NearopharmaeologyVol. 31, No. 1, pp.33-38, 1992
0028-3908/92 $5.00 + 0.00 Pergamon Press plc
Printed in Great Britain
EFFECTS OF ADINAZOLAM ON PLASMA CATECHOLAMINE, HEART RATE AND BLOOD PRESSURE RESPONSES IN STRESSED AND NON-STRESSED RATS M. J. KRIEMAN,D. M. HERSHOCK,I. J. GREENBERGand W. H. VOGEL* Department of Pharmacology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust St, Philadelphia, PA 19107, U.S.A. and The Upjohn Company, Kalamazoo, Michigan, U.S.A. (Accepted 21 June f99i)
Summary-Adinazolam (ADI) is a new benzodiazepine with anxiolytic and antidepressant properties. To assess its effects on the acute stress response, rats were given a single intra~riton~l injection of 2.5 or 5.0 mg/kg of AD1 and stressed for 1 hr by restraint. Neither dose of ADI had any effect on heart rate, blood pressure or norepinephrine (NE) and epinephrine (EP) in plasma in the resting rats. In the stressed animal, 2.5 and 5.0 mg/kg of ADI did not affect stress-induced increases in heart rate or blood pressure but both significantly reduced the stress-induced increases in plasma NE and EP. During certain stressful experiences in patients with abno~ally-increase sympathetic drive, AD1 may he thera~uti~ily useful
in reducing high levels of catecholamines. Key words-adinazolam, stress, norepinephrine, epinephrine, heart rate, blood pressure.
It is well-established that a wide variety of stresses causes significant biochemical, physiological and behavioral changes (Natelson, Creighton, McCarty, Tapp, Pittman and Ottenweiller, 1987; Livesey, Miller and Vogel, 1985; Blizard and Morris, 1987). These changes include marked increases in catecholamines in plasma, heart rate and blood pressure (Livesey et al., 1985; Taylor, Harris, Krieman and Vogel, 1989). Treatment with certain anxiolytic drugs or alcohol can attenuate the stress-induced increases of norepinephrine and epinephrine in plasma, heart rate and blood pressure in animals and humans (Taylor et of., 1989; Stratton and Halter, 1985; Graffy Sparrow, Roggendorf and Vogel, 1987). Among the anxiolytics, alprazolam has been found to significantly decrease levels of norepinephrine and epinephrine in plasma and mean arterial blood pressure during stress (Vogel, Miller, DeTurck and Routzahn, 1984; Graffy Sparrow, 1987). Adinazolam, a derivative of alprazolam, is a new triazolobenzodiazepine possessing both anxiolytic and antidep~s~nt activity (Lahti, Sethy, Barsuhn and Hester, 1983). It was the objective of this study to determine if adinazolam would also reduce stress-induced increases in catecholamines in plasma, heart rate and blood pressure. METHODS Male Sprague-Dawley rats, 300-350 g, were obtained from Zivic Miller Laboratories (Zelienople, *Correspondence and reprint requests to: Dr Wolfgang H. Vogel, Department of Pharmacology, Thomas Jefferson University, 1020 Locust St, Philadelp~a, PA 19107, U.S.A. 33
Pennsylvania), individually housed and given food and water ad fibitum.After a period of acclimatization of 1 week, an aortic catheter was surgically implanted in each animal, as described previously (Taylor et al., 1989) but with the following modifications: the catheter was not sutured to the psoas muscle, nor was the trochar passed through the psoas muscle. Instead, the catheter was run on top of the mnscie to the back of the animal. The trochar, with catheter attached, was then run along the back of the animal under the skin and brought out through an incision at the back of the neck. This modification was performed to lessen muscle trauma and avoid damage to the plexus of nerves that traverse the psoas muscle. In addition, not anchoring the catheter to the muscle allowed freer movement of the animal, without fear of ripping the canula out of the aorta. Another change was that the catheter was not attached to the spring-pulley apparatus. Instead, the closed end of a plastic needle cap was cut off and the remaining rigid tube was used to protect the catheter. Approximately 3 cm of the catheter were left at the back of the neck. The catheter was threaded through the plastic tube and one end of the tube was fixed with cyanoacrylate glue (Su~rGIue~) to the skin on the back of the neck. This procedure also allowed for unimpeded movement of the animal within the home cage. The catheters were flushed daily with heparinized saline (1000 U/ml). After a recovery period of 48 hr baseline (time - 15) blood samples were drawn and heart rate and blood pressure recordings were taken using a Grass Model 7 polygraph and Gould P23XL transducers. Animals were then given an intraperitoneal injection of 2.5 mg/kg adinazolam
M.
34
J. &IEMAN
mesylate (AD12.5) (Upjohn, Kalamazoo, Michigan), 5.0 mg/kg adinazolam (ADI5.0) or 1 ml/kg vehicle (0.9% saline). Fifteen minutes after the injection (time 0), blood samples, heart rate and blood pressure measurements were taken. Animals in control or treatment groups were then stressed by immobilization for 1 hr and blood samples, heart rate and blood pressure recordings taken at time 15, 30 and 60 min, during stress. Immobilization was performed by taping the legs of the animals to the laboratory bench. After the stress period of 1 hr, the animals were released, returned to the home cage for recovery and 1 hr post-stress samples and recording were taken. Non-stress adinazolam- and vehicle-treated animals remained in the home cage for the entire test period and were sampled in the same manner as above. Approximately 0.3 ml of blood was withdrawn for each sample and an equal amount of 0.9% saline was reinfused to prevent changes in blood volume. All blood samples were drawn into syringes containing an EGTA/GSH solution and transferred immediately to cooled microcentrifuge tubes. The tubes were spun at 2500 rpm, 4°C for 15 min. Plasma was drawn off, placed into another set of labeled microcentrifuge tubes and frozen at -70°C until assay. Determinations of NE and EP in plasma were made using a radioenzymatic assay (Cat-A-Kit, Amersham Corp.). Heart rate and systolic and diastolic blood pressure were determined directly from the polygraph tracings. Mean arterial blood pressure was calculated from the systolic and diastolic pressures, using the following equation: mean arterial pressure = diastolic + 1/3(systolic - diastolic pressure) (Beme and Levy, 1972). The data were analyzed using a three-way analysis of variance (ANOVA) with repeated measures, two-way ANOVA, Students t-test, Student-Newman-Keuls
et
al.
and the Trapezoidal rule for the area under the curve, where indicated. All studies were approved by the Institutional Animal Care and Use Committee (No. 120 CS). RESULTS
Global behavior
At time - 15, the global behavior of all animals was normal and they either groomed or slept. Fifteen minutes after the injection (time 0), the vehicletreated controls, animals treated with 2.5 mg/kg adinazolam and some of the group given 5 mg/kg adinazolam, were sleeping curled up under the shavings with eyes closed. Some of the animals treated with 5.0 mg/kg adinazolam appeared to be slightly sedated; they lay on top of the shavings with their eyes open. During stress, behavioral differences between the vehicle-treated and drug-treated animals became more apparent. The vehicle-treated animals vocalized, chattered their teeth, struggled and chewed at the restraints. The animals given 2.5 and 5.0 mg/kg ADI, on the other hand, struggled and vocalized less, although they did attempt to chew at the restraints. One hour after the stress, all animals were resting under the shavings, drinking water or eating lab chow. Catecholamines in plasma
The results for catecholamines in plasma are summarized in Table 1. At time - 15, there were no differences in levels of norepinephrine (NE) or epinephrine (EP) in plasma, between any of the groups. Fifteen minutes after injection, at time 0, the levels of both catecholamines in plasma remained unchanged in all the groups.
Table I. Levels of norepinephrine and epinephrine in plasma in stressed and non-stressed male Sprague-Dawley rats, pm-treated with vehicle or 2 different doses of adinazolam Time (min) -15
0
15
142 f 69 112*40 130 f 35 113f75 137 * 74 156 k 135
132i66 556 k 424’ 104*25 307 f 97* 104537 350 * 221
153 + 420 + 123 f 311* 149 f 292 +
58 187’ 81 105 35 199
148+52 494 f 220’ 119*40 294 * 1027 169k59 213 + 116t
189 k 87 141*44 88 f 33 93 * 29 17Ok92 146*70
195 * 84 693 f 468* 68 + 38 470 f 75’ 140*71 505 f 288*
197 f 51 587 & 257. SO+100 452 + 147* 162 k 48 438 f 280
205 f 98 670 + 386’ 94 * 71 314 * 147t 2OOk93 469 f 191
30
60
PS 1
AUC
Norepinephrine (pglml)
VEH VEH ADI ADI AD1 ADI
NS S 2.5 NS 2.5 S 5.0 NS 5.0 S
103 * 34 look44 100 f 24 97 f 22 74 f 23 130 * 53
160*81 206+91
194 f 130 237 f 456
2082 f 21132 f 959 f 9368 f 3338 f 6548 f
1537 7145. 1639 4770.t 2456 8385*t
- 10 f 30458 f - 1977 f 9508 f 11348 f 12116 f
3058 14668, 4243 757l.t 13697 8332.7
Epinephrine (pg/ml)
VEH VEH AD1 AD1 AD1 AD1
NS S 2.5 NS 2.5 S 5.0 NS 5.0 S
226 f 82 188 * 53 127 f 71 198 f 92 117f66 150*40
170 + 71 264+ 150
113*67 571 f 130
Values are means + SD; N = 6-10; VEH = Vehicle; AD1 2.5 = 2.5 mg/kg adinazolam; AD1 5.0 = 5.0 mg/kg adinazolam; NS = non-stressed; S = stressed. *P < 0.05 compared to VEH NS. tP < 0.05 compared to VEH S. Animals received 2.5 mg/kg adinazolam, 5.0 mg/kg adinazolam or 1 ml/kg vehicle immediately at - 15 min; stress or control period began immediately after 0 min and ended after 60 min; PS 1 is 1 hr after stress or control period; AUC is the area under the stress curve from 15-60 min, minus the - 15 min baseline. Statistics: 3-way ANOVA with repeated measures and SNK for 15-60 min, 2-way ANOVA for - 15 and 0 min. and PS 1; Student’s r-test for each adinazolam-treated group vs VEH S and VEH NS groups for AUC.
Adinazolam
and acute stress response
The tevelsof NE and EF in plasma did not change from baseline in non-stress vehicle-treated groups and 2.5 or 5.0 mg/kg AD1 groups, over the entire test period. During stress, however, levels of NE and EPI in plasma rose significantly from baseiine in the vehicle-stress group. fn the groups given 2.5 and %Omg/kg AD1 and stress, the levels of NE and EP also rose from baseline but these increases were less pronounced, compared to those of the vehide-stress group, at the individual times (time 15, 30 and 60). However, the overall stress responses or the areas under the stress curves for NE and EP were significantly smaller for both adin~olam-treaty groups, as compared to those for NE and EP of the vehielestress group. There was no statistical difference in the response of catecholamines during stress between the two doses of adinazolam, although the area under the curve for NE and EP in both adin~olam-trots stress groups was significantly greater than the area under the curve for the vehicle-treated non-stress group. One hour after stress Ievels of NE and EP in plasma had returned to baseline in the groups given 5.0 mg/kg AD1 and vehicle plus stress. No statistically significant differences in levels of NE and EP in plasma were noted between any of these groups. Levels were not determined in the groups given 2.5 mg/kg ADI.
35
In the non-stressed animals, heart rate and blood pressure in both 2.5 and S.Omg/kg ADI vehicletreated groups remained essentially unchanged during the entire test period, although blood pressure did appear to transientiy and slightly decrease in AD&treated non-stressed animals. In the stressed animals, heart rate and mean arterial blood pressure increased dramatically in 2.5 and 5.Omgfkg AD1 and vehicle-stress groups and were significantly greater, as compared to the paired nonstressed groups. Arterial pressure rose between 14-18 mmHg in all stressed groups, with no significant differences between either of the adinazolamtreated groups or the vehicle-treated group. The heart rate of all stressed groups rose between 108-148 beats per min, during the stress period and were significantly different from the respective pre-stress baseline and time~mat~h~ vehicle-treated non-stress group. However, again there were no differences between either of the adinazolam-treated stress groups and the vehicle-treated stress group. One hour after stress, the heart rate in the 2.5 mg/kg AD1 plus stress group, remained elevated but was returning to baseline. The heart rates in both vehicle and 5.Omg/kg ADI stressed groups were no longer signiticantly higher than either the non-stress or pre-stress values. Arterial pressure for all 3 stressed groups had decreased to pre-stress levels by 1 hr after stress and no statistical differences were found between any of the groups.
The data for heart rate and btood pressure for all groups are summarized in Table 2. At the pretreatment baseline (time - 15), there were no differences in heart rate or mean arterial blood pressure between any of the groups, By time 0, neither 2.5 mg/kg adinazolam, 5.0 mg/kg adinazolam or 1 ml/kg vehicle, had resulted in any change in heart rate or blood pressure.
DISCUSSION
A~nazol~~ a t~a~olo~~odi~epine~ is a novel therapeutic agent, said to possess both anxiolytic and antidepressant actions. Its anxiolytic properties are based on its ability to suppress release of the anxiogenie compound, corticosterone during stress (Lahti
Table 2. Heart rate and mean arterial blood pressure in stressed and non-stressed male Sprague-Dawley 2 different doses of adinaaolam
rats, pm-treated with vehicle or
Time (n-&r)
Heart
-15
0
15
30
69
PS 1
342 f 22 333 + 32 34O*f2 332 i 26 338 + 38 347 * 18
350 + 21 346125 3692 15 386 k 62 378 * 39 352 k 33
337 f I5 488 f 31* 355 & 16 498 * 39’ 361 j: 34 499 * 208
350 i 32 491* 24* 34629 4%f8* 366 f 31 500 + 22*
353*20 486 i 24* 348*x3 494 * 20* 360*28 4% f 20*
341218 399 f 27 353 f 37 424 f 45, 305 f 25 375140
92i6 i10+g* 8857 111 f6* 89 + 8
%f6 95flO 8’?& 10 101 f 8 98 f 3 99+5
rate @pm)
VEH NS VEH S AD1 2.5 NS ADI 2.5 S AD1 5.0 NS ADI 5.0 S Mean art&cl &ad VEH NS VEH S ADI 2.5 ris AD1 2.5 S ADI 5.0 NS AD1 5.0 S
pressure
(mmHg)
9555 9szbtfr g9+4 94 f 5 94*8 92+IO
112fS 88 f 7 108 f 6”
106T7*
ADI 5.0 = 5.0 mgfkg a&nazolam; NS = non-stmsseeh Results am means f SD; N = 610; VEN = vebiile; AD1 2.5 = 2.5 m&kg adinaeoti, !I = stressed. lP e O.O$compared to VEH NS. Animab received 5 m&g adinazolam, 2.5 mg/kg adinazolam or 1 ml/kg vehicle at - IS min; stressed or non4tressad control period began immediately after Omits and ended after 60min; PS 1 is the timepoint 1 hr afterstress or control period. Statistics: Sway ANOVA with repeated measures and Student-Neumau-Keuls (SNK) for l%Omin; Z-way ANOYA with SNK for -ISmin, Omin and PS I.
36
M. J. KRIEMAN
et al., 1983). However, little is known about its effects on the sympathetic or cardiovascular components of the stress response. Thus, the purpose of this study was to determine the effects of various doses of adinazolam on catecholamines, heart rate and mean arterial blood pressure, during stress. In non-stressed animals, the acute administration of 5 mg/kg adinazolam but not the smaller dose of 2.5 mg/kg, caused some behavioral sedation. Neither dose, however, produced any significant changes in heart rate, mean arterial blood pressure (Table 2) or levels of catecholamines in plasma (Table 1). The larger dose of adinazolam appeared to slightly decrease the blood pressure, with a drop of 10mmHg from the pre-treatment baseline (time - 15) at time 30 but this was transient and not statistically significant. Preliminary data from a group given 10 mg/kg (not included in the statistical analysis), also showed no significant effects of this larger dose on heart rate, mean arterial blood pressure or catecholamines in plasma in non-stressed animals. Overall, different doses of adinazolam had no effects on hemodynamic parameters and catecholamines in plasma in animals at rest. During stress, a great increase from the pretreatment baseline in heart rate, mean arterial blood pressure and NE and EP in plasma was seen in groups given vehicle, 2.5 and 5.0 mg/kg AD1 and stressed. Pre-treatment with adinazolam with either dose did not protect against the stress-induced increase in heart rate or mean arterial blood pressure, both parameters showed similar increases, compared to vehicle-stress values. This also held true for the small number of animals tested with 10 mg/kg adinazolam. However, both 2.5 and 5.0 mg/kg adinazolam did have significant effects on the level of catecholamines in plasma during stress. Both doses of adinazolam markedly suppressed the levels of both NE and EP during the entire period of stress (AUC from Table 1) but there was no difference in the response between the doses, 2.5 mg/kg was just as effective as the 5.0 mg/kg dose. Although the levels of both NE and EP, with both doses of adinazolam during stress, were lower than those in vehicle-treated stressed animals, they were still significantly higher than nonstressed animals at several times (Table 1, time 15-60) and the overall response to stress (Table 1, AUC). Additional preliminary data from the group given 10 mg/kg indicated an even greater suppression of levels of catecholamines during stress but this benefit may be offset by greater behavioral effects. The end results were that various doses of adinazolam reduced the stress-increased levels of catecholamines in plasma but had no effect on the stress-increased heart rate or mean arterial blood pressure. In comparison with the effects of adinazolam in the non-stressed animal, other studies with benzodiazepines show that diazepam (Vogel et al., 1984), alprazolam (Vogel et al., 1984) and chlordiazepoxide (de Boer, Slangen and van der Gugten, 1991) do not
et al.
affect levels of catecholamines in plasma. In contrast, buspirone, pharmacologically and structurally different from the benzodiazepines, causes a rise in levels of norepinephrine and epinephrine in plasma in resting animals (Taylor et al., 1989). In this study, adinazolam showed no effect on resting heart rate or blood pressure. In contrast, diazepam (Conahan and Vogel, 1986) and alprazolam (Graffy Sparrow, 1987) can increase heart rate, while buspirone decreases heart rate (Taylor et al., 1989). Blood pressure is unaffected by diazepam (Conahan and Vogel, 1986) and buspirone (Taylor et al., 1989) but slightly reduced by alprazolam (Charney, Breier, Jatlow and Heninger, 1986). Thus, these anxiolytic drugs exhibit quite different pharmacological profiles in the nonstressed animal. In the stressed animal, 2.5 and 5.0 mg/kg of adinazolam markedly reduced the stress response of both catecholamines in plasma but were without effect on the responses of heart rate and blood pressure to stress. Alprazolam has also been shown to antagonize the stress-induced increases in catecholamines in plasma (Stratton and Halter, 1985; Vogel et al., 1984) and to have no effect on the increased heart rate but effectively decreases the response of blood pressure to stress (Graffy Sparrow, 1987). Similarly, diazepam can reduce stress-induced increases in NE in plasma and blood pressure but not affect heart rate and responses of EP in plasma (Conahan and Vogel, 1986; Vogel et al., 1984). Chlordiazepoxide mainly reduces stress-induced increases in EP (de Boer et al., 1991). In contrast, the non-benzodiazepine, buspirone, increases levels of catecholamines in plasma even further during stress but markedly reduces blood pressure and changes in heart rate (Taylor et al., 1989). Thus, the effects of these anxiolytic drugs are more pronounced during stress. The benzodiazepine anxiolytics have been shown to reduce one or more catecholamine in plasma during stress but vary in their cardiovascular effects. This study, with adinazolam, a new benzodiazepine, indicates that it also fits this pattern. At present, it is uncertain how adinazolam antagonizes stress-induced increases in catecholamines in plasma. There is evidence that adinazolam reduces noradrenergic activity in the locus ceruleus and other areas of the brain (Cardoni, 1990; Chamey, Heninger and Breier, 1984; File and Pellow, 1985).This inhibition could lead to reduced sympathetic nervous activity and less activation of the adrenal medulla during stress, thereby decreasing release of both norepinephrine and epinephrine and thus the levels of catecholamines in plasma. Adinazolam may also decrease levels of catecholamines during stress, through an action on Type I benzodiazepine receptors (Maiewski, Larscheid, Cook and Mueller, 1985). Type I benzodiazepine receptors are coupled to the y-aminobutyric acid-chloride (GABA-Cl)ionophore and are found throughout the cerebellum, brainstem and cerebral cortex (Corda, Giorgi, Longoni, Ongini,
Adinazolam and acute stress response
Bamett, Montaldo and Biggio, 1988). Stimulation by AD1 at these inhibitory-type receptor complexes may, in turn, depress sympathetic outflow in these areas of the brain. Cardiovascular effects of some benzodiazepines may be the result of central mechanisms that regulate heart rate and blood pressure. The locus ceruleus, hypothalamus, medulla and other areas of the brain may have GABA-benzodiazepine receptor complexes impinging upon them (Chamey et al., 1986). These areas are also major control areas for heart rate and blood pressure (Johnson, Lambie and Spalding, 1984) and this may be one reason why alprazolam (Graffy Sparrow, 1987; Stratton and Halter, 1985) and diazepam (Conahan and Vogel, 1986; Vogel et al., 1984) are able to antagonize increases in mean arterial blood pressure during stress. Since neither dose of adinazolam had any effect on stress-increased mean arterial blood pressure in this study, it may not be able to affect these control centers to the same degree as diazepam and alprazolam. One explanation may be structural/functional dissimilarities of the benzodiazepines. Adinazolam has a bulky alkyl side chain, compared to diazepam and alprazolam (Cardoni, 1990), which could be responsible for different central cardiovascular actions. Additionally, in a test for antidepressant activity, adinazolam and imipramine (a tricyclic antidepressant) have been shown to potentiate the effects of norepinephrine on blood pressure (Lahti et al., 1983). Diazepam and alprazolam are known to have little to no antidepressant activity, as well as little to no effects on this pressor response. This functional dissimilarity may also partially explain why adinazolam did not affect the stress-increase blood pressure in this study, while alprazolam and diazepam have both been shown to reduce stress-induced increases in blood pressure. Patients with panic attacks often show increased sympathetic tone and increased levels of catecholamines in plasma (Chamey et al., 1984; Villacres, Hollifield, Katon, Wilkinson and Veith, 1987; Nesse, Cameron, Curtis, McCann and Huber-Smith, 1984), although the casual relationship between sympathetic activity and the clinical manifestations of panic are unclear. Adinazolam has recently been shown to be effective in treating panic and phobic disorders (Pyke and Greenberg, 1989). Catecholamines in plasma can also be elevated in patients with myocardial infarction, congestive heart failure or pheochromocytoma (Karl&erg, Bryer and Roberts, 1981; Goldstein, 1984; Thomas and Marks, 1978; Cruickshank, NeilDwyer, Degaute, Hayes, Kuume, Kytta, Vincent, Carruthers and Patel, 1987). It has been postulated that high levels of endogenous catecholamines may cause myocardial damage (Cruickshank et al., 1987; Evans, Downing and Chen, 1985). Since adinazolam reduces elevated levels of catecholamines in plasma it is possible that this drug may be beneficial in these clinical conditions.
31
Acknowledgements-We
gratefully acknowledge the support of this study by PHS Grant AA 06107 and The Upjohn Company.
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Karl&erg R. P., Cryer P. E. and Roberts R. (1981) Serial plasma catecholamine response early in the course of clinical acute myocardial infarction: relationship to infarct extent and-mortality. Am. Heart. J. 102: 24-29. Lahti R. A.. Sethv V. H.. Barsuhn C. and Hester J. B. (1983) Pharmacologi&l pro&e of the antidepressant adinazoi lam, a triazolobenzodiazepine. Neuropharmacology 22: (1 l), 1277-1282. Livesey G. T., Miller J. M. and Vogel W. H. (1985) Plasma norepinephrine, epinephrine and corticosterone stress responses to restraint in individual male and female rats, and their correlations. Neurosci. L&t. 62: 51-56.
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0028-3908/92 $5.00 + 0.00 Pergamon Press plc
Printed in Great Britain
EFFECTS OF ADINAZOLAM ON PLASMA CATECHOLAMINE, HEART RATE AND BLOOD PRESSURE RESPONSES IN STRESSED AND NON-STRESSED RATS M. J. KRIEMAN,D. M. HERSHOCK,I. J. GREENBERGand W. H. VOGEL* Department of Pharmacology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust St, Philadelphia, PA 19107, U.S.A. and The Upjohn Company, Kalamazoo, Michigan, U.S.A. (Accepted 21 June f99i)
Summary-Adinazolam (ADI) is a new benzodiazepine with anxiolytic and antidepressant properties. To assess its effects on the acute stress response, rats were given a single intra~riton~l injection of 2.5 or 5.0 mg/kg of AD1 and stressed for 1 hr by restraint. Neither dose of ADI had any effect on heart rate, blood pressure or norepinephrine (NE) and epinephrine (EP) in plasma in the resting rats. In the stressed animal, 2.5 and 5.0 mg/kg of ADI did not affect stress-induced increases in heart rate or blood pressure but both significantly reduced the stress-induced increases in plasma NE and EP. During certain stressful experiences in patients with abno~ally-increase sympathetic drive, AD1 may he thera~uti~ily useful
in reducing high levels of catecholamines. Key words-adinazolam, stress, norepinephrine, epinephrine, heart rate, blood pressure.
It is well-established that a wide variety of stresses causes significant biochemical, physiological and behavioral changes (Natelson, Creighton, McCarty, Tapp, Pittman and Ottenweiller, 1987; Livesey, Miller and Vogel, 1985; Blizard and Morris, 1987). These changes include marked increases in catecholamines in plasma, heart rate and blood pressure (Livesey et al., 1985; Taylor, Harris, Krieman and Vogel, 1989). Treatment with certain anxiolytic drugs or alcohol can attenuate the stress-induced increases of norepinephrine and epinephrine in plasma, heart rate and blood pressure in animals and humans (Taylor et of., 1989; Stratton and Halter, 1985; Graffy Sparrow, Roggendorf and Vogel, 1987). Among the anxiolytics, alprazolam has been found to significantly decrease levels of norepinephrine and epinephrine in plasma and mean arterial blood pressure during stress (Vogel, Miller, DeTurck and Routzahn, 1984; Graffy Sparrow, 1987). Adinazolam, a derivative of alprazolam, is a new triazolobenzodiazepine possessing both anxiolytic and antidep~s~nt activity (Lahti, Sethy, Barsuhn and Hester, 1983). It was the objective of this study to determine if adinazolam would also reduce stress-induced increases in catecholamines in plasma, heart rate and blood pressure. METHODS Male Sprague-Dawley rats, 300-350 g, were obtained from Zivic Miller Laboratories (Zelienople, *Correspondence and reprint requests to: Dr Wolfgang H. Vogel, Department of Pharmacology, Thomas Jefferson University, 1020 Locust St, Philadelp~a, PA 19107, U.S.A. 33
Pennsylvania), individually housed and given food and water ad fibitum.After a period of acclimatization of 1 week, an aortic catheter was surgically implanted in each animal, as described previously (Taylor et al., 1989) but with the following modifications: the catheter was not sutured to the psoas muscle, nor was the trochar passed through the psoas muscle. Instead, the catheter was run on top of the mnscie to the back of the animal. The trochar, with catheter attached, was then run along the back of the animal under the skin and brought out through an incision at the back of the neck. This modification was performed to lessen muscle trauma and avoid damage to the plexus of nerves that traverse the psoas muscle. In addition, not anchoring the catheter to the muscle allowed freer movement of the animal, without fear of ripping the canula out of the aorta. Another change was that the catheter was not attached to the spring-pulley apparatus. Instead, the closed end of a plastic needle cap was cut off and the remaining rigid tube was used to protect the catheter. Approximately 3 cm of the catheter were left at the back of the neck. The catheter was threaded through the plastic tube and one end of the tube was fixed with cyanoacrylate glue (Su~rGIue~) to the skin on the back of the neck. This procedure also allowed for unimpeded movement of the animal within the home cage. The catheters were flushed daily with heparinized saline (1000 U/ml). After a recovery period of 48 hr baseline (time - 15) blood samples were drawn and heart rate and blood pressure recordings were taken using a Grass Model 7 polygraph and Gould P23XL transducers. Animals were then given an intraperitoneal injection of 2.5 mg/kg adinazolam
M.
34
J. &IEMAN
mesylate (AD12.5) (Upjohn, Kalamazoo, Michigan), 5.0 mg/kg adinazolam (ADI5.0) or 1 ml/kg vehicle (0.9% saline). Fifteen minutes after the injection (time 0), blood samples, heart rate and blood pressure measurements were taken. Animals in control or treatment groups were then stressed by immobilization for 1 hr and blood samples, heart rate and blood pressure recordings taken at time 15, 30 and 60 min, during stress. Immobilization was performed by taping the legs of the animals to the laboratory bench. After the stress period of 1 hr, the animals were released, returned to the home cage for recovery and 1 hr post-stress samples and recording were taken. Non-stress adinazolam- and vehicle-treated animals remained in the home cage for the entire test period and were sampled in the same manner as above. Approximately 0.3 ml of blood was withdrawn for each sample and an equal amount of 0.9% saline was reinfused to prevent changes in blood volume. All blood samples were drawn into syringes containing an EGTA/GSH solution and transferred immediately to cooled microcentrifuge tubes. The tubes were spun at 2500 rpm, 4°C for 15 min. Plasma was drawn off, placed into another set of labeled microcentrifuge tubes and frozen at -70°C until assay. Determinations of NE and EP in plasma were made using a radioenzymatic assay (Cat-A-Kit, Amersham Corp.). Heart rate and systolic and diastolic blood pressure were determined directly from the polygraph tracings. Mean arterial blood pressure was calculated from the systolic and diastolic pressures, using the following equation: mean arterial pressure = diastolic + 1/3(systolic - diastolic pressure) (Beme and Levy, 1972). The data were analyzed using a three-way analysis of variance (ANOVA) with repeated measures, two-way ANOVA, Students t-test, Student-Newman-Keuls
et
al.
and the Trapezoidal rule for the area under the curve, where indicated. All studies were approved by the Institutional Animal Care and Use Committee (No. 120 CS). RESULTS
Global behavior
At time - 15, the global behavior of all animals was normal and they either groomed or slept. Fifteen minutes after the injection (time 0), the vehicletreated controls, animals treated with 2.5 mg/kg adinazolam and some of the group given 5 mg/kg adinazolam, were sleeping curled up under the shavings with eyes closed. Some of the animals treated with 5.0 mg/kg adinazolam appeared to be slightly sedated; they lay on top of the shavings with their eyes open. During stress, behavioral differences between the vehicle-treated and drug-treated animals became more apparent. The vehicle-treated animals vocalized, chattered their teeth, struggled and chewed at the restraints. The animals given 2.5 and 5.0 mg/kg ADI, on the other hand, struggled and vocalized less, although they did attempt to chew at the restraints. One hour after the stress, all animals were resting under the shavings, drinking water or eating lab chow. Catecholamines in plasma
The results for catecholamines in plasma are summarized in Table 1. At time - 15, there were no differences in levels of norepinephrine (NE) or epinephrine (EP) in plasma, between any of the groups. Fifteen minutes after injection, at time 0, the levels of both catecholamines in plasma remained unchanged in all the groups.
Table I. Levels of norepinephrine and epinephrine in plasma in stressed and non-stressed male Sprague-Dawley rats, pm-treated with vehicle or 2 different doses of adinazolam Time (min) -15
0
15
142 f 69 112*40 130 f 35 113f75 137 * 74 156 k 135
132i66 556 k 424’ 104*25 307 f 97* 104537 350 * 221
153 + 420 + 123 f 311* 149 f 292 +
58 187’ 81 105 35 199
148+52 494 f 220’ 119*40 294 * 1027 169k59 213 + 116t
189 k 87 141*44 88 f 33 93 * 29 17Ok92 146*70
195 * 84 693 f 468* 68 + 38 470 f 75’ 140*71 505 f 288*
197 f 51 587 & 257. SO+100 452 + 147* 162 k 48 438 f 280
205 f 98 670 + 386’ 94 * 71 314 * 147t 2OOk93 469 f 191
30
60
PS 1
AUC
Norepinephrine (pglml)
VEH VEH ADI ADI AD1 ADI
NS S 2.5 NS 2.5 S 5.0 NS 5.0 S
103 * 34 look44 100 f 24 97 f 22 74 f 23 130 * 53
160*81 206+91
194 f 130 237 f 456
2082 f 21132 f 959 f 9368 f 3338 f 6548 f
1537 7145. 1639 4770.t 2456 8385*t
- 10 f 30458 f - 1977 f 9508 f 11348 f 12116 f
3058 14668, 4243 757l.t 13697 8332.7
Epinephrine (pg/ml)
VEH VEH AD1 AD1 AD1 AD1
NS S 2.5 NS 2.5 S 5.0 NS 5.0 S
226 f 82 188 * 53 127 f 71 198 f 92 117f66 150*40
170 + 71 264+ 150
113*67 571 f 130
Values are means + SD; N = 6-10; VEH = Vehicle; AD1 2.5 = 2.5 mg/kg adinazolam; AD1 5.0 = 5.0 mg/kg adinazolam; NS = non-stressed; S = stressed. *P < 0.05 compared to VEH NS. tP < 0.05 compared to VEH S. Animals received 2.5 mg/kg adinazolam, 5.0 mg/kg adinazolam or 1 ml/kg vehicle immediately at - 15 min; stress or control period began immediately after 0 min and ended after 60 min; PS 1 is 1 hr after stress or control period; AUC is the area under the stress curve from 15-60 min, minus the - 15 min baseline. Statistics: 3-way ANOVA with repeated measures and SNK for 15-60 min, 2-way ANOVA for - 15 and 0 min. and PS 1; Student’s r-test for each adinazolam-treated group vs VEH S and VEH NS groups for AUC.
Adinazolam
and acute stress response
The tevelsof NE and EF in plasma did not change from baseline in non-stress vehicle-treated groups and 2.5 or 5.0 mg/kg AD1 groups, over the entire test period. During stress, however, levels of NE and EPI in plasma rose significantly from baseiine in the vehicle-stress group. fn the groups given 2.5 and %Omg/kg AD1 and stress, the levels of NE and EP also rose from baseline but these increases were less pronounced, compared to those of the vehide-stress group, at the individual times (time 15, 30 and 60). However, the overall stress responses or the areas under the stress curves for NE and EP were significantly smaller for both adin~olam-treaty groups, as compared to those for NE and EP of the vehielestress group. There was no statistical difference in the response of catecholamines during stress between the two doses of adinazolam, although the area under the curve for NE and EP in both adin~olam-trots stress groups was significantly greater than the area under the curve for the vehicle-treated non-stress group. One hour after stress Ievels of NE and EP in plasma had returned to baseline in the groups given 5.0 mg/kg AD1 and vehicle plus stress. No statistically significant differences in levels of NE and EP in plasma were noted between any of these groups. Levels were not determined in the groups given 2.5 mg/kg ADI.
35
In the non-stressed animals, heart rate and blood pressure in both 2.5 and S.Omg/kg ADI vehicletreated groups remained essentially unchanged during the entire test period, although blood pressure did appear to transientiy and slightly decrease in AD&treated non-stressed animals. In the stressed animals, heart rate and mean arterial blood pressure increased dramatically in 2.5 and 5.Omgfkg AD1 and vehicle-stress groups and were significantly greater, as compared to the paired nonstressed groups. Arterial pressure rose between 14-18 mmHg in all stressed groups, with no significant differences between either of the adinazolamtreated groups or the vehicle-treated group. The heart rate of all stressed groups rose between 108-148 beats per min, during the stress period and were significantly different from the respective pre-stress baseline and time~mat~h~ vehicle-treated non-stress group. However, again there were no differences between either of the adinazolam-treated stress groups and the vehicle-treated stress group. One hour after stress, the heart rate in the 2.5 mg/kg AD1 plus stress group, remained elevated but was returning to baseline. The heart rates in both vehicle and 5.Omg/kg ADI stressed groups were no longer signiticantly higher than either the non-stress or pre-stress values. Arterial pressure for all 3 stressed groups had decreased to pre-stress levels by 1 hr after stress and no statistical differences were found between any of the groups.
The data for heart rate and btood pressure for all groups are summarized in Table 2. At the pretreatment baseline (time - 15), there were no differences in heart rate or mean arterial blood pressure between any of the groups, By time 0, neither 2.5 mg/kg adinazolam, 5.0 mg/kg adinazolam or 1 ml/kg vehicle, had resulted in any change in heart rate or blood pressure.
DISCUSSION
A~nazol~~ a t~a~olo~~odi~epine~ is a novel therapeutic agent, said to possess both anxiolytic and antidepressant actions. Its anxiolytic properties are based on its ability to suppress release of the anxiogenie compound, corticosterone during stress (Lahti
Table 2. Heart rate and mean arterial blood pressure in stressed and non-stressed male Sprague-Dawley 2 different doses of adinaaolam
rats, pm-treated with vehicle or
Time (n-&r)
Heart
-15
0
15
30
69
PS 1
342 f 22 333 + 32 34O*f2 332 i 26 338 + 38 347 * 18
350 + 21 346125 3692 15 386 k 62 378 * 39 352 k 33
337 f I5 488 f 31* 355 & 16 498 * 39’ 361 j: 34 499 * 208
350 i 32 491* 24* 34629 4%f8* 366 f 31 500 + 22*
353*20 486 i 24* 348*x3 494 * 20* 360*28 4% f 20*
341218 399 f 27 353 f 37 424 f 45, 305 f 25 375140
92i6 i10+g* 8857 111 f6* 89 + 8
%f6 95flO 8’?& 10 101 f 8 98 f 3 99+5
rate @pm)
VEH NS VEH S AD1 2.5 NS ADI 2.5 S AD1 5.0 NS ADI 5.0 S Mean art&cl &ad VEH NS VEH S ADI 2.5 ris AD1 2.5 S ADI 5.0 NS AD1 5.0 S
pressure
(mmHg)
9555 9szbtfr g9+4 94 f 5 94*8 92+IO
112fS 88 f 7 108 f 6”
106T7*
ADI 5.0 = 5.0 mgfkg a&nazolam; NS = non-stmsseeh Results am means f SD; N = 610; VEN = vebiile; AD1 2.5 = 2.5 m&kg adinaeoti, !I = stressed. lP e O.O$compared to VEH NS. Animab received 5 m&g adinazolam, 2.5 mg/kg adinazolam or 1 ml/kg vehicle at - IS min; stressed or non4tressad control period began immediately after Omits and ended after 60min; PS 1 is the timepoint 1 hr afterstress or control period. Statistics: Sway ANOVA with repeated measures and Student-Neumau-Keuls (SNK) for l%Omin; Z-way ANOYA with SNK for -ISmin, Omin and PS I.
36
M. J. KRIEMAN
et al., 1983). However, little is known about its effects on the sympathetic or cardiovascular components of the stress response. Thus, the purpose of this study was to determine the effects of various doses of adinazolam on catecholamines, heart rate and mean arterial blood pressure, during stress. In non-stressed animals, the acute administration of 5 mg/kg adinazolam but not the smaller dose of 2.5 mg/kg, caused some behavioral sedation. Neither dose, however, produced any significant changes in heart rate, mean arterial blood pressure (Table 2) or levels of catecholamines in plasma (Table 1). The larger dose of adinazolam appeared to slightly decrease the blood pressure, with a drop of 10mmHg from the pre-treatment baseline (time - 15) at time 30 but this was transient and not statistically significant. Preliminary data from a group given 10 mg/kg (not included in the statistical analysis), also showed no significant effects of this larger dose on heart rate, mean arterial blood pressure or catecholamines in plasma in non-stressed animals. Overall, different doses of adinazolam had no effects on hemodynamic parameters and catecholamines in plasma in animals at rest. During stress, a great increase from the pretreatment baseline in heart rate, mean arterial blood pressure and NE and EP in plasma was seen in groups given vehicle, 2.5 and 5.0 mg/kg AD1 and stressed. Pre-treatment with adinazolam with either dose did not protect against the stress-induced increase in heart rate or mean arterial blood pressure, both parameters showed similar increases, compared to vehicle-stress values. This also held true for the small number of animals tested with 10 mg/kg adinazolam. However, both 2.5 and 5.0 mg/kg adinazolam did have significant effects on the level of catecholamines in plasma during stress. Both doses of adinazolam markedly suppressed the levels of both NE and EP during the entire period of stress (AUC from Table 1) but there was no difference in the response between the doses, 2.5 mg/kg was just as effective as the 5.0 mg/kg dose. Although the levels of both NE and EP, with both doses of adinazolam during stress, were lower than those in vehicle-treated stressed animals, they were still significantly higher than nonstressed animals at several times (Table 1, time 15-60) and the overall response to stress (Table 1, AUC). Additional preliminary data from the group given 10 mg/kg indicated an even greater suppression of levels of catecholamines during stress but this benefit may be offset by greater behavioral effects. The end results were that various doses of adinazolam reduced the stress-increased levels of catecholamines in plasma but had no effect on the stress-increased heart rate or mean arterial blood pressure. In comparison with the effects of adinazolam in the non-stressed animal, other studies with benzodiazepines show that diazepam (Vogel et al., 1984), alprazolam (Vogel et al., 1984) and chlordiazepoxide (de Boer, Slangen and van der Gugten, 1991) do not
et al.
affect levels of catecholamines in plasma. In contrast, buspirone, pharmacologically and structurally different from the benzodiazepines, causes a rise in levels of norepinephrine and epinephrine in plasma in resting animals (Taylor et al., 1989). In this study, adinazolam showed no effect on resting heart rate or blood pressure. In contrast, diazepam (Conahan and Vogel, 1986) and alprazolam (Graffy Sparrow, 1987) can increase heart rate, while buspirone decreases heart rate (Taylor et al., 1989). Blood pressure is unaffected by diazepam (Conahan and Vogel, 1986) and buspirone (Taylor et al., 1989) but slightly reduced by alprazolam (Charney, Breier, Jatlow and Heninger, 1986). Thus, these anxiolytic drugs exhibit quite different pharmacological profiles in the nonstressed animal. In the stressed animal, 2.5 and 5.0 mg/kg of adinazolam markedly reduced the stress response of both catecholamines in plasma but were without effect on the responses of heart rate and blood pressure to stress. Alprazolam has also been shown to antagonize the stress-induced increases in catecholamines in plasma (Stratton and Halter, 1985; Vogel et al., 1984) and to have no effect on the increased heart rate but effectively decreases the response of blood pressure to stress (Graffy Sparrow, 1987). Similarly, diazepam can reduce stress-induced increases in NE in plasma and blood pressure but not affect heart rate and responses of EP in plasma (Conahan and Vogel, 1986; Vogel et al., 1984). Chlordiazepoxide mainly reduces stress-induced increases in EP (de Boer et al., 1991). In contrast, the non-benzodiazepine, buspirone, increases levels of catecholamines in plasma even further during stress but markedly reduces blood pressure and changes in heart rate (Taylor et al., 1989). Thus, the effects of these anxiolytic drugs are more pronounced during stress. The benzodiazepine anxiolytics have been shown to reduce one or more catecholamine in plasma during stress but vary in their cardiovascular effects. This study, with adinazolam, a new benzodiazepine, indicates that it also fits this pattern. At present, it is uncertain how adinazolam antagonizes stress-induced increases in catecholamines in plasma. There is evidence that adinazolam reduces noradrenergic activity in the locus ceruleus and other areas of the brain (Cardoni, 1990; Chamey, Heninger and Breier, 1984; File and Pellow, 1985).This inhibition could lead to reduced sympathetic nervous activity and less activation of the adrenal medulla during stress, thereby decreasing release of both norepinephrine and epinephrine and thus the levels of catecholamines in plasma. Adinazolam may also decrease levels of catecholamines during stress, through an action on Type I benzodiazepine receptors (Maiewski, Larscheid, Cook and Mueller, 1985). Type I benzodiazepine receptors are coupled to the y-aminobutyric acid-chloride (GABA-Cl)ionophore and are found throughout the cerebellum, brainstem and cerebral cortex (Corda, Giorgi, Longoni, Ongini,
Adinazolam and acute stress response
Bamett, Montaldo and Biggio, 1988). Stimulation by AD1 at these inhibitory-type receptor complexes may, in turn, depress sympathetic outflow in these areas of the brain. Cardiovascular effects of some benzodiazepines may be the result of central mechanisms that regulate heart rate and blood pressure. The locus ceruleus, hypothalamus, medulla and other areas of the brain may have GABA-benzodiazepine receptor complexes impinging upon them (Chamey et al., 1986). These areas are also major control areas for heart rate and blood pressure (Johnson, Lambie and Spalding, 1984) and this may be one reason why alprazolam (Graffy Sparrow, 1987; Stratton and Halter, 1985) and diazepam (Conahan and Vogel, 1986; Vogel et al., 1984) are able to antagonize increases in mean arterial blood pressure during stress. Since neither dose of adinazolam had any effect on stress-increased mean arterial blood pressure in this study, it may not be able to affect these control centers to the same degree as diazepam and alprazolam. One explanation may be structural/functional dissimilarities of the benzodiazepines. Adinazolam has a bulky alkyl side chain, compared to diazepam and alprazolam (Cardoni, 1990), which could be responsible for different central cardiovascular actions. Additionally, in a test for antidepressant activity, adinazolam and imipramine (a tricyclic antidepressant) have been shown to potentiate the effects of norepinephrine on blood pressure (Lahti et al., 1983). Diazepam and alprazolam are known to have little to no antidepressant activity, as well as little to no effects on this pressor response. This functional dissimilarity may also partially explain why adinazolam did not affect the stress-increase blood pressure in this study, while alprazolam and diazepam have both been shown to reduce stress-induced increases in blood pressure. Patients with panic attacks often show increased sympathetic tone and increased levels of catecholamines in plasma (Chamey et al., 1984; Villacres, Hollifield, Katon, Wilkinson and Veith, 1987; Nesse, Cameron, Curtis, McCann and Huber-Smith, 1984), although the casual relationship between sympathetic activity and the clinical manifestations of panic are unclear. Adinazolam has recently been shown to be effective in treating panic and phobic disorders (Pyke and Greenberg, 1989). Catecholamines in plasma can also be elevated in patients with myocardial infarction, congestive heart failure or pheochromocytoma (Karl&erg, Bryer and Roberts, 1981; Goldstein, 1984; Thomas and Marks, 1978; Cruickshank, NeilDwyer, Degaute, Hayes, Kuume, Kytta, Vincent, Carruthers and Patel, 1987). It has been postulated that high levels of endogenous catecholamines may cause myocardial damage (Cruickshank et al., 1987; Evans, Downing and Chen, 1985). Since adinazolam reduces elevated levels of catecholamines in plasma it is possible that this drug may be beneficial in these clinical conditions.
31
Acknowledgements-We
gratefully acknowledge the support of this study by PHS Grant AA 06107 and The Upjohn Company.
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