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DM235 (sunifiram), a new compound struc-turally related to piracetam, prevented the amnesia in-duced by scopolamine (1.5 mg kg
i.p.), after intraperi-toneal (0.001–0.1 mg kg
) or oral (0.01–0.1 mg kg
) ad-ministration, as shown by a passive avoidance test inmice. The antiamnesic effect of DM235 was comparableto that of well-known nootropic drugs such as piracetam(30–100 mg kg
i.p.), aniracetam (100 mg kg
p.o.) orrolipram (30 mg kg
p.o.). DM235 also preventedmecamylamine (20 mg kg
i.p.)-, baclofen (2 mg kg
i.p.)- and clonidine (0.125 mg kg
i.p.)-induced amnesiain the same test. In the Morris water maze test with rats,scopolamine (0.8 mg kg
i.p.) inhibited the reduction of escape latency in both acquisition and retention/retrainingtests. DM235 (0.1 mg kg
i.p.), 20 min before each dailyacquisition training, prevented the scopolamine-inducedmemory impairment. DM235 (1 mg kg
i.p.) also re-duced the duration of pentobarbitone-induced hypnosis inmice without modifying the induction time of hypnosis.At the highest effective doses, the investigated compoundneither impaired motor coordination (rota-rod test), normodified spontaneous motility and inspection activity(Animex and hole board tests).These results indicate that DM235, a compound struc-turally related to piracetam, is a novel nootropic endowedwith the capability to prevent cognitive deficits at verylow doses. Indeed, its potency is about 1,000 times higherthan that of the most active piracetam-like compounds.
DM235 · Sunifiram · Nootropic drugs ·Piracetam · Learning and memory · Passive avoidance ·Morris water maze
The so-called nootropic compounds are a group of phar-macologically active pyrrolidone derivatives that, in somerespects, occupy a special position in the pharmacology of the central nervous system. The first pyrrolidone to cometo the attention of clinicians was piracetam. This com-pound was developed in the late 1960s after pioneeringresearch by Giurgea who also coined the term “no-otropic”, meaning enhancement of learning and memory.Since then, there has been much pharmaceutical interestin a broad range of indications and in new compounds(aniracetam, oxiracetam, pramiracetam, nefiracetam, ne-bracetam, fasoracetam, levetiracetam, etc.). A wide rangeof animal models has been used to show improvements incognitive function. These tests include maze and spatiallearning, passive avoidance, matching-to-sample, activeavoidance, choice reaction, conditional avoidance andmotor tasks that show definitely positive effects on reten-tion performance in laboratory animals (Verloes et al.1988; Sarter 1991; Gouliaev and Senning 1994). No-otropic drugs also facilitate the transcallosal, interhemi-
spheric transfer of information (Okuyama and Aihara 1988)
and enhance long-term potentiation (LTP) in guinea-pighippocampal slices (Satoh et al. 1986; Pugliese et al.1989). The members of this class show very low toxicity,have no sedative or stimulatory effects and lack the seri-ous side effects of psychostimulants (Heise 1987). Thisfavourable pharmacological profile has stimulated inves-tigation of the potential antiamnesic activity of nootropicsin human neurodegenerative conditions. Clinical studieshave focused on cognition enhancement and memory im-provement by nootropic drugs. Some pyrrolidone deriva-tives, such as piracetam, aniracetam and oxiracetam, ame-liorate the condition of elderly patients suffering frommild to moderate mental deterioration (Chouinard et al.
C. Ghelardini · N. Galeotti · F. Gualtieri·M. N. Romanelli · C. Bucherelli · E. Baldi·A. Bartolini
DM235 (sunifiram): a novel nootropic with potentialas a cognitive enhancer 
Naunyn-Schmiedeberg’s Arch Pharmacol (2002) 365:419–426DOI 10.1007/s00210-002-0577-3Received: 20 December 2001 / Accepted: 8 April 2002 / Published online: 15 May 2002
C. Ghelardini (
) · N. Galeotti · A. BartoliniDepartment of Preclinical and Clinical Pharmacology,University of Florence,Viale G. Pieraccini 6, 50139 Florence, Italye-mail: ghelard@pharm.unifi.it,Tel.: +39-55-4271312, Fax: +39-55-4271280F. Gualtieri · M.N. RomanelliDepartment of Pharmaceutical Sciences, University of Florence,Via G. Capponi 6, 50121 Florence, ItalyC. Bucherelli · E. BaldiDepartment of Physiological Sciences, University of Florence,Viale G.B. Morgagni 63, 50134 Florence, Italy©Springer-Verlag 2002
1983; Maina et al. 1989; Nicholson 1990; Vernon andSorkin 1991; Lee and Benfield 1994), of geriatric patientswith cerebrovascular insufficiency (Foltyn et al. 1983), inAlzheimer’s disease (Senin et al. 1991; Croisile et al.1993; Parnetti et al. 1997) and are useful in the treatmentof cognitive deficits in early Parkinsonism (Oepen et al.1985).Preliminary pharmacological studies have shown that1,4-diazabicyclo[4.3.0]nonan-9-ones, structurally relatedto piracetam, could represent a class of nootropic agents(Manetti et al. 2000a, 2000b). Among them, the com-pound DM235 (sunifiram; Fig.1) appears to be endowedwith the best pharmacological profile. The aim of the pre-sent study was to investigate further the ability of DM235to ameliorate impaired or unimpaired memory functionsin mice and rats.
Materials and methods
Male Swiss albino mice (23–30 g) and 70–day-old malehooded Long-Evans (average body weight 270 g) from Morini(San Polo d’Enza, Italy) were used. Mice were housed 15 per cage;the rats were housed individually in stainless-steel cages. Foradaptation, the cages were placed in the experimental room 24 hprior to tests. Animals always had free access to a standard labora-tory diet (TRM, Harlan, Padua, Italy) and tap water and were keptat 23±1°C with a 12-h light/dark cycle (light on at 7 a.m.). All ex-periments were carried out according to the guidelines of the Eu-ropean Community Council for experimental animal care. All ex-periments were performed blind.
Passive-avoidance test.
The test was performed according to thestep-through method described by Jarvik and Kopp (1967). Theapparatus consists of a two-compartment acrylic box with a lightedcompartment connected to a darkened one by a guillotine door.Mice, as soon as they entered the dark compartment, received apunishing electrical shock (0.3 mA, 1 s). The latency times for en-tering the dark compartment were measured in the training test and24 h later in the retention test. The maximum entry latencies al-lowed in the training and retention sessions were 60 and 180 s re-spectively.
Spatial reference memory in the Morris water maze.
Spatial learn-ing was assessed in an open-field water maze (Morris 1984), con-sisting of a large, circular, transparent tank (diameter 1.5 m; depth0.6 m) containing water at 24±1°C at a depth of 0.3 m. The rats’task was to escape from the water by locating a hidden, transparentescape platform (diameter 14 cm) submerged 1.5 cm below thesurface of the water. The water was made partially opaque by theaddition of 3 l semi-skimmed milk that prevented the animals fromseeing the platform. The pool was located on the floor in the cen-tre of an acoustically insulated room (4
4 m) kept at a constanttemperature (24±1°C). Illumination inside the room, containingvarious prominent cues, was 60 lux. The swim paths taken by theanimals in the pool were monitored by a video camera mounted inthe ceiling. The resulting video signal was relayed to a videorecorder.All rats were trained to find a hidden escape platform, in a fixedlocation. They received 5 days of training with a ten-trial block perday. The platform was located in the centre of a chosen quadrant of the pool. The rats were placed into the pool facing the side wall ata position chosen randomly (the start points were chosen ran-domly, always starting from the external edge) across trials and al-lowed to swim until they found the platform, or for a maximum of 60 s. Any rat that failed to find the platform in time was guided toits location by the experimenter. The rats were then allowed to re-main on the platform for 20 s. They were then removed gentlyfrom the platform and placed for 20 s in a cage on the floor of thesame room before commencing the next trial. On completion of behavioural testing the rats were returned to their home cageswhere they were warmed briefly under a heating lamp. Then, 96 hafter the last acquisition training, the rats were again subjected tothe same behavioural procedure (retention/retraining test). The la-tencies for reaching the platform were recorded blindly using astopwatch. Data reported for each day’s training were the means of ten trials.
Pentobarbitone-induced hypnosis.
After mice had been given pen-tobarbitone sodium (60 mg kg
i.p.), the loss of the righting reflexwas measured. The duration of hypnosis was taken as the time re-quired to regain the righting reflex. Mice were pretreated withDM235 (0.1–1 mg kg
i.p.), or piracetam (30 mg kg
i.p.) 20 or30 min respectively before the injection of pentobarbitone.
 Hole board test.
The hole board test utilizes a 40-cm square planewith 16 flush-mounted cylindrical holes (diameter 3 cm) distrib-uted 4-by-4 in an equidistant, grid-like manner. The plane of thehole board is made of black metal; the separation of the holes fromeach other is 5.5 cm; the distance of the outermost holes from theedge of the board is 5 cm. The mice were placed in the centre of the board one by one and left to move about freely for a period of 5 min each. Two photoelectric beams, crossing the plane frommid-point to mid-point of opposite sides, thus dividing the planeinto four equal quadrants, automatically signalled the movement of the animals on the surface of the plane. Miniature photoelectriccells, in each of the 16 holes, recorded the exploration of the holes(head plunging activity) by the mice.
 Rota-rod test.
The apparatus consists of a base platform and a ro-tating rod of 3 cm diameter with a non-skid surface made of black plastic. The rod was placed at a height of 15 cm from the base. Therod, 30 cm in length, was divided into five equal sections by sixdisks, thus allowing up to five mice to be tested simultaneously,with a rod rotation speed of 16 rpm. The integrity of motor coordi-nation was assessed as the number of falls from the rod in 30 s, ac-cording to Vaught et al. (1985). Performance time was measuredbefore and 15, 30 and 45 min after intraperitoneal administrationof the drugs.
Spontaneous activity meter (Animex).
Locomotor activity in ratswas quantified using an Animex activity meter Type S (LKB,Farad, Sweden) set to maximum sensitivity. Every movement of rats, which were placed on the top of the Animex activity meter,produced a signal due to variation in inductance and capacity of the apparatus resonance circuit. Signals were converted automati-cally to numbers. On the day of the experiment the rats weretreated and the cage, containing three rats, then put on the measur-ing platform. Activity counts were made for 5 min at 15-min inter-vals for 45 min (total of three sessions) starting immediately afterinjection of the drug. Because of the arbitrary scale adopted toquantify movements, drug-treated rats were always compared withsaline-treated ones.420
DM235 (sunifiram):1-benzoyl-4-propionylpiper-azine
The following drugs were used: DM235 (sunifiram) pre-pared in the Department of Pharmaceutical Sciences of Universityof the Florence according to the method described by Manetti et al.(2000a); scopolamine hydrobromide, piracetam, (±)-baclofen(Sigma); mecamylamine hydrochloride, clonidine hydrochloride,(±)-rolipram (RBI); nicotine hydrogentartrate (Fluka); aniracetam(A. Menarini Industrie Farmaceutiche Riunite); pentobarbitone(Sagatal). Drugs were dissolved in isotonic (NaCl 0.9%) saline so-lution, for i.p. injection, or dispersed in sodium carboxymethyl cel-lulose 1%, for p.o. administration, immediately before use. Drugconcentrations were such that the necessary dose could be admin-istered in volumes of 10 ml kg
(i.p. or p.o.) for mice and 3 ml kg
(i.p.) for rats.
Pharmacological treatments.
For memory disruption in the pas-sive avoidance test, mice were injected i.p. with amnesic drugs im-mediately after termination of the training trial (scopolamine, bac-lofen, mecamylamine) or 60 min before the training trial (cloni-dine). DM235, piracetam, aniracetam, rolipram and nicotine wereinjected 20 (i.p.) or 30 (p.o.) min before the training trial. In thewater maze experiments, rats were injected i.p. with DM235and/or scopolamine 20 min before each daily acquisition training.The day of the retention/retraining rats were all injected i.p. withsaline solution 20 min before the test.
Statistical analysis.
All experimental results are given as
means±SEM. Analysis of variance (ANOVA), followed by Fisher’s
pro-tected least significant difference (PLSD) procedure for post-hoccomparison, was used to verify significance of differences be-tween means in mouse behavioural data. Mixed ANOVAs withpharmacological treatments as a between-subjects variable and the
training days as a within-subjects variable and Newman-Keuls mul-
tiple comparisons test were used for rat behavioural experiments.Data were analysed using StatView software on a Macintosh com-puter.
<0.05 (2-tailed) was considered significant.
In the passive avoidance test in mice, pretreatment withDM235 prevented the amnesia induced by the administra-tion of the antimuscarinic drug scopolamine (1.5 mg kg
i.p.) after i.p. (0.001–0.1 mg kg
, Fig.2A) or p.o.(0.01–0.1 mg kg
, Fig.2B) injection. The maximal anti-amnesic effect of DM235 was obtained with the dose of 0.001 mg kg
i.p. and maintained up to 0.1 mg kg
i.p.The DM235-induced antiamnesic effect was of the sameintensity as that exerted by the well-known nootropicdrugs piracetam (30 mg kg
i.p.), aniracetam (100 mgkg
p.o.) or rolipram (30 mg kg
p.o.) (Fig.2A and B).Lower doses of DM235 (0.0001 mg kg
i.p.), piracetam(10 mg kg
i.p.) aniracetam (50 mg kg
p.o.) or rolipram(10 mg kg
p.o.) (Fig.2A and B) were devoid of anyameliorative effect on scopolamine-induced amnesia. At0.1 mg kg
i.p., the entrance latency value of the DM235-treated group in the retention session was comparable tothat produced by control mice.The administration of DM235 (0.01–0.1 mg kg
antagonized the memory disruption produced by mecamyl-amine
(20 mg kg
i.p.), similarly to the antiamnesic ef-fect produced by nicotine (5 mg kg
i.p.) and piracetam(30 mg kg
i.p.). DM235 at 0.001 mg kg
i.p. was com-pletely ineffective (Fig.3).DM235 at (0.01 and 0.1 mg kg
i.p.) also preventedthe amnesia induced by baclofen (2 mg kg
i.p.), but at
Dose/response curve for DM 235 administered either i.p. incomparison with piracetam (
) or orally in comparison withaniracetam and rolipram (
) on amnesia induced by scopolamine(1.5 mg kg
i.p.) in a passive avoidance test in mice. DM235,piracetam, aniracetam and rolipram were administered 20 min(i.p.) or 30 min (p.o.) before the training session while scopol-amine was injected immediately after. The
in the
indicate the numbers of mice tested. **
<0.01, *
<0.05 vs. scopol-amine-treated mice
Dose/response curve for DM235 (administered i.p.) incomparison with nicotine and piracetam on amnesia induced bymecamylamine (20 mg kg
i.p.) in a passive avoidance test inmice. DM235, nicotine and piracetam were administered 20 minbefore training session while mecamylamine was injected immedi-ately after. **
<0.01; *
<0.05 vs. mecamylamine-treated mice

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