Effects of the CCKB antagonist CI-988 on responses to mCPP in generalized anxiety disorder

Psychiatry Research 85 Ž1999. 225]240

Effects of the CCK B antagonist CI-988 on responses to mCPP in generalized anxiety disorder Andrew W. Goddarda,U , Scott W. Woodsa , Roy Money a , Atul C. Pande b, Dennis S. Charney c , Wayne K. Goodmand, George R. Heninger a , Lawrence H. Price e a

Yale Uni¨ ersity Department of Psychiatry, Connecticut Mental Health Center, 34 Park St, New Ha¨ en, CT 06519, USA b Parke-Da¨ is Pharmaceutical Research Di¨ ision of Warner-Lambert Co. Clinical Research, CNS, 2800 Plymouth Rd, Ann Arbor, MI 48015, USA c Yale Uni¨ ersity Department of Psychiatry, 25 Park St, New Ha¨ en, CT 06519, USA d Uni¨ ersity of Florida College of Medicine, Department of Psychiatry, PO Box 100256, Gaines¨ ille, FL 32610-0256, USA e Brown Uni¨ ersity Department of Psychiatry and Human Beha¨ ior, Butler Hospital, 345 Blackstone Bl¨ d., Pro¨ idence, RI 02906, USA Received 8 August 1997; received in revised form 28 December 1998; accepted 31 January 1999

Abstract In order to evaluate the effect of the CCK B antagonist CI-988 on behavioral, neuroendocrine, and physiologic responses to the mixed, post-synaptic serotonin Ž5-HT. agonistrantagonist mCPP, 16 patients with a principal DSM-III-R diagnosis of generalized anxiety disorder ŽGAD. were enrolled in a study that involved two challenge tests. On one day, patients received a single oral dose of CI-988 followed 30 min later by an i.v. infusion of 0.1 mgrkg mCPP. On a second test day patients received placebo CI-988 followed 30 min later by active i.v. mCPP. The sequence of CI-988 was randomly assigned and the testing was conducted in double-blind fashion. In an initial dose-finding phase Ž N s 6. with a dose of CI-988 of 25 mg, there were no significant between-test differences in behavioral response to mCPP. Accordingly, the second phase of the study was conducted with a CI-988 dose of 100 mg in another of patients Ž N s 10.. CI-988 Ž100 mg. was well tolerated and had no significant effects on pretest anticipatory anxiety. There was no significant blunting of the anxiety response to mCPP as a result of CI-988 administration, nor did CI-988 affect physiologic or neuroendocrine measures. Correlations between peak changes in plasma levels of CI-988 and mCPP-induced anxiety in the high-dose patient group were not significant. Overall, these findings did not provide evidence of anxiolytic effects of CI-988 in patients with GAD. The lack of effect of CI-988 on neuroendocrine and physiological measures further suggests that CI-988’s pharmacological effects could

U

Corresponding author. Fax: q1 203 7645963; e-mail: [email protected]

0165-1781r99r$ - see front matter Q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 5 - 1 7 8 1 Ž 9 9 . 0 0 0 1 5 - 3

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be independent of 5-HT function. However, follow-up studies using higher doses of CI-988 are indicated to confirm this preliminary finding as are studies more closely evaluating the interrelationship between CCK and 5-HT function in GAD. Q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Anxiety; Serotonin; Cholecystokinin; m-Chlorophenylpiperazine

1. Introduction Generalized anxiety disorder ŽGAD. is a prevalent, disabling psychiatric condition that often runs a chronic course ŽWittchen et al., 1994. and appears to be genetic in origin ŽKendler et al., 1992.. Several lines of evidence indicate involvement of the serotonin Ž5-HT. system in GAD. For instance, certain medications with documented clinical efficacy for GAD influence 5-HT function. These include buspirone, a 5-HT1A receptor partial agonist ŽGammans et al., 1992., imipramine ŽRickels et al., 1993., which acutely blocks 5-HT reuptake and decreases the sensitivity of post-synaptic 5-HT2A receptors with chronic administration, and ritanserin, a 5-HT2r1C antagonist ŽCeulemans et al., 1985.. More recently, the novel anxiolytic serazapine, a selective 5-HT2 antagonist, has shown clinical efficacy in GAD in a multicenter trial ŽKatz et al., 1993.. The beneficial effects of serazapine were primarily seen on the psychic symptoms of GAD. In addition, a role for the 5-HT system in the pathophysiology of GAD is suggested by two clinical neurobiologic studies. GAD patients were abnormally sensitive to the mixed post-synaptic 5-HT agonistrantagonist mchlorophenylpiperazine Ž mCPP. ŽGermine et al., 1992., a well-validated anxiogenic agent in both animal and human models of anxiety ŽKahn and Wetzler, 1991.. These data are consistent with post-synaptic 5-HT2C , 5-HT2A ŽFiorella et al., 1995. or possibly 5-HT3 receptor ŽKahn and Wetzler, 1991. hypersensitivity in GAD. Recent data also suggest that mCPP binds to the 5-HT transporter, facilitating 5-HT release ŽBaumann et al., 1995.. This mechanism may also contribute to anxiogenic effects observed with mCPP. In another clinical study with GAD patients, elevated urinary levels of the 5-HT metabolite 5-hydroxyindoleacetic acid Ž5-HIAA. predicted higher anxiety levels in GAD patients ŽGarvey et al.,

1995., indicating a tendency towards increased 5-HT metabolism in more anxious GAD patients. These clinical data support the hypothesis of 5-HT dysfunction in GAD, which might be corrected by 5-HT-directed pharmacotherapies. Peptide neurotransmitters such as cholecystokinin ŽCCK. may also be involved in the pathophysiology and treatment of GAD. Recent preclinical and clinical studies have implicated CCK in the mediation of fear behaviors in animals and humans ŽHarro et al., 1992; Bradwejn and Koszycki, 1994; Crawley and Corwin, 1994; Rex et al., 1994; Fendt et al., 1995.. CCK B receptor agonists such as CCK-4 are anxiogenic in normal control subjects ŽDeMontigny, 1989. and panic patients ŽBradwejn et al., 1991., with patients much more sensitive in their responses to these probes. Furthermore, GAD patients were abnormally sensitive to administration of the CCK B agonist, pentagastrin, suggesting that CCK B receptor hypersensitivity could play a role in the pathophysiology of this disorder ŽBrawman-Mintzer et al., 1997.. The precise mechanism of CCK-induced anxiety, however, remains unclear. In response to the work implicating CCK in anxiety and fear behaviors, recent anxiolytic drug development efforts have focused on the therapeutic potential of CCK B receptor antagonists. Potent and selective CCK B antagonist compounds, such as CI-988 ŽParke-Davis. and L-365,260 ŽMerck. have shown considerable promise as novel anxiolytic agents in some standard preclinical ŽSingh et al., 1991; Hughes and Woodruff, 1992; Dooley and Klamt, 1993; Belzung et al., 1994; Josselyn et al., 1995. and clinical tests ŽBradwejn et al., 1994.. However, clinical trial results with CI-988 in GAD have been disappointing ŽBammert-Adams et al., 1995. and the ability of CI-988 to block anxiety in other paradigms of human anxiety such as lactate-induced panic ŽCowley et al., 1996. and

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CCK-4 induced panic Žvan Megen et al., 1997. has been limited. These findings may be due to the poor systemic availability of this class of compounds, and of CI-988 in particular ŽParke-Davis, unpublished data.. Nonetheless, if agents from this class of compounds were eventually found to be clinically effective for GAD, they would have major advantages over the benzodiazepine anxiolytics that are widely used for GAD, as they are non-sedating and have minimal abuserdependence liability ŽWettstein et al., 1994.. Also, in preclinical studies, they appear to have a rapid onset of action, a feature that would be an advantage over pharmacotherapies that modulate 5-HT function Žtricyclics, SSRIs, buspirone.. Functional interactions between 5-HT and CCK may be of relevance to understanding the pathophysiology and treatment of human anxiety disorders such as GAD. There is preliminary evidence of functional interactions between the CCK and 5-HT systems, which may have a role in fear behaviors in animals. For example, stress-induced increases in 5-HT levels in the lateral prefrontal cortex in guinea pigs were markedly potentiated by acute i.p. administration of CCK-4, and attenuated by administration of a CCK B antagonist ŽRex et al., 1994b.. Also, prior administration of the CCK B antagonist, L-365,260, dose dependently reversed citalopram-induced decreases in exploratory behavior in rats ŽMatto et al., 1996.. Together these observations suggest that endogenous CCK release enhances or amplifies stress responses that are 5-HT-mediated. However, another group observed more evidence of CCK A receptor involvement in a similar model of SSRI-induced anxiety in rats ŽBickerdike et al., 1994.. Another potential link between 5-HT and CCK may be via the 5-HT3 receptor. Preclinical anatomical and pharmacological data suggest that 5-HT via a presynaptic 5-HT3 heteroreceptor on CCK neurons facilitates CCK release ŽPaudice and Raiteri, 1991; Raiteri et al., 1993.. Also blockade of the 5-HT3 receptor with ondansetron, resulted in a decrease in CCK B agonist-induced fear behavior in one animal study ŽVasar et al., 1993.. This result, however, was not reproduced in a recent study of humans with panic disorder ŽMcCann et al., 1997.. In summary, then, there

227

may be reciprocal stimulatory effects of 5-HT on CCK function that serve to perpetuate stress-related behaviors in animals. The functional relationship between these systems merits further investigation to determine its significance for fear and anxiety behaviors in humans. The purpose of the present investigation was to assess the anxiolytic potential of CI-988 in GAD utilizing the mCPP challenge test as a model of human anxiety. The mCPP test has been recently proposed as a valid model to screen potential anxiolytic compounds with a variety of mechanisms in the preclinical laboratory ŽBilkei-Gorzo et al., 1998.. We chose to utilize the mCPP test in GAD patients as our previous work suggested that this group was abnormally sensitive to mCPP ŽGermine et al., 1992., in contrast to other patient groups we have studied, such as panic disorder, in which mCPP behavioral effects have been similar to effects in control subjects ŽCharney et al., 1987.. An additional aim of the study was to gather further evidence of functional CCKr5-HT interactions of potential relevance to human anxiety. From the brief review above, it is conceivable, though speculative, that part of the mechanism of mCPP-induced anxiety is related to enhancement of CCK release, possibly via stimulation of the 5-HT3 receptor. mCPP could do this by increasing release of 5-HT in general ŽBaumann et al., 1995., or by direct agonism of the 5-HT3 receptor ŽKahn and Wetzler, 1991., which receptor may be hypersensitive in GAD. Some animal ŽKennett et al., 1989. and human data ŽBroocks et al., 1997. support a role for the 5-HT3 receptor in mCPP’s anxiogenic effects, while other results have been negative ŽSilverstone and Cowen, 1994.. If mCPP-induced anxiety in GAD patients is partly due to increased brain CCK release, then agents that block the post-synaptic CCK B receptors, such as CI-988, could attenuate mCPP’s anxiogenic effects. We selected the CCK B antagonist, CI-988, for this study, because, in spite of its potential pharmacokinetic limitations, it was the only agent of this class available to us at the inception of the study. However, because of its novel action and potential clinical utility, further investigation of CI-988 appeared indicated. Accordingly, we examined the effect of two different doses of CI-988

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Ž25 and 100 mg. on mCPP-induced anxiety in unmedicated GAD patients. These doses were selected based on preclinical evidence of anxiolytic effects in this range ŽSingh et al., 1991.. Also there were no toxicology data available on doses above 100 mg. Patients participated in two neurobiologic challenge tests, with oral CI-988 administered on one test day and placebo given on the second day. The sequence of CI-988 was randomly assigned and the testing was conducted in double-blind fashion. CI-988 or placebo was given prior to an intravenous Ži.v.. infusion of 0.1 mgrkg of mCPP, which we have previously shown to provoke excessive anxiety and anger in GAD patients ŽGermine et al., 1992.. A positive finding Žattenuation of mCPP-induced anxiety in GAD by CI-988. would indicate that CI-988 is likely to be a clinically significant anxiolytic in GAD patients, possibly acting via CCKr5-HT neuronal interactions. A negative result would indicate that either CI-988 is not anxiolytic in GAD or acts via mechanisms unrelated to CCKr5-HT interactions in this disorder. 2. Materials and methods 2.1. Patients Sixteen consecutively admitted patients Ž10 males, six females. with a principal diagnosis of GAD were enrolled in the study. Their mean " S.D. age was 41 " 11 years, and mean weight was 80 " 18 kg, with a mean duration of illness of 16 " 16 years. Patients were recruited into the clinic by advertisements in local print and broadcast media. All patients gave voluntary written informed consent to participate. The diagnosis of GAD was confirmed by semi-structured interview based on DSM-III-R ŽSCID-NP. ŽSpitzer et al., 1990., administered by an experienced clinician. GAD was diagnosed only if chronic generalized anxiety was the patient’s chief complaint and the GAD antedated any episode of major depression. Patients were required to have a Hamilton Depression Scale ŽHAM-D. ŽHamilton, 1960. score of - 18 at intake. Additional current psychiatric co-morbidity in the study sample included panic disorder with Ž N s 3. and without agoraphobia

Ž N s 2. in partial remission, agoraphobia without a history of panic Ž N s 1., generalized social phobia Ž N s 2., simple phobia Ž N s 1., dysthymia Ž N s 2., and major depression Ž N s 4.. Eleven of the 16 patients had at least one co-morbid diagnosis, and four of these 11 carried two additional diagnoses. Most patients were free of psychotropic medications for at least 4 weeks prior to testing. However, two patients who were taking buspirone on a regular basis were discontinued 9 and 8 days, respectively, before their first test. No patients were taking fluoxetine or standing regimens of benzodiazepines. Two patients were taking occasional PRN benzodiazepine doses: one discontinued PRN alprazolam use over 1 week before the study and the other took a single dose of 5 mg of diazepam 2 days before the first test. All patients were in good physical health, ascertained by a careful medical history, a full physical exam including neurological exam, EKG, urinalysis, and other laboratory tests including electrolytes, glucose, liver function tests, renal function tests, full blood count and differential, thyroid functions, and RPR. All patients had a negative urine toxicology screen before the start of the protocol, except for the patient who took diazepam. All females had a negative serum pregnancy test. The first six patients participated in a low-dose protocol where a CI-988 dose of 25 mg was used. The last 10 patients were in a higherdose protocol with a CI-988 dose of 100 mg. One of the six patients in the low-dose group who underwent testing completed only 1 test day because of severe anxiety during the procedure. 2.2. Testing protocol Patients received two challenge tests separated by approximately 1 week Žmean time between challenges, 7 " 3 days.. They fasted from 12 midnight the day before each test except for sips of water. On the patients’ arrival at the Clinical Neuroscience Research Unit at 08.00 h, baseline behavioral ratings and vital signs were taken. An intravenous cannula was inserted in an antecubital vein in preparation for the mCPP infusion and for blood sampling. Active CI-988 capsules Žone 25-mg capsules for low-dose patients; two 50-mg

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capsules for high-dose patients. or placebo capsules were given at 09.30 h on each test day. CI-988 was administered in a double-blind, randomly assigned manner, with the blind maintained until completion of the entire study. In the low-dose Ž25 mg. phase of the study, three patients received active CI-988 first in the testing sequence Žone of these patients dropped out. and three received it second. In the high-dose Ž100 mg. phase, five patients received active CI-988 during the first test and five during the second. Thirty minutes after administration of CI-988, 0.1 mgrkg of open-label active mCPP was given as an i.v. infusion over 20 min via a Harvard pump. 2.2.1. Beha¨ ioral measures At 08.00 h on the morning of each test, an experienced clinician administered the Hamilton Anxiety Scale ŽHAM-A. ŽHamilton, 1959. assessment to the patients. Patients serially rated their subjective mood states on a 0]100-mm visual analog scale ŽVAS.. The ratings covered 13 mood states, including talkative, happy, drowsy, nervous, sad, calm, depressed, anxious, energetic, fearful, mellow, high, angry. VAS ratings were at y15 min Žbaseline . and y1 min prior to CI-988 ingestion, and then at 25, 60, 90, 120, 180 and 240 min post-CI-988 ingestion. The HAM-A was repeated at 90 min post-CI-988 ingestion Žor 60 min after the start of the mCPP infusion.. Thus, the HAMA was used as a within-challenge measure in the study, with patients responding to the questions based on their current symptomatology at the time of interview. 2.2.2. Physiologic measures Vital signs, including sitting systolic and diastolic blood pressure ŽBP. and sitting heart rate, were measured at the same time points as the VAS ratings. Because of missing data during the low-dose phase of the study, the y15-min time point was dropped from the data analysis, and the y1-min value used as the baseline for these parameters. 2.2.3. Biochemical measures Blood samples for hormone analysis Žplasma cortisol, prolactin and growth hormone. were

229

drawn at y15 and y1 min prior to CI-988 ingestion and then at 25, 60, 90, 120, 180 and 240 min post-CI-988, paralleling the VAS behavioral ratings. Blood samples for CI-988 plasma level analysis were drawn at y15 min prior to CI-988 ingestion, and then at 25, 60, 90, 120, 180 and 240 min post-CI-988. Plasma hormone levels were determined by radioimmunoassay ŽRIA.. Plasma cortisol was measured with an RIA kit from INCSTAR ŽStillwater, MN., with intra- and inter-assay coefficients of variation ŽC.V.s. of 3% and 5%, respectively. Prolactin was assayed using a kit from the same manufacturer, with intra- and inter-assay C.V.s of 3% and 6%. Growth hormone was determined by a monoclonalrpolyclonal antibody RIA kit from Nichols Institute Diagnostic ŽSan Juan Capistrano, CA., with intra- and inter-assay C.V.s of 3% and 4%. Plasma CI-988 concentrations were assayed by a highly sensitive HPLC method with fluorescence detection, at AvTech Laboratories Inc. ŽKalamazoo, MI.. 2.3. Data analysis Data were analyzed by Statistical Analysis System ŽSAS Institute, Cary, NC. and Biomedical Programs ŽBMDP. statistical packages. Principal outcome measures were VAS anxiety, nervousness, fear, calm, and anger. Within-challenge HAM-A scores were a secondary outcome measure. We predicted that if CI-988 was an effective anxiolytic it would attenuate the expected increases in anxiety and related mood states triggered by i.v. mCPP administration. We also anticipated that such a finding would be dose-related. Analyses of other secondary measures Žphysiologic and neuroendocrine. were also directed toward detection of attenuating effects of CI-988 on responses to mCPP. Analysis of variance ŽANOVA. with repeated measures was used to examine the whole data set. Analyses were performed on data from patients who completed the protocol Ž N s 15.. The main question of the data analysis, whether or not CI-988 blunted mCPPinduced anxiety, was tested by examination of the ANOVA interaction of drug ŽCI-988 vs. placebo. = time Žwithin-challenge .. The effect of CI-988

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230

dose was ascertained by inspection of the drug= time = dose Žlow vs. high. interaction. Thus, there were two within-group factors Ždrug and time. and one between-groups factor Ždose. in the analysis. Follow-up paired t-tests were performed when ANOVAs were statistically significant. For the high-dose group, Pearson correlational analyses were performed between mean peak change from baseline scores for VAS anxiety and nervousness, and mean peak change from baseline scores for plasma CI-988 levels and all neuroendocrine and physiologic measures. These correlations were performed separately for the placebo and active CI-988 tests. Significance was set at P- 0.05 Žtwo-tailed.. 3. Results 3.1. Beha¨ ioral measures Baseline Žpre-test. levels of most VAS mood variables were similar between tests and between

groups. However, in the high-dose CI-988 group, baseline levels of VAS fear were significantly lower on the placebo test day than on the active test day Žpaired t s 3.1, d.f.s 9, P- 0.05. ŽTable 2.. Baseline levels of VAS sad feelings were also lower on the placebo test day than on the active day in the high-dose group Ž9 " 9 vs. 19 " 19 mm; paired t s 2.7, d.f.s 9, P- 0.05.. In the low-dose group, patients were less happy at baseline on the placebo test day than on the active day Ž17 " 22 vs. 26 " 18 mm; paired t s 3.5, d.f.s 4, P- 0.05.. Other VAS measures were similar between tests, within each group. There were between-group baseline differences only for VAS drowsiness and VAS calm feelings. The high-dose group was significantly more drowsy Ž24 " 12 vs. 10 " 8 mm; Student’s t s 2.3, d.f.s 13, P - 0.05. and calm Ž43 " 10 vs. 19 " 16 mm; t s 3.7, d.f.s 13, P0.01. than the low-dose group at baseline. As expected, there were robust increases in anxiety-related mood variables due to mCPP infusion Žsee Tables 1 and 2.. There were signifi-

Table 1 Effects of low-dose CI-988 Ž25 mg p.o.. on subjective mood responses to 0.1 mgrkg i.v. mCPP in GAD patients Ž N s 5. VAS Mood state Žmm.

Drug ŽCI-988.

Time Žmin.a Baseline Žy15 min.

Anxiety

Active Placebo

32 " 24 38 " 36

y7 " 37 y5 " 15

y19 " 26 y5 " 16

27 " 50 31 " 35

16 " 40 9 " 43

Fear

Active Placebo

21 " 25 30 " 34

y6 " 22 y1 " 12

y12 " 19 2 " 11

15 " 66 32 " 34

Nervousness

Active Placebo

32 " 32 32 " 30

y10 " 37 y5 " 12

y18 " 34 y2 " 8

33 " 39 32 " 38

Calm

Active Placebo

14 " 17 24 " 19

12 " 25 4 " 30

11 " 37 1 " 34

Anger

Active Placebo

1"1 11 " 22

4 " 12 y7 " 14

Depression

Active Placebo

12 " 12 9 " 18

3 " 23 2"2

a

Change from baseline y1

q25

q180

q240

0 " 29 y1 " 54

y15 " 32 y15 " 37

y22 " 24 y18 " 35

4 " 54 10 " 47

y6 " 43 y6 " 46

y9 " 32 y15 " 37

y16 " 29 y13 " 35

11 " 53 12 " 14

1 " 35 2 " 40

y25 " 33 y13 " 27

y24 " 39 y17 " 21

2 " 33 y19 " 15b

2 " 31 y1 " 27

20 " 17 4 " 29

7 " 40 12 " 25

0"3 y8 " 17

0"3 y6 " 12

1"3 y3 " 7

1"3 y6 " 13

0"3 y4 " 9

1"4 y8 " 18

y2 " 23 3"6

0 " 29 y7 " 16

y7 " 18 9 " 12

3 " 31 14 " 15

y3 " 22 5"6

y9 " 15 y1 " 6

CI-988 given at Times 0 min; mCPP given at Times 30 min. P - 0.05, paired t-test, baseline vs. time point. All values are mean " S.D.

b

q60

q90

q120

44 " 29b 6 " 28

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231

Table 2 Effects of high-dose CI-988 Ž100 mg p.o.. on subjective mood response to 0.1 mgrkg i.v. mCPP in GAD patients Ž N s 10. VAS Mood state Žmm.

Drug ŽCI-988.

Time Žmin.a Baseline Žy15 min.

Anxiety

Active Placebo

41 " 19 31 " 23

y7 " 9d y2 " 11

y12 " 17 1 " 17

24 " 23b 29 " 30d

Fear

Active Placebo

30 " 24e 13 " 19e

y11 " 17 6"8

y11 " 17 4 " 11

Nervousness

Active Placebo

37 " 20 32 " 21

y6 " 18 y2 " 14

y6 " 13 1 " 11

Calm

Active Placebo

42 " 18 45 " 10

y13 " 14d y16 " 13c

Anger

Active Placebo

6 " 11 6"9

Depression

Active Placebo

18 " 19 12 " 14

Change from baseline y1

q25

q60

q90

q120

q180

q240

y1 " 21 7 " 22

y20 " 19 1 " 22

y21 " 19 y6 " 18

y20 " 19 y11 " 18

16 " 32 27 " 36d

y13 " 26 5 " 30

y22 " 19 2 " 21

y27 " 23 y7 " 15

y23 " 18 y6 " 18

24 " 15b 18 " 32

y5 " 25 6 " 21

y17 " 18 y5 " 11

y18 " 18 y10 " 15

y17 " 18 y13 " 16

y6 " 17 y18 " 12b

y28 " 22c y26 " 19c

y11 " 25 y10 " 33

y2 " 28 y4 " 25

4 " 20 y9 " 25

3 " 19 y7 " 23

0"2 0"1

y2 " 5 y1 " 4

y1 " 19 17 " 32

y5 " 10 1 " 18

y5 " 11 y4 " 7

y5 " 11 y4 " 7

y5 " 10 y4 " 7

y7 " 17 y1 " 17

y8 " 14 0 " 18

1 " 15 19 " 24

y11 " 14 8 " 18

y14 " 17 2 " 26

y13 " 15 1 " 26

y12 " 14 y3 " 23

a

CI-988 given at Times 0 min; mCPP given at Times 30 min. P - 0.001, paired t-test, baseline vs. time point. c P - 0.01, paired t-test, baseline vs. time point. d P- 0.05, paired t-test, baseline vs. time point. e P - 0.05, paired t-test, baseline vs. baseline. All values are mean " S.D. b

cant ANOVA main effects of time for VAS anxiety Ž F s 10.42, d.f.s 7,91, P- 0.001., fear Ž F s 6.45, d.f.s 7,91, P- 0.001., nervousness Ž F s 3.17, d.f.s 7, 91, P- 0.005., and calm feelings Ž F s 5.85, d.f.s 7,91, P- 0.001.. Peak changes Žincreases in VAS anxiety, nervousness, fear; decreases in calm feelings. in these variables were noted approximately 30 min following the start of the mCPP infusion Ž60 min post-CI-988. for both groups of patients ŽTables 1 and 2.. The time course of anxiety symptoms in these GAD patients was similar to that reported previously by our group ŽGermine et al., 1992.. In patients receiving the 100-mg CI-988 dose there was a non-significant trend for a more rapid return to baseline anxiety levels with the active compared to the placebo CI-988 test. There were no significant drug= time interactions with these or any other VAS variables, nor were there any significant drug= time = dose interactions, indicating that

neither low nor high-dose CI-988 attenuated mCPP-induced anxiety symptoms compared to placebo CI-988. The ANOVA drug= time interaction for the VAS calm variable reached trendlevel significance Ž F s 1.81, d.f.s 7,91, P- 0.1., mainly due to patients remaining more calm during the active, low-dose CI-988 test relative to the control test ŽTable 1.. In contrast to our previous report ŽGermine et al., 1992., there were no statistically significant changes in the anxiety-associated VAS mood states of anger and depression following mCPP administration during either placebo or active CI-988 tests ŽTables 1 and 2.. There was a non-significant increase in mCPPinduced anger Ž17 " 32 mm. in the high-dose patient group on the placebo CI-988 test day ŽTable 2, 60 min post-CI-988 time point.. This score was accounted for by data from 4r10 patients in the group that had mCPP-induced

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anger responses of ) 40 mm above baseline levels. In contrast, there was no appreciable anger response at the same time point on the high-dose, active CI-988 test day Žy1 " 19 mm.. There were mCPP-induced changes in other VAS mood states not directly associated with anxiety. Significant ANOVA main effects of time were observed for VAS happiness Ž F s 3.34, d.f.s 7, P- 0.01., drowsiness Ž F s 2.58, d.f.s 7, P- 0.05., and high feelings Ž F s 4.93, d.f.s 7, P - 0.01.. Decreases in VAS happy feelings peaked approximately 30 min post-mCPP infusion Ž60 min post-CI-988.. The mean change from baseline at this time was significant in the low-dose group on the placebo test day only Žy14 " 9 mm; paired t s 3.4, d.f.s 4, P- 0.05.. Increases in VAS drowsiness were generally maximal at 30 min post-mCPP, but none were significantly different from baseline scores by paired t-test. Increases in VAS high feelings peaked 30 min post-mCPP infusion. These changes were significant in the high-dose group on the active Ž26 " 35 mm; t s 2.4, d.f.s 9, P- 0.05. and placebo Ž29 "

34 mm, t s 2.7, d.f.s 9, P - 0.05. test days, with a trend in the low-dose group on the placebo day Ž18 " 18 mm, t s 2.3, d.f.s 4, P- 0.1.. These changes are consistent with previous observations of anxiety patients’ self-report of mCPP effects ŽSimeon et al., 1995.. Drug= time and drug= dose= time interactions were not significant for these variables, indicating no effect of CI-988 on these mCPP-induced mood changes. There was no discernible effect of mCPP on total HAM-A scores, which appeared to be an insensitive measure of state anxiety in the context of the challenge paradigm. Mean baseline HAMA scores for the high-dose group were 11 " 5 Žactive CI-988 test. and 11 " 4 Žplacebo test. and for the low-dose group were 12 " 6 Žactive CI-988 test. and 10 " 6 Žplacebo test.. 3.2. Physiologic measures Mean baseline and change from baseline values for all physiologic measures are summarized in Table 3. The table separates patients receiving

Table 3 Effects of low-dose Ž25 mg. and high-dose Ž100 mg. CI-988 on physiological responses to 0.1 mgrkg i.v. mCPP in GAD patients Drug ŽCI-988.

Time Žmin.a Baseline Žy1 min.

Active Placebo

118 " 22 115 " 22

4"9 6"4

6 " 10 12 " 15

6"8 5 " 12

3"7 3"9

2"6 0"9

y4 " 12 1"8

Sitting diastolic BP ŽmmHg.

Active Placebo

80 " 11 74 " 15

1"6 4"4

5"4 6"8

6"8 3"7

1"4 2 " 10

2"4 0 " 13

y2 " 7 y2 " 8

Sitting pulse rate Žb.p.m..

Active Placebo

80 " 5 80 " 3

2"5 y4 " 10

4"3 y2 " 7

0"6 y2 " 5

1"5 y4 " 3

4"5 y4 " 7

High dose Ž N s 10. Sitting systolic BP ŽmmHg.

Active Placebo

120 " 16 123 " 19

7"6 y1 " 5

3"9 7"9

5 " 10 y2 " 12

1"7 y3 " 11

2"8 y1 " 10

Sitting diastolic BP ŽmmHg.

Active Placebo

82 " 9 81 " 12

4"6 2"6

3"5 4 " 10

3"4 y1 " 7

0"4 1"5

y2 " 4 1"3

Sitting pulse rate Žb.p.m..

Active Placebo

70 " 7 71 " 19

2"7 6 " 17

3"9 7 " 20

Variable

Low dose Ž N s 5. Sitting systolic BP ŽmmHg.

a

Change from baseline q 25

CI-988 given at Times 0 min; mCPP given at Times 30 min. All values are mean " S.D.

q60

q90

3 " 10 5 " 21

q120

8"7 3 " 19

q180

6"7 5 " 21

q240

3"8 y5 " 10

0"6 1 " 11 y3 " 4 2"9 2"5 6 " 18

A.W. Goddard et al. r Psychiatry Research 85 (1999) 225]240

low-dose CI-988 from those receiving the higher dose. Baseline scores for blood pressure ŽBP. and heart rate were similar between tests and between groups, as confirmed by appropriate t-test analyses. The ANOVA main effect of time was not significant for BP Žsystolic and diastolic. or heart rate, indicating no appreciable effect of mCPP on these measures. The drug= time and drug= time = dose interaction terms were also not statistically significant, consistent with no effect of either dose of CI-988 on physiologic measures during the study.

3.3. Neuroendocrine measures

mCPP caused increases in plasma growth hormone ŽFig. 1., cortisol ŽFig. 2., and prolactin ŽFig. 3., consistent with previous work in anxiety disorder patients ŽCharney et al., 1987; Germine et al., 1994.. Mean baseline scores for neuroendocrine measures were generally similar between tests and between groups. One exception was

233

plasma prolactin, which was significantly higher in the high-dose group on the placebo test day Ž9.6 " 3.9 ngrdl. than on the active day Ž7.3" 3.2 ngrdl; paired t s 2.3, d.f.s 9, P- 0.05.. The ANOVA detected significant main effects of time for growth hormone Ž F s 4.58, d.f.s 7,91, P0.001., cortisol Ž F s 20.06, d.f.s 7,91, P - 0.001., and prolactin Ž F s 5.84, d.f.s 7,91, P- 0.001.. Growth hormone levels peaked 90 min after the start of the mCPP infusion, while cortisol and prolactin levels peaked after 60]90 min. There were no significant drug= time interactions for any of the neuroendocrine measures, demonstrating no effect of CI-988 on neuroendocrine responses to mCPP.

3.4. CI-988 plasma concentrations

Plasma concentrations of CI-988 peaked 90 min following ingestion of 100 mg of CI-988 and at 60 min in the case of 25 mg of CI-988 ŽFig. 4.. The main effect of time reached a trend-level of sig-

Fig. 1. Effects of low-dose Ž25 mg. and high-dose Ž100 mg. CI-988 on plasma growth hormone responses to 0.1 mgrkg i.v. mCPP in GAD patients. B, High-dose Ž N s 10., active CI-988; I, high-dose Ž N s 10., placebo; ', low-dose Ž N s 5., active CI-988; and ^, low-dose Ž N s 5., placebo.

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Fig. 2. Effects of low-dose Ž25 mg. and high-dose Ž100 mg. CI-988 on mean plasma cortisol responses to 0.1 mgrkg i.v. mCPP in GAD patients. B, High-dose Ž N s 10., active CI-988; I, high-dose Ž N s 10., placebo; ', low-dose Ž N s 5., active CI-988; and ^, low-dose Ž N s 5., placebo.

nificance Ž F s 1.92, d.f.s 6,78, P- 0.09. when 15 patients were used in the analysis Žwith the one drop-out excluded.. With all 16 patients included, the main effect of time was significant for CI-988 concentrations Ž F s 2.26, d.f.s 6,84, P- 0.05.. The dose= time interaction term was not significant, due to substantial variability in plasma concentrations at the 100-mg dose. 3.5. Correlational analyses

Pearson correlations were performed between peak change from baseline anxiety and nervousness scores, and peak changes in other variables, including sitting systolic and diastolic BP, sitting pulse rate, and plasma levels of prolactin, cortisol, growth hormone, and CI-988. These analyses were performed for the high-dose group of patients Ž N s 10. for the active CI-988 and placebo CI-988 tests. Since the ANOVAs had detected little impact of CI-988 on mCPP responses, we reasoned that if trends were present in the correlational analysis we would be most likely to observe them in the high-dose patients. However, none of the above correlations were significant.

4. Discussion We did not find an effect of preadministration of a single oral dose of 25 mg of CI-988 or 100 mg on GAD patients’ responses Žbehavioral, physiological, or neuroendocrine. to the mCPP challenge test. Furthermore, we did not observe significant effects of CI-988, at these doses, on prechallenge test anticipatory anxiety. The results were counter to our hypothesis that the anxiolytic action of CI-988 would attenuate responses to mCPP in this patient population. The mCPP test in this protocol was functioning in a manner consistent with our earlier work in GAD ŽGermine et al., 1992., except that the robust anger response to mCPP was not observed in the present patient sample. These data suggest that purported anxiolytic properties of CI-988 might be independent of the 5-HT system, a system that has been implicated in the anxiolytic action of several standard pharmacotherapies used for GAD, including tricyclic antidepressants and buspirone. Several factors must be considered in interpreting our data. One problem is the possibility that the CI-988 doses used were inadequate to block the effects of mCPP because of CI-988’s poor

A.W. Goddard et al. r Psychiatry Research 85 (1999) 225]240

235

Fig. 3. Effects of low-dose Ž25 mg. and high-dose Ž100 mg. CI-988 on mean plasma prolactin responses to 0.1 mgrkg i.v. mCPP in GAD patients. Ž1. P- 0.05, paired t-test, baseline vs. baseline. B, High-dose Ž N s 10., active CI-988; I, high-dose Ž N s 10., placebo; ', low-dose Ž N s 5., active CI-988; and ^, low-dose Ž N s 5., placebo.

systemic availability. This characteristic of CI-988 may account for its uncertain clinical efficacy in GAD patients even at a 300-mgrday total dose ŽBammert-Adams et al., 1995., as well as the modest effect of 100 mg of CI-988 on CCK-4-induced anxiety in healthy control subjects ŽBrad-

wejn et al., 1995. and trend effects in panic patients Žvan Megen et al., 1997.. In contrast, the CCK B antagonist L-365,260, which is thought to be similar to CI-988 in its affinity for the CCK B receptor ŽWettstein et al., 1994., has been reported to have marked effects on CCK-4-induced

Fig. 4. Mean " S.E. CI-988 levels in GAD patients receiving low-dose Ž25 mg. and high-dose Ž100 mg. CI-988 prior to challenge with mCPP. Ž1. P- 0.05, paired t-test, baseline vs. time point. Ž2. P- 0.01, paired t-test, baseline vs. time point. B, High-dose CI-988 Ž N s 10.; and v, low-dose CI-988 Ž N s 6..

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anxiety in control subjects ŽLines et al., 1993. and panic patients ŽBradwejn et al., 1994., suggesting that it is more bioavailable than CI-988. The pharmacokinetic data from the present study are consistent with the limited bioavailability of oral CI-988, and also suggest considerable inter-individual variability in CI-988 plasma levels following a given dose. An oral CI-988 dose of 200]300 mg may be required to detect anxiolytic effects with the mCPP paradigm. No human data are currently available on the effect of CI-988 in this higher dose range. The timing of CI-988 administration in the present study also appears to have been sub-optimal. At the time that we began the study, available pharmacokinetic data suggested that peak CI-988 blood levels would occur within 1 h of administration ŽParke-Davis, unpublished data.. However, we observed that peak CI-988 blood levels, at least for the 100-mg dose, did not occur until 90 min after oral administration ŽFig. 4.. Peak mCPP-induced behavioral effects were observed 60 min post-CI-988 Ž30 min after the start of the mCPP infusion. ŽTables 1 and 2.. Thus, administration of CI-988 60 min Žrather than 30 min. before the start of the mCPP infusion may have had a greater impact on mCPP-induced behavioral changes, and perhaps the other variables studied. The observation of a non-significant tendency for high-dose patients to have a more rapid return to baseline anxiety levels during the active compared to the placebo CI-988 test ŽTables 1 and 2. is consistent with this view. However, the mean plasma concentrations at the end of the mCPP infusion were s 2.1 ngrml with 2r10 patients having no measurable blood level at this time. In contrast, mean plasma CI-988 concentrations at the time of CCK-4 challenge Ž120 min after a 100-mg oral dose of CI-988. were 1.87 ngrml in one study ŽBradwejn et al., 1995. and 1.39 ngrml in another Žvan Megen et al., 1997.. In the former study mild anxiolytic effects of CI-988 were observed, while in the second only non-significant trends towards anxiolysis were reported. Thus, despite the suboptimal timing of the CI-988 dose in the present study, the plasma levels were in the range at which anxiolytic effects could be expected. Unfortunately, there are no

published or available unpublished ŽDr. Pande, personal communication. animal data correlating blood levels of CI-988 with its anxiolytic effects to add to the interpretation of the human data. Limitations of the mCPP paradigm itself may have had a bearing on the present findings. There has been debate in the mCPP literature about the appropriate dose and route of administration to employ in human studies with control subjects and anxiety patients ŽCharney et al., 1987; Kahn et al., 1988; Murphy et al., 1989; Pigott et al., 1993; Germine et al., 1994; Goodman et al., 1995.. The i.v. dose of 0.1 mgrkg has been criticized for being ‘overstimulating’ for both panic patients and control subjects, making separation between these two groups in terms of mCPP test responses more difficult ŽKahn et al., 1988.. It is possible that such an overstimulation effect reduced the sensitivity of the present study for detecting an anxiolytic effect of CI-988. However, our previous work with mCPP at a dose of 0.1 mgrkg i.v. suggested that on several behavioral measures there was good discrimination between control subjects and GAD patients ŽGermine et al., 1992.. Also, the challenge procedure was well tolerated in 15r16 subjects, with behavioral responses being of mild-moderate intensity, which argues against the hypothesis of excessive stimulation. There were other potential difficulties with our study design. Test]retest effects with mCPP and pharmacokinetic interactions between CI-988 and mCPP were potential confounds. However, these appeared not to pose major problems in the present data set, judging by the similar between-test and between-group mCPP responses. Another problem area was the type of CCKr5-HT interaction being tested by the present paradigm. The mCPP test is generally accepted as a measure of post-synaptic 5-HT Žespecially 5-HT2C . function ŽFiorella et al., 1995., although its 5-HT effects are more complex than this ŽKahn and Wetzler, 1991; Baumann et al., 1995.. Consequently, potentially relevant CCKr5-HT interactions occurring presynaptically ŽBoden et al., 1991. were not carefully tested by the present method. In addition, the preclinical literature has provided evidence of 5-HT3rCCK ŽPaudice and Raiteri, 1991; Raiteri et al., 1993; Vasar et al., 1993; Morales

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and Bloom, 1997., rather than 5-HT2rCCK interactions, which could mediate fear and anxiety behaviors. Thus mCPP is arguably not the best tool with which to examine such interactions. However, it is possible that part of the anxiogenic effect of mCPP is due to 5-HT3 receptor stimulation, an idea supported by data from some studies ŽKennett et al., 1989; Broocks et al., 1997. but not others ŽSilverstone and Cowen, 1994.. Factors related to the study sample could also have influenced our outcome. There was significant psychiatric co-morbidity in our patient sample, with other anxiety and mood disorders accounting for all of these additional diagnoses. It is difficult to say, therefore, how a ‘pure’ GAD population would have responded to our protocol. However, the pattern of co-morbidity observed is not atypical for GAD patients in general ŽAngst, 1993; Wittchen et al., 1994; Goisman et al., 1995.. Data from the US National Co-morbidity Survey ŽWittchen et al., 1994. indicated that 65% of GAD patients had concurrent psychiatric comorbidity, primarily with mood and other anxiety disorders. This figure is similar to the co-morbidity rate of 69% in our study sample. While the presence of other anxiety diagnoses such as panic disorder in partial remission and social phobia may have confounded responses to mCPP in this sample, co-morbid depression is not likely to have had a significant impact, as patients with major depression do not appear to have abnormal behavioral responses to mCPP ŽAnand et al., 1994.. Our GAD sample did appear to have mild levels of illness severity on the basis of the within-challenge HAM-A scores. However, the severity levels may be somewhat underestimated by our utilizing the HAM-A as a state-anxiety measure. Nonetheless, in the present context, a mildly ill patient group might be expected to exhibit decreased responsiveness to mCPP, thereby predisposing to a type I rather than type II error, which would be the main concern with the present data set. Lower levels of GAD symptom severity are consistent with the reduced anger responses we observed in the present sample, a result which did not concur with our previous GADrmCPP findings ŽGermine et al., 1992.. Lingering effects of

237

prestudy medication treatments did not seem to be a major source of error in the present investigation, as most patients were medication-free on admission. In the low-dose group the patient who had recently taken a single dose of diazepam received 25 mg of active CI-988 on the first test day and placebo CI-988 on the second. The diazepam might have been expected to bias toward a positive effect of CI-988 on mCPP-induced anxiety responses, an outcome that was not observed. In summary we did not observe anxiolytic effects of CI-988 utilizing the mCPP chemical model of anxiety. Thus, CI-988, in the doses used, is unlikely to be a useful anxiolytic in GAD. This result is consistent with the data from other clinical studies with CI-988 documenting weak or no anxiolytic effects. However, these data should be interpreted cautiously because of methodological limitations in the present study Žsmall sample size, co-morbidity, and timing of the CI-988 dose.. Despite these methodological limitations, patients Žreceiving the 100-mg CI-988 dose. had mean plasma levels of CI-988 that were similar to those achieved in other clinical studies examining the impact of CI-988 on anxiogenic challenges, and the mCPP test was functioning as expected in our patient sample. This suggests that the limited bioavailability of CI-988 Ž- 1% CNS penetrance . could be a major factor contributing to the negative outcome. The fact that one human study observed mild anxiolytic effects ŽBradwejn et al., 1995. and others Žvan Megen et al., 1997. have observed trend-level anxiolytic effects is quite consistent with this view. Further evaluation of the present study paradigm with higher doses of CI-988 or another compound with better systemic availability Že.g. L-365,260. is required before a definitive judgment can be made on the ability of CCK B antagonists to attenuate mCPP-induced anxiety. In this regard, newer CCK antagonist compounds with greater CNS penetrance have been already been synthesized ŽTrivedi et al., 1998.. In follow-up work selecting a GAD group without co-morbid anxiety disorders would be of value as these co-morbid diagnoses could contribute to overstimulation effects with mCPP.

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However, if follow-up studies provided confirmation of the present negative findings, this would indicate that CCK B antagonists, as a class, are not especially anxiolytic in GAD. An alternative explanation of a negative result with the mCPP test is that CCK B antagonists have a novel anxiolytic mechanism, possibly independent of the classical monoamine neurotransmitter systems such as 5-HT. Follow-up studies examining the effect of CCK B antagonists on clinical probes of presynaptic 5-HT function Že.g. acute SSRI or fenfluramine challenge. may be of value in establishing the independence of their actions from 5-HT in this patient population. Acknowledgements The study was supported by a grant from Parke-Davis, and also by Public Health Service Grants MH-36229, MH-30929, MH-40140, MH01322 Žto AWG. and the State of Connecticut. We thank Deborah Mordowanec, RN, and Candy Hill, MS, for expert assistance, Francine Augeri, RN, Mark Germine, MD, and Diane Sholomskas, Ph.D., for their clinical expertise, and Beth Ruff for her computer graphics work. References Anand, A., Charney, D.S., Delgado, P.L., McDougle, C.J., Heninger, G.R., Price, L.H., 1994. Neuroendocrine and behavioral responses to intravenous m-chlorophenylpiperazine Ž mCPP. in depressed patients and healthy comparison subjects. American Journal of Psychiatry 151, 1626]1630. Angst, J., 1993. Comorbidity of anxiety, phobia, compulsion and depression. International Clinical Psychopharmacology 1, 21]25. Bammert-Adams, J., Pyke, R.E., Costa, J., Cutler, N.R., Schweizer, E., Wilcox, C.S., Wisselink, P.G., Greiner, M., Pierce, M.W., Pande, A.C., 1995. A double-blind, placebocontrolled study of a CCK-B receptor antagonist, CI-988, in patients with generalized anxiety disorder. Journal of Clinical Psychopharmacology 15, 428]434. Baumann, M.H., Mash, D.C., Staley, J.K., 1995. The serotonin agonist m-chlorophenylpiperazine Ž mCPP. binds to serotonin transporter sites in human brain. Neuroreport 6, 2150]2152. Belzung, C., Pineau, N., Beuzen, A., Misslin, R., 1994. PD135158, a CCK-B antagonist, reduces ‘state’, but not

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