The Lilly Research Laboratories, Eli Lilly and Company Indianapolis, Indiana 46206 (Received in final form August 22, 1979) SUMMARY The combination of fluoxetine (10 mg/kg) and L-5-hydroxytryptophan (5-HTP) (10 mg/kg) significantly lowered blood pressure in spontaneously hypertensive rats and in rats made hypertensive by treatment with deoxycorticosterone (DOCA) and saline . Fluoxetine alone also had a significant effect on blood pressure in DOCA hypertensive rats, but not as great an effect as the combination. Since fluoxetine is an inhibitor of serotonin reuptake and 5-HTP is the serotonin precursor, the antihypertensive effect of this drug combination strengthens previous evidence that serotonin neurons have a role in the central regulation of blood pressure . A role of serotonin neurons in the central regulation of blood pressure has been suggested (1) . Some evidence supports the idea that enhancement of central serotoninergic function lowers blood pressure . For instance, the serotonin precursor 5-hydroxytryptophan (5-HTP) caused a fall in blood pressure in conscious rats (2), in anesthetized dogs pretreated with a monoamine oxidase inhibitor (3), and in anesthetized cats with or without inhibition of monoamine oxidase or peripheral decarboxylase (4,5) . Either 5-HTP or serotonin injected directly into the cerebrospinal fluid in dogs lowered blood pressure (6) . In cats, 5-HTP injected intracerebroventricularly lowered blood pressure, and the effect was prevented by decarboxylase inhibition (7) . The hypotensive effects of systemically-administered 5-HTP in dogs pretreated with a monoamine oxidase inhibitor were abolished or attenuated by serotonin antagonists--methysergide and yohimbine (8) . Other results consistent with a negative influence of central serotonin neurons on blood pressure include an elevation of blood pressure in rats after p-chlorophenylalanine, an inhibitor of serotonin synthesis (9) . Recently ß-endorphin was reported to lower blood pressure by a mechanism suggested to involve enhanced central serotonin activity inasmuch as the effect was reduced in rats treated with p-chlorophenylalanine or with serotonin antagonists (cyproheptadine, mianserin and metergoline) or enhanced by fluoxetine, a serotonin uptake inhibitor (10) .

0024-3205/79/141237-06$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd

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Some pharmacologic studies, however, have yielded opposite conclusions, that suppression of central serotoninergic function lowers blood pressure and that enhanced serotonin activity increases blood pressure . For example, p-chlorophenylalanine has been reported to lower blood pressure in normotensive rabbits (11) and in normotensive or hypertensive rats (12,13) . 5,6-Dihydroxytryptamine, which causes lesions in serotonin neurons, has been reported to lower blood pressure in normotensive rabbits (14) and in hypertensive rats (15,16) . Dunkley et _al (17) reported a hypertensive response to 5-HTP in conscious dogs_ A possible reason for some of the inconclusive or contradictory results may be the relative lack of specificity of many drugs used to manipulate central serotonin neurons . Even 5-HTP, the physiologic precursor to serotonin, may affect nonserotonergic neurons at high doses . One means of overcoming this problem with specificity of 5-HTP is to administer it along with a specific inhibitor of the uptake pump on serotonin neurons . Any effect of 5-HTP that is mediated by serotonin neurons would be potentiated by such an uptake inhibitor, whereas an effect not mediated by serotonin neurons would not be potentiated . Our studies have shown that fluoxetine, a specific inhibitor of the uptake pump on serotonin neurons (18,19), potentiates the hypotensive effect of 5-HTP . We are reporting here that treatment with fluoxetine, and especially combined treatment with fluoxetine and 5-HTP, decreases blood pressure in DOCA hypertensive rats . The combined treatment also decreases blood pressure in spontaneously hypertensive rats . MATERIALS AND METHODS Fluoxetine hydrochloride (LY110140) was synthesized in the Lilly Research Laboratories . L-5-Hydroxytryptophan was purchased from Sigma . Studies on SHR. Male SHR were supplied by Taconic Farms, Germantown, N.Y . Rats were acclimated to our environment for about three months . Purina laboratory chow and water were available ad libitum. Lights were on twelve hours a day. At the time of experimentation, rats were 7 to 8 months o£ age and weighed 380 to 430 g. Systolic blood pressure was determined indirectly at 250C with an automated, 24-rat system using photoelectric sensors (20,21) . On the day of the experiment, rats were transferred to individual holders and their before-treatment blood pressures were determined at least 30 min thereafter to minimize the stress from handling . Rats were kept in their holders throughout the experiment . Multiple measurements were made on each rat at each time period . Heart rates were calculated by manually counting the excursions of the blood pressure tracings . All compounds were given subcutaneously to the rats while they were in the holders . Studies on DOCA hypertensive rats . These studies were done by Pharmakon Laboratories, Scranton, Pennsylvania, under contract . We are grateful to Robert E. Panasevich who directed the study . Male Sprague Dawley rats (weighing 200 to 320 g at the time of the studies) from The Charles River Laboratories, Wilmington, Massachusetts, were used . rats were made hypertensive by subcutaneous injection of deoxycorticosterone (10 mg/rat/day, five days per week) over a period of 3 weeks . An aortic cannula was placed in Saline (0 .9 ") was available ad libitum. the abdominal aorta of each animal 24 hrs before the test substances were

Fluoxetine & 5HTP in Hypertensive Rats

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administered intraperitoneally (22) . Systolic and diastolic blood pressures were measured by means of a Statham P23Db pressure transducer Heart rate was monitored using a Narco and a Grass Model 7 Polygraph . Biotachometer BR1200 . Statistical Methodology. The changes in systolic blood pressure from Duncan's multiple range pre-drug values were calculated for each rat. test (P- .05) was used to compare these changes in systolic pressure among Treatment groups treatment groups at each post-drug observation time . significantly different from the vehicle or combination group are indicated in Tables 1 and 2 . RESULTS Table 1 shows the effects of fluoxetine and 5-HTP given singly or in combination on blood pressure in spontaneously hypertensive rats . The combination caused a significantly greater drop in blood pressure as compared to the vehicle group, achieving a maximum average lowering of blood pressure of 59 mm Hg . The effects persisted throughout the 5 hour Although rats receiving fluoxetine alone or 5-HTP alone experiment . exhibited a drop in blood pressure, this was not significantly different from the drop seen in the vehicle group . Heart rate (not shown) was not changed significantly by any of the treatments . TABLE 1 Antihypertensive effect of fluoxetine and L-5-hydroxytryptophan (5-HTP) in spontaneously hypertensive rats . Systolic blood pressure Time, hours 0 1 3 4 5

Fluoxetine 193 170 167 160 153

*The change from change observed tThe change from change observed

+ + + + +

6 8 14 9 10

5-HTP 186 163 173 161 164

+ + + + +

3 3 5t 3 3t

(as. Hg), mean + s .e .m . for 6 rats Fluoxetine + 5-HTP 197 149 138 150 141

+ + + + +

8 13* 10* 9* 8*

Vehicle 201+8 192 + 10 188 + 8 188 + 5 173 + 5

the pre-drug value is significantly different from the in the vehicle group . the pre-drug value is significantly different from the in the fluoxetine + 5-HTP group.

All comparisons were made using Duncan's multiple range test (P - .05) . Fluoxetine and 5-HTP were injected s .c . at 10 mg/kg at zero time . The antihypertensive effect of the fluoxetine/5-HTP combination in DOCA hypertensive rats is shown in Table 2. In this study fluoxetine alone significantly lowered blood pressure compared to vehicle with a maximum drop of 15 ma Hg . The effect persisted at 4 hours but was not significant at 24 hours . However 5-HTP alone had only a slight effect on

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blood pressure compared to the vehicle and this effect was significant only at 4 hours . The combination of fluoxetine and 5-HTP lowered blood pressure rapidly . The effect was significant compared to vehicle and persisted at 4 hours . By 24 hrs there no longer was a significant reduction in blood pressure . The maximum average reduction in blood pressure produced by this drug combination was 37 mm Hg at 1 hr . Diastolic blood pressures showed similar trends . Heart rate (not shown) was not significantly altered by any of the treatments . DISCUSSION The combination of fluoxetine and 5-HTP produced distinct antihypertensive effects both in spontaneously hypertensive rats and in DOCA hypertensive rats . This finding is consistent with previous literature cited in the Introduction in which 5-HTP alone or in combination with monoamine oxidase inhibitors or peripheral decarboxylase inhibitors lowered blood pressure in normotensive or hypertensive animals of various species . To our knowledge, this is the first report of antihypertensive effects of 5-HTP given in combination with an inhibitor of serotonin uptake . TABLE 2 Antihypertensive effect of fluoxetine and L-5-hydroxytryptophan (5-HTP) in ROCA hypertensive rats . Systolic blood pressure Time, Hours 0 1 2 4 24

Fluoxetine

211 196 198 199 221

+ 9 10* t + 10* t + 9* + 9t

+

*The change from change observed Me change from change observed

(mm Hg), mean + s.e .m . for 6 rats

5-HTP

214 204 210 203 213

+ + + + +

7 7t 8t 7* 10

Fluoxetine + 5-HTP

210 173 180 188 199

+ + + + +

6 6* 7* 7* 6

Vehicle

215 213 218 221 216

+ + + + +

10 8 9 11 9

the pre-drug value is significantly different from the in the vehicle group . the pre-drug value is significantly different from the in the fluoxetine + 5-HTP group.

All comparisons made using Duncan's multiple range test (P - .05) . Fluoxetine and 5-HTP were injected i .p . at 10 mg/kg at zero time . The mechanism by which enhancement of serotonin neurotransmission by fluoxetine and 5-HTP leads to blood pressure lowering is not certain but may be related to diminished central sympathetic neural outflow thought to accompany enhancement of central serotonin function (see 23) . Fenfluramine, an agent that stimulates central serotonin receptors by releasing

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endogenous serotonin, has been reported to lower blood pressure in hypertensive patients (24) and to lower plasma norepinephrine and dopamine ß-hydroxylase levels (25) . One concern about the use of 5-HTP as a pharmacologic means of enhancing serotonin neural activity is that 5-HTP can be converted to serotonin in cells other than serotonin neurons . Only the aromatic L-amino acid decarboxylase is required for converting 5-HTP to serotonin. This enzyme can convert 5-HTP to serotonin in peripheral tissues and in non-neuronal cells . Serotonin formed from 5-HT? may enter monoaminergic neurons other than serotonin neurons or may be formed there and cause the release of other monoamines from storage granules . Thus 5-HTP, especially at high doses, does not affect serotonin neurons specifically . Fluoxetine, however, appears to be a highly selective inhibitor of the uptake pump on serotonin neurons with no effects in vivo on other uptake mechanisms (18,26,27) . Thus the ability of fluoxetine to enhance the effect of 5-HTP or the ability of the fluoxetine/5-HTP combination to produce effects not seen with either drug alone strongly supports the idea that the effects are mediated by actions on serotonin neurons . Doses of 5-HTP as low as 10 mg/kg appear to act with reasonable specificity on serotonin neurons (28), and the combination with fluoxetine further assures selective effects on serotonin neurons . Thus we regard the antihypertensive action of the fluoxetine/5-HTP combination as reasonably strong evidence that enhancement of serotonin neuronal activity leads to blood pressure lowering . The possibility that enhancement of serotoninergic neurotransmission in this way or in some other way might be clinically useful in the treatment of hypertension deserves additional consideration . REF RENCES 1. M. J . ANTONACCIO . pp . 131-16 5 in M. J. Antonaccio (editor), CARDIOVASCULAR PHARMACOLOGY . Raven Press, New York, 1977 . 2. M. HENNING AND A. RUBENSON . Acta Pharmacol . Toxicol . _29, 145-154 (1971) . 3. M. J . ANTONACCIO AND R. D. ROBSON . J . Pharm. Pharmacol . _25, 495-497 (1973) . 4. J . FLOREZ AND J. A. ARMIJO . Bur . J. Pharmacol . 26, 108-110 (1974) . 5. T . BAUM AND A. T. SHROPSHIRE . Neuropharmacol . _14, 227-233 (1975) . 6. J . W. MCCUBBIN, Y. KANEKO AND I . H. PAGE . Circ . Res . _8, 849-858 (1960) . 7. A. S . TADEPALLI, E . MILLS AND S . M. SCHANBERG . J. Pharmacol . Exp. Ther . 202, 310-319 (1977) . 8 . M. J. ANTONACCIO AND R. D. ROBSON . Arch . int . Pharmacodyn . 213, 200-210 (1975) . 9 A. ITO ANDS . M. SCHANBERG . J. Pharmacol . Exp . Ther . 181, 65-74 (1972) . 10 . I . LEMAIRE, R . TSENG AND S . LEMAIRE. Proc . Nat. Acad . Sci . _75, 6240-6242 (1978) . 11 . L. M. H. WING AND J . P . CHALMERS . Clin . Exp. Pharmacol . Physiol . _ 1, 219-229 (1974) . 12 . . JARROTT, A . MCQUEEN, L. GRAF AND W. J . LOUIS. Clin . Exp . Pharmacol . Physiol . Suppl . 2, 201-205 (1975) . 13 . W. DE JONG, F . P. NIJKAMP AND B . BONUS . Arch . int. Pharmacodyn. 213, 272-284 (1975) .

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14 . L . M . H . WING AND J . P . CHAL1dSRS . Circ . Res . _35, 504-513 (1974) . 15 . L . FINCH . Clin . Exp . Pharmacol . Physiol . _2, 503-508 (1975) . 16 . R . 8 . BUCKINGHAM, T . C . HAMILTON AND D . ROBSON . Sur . J . Pharmacol . 36, 431-437 (1976) . 17 . H . DUNKLEY, I . SANGHVI, 8 . FRIEDMAN AND S . GERSHON . Psychopharmacologia 26, 161-172 (1972) . 18 . D . T . WONG, F . P . BYbWTER, J . S . HORNG AND B . B . MOLLOY . J . Pharmacol . Exp . Ther . 193, 804-811 (1975) . 19 . R . W . FULLER, K . W . PERKY AND B . B . MOLLOY . J . Pharmacol . Exp . Ther . 193, 796-803 (1975) . 20 . T . T . YEN, C . E . POWELL AND D . C . PEARSON . Proc . Secon d International Symposium on the Spontaneously Hypertensive Rat . DREW Publ . No . (NIH) 77-1179, 1977, pp . 486-490 . 21 . T . T . YEN, D . V . PEARSON, C . E . POWELL AND G . L . KIRSCHNER . Life _Sci . _22, 359-362 (1978) . 22 . J . R . WEEKS AND J . A . JONES . Proc . Soc . Exp . Biol . Ned . 104, 646648 (1960) . 23 . C . M . HIATT, S . H . RABINOWITZ AND B . LOWN . Circ . Rea . _44, 723-730 (1979) . 24 . J . TUOMILEHTO, H . SILTANSN AND S . JESPERSEN . Curr . Ther . Res . 22, 821-827 (1977) . 25 . C . E . DS LA VEGA, S . SLATER, M . G . ZIEGLER, C . R . LAKE AND D . L . MURPHY . Clin . Pharmacol . Ther . 21, 216-221 (1977) . 26 . R . W . FULLER AND D . T . WONG . Fed. Proc . _36, 2154-2158 (1977) . 27 . R . W . FULLER AND K . W . PERRY . J . Pharm . Pharmacol . 29, 710-711 (1977) . 28 . K . FUXE, L . L . BUTCHER AND J . ENGEL . J . Pharm . Pharmacol . _23, 420424 (1971) .