Combined Nicotinic and Muscarinic Blockade in Elderly Normal Volunteers: Cognitive, Behavioral, and Physiologic Responses John T. Little, M.D., Douglas N. Johnson, Ph.D., Marcia Minichiello, M.A., Herb Weingartner, Ph.D., and Trey Sunderland, M.D.

Establishing a pharmacologic model of the memory deficits scopolamine alone. Increased impairment was also seen for of Alzheimer’s disease could be an important tool in the mecamylamine ϩ scopolamine condition as compared to understanding how memory fails. We examined the scopolamine alone in selected behavioral ratings. Pupil size combined effects of the muscarinic antagonist scopolamine increased when mecamylamine was added to scopolamine, and the nicotinic antagonist mecamylamine in eight normal while systolic blood pressure and pulse changed in elderly volunteers (age 61.9 Ϯ 8.3 yrs, SD). Each received concordance with ganglionic blockade. These data together four separate drug challenges (scopolamine (0.4 mg IV), with previous brain-imaging results suggest that this mecamylamine (0.2 mg/kg up to 15 mg PO), muscarinic–nicotinic drug combination may better model mecamylamine ϩ scopolamine, and placebo). There was a Alzheimer’s disease than either drug alone. trend toward increased impairment in explicit memory for [Neuropsychopharmacology 19:60–69, 1998] the mecamylamine ϩ scopolamine condition as compared to Published by Elsevier Science Inc.

KEY WORDS: Scopolamine; Mecamylamine; Cognitive; al. 1985; Shimohama et al. 1986; Whitehouse and Au Geriatrics; Muscarinic antagonist; Nicotinic antagonist 1986; D’Amato et al. 1987; Zubenko et al. 1988), many cognitive dysfunction modeling studies have focused Given the limited animal models of Alzheimer’s disease on this system (Beatty et al. 1986; Sunderland et al. 1987; (AD) and other cognitive disorders, pharmacologic Broks et al. 1988; Dunne 1990; Flicker et al. 1990; Sun- modeling of memory deficits in humans has been pro- derland et al. 1990; Molchan et al. 1992). An earlier re- posed as an approach that can be used to probe the neu- port of a potential noninvasive test for AD using the rochemistry of memory (Sunderland et al. 1986). Be- cholinergic antagonist tropicamide (Scinto et al. 1994) cause there is abundant evidence that the cholinergic and the ongoing clinical use of the anticholinesterase in- system is of special importance in AD (Davies and hibitors tacrine and donepezil (Davis et al. 1992; Farlow Verth 1978; Bowen et al. 1983; Coyle et al. 1983; Mash et et al. 1992; Knapp et al. 1994; Rogers et al. 1996) for the treatment of AD further highlight the importance of From the Geriatric Psychiatry Branch (JTL, MM, TS), National Institute of Mental Health, Bethesda, Maryland; and Section of Cog- cholinergic neurons in AD. nitive Neurosciences (HW), National Institute on Alcohol Abuse Modeling studies involving the cholinergic system in and Alcoholism, Bethesda, Maryland; and Department of Psychol- humans have focused principally on the muscarinic sys- ogy (DNJ), Colgate University, Hamilton, New York, USA. Address correspondence to: John T. Little, M.D., Western Psychiat- tem, but the nicotinic system is also important in cognition ric Institute and Clinic, 3811 O’Hara Street, Pittsburgh, PA 15213, USA. and AD (Whitehouse et al. 1986; Newhouse et al. 1992; Address reprint requests to: Trey Sunderland, M.D., NIMH, 10/ Newhouse et al. 1994). In human autopsy studies, for 3N228 MSC 1275, Bethesda, MD 20892, USA. Received 10 December 1996; revised 8 October 1997; accepted 14 example, there is a marked reduction of cortical nico- November 1997. tinic receptors in AD patients as compared to matched

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NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 1 Nicotinic and Muscarinic Blockade in Elderly Normal Volunteers 61

controls, in contrast to the findings with muscarinic re- were completed to rule out medical and specific neuro- ceptors (Whitehouse et al. 1986; Nordberg et al. 1990; psychiatric disorders. Individuals with a history of hyper- Perry et al. 1990; Nordberg et al. 1992; Kellar et al. 1987; tension or cardiac illness were excluded from participa- Flynn and Mash 1986). Hence, we became interested in tion. Laboratory tests included electrocardiogram, chest the combined effects of these two systems, specifically X-ray, hematology and chemistry panels, urinalysis, HIV, employing scopolamine as a centrally active muscarinic hepatitis screen, rapid plasma reagin (RPR) tests, and thy- antagonist and mecamylamine as a nicotinic antagonist. roid function tests. Subjects were drug-free for at least 2 To our knowledge, mecamylamine and scopolamine weeks before beginning the study except for one subject have only been combined in one previous human study on stable, routine clinical doses of levothyroxine and one (Gitelman and Prohovnik 1992), which showed with subject on stable postmenopausal doses of estrogen. All 133⌾e inhalation methods that the nicotinic antagonist subjects were nonsmokers and were caffeine and alcohol mecamylamine decreased perfusion in parietotemporal free for at least 24 hours before study days. cortex, while the addiction of scopolamine reduced blood flow in frontal cortex. Because central blood flow Procedures in parietal and temporal cortex are known to be affected by AD (Haxby et al. 1988; Prohovnik et al. 1988; Fried- There were 4 study days separated by at least 72 hours for land et al. 1989; Kumar et al. 1991), the blood flow pat- each subject. Subjects received four separate challenges: tern induced by scopolamine and mecamylamine to- placebo, scopolamine, mecamylamine, and scopolamine ϩ gether may more closely model AD than scopolamine mecamylamine. As a safety measure against such possi- alone. Because cognitive testing was limited, behavioral ble medication side effects as hypotension, the first 2 study measurements were absent, and there was no placebo days included only placebo or mecamylamine alone. or scopolamine-alone condition in the Gitelman and Only after subjects safely tolerated mecamylamine alone Prohovnik study, speculations about the cognitive were they exposed to the combination of mecamyl- modeling of this combination were limited. Hence, the amine ϩ scopolamine. Hence, subjects received mecamyl- current study was initiated in normal elderly volun- amine or placebo for days 1 and 2, and scopolamine or teers to determine whether combined muscarinic and scopolamine ϩ mecamylamine for days 3 and 4. The or- nicotinic blockade would more closely mimic the cogni- der of the medication administration within those pa- tive, physiological, and behavioral deficits found in AD. rameters was randomized and balanced across subjects. Several subjects exposed to mecamylamine developed prolonged orthostatic hypotension and so were kept METHODS overnight on the research unit; for those subjects, the orthostatic blood pressure was normalized for least 72 Subjects hours before any further study days occurred. Written informed consent was obtained, and subjects After overnight fast, subjects reported to clinic at 8:00 were paid for their participation. Procedures were in ac- A.M. for each study day. An intravenous catheter was cord with the ethical standards of the committee on hu- placed in an antecubital vein in the nondominant arm at man experimentation of the National Institute of Men- least 20 min before the first medication was administered. tal Health (NIMH). Eight elderly normal volunteers Medications were given orally or intravenously under completed the study (five females and three males, double-blind randomized conditions. The dosing and mean age Ϯ standard deviation [SD] ϭ 61.9 Ϯ 8.3 years, timing of medications were chosen to optimize any pos- mean weight Ϯ SD ϭ 80.8 Ϯ 7.8 kg). Three additional sible cognitive effects, while minimizing physical side ef- normal volunteers were withdrawn from the study af- fects. Oral mecamylamine 0.2 mg/kg up to a maximum ter they developed significant postural hypotension from dose of 15 mg (seven subjects received 15 mg and one sub- mecamylamine (one female and two males, mean age Ϯ ject received 13.75 mg), or placebo was given after base- SD ϭ 70 Ϯ 5.6 years, mean weight Ϯ SD ϭ 61.2 Ϯ 9.4 line vital signs and behavioral ratings had been deter- kg; one completed only the first study day, and two mined (at t1 as shown in Figure 1). Sixty min after the completed 2 study days). The eight subjects completing PO medication (t2 in Figure 1), scopolamine 400 mcg or the study were well educated (years of education Ϯ SD ϭ 0.9% sodium chloride placebo was infused over 1 min. 15.5 Ϯ 3.5), of superior intelligence (estimated verbal in- telligence quotient Ϯ SD ϭ 121.0 Ϯ 4.2 [Grober and Sli- Cognitive Testing winski 1991], and Wechsler Memory Scale-Revised Ϯ SD ϭ 121.5 Ϯ 15.7, [Wechsler 1987]), and without evi- The cognitive measures were chosen to examine cognitive dence of cognitive dysfunction as assessed by a stan- domains known to be impaired in Alzheimer’s disease; dard dementia screening instrument (Mattis Dementia namely, explicit memory and learning, access to categor- Rating Scale score Ϯ SD ϭ 142.1 Ϯ 1.0) (Mattis 1976). ical knowledge in long-term memory, and simple reaction Medical histories were obtained, physical and mental time (see Nebes 1992 for review). Testing began 75 min- status exams were performed, and laboratory evaluations utes after the scopolamine/placebo infusion (135 min

62 J.T. Little et al. NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 1

Figure 1. Study design for drugs and testing in combination challenge paradigm in elderly normal volun- teers.

after mecamylamine/placebo administration) and lasted Semantic Memory. Access to categorical (structured) about 30 min. Five different versions of each task were cre- knowledge in long-term memory was tested by asking ated. Test versions were equated for word and name fre- subjects to generate as many exemplars as possible quency (word list learning, [Kucera and Francis 1967] and within 60 sec to a letter or category name. Subjects did the number of words retrieved (fluency tasks [Borkowski this fluency task four times each session (two letters et al. 1967}) based on published normative data. The and two category names, such as parts of a building or different versions were randomized to create five unique types of fruit). One letter and category was repeated test packets. Each subject participated in a practice session across sessions (fixed) and one was changed from ses- as well as the four drug conditions outlined above. The sion to session (variable). assignment of test packets was counterbalanced across drug administration days to avoid the possibility of form Simple Reaction Time. This task consisted of 40 trials, differences confounding the drug effects. The tests were wherein subjects were instructed to maintain visual fixa- administered in a fixed order for all sessions. tion on a fixation cross, and then to press a button as Explicit Memory and Learning. This was a prompted- quickly as possible when a large red disk appeared on the recall, selective-reminding learning and memory procedure computer display. Each trial began with a fixation point that has been frequently used as a method for assessing that remained on the screen for a random period between explicit memory functions (Buschke 1973). Each subject 300 and 1700 milliseconds (ms). On 35 trials, the fixation was presented a list of 12 unrelated common English point was replaced by a filled red circle that remained on words. The subject was then asked to remember as many the screen until a response was made or 3,500 ms elapsed. of the words as possible. Words that were not remem- Five of the trials were catch trials, where no filled red cir- bered were repeated by the examiner and the subject was cle (imperative stimulus) was presented. The dependent again asked to remember all of the words on the original measures were reaction time to the imperative stimulus, list. Thus, the subject had to recall those words that were omission error rate (percentage of trials that subjects did just read (the selectively prompted words) as well as the not respond to the disk), and commission error rate (per- words previously recalled that were not presented again. centage of trials that subjects responded during the catch This procedure was repeated for seven trials unless all of trials or before the presentation of the disk). the words were successfully remembered for two consec- utive trials. In this manner, the list learning mean (mean Behavioral Measures number of words recalled across trials), and the list learn- ing mean consistency (mean consistency scores across tri- Behavioral and physiologic ratings were obtained at als) were calculated. The list learning consistency between baseline and then throughout the study, as shown in two trials was defined as the number of words in a given Figure 1. Rating scales included: the National Institute trial correctly recalled without prompting divided by the of Mental Health Self-Rating Scale (Van Kammen and number of words correctly recalled in the previous trial. Murphy 1975; Murphy et al. 1989), a modified version

NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 1 Nicotinic and Muscarinic Blockade in Elderly Normal Volunteers 63

of the Brief Psychiatric Rating Scale (BPRS) (Overall and other cognitive tasks (semantic memory, lexical search Gorham 1962; Overall and Beller 1984), a physical and retrieval, and processing speed), performance symptom checklist containing 25 items rated on a scale scores were generally lower with the addition of of 0 to 3 for severity, and a visual analog scale for mood mecamylamine to scopolamine as compared to scopol- in which subjects indicated their overall mood on a 100- amine alone, but these differences were not statistically mm scale in which 0 reflected the best mood ever. significant (see Table 1). Explicit Memory. Mean list learning and consistency Physiologic Measures performance changed across drug conditions for the eight subjects, as shown in Table 1. As expected, scopol- For clinical monitoring, vital signs were measured at base- amine alone produced a significant impairment in line and then every 30 min during each study day. Figure mean list learning and consistency (p Ͻ .01). The addi- 1 illustrates the hourly time points used for data analy- tion of mecamylamine to scopolamine for the eight sub- sis. Blood pressure and pulse were measured on an au- jects produced a tend toward increased impairment for tomated monitor. Oral temperature was measured on list learning consistency compared to scopolamine an automated digital thermometer, and pupil size was alone (p Ͻ .1, see Figure 2a). When all 10 subjects were measured with a pupil gauge card. Patients were not al- considered who completed the first 2 study days, epi- lowed to get out of bed for the first 5 hours of the study, sodic memory was significantly impaired after subjects and then only if there were no clinically significant pos- received mecamylamine alone as compared to placebo tural changes in blood pressure. It is noteworthy that for list-learning mean (F[1,9] ϭ 6.74, p ϭ .029, not some subjects developed significant postural changes in shown in Table 1), and for list-learning consistency blood pressure at increments of 30Њ (particularly after (F[1,9] ϭ 5.21, p ϭ .048, not shown in Table 1). eating), so blood pressures were obtained whenever a position toward sitting up was increased by 30Њ or Semantic Memory. The overall ANOVAs for fixed more, and patients were closely observed when making and variable category fluency were not significant for positional changes away from being completely supine. the eight subjects completing all 4 study days. How- ever, a separate analysis including all 10 subjects com- pleting the first 2 study days did show differences be- Data Analysis tween mecamylamine alone and placebo. Namely, fixed Data were analyzed for overall treatment effect (scopol- category fluency was significantly reduced (F[1,9] ϭ amine vs. placebo, mecamylamine vs. placebo, mecamyl- 5.41, p ϭ .045), and variable category preservations in- amine ϩ scopolamine vs. scopolamine) across time by creased (F[1,9] ϭ 6.00, p ϭ .037) after subjects received repeated-measures analysis of variance (ANOVA) with mecamylamine alone as compared to placebo (not time ϫ drug contrasts determined a priori. Statistical shown in Table 1). comparisons were limited, because paired comparisons Lexical Search and Retrieval. Overall ANOVAs were were only examined when overall ANOVAs for vari- not significant for fixed or variable letter fluency tasks ables were significant. Carryover effects between treat- with the eight subjects completing all 4 study days. ment conditions were initially tested by ANOVA and However, when the 10 subjects completing the first 2 ruled out; therefore, order was dropped from further study days were analyzed, there was a trend toward in- analysis. To remove any effects of baseline differences, creased preservations (F[1,9] ϭ 3.86, p ϭ .081) after delta values (timeϫ Ϫ baseline) were used for statistical mecamylamine was given alone as compared to pla- analysis. For the sake of simplicity of explanation, mean cebo (not shown in Table 1). values are given in the tables that follow from the #4 test battery (t4 ϭ 120 minutes after scopolamine infu- Processing Speed. Scopolamine significantly increased sion), as shown in Figure 1. The SAS GLM program simple reaction time as compared to placebo (p Ͻ .01) (SAS Institute, Cary, NC) was utilized for data analysis. and created a trend toward increased omission errors (p Ͻ .1), as shown in Table 1.

RESULTS Behavioral Ratings Cognition As anticipated, significant behavioral effects of scopol- As expected, scopolamine impaired cognitive function- amine as compared to placebo were seen across a num- ing on multiple tests such as list learning and process- ber of measures. Further behavioral impairment was ing speed. Of more interest is the finding of a trend to- found with the addition of mecamylamine to scopol- ward increased impairment in episodic memory with amine in such selected measures as the BPRS 24-item the addition of mecamylamine to scopolamine as com- subscale and the NIMH-SRS anxiety subscale, as de- pared to scopolamine alone for these eight subjects. For scribed below. Table 2 summarizes behavioral data by

64 J.T. Little et al. NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 1

Table 1. Cognitive Effects in Eight Normal Elderly Volunteers Following Drug Administration on 4 Separate Days

Drug Conditions Overall ANOVA SCO MEC SCO ϩ MEC Cognitive Task F(3,21) p value PLA (vs. PLA) (vs. PLA) (vs. SCO)

Episodic memory List learning mean (7 trials) 19.13 .001 9.5 Ϯ 0.4 6.4 Ϯ 0.7** 9.0 Ϯ 0.6† 5.7 Ϯ 0.4 List learning mean consistency 19.43 .001 0.84 Ϯ 0.03 0.51 Ϯ 0.07** 0.76 Ϯ .05† 0.33 Ϯ 0.07† Semantic memory Fluency, variable category 0.67 NS 14.3 Ϯ 1.5 13.9 Ϯ 0.8 12.1 Ϯ 1.6 12.4 Ϯ 1.8 Fluency, fixed category 1.58 NS 16.0 Ϯ 2.0 16.3 Ϯ 1.9 14.5 Ϯ 2.0 15.1 Ϯ 2.1 Lexical search and retrieval Fluency, variable letter 1.17 NS 14.6 Ϯ 1.4 14.6 Ϯ 1.7 15.6 Ϯ 1.5 12.9 Ϯ 1.9 Fluency, fixed letter 0.52 NS 17.6 Ϯ 1.3 15.9 Ϯ 2.0 16.8 Ϯ 1.5 16.5 Ϯ 1.4 Processing speed Simple reaction time (ms) 15.27 .001 288 Ϯ 16 355 Ϯ 18** 299 Ϯ 24 362 Ϯ 15 Omission errors 3.32 .046 0.0 Ϯ 0.0 1.1 Ϯ 0.5† 0.0 Ϯ 0.0 1.1 Ϯ 0.5 Commission errors 2.13 NS 0.0 Ϯ 0.0 2.1 Ϯ 2.1 0.0 Ϯ 0.0 8.3 Ϯ 5.5

Values are mean Ϯ SEM. PLA ϭ placebo, SCO ϭ scopolamine, MEC ϭ mecamylamine. By repeated-measures analysis of variance, ** ϭ p Ͻ .01, * ϭ p Ͻ .05, and † ϭ p Ͻ .1. Significance levels are noted for contrasts in: SCO column for SCO vs. PLA, MEC column for MEC vs. PLA, SCO ϩ MEC column for SCO ϩ MEC vs. SCO. providing means at a consistent time (test battery #4, or vidually and in combination, as described below. Table t4, as shown in Figure 1) across drug conditions for 3 summarizes these by providing means at a consistent each measurement. Only those subscales that showed time (test battery #4, or t4) across drug conditions for significant changes are presented in the table. each measurement. Only those subscales that showed significant changes are presented in the table. BPRS Factors. As shown in Table 2, scopolamine as compared to placebo increased impairment, and the total score 24 items (p Ͻ .01), as well as anxiety/depression, Vital Signs total score 18 items, and mania (p Ͻ .05), and anergia at a trend level (p Ͻ .1). Interestingly, there were further in- Pupil Size. This was significantly increased after sco- creases in impairment for the mecamylamine ϩ scopol- polamine infusion as compared to placebo (p Ͻ .01) at all amine condition as compared to scopolamine alone for time points measured. A further increase in pupil diam- the total score 24 items (p Ͻ .01, see Figure 2b), and at eter resulted from the addition of mecamylamine to sco- trend levels for impairment, anxiety/depression, and to- polamine as compared to scopolamine alone at t4 (p Ͻ tal score 18 items (p Ͻ .1). At t3 (test battery #3 as shown .01, see Figure 2d). A trend toward increase (p Ͻ .1) in in Figure 1), the mania subscale was increased with pupil size also occurred with mecamylamine alone as mecamylamine plus scopolamine as compared to sco- compared to placebo at the same time point. polamine alone (p Ͻ .05, not shown in Table 2). Mecamylamine alone as compared to placebo also pro- Systolic Blood Pressure. This decreased after the ad- duced a trend increase on total score 24 items at t4 (p Ͻ .1). ministration of mecamylamine as compared to placebo at t5 and t6 (p Ͻ .01, not in Table 3) and at a trend level NIMH Self-Rating Scale. As shown in Table 2, scopol- at t3 (p Ͻ .1, not in Table 3). Systolic blood pressure was amine produced a significant increase (p Ͻ .05) in func- significantly lower with the combination of mecamy- tional deficit score as compared to placebo, which further lamine and scopolamine as compared to scopolamine increased at a trend level (p Ͻ .1) when mecamylamine alone at all time points (p Ͻ .01 at t3 and t5, and p Ͻ .05 was added. Anxiety also increased with the addition of at t4 and t6). Systolic blood pressures for all drug condi- mecamylamine to scopolamine as compared to scopola- tions at t4–6 are shown in Figure 2c. mine alone (p Ͻ .05). Visual Analog Mood Scale. The overall ANOVA was Diastolic Blood Pressure. This decreased with scopol- not significant, so individual drug comparisons were amine as compared to placebo (p Ͻ .01, shown in Table not considered. 3), and at a trend level at t3 (p Ͻ .1, not shown in Table 3). Diastolic blood pressure did not change with mecamylamine alone as compared to placebo, but did Physiologic Measures decrease with the combined mecamylamine and scopol- A variety of physiologic changes were seen when sco- amine condition as compared to scopolamine at t3 (p Ͻ polamine and mecamylamine were administered indi- .05, not shown in Table 3). NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 1 Nicotinic and Muscarinic Blockade in Elderly Normal Volunteers 65

Figure 2. Cognitive, behavioral, and physiologic variables in eight elderly normal volunteers across four drug conditions: Placebo (PLA) PO and IV, Mecamylamine (MEC) 0.2mg/kg up to 15mg PO, Scopolamine (SCO) 0.4mg IV, and SCO ϩ MEC. Values are mean Ϯ SEM. p values († ϭ p Ͻ .1, * ϭ p Ͻ .05, ** ϭ p Ͻ .01) are given for drug condition contrasts (MEC vs. PLA, SCO vs. PLA, SCO ϩ MEC vs. SCO) for: (a) list learning mean consistency %; (b) total score 24 item of BPRS 3 hours after study day onset (t4) (c) systolic blood pressure 3–5 h after study day onset (d) pupil diameter in mm 3 h after study day onset (t4).

Pulse. This decreased after scopolamine was given as were increased (p Ͻ .05). While scores for these symp- compared to placebo at all time points (p Ͻ .05). Con- toms increased further with the addition of mecamyl- versely, pulse increased after mecamylamine was given amine to scopolamine, the differences were not statisti- as compared to placebo at t3 (p Ͻ .01, not shown in Ta- cally significant. At t3 (not shown in Table 3), there was ble 3) and t4 (p Ͻ .05). Pulse also increased with the a trend toward increase (p Ͻ .1) in weakness with sco- combination of mecamylamine and scopolamine as polamine as compared to placebo, and a trend toward compared to scopolamine alone at t3-5 (p Ͻ .01), and at decrease in weakness with mecamylamine ϩ scopol- t6 (p Ͻ .05, 5 h after the study began). (The overall amine as compared to scopolamine alone (p Ͻ .1). ANOVAs for temperature and respiration were not signif- icant.) DISCUSSION Physical Symptoms By combining the centrally active muscarinic blocker The means for self-reported physical symptoms at t4 are scopolamine with the nicotinic blocker mecamylamine, given in Table 3. After scopolamine infusion as com- we have successfully generated a profile of cognitive pared to placebo, dry mouth, tiredness, and drowsiness impairments that may better mimic the cognitive pro- 66 J.T. Little et al. NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 1

Table 2. Behavioral Ratings in Eight Normal Elderly Volunteers Across Three Different Scales Following Drug Administration on 4 Separate Days

Overall ANOVA Drug Conditions

F(12,84) SCO MEC SCO ϩ MEC Subscale Drug ϫ Time p value PLA (vs. PLA) (vs. PLA) (vs. SCO)

BPRS factors Anxiety/depression 2.28 .026 1.0 Ϯ 0.0 1.1 Ϯ 0.1* 1.1 Ϯ 0.0 1.3 Ϯ 0.1† Anergia 6.15 .001 1.0 Ϯ 0.0 1.3 Ϯ 0.1† 1.1 Ϯ 0.1 1.5 Ϯ 0.1 Impairment 15.35 .001 1.0 Ϯ 0.0 1.5 Ϯ 0.0** 1.0 Ϯ 0.0 1.8 Ϯ 0.2† Mania 7.84 .001 1.0 Ϯ 0.0 1.2 Ϯ 0.1* 1.0 Ϯ 0.0 1.3 Ϯ 0.1 Total score (18 items) 6.62 .001 18.1 Ϯ 0.1 20.3 Ϯ 0.8* 18.8 Ϯ 0.3 21.4 Ϯ 0.8† Total score (24 items) 13.77 .001 24.1 Ϯ 0.1 27.8 Ϯ 1.0** 24.9 Ϯ 0.3† 30.3 Ϯ 1.2** NIMH-SRS subscales Anxiety 3.82 .008 0.3 Ϯ 0.3 1.3 Ϯ 0.9 0.3 Ϯ 0.3 4.4 Ϯ 1.3* Functional deficit 8.69 .001 0.4 Ϯ 0.4 11.3 Ϯ 2.4** 1.7 Ϯ 1.3 17.9 Ϯ 3.8† Visual analogue Ϯ Ϯ Ϯ Mood scale 2.29 .104 29.0 5.9 39.0 6.5 35.0 6.7 42.0 Ϯ 8.2

All values are mean Ϯ SEM from the t4 point (180 min after MEC/PLA and 120 min after SCO/PLA). BPRS ϭ Brief Psychiatric Rating Scale. NIMH-SRS: National Institute of Mental Health Self-Rating Scale. PLA ϭ placebo, SCO ϭ scopolamine, MEC ϭ mecamylamine. By repeated-mea- sures analysis of variance, † ϭ p Ͻ .1, * ϭ p Ͻ .05, and ** ϭ p Ͻ .01. Significance levels are noted for contrasts in: SCO column for SCO vs. PLA, MEC column for MEC vs. PLA, and SCO ϩ MEC column for SCO ϩ MEC vs. SCO.

file of AD than either agent alone. Furthermore, behav- statistical comparisons were, in fact, quite limited, be- ioral and physiologic effects were also consistent with cause contrasts were determined a priori and were ex- enhanced effects of the combination over either individ- amined only when overall ANOVAs were significant. ual drug. Although previous work has demonstrated Most importantly, our data suggest that mecamyl- the blood flow effect of this combination (Gitelman and amine ϩ scopolamine generates additional cognitive Prohovnik 1992), this paper presents the first double- impairment when compared to scopolamine alone and blind human study in which the cognitive, behavioral, may, therefore, serve as a better model of AD than sco- and physiologic effects of combined scopolamine and polamine alone. As reviewed by Molchan et al. (1992), mecamylamine administration were compared to the scopolamine alone can produce deficits in elderly con- effects of the drugs individually. trols that overlap with some of the deficits seen in AD, A limitation of the study is the small sample size of such as impaired episodic memory, semantic memory, eight subjects who completed all four drug conditions. attention, and vigilance. In addition to replicating the This limitation surfaces, for example, when considering bulk of those findings, we also found that mecamyl- the trend level finding of increased impairment in list amine alone can impair cognitive functioning in do- learning mean consistency with the addition of mecamy- mains that are also affected in AD. Namely, with 10 lamine to scopolamine as compared to scopolamine subjects receiving mecamylamine alone as compared to alone. Rather than discarding this observation, how- placebo, we found deficits in episodic and semantic ever, we believe that the implications of this trend ob- memory tasks. Although others have also found that servation are important and should be considered for mecamylamine alone produces cognitive impairment in several reasons. First, this is a pilot study in a rather un- normal volunteers (Stolerman et al. 1973; Newhouse et charted area. Second, as discussed later, both animal al. 1992) that increases with age (Newhouse et al. 1994), and the previous human data are consistent with the the previous human study examining the combined finding of increased cognitive impairment when nico- drugs found diminished performance on the Buschke tinic blockade is added to muscarinic blockade. Further- Selective Reminding Test after scopolamine was added more, increased impairment is also seen with the addi- to mecamylamine (Gitelman and Prohovnik 1992). Un- tion of mecamylamine to scopolamine across a number fortunately, the lack of a scopolamine-alone test condi- of other variables (behavioral and physiological), sug- tion in that experiment prevented the determination of gesting a broader, consistent effect of this drug combi- whether the effect was attributable to scopolamine nation as compared to scopolamine alone. Another pos- alone or the combined condition of scopolamine ϩ sible concern regarding our study is that the data mecamylamine. Our results, consistent with animal analysis considers a large number of variables without studies in which the co-administration of mecamyl- Bonferonni corrections. However, although a wide vari- amine and scopolamine increased memory impairment ety of dependent variables are considered in this study, (Levin et al. 1989, 1990; Riekkinen et al. 1990; Cozzolino NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 1 Nicotinic and Muscarinic Blockade in Elderly Normal Volunteers 67

Table 3. Physiologic Responses in Eight Normal Elderly Volunteers Following Drug Administration on 4 Separate Days

Drug Conditions Overall ANOVA SCO MEC SCO ϩ MEC Subscale Drug ϫ Time p value PLA (vs. PLA) (vs. PLA) (vs. SCO)

Vital signs F(15,105) Pupil size (mm) 9.41 .001 2.9 Ϯ 0.2 3.7 Ϯ 0.3** 3.4 Ϯ 0.3† 4.4 Ϯ 0.2** Systolic BP 2.11 .018 128.0 Ϯ 3.6 121.0 Ϯ 4.3 120.0 Ϯ 2.9 116.0 Ϯ 6.4* Diastolic BP 1.80 .065 74.0 Ϯ 2.1 69.0 Ϯ 2.3** 69.0 Ϯ 3.0 65.0 Ϯ 2.4 Pulse/min 3.12 .002 64.0 Ϯ 2.4 59.0 Ϯ 1.8* 70.0 Ϯ 2.4* 70.0 Ϯ 2.4** Physical symptoms checklist F(12,84) Dry mouth 5.97 .001 0.3 Ϯ 0.2 1.4 Ϯ 0.3* 0.6 Ϯ 0.3 1.8 Ϯ 0.3 Tiredness 3.57 .005 0.0 Ϯ 0.0 0.6 Ϯ 0.3* 0.6 Ϯ 0.3 0.9 Ϯ 0.3 Ϯ Ϯ Ϯ Drowsiness 5.66 .001 0.0 0.0 1.1 0.4* 0.3 0.2 1.3 Ϯ 0.3

All values are mean Ϯ SEM from the t4 time point (180 min after MEC/PLA and 120 min after SCO/PLA). PLA ϭ placebo, SCO ϭ scopolamine, MEC ϭ mecamylamine. By repeated-measures analysis of variance, ** ϭ p Ͻ .01, * ϭ p Ͻ .05, and † ϭ p Ͻ .1. Significance levels are noted for contrasts in: SCO column for SCO vs. PLA, MEC column for MEC vs. PLA, and SCO ϩ MEC column for SCO ϩ MEC vs. SCO.

et al. 1994), suggest that at least episodic memory may ity is not fully elucidated. It has been hypothesized that be more impaired with the drug combination than with nicotinic and muscarinic receptors may interact through either drug alone. synapses containing presynaptic nicotinic receptors and Interestingly, the behavioral and physiologic effects postsynaptic muscarinic receptors (Levin 1992). Fur- of combined mecamylamine and scopolamine adminis- thermore, there is evidence for nicotinic presynaptic au- tration as compared to scopolamine alone paralleled the toreceptors on cholinergic neurons (Lapchak et al. 1989a, cognitive effects, as shown in Figure 2. Behaviorally, b). This might account for the finding that mecamylamine significant increases were found for the BPRS total produced a dose-dependent inhibition of brain [3H]ace- score (24 item) and NIMH-SRS anxiety subscale as well tylcholine synthesis that correlated with amnestic ef- as trends for the BPRS factors of anxiety/depression, fects in rats, leading Elrod and Buccafusco (1991) to impairment, total score (18 item), and the NIMH-SRS postulate that presynaptic nicotinic receptors may exert functional deficit subscale, thus demonstrating that the modulatory influence on cholinergic neurons. addition of nicotinic blockade to muscarinic blockade In summary, this study was the first in humans to ex- increases behavioral impairment. At the same time, pu- amine, in a controlled design, the cognitive, behavioral, pil size increased with both anticholinergic agents. Con- and physiologic effects of the combined administration sidering the finding of increased pupil dilation in re- of scopolamine and mecamylamine as compared to the sponse to the topical anticholinergic agent tropicamide individual drugs alone. Our cognitive, behavioral, and in patients with probable AD vs. controls (Scinto et al. physiologic data are consistent with an improved 1994), our finding of a further increase in pupil size model for AD with this drug combination as compared with the addition of mecamylamine to scopolamine in to scopolamine alone and are consistent with preclini- healthy elderly controls models the cholinergic pupil- cal, human, and autopsy studies demonstrating the im- lary sensitivity seen in the AD patients. From a physio- portant role that the nicotinic system plays in normal logic perspective, the changes in blood pressure and cognition and in such disease states as AD. Given the pulse with mecamylamine were not unexpected, con- considerable molecular diversity of the nicotinic and sidering its earlier development as a potent antihyper- muscarinic receptors (Decker et al. 1995; Brann et al. tensive agent (Freis 1955; Freis and Wilson 1956; Moyer 1993; Wang et al. 1992) and the relative nonselectivity of et al. 1957; Smirk and McQueen 1957). Of importance mecamylamine and scopolamine, future modeling here, however, is that the subjects in this study main- studies will benefit from the use of more selective phar- tained a supine position at least during the first 5 hours macologic agents. of the study (including during all cognitive testing) and that any blood pressure decreases while supine were not clinically significant. ACKNOWLEDGMENTS Although mecamylamine is known to be a centrally acting nicotinic receptor antagonist with both competi- The authors thank Wilma Davis, Kathleen Dietrich, R.N., tive and noncompetitive properties (Martin et al. 1989; Charyl Staso, R.N., Julie Jaffe, Jeanne Radcliffe, R.N., and the Takayama et al. 1989; Martin et al. 1990), the mecha- nursing staff on NIH Clinical Center Nursing Unit 3 East for nism for nicotinic modulation of other cholinergic activ- their help with this project. 68 J.T. Little et al. NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 1

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