The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1.

The study was approved by the cantonal ethics committee (No. 152/09), registered in the Clinical Trials Protocol Registration System (NCT01011036), and performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.

This is a randomized double-blind crossover study, using pain assessment methods that explore different nociceptive mechanisms. Clobazam 20 mg, clonazepam 1 mg and tolterodine 1 mg (active placebo) were compared. At the end of each session the benzodiazepine antagonist flumazenil 0.2 mg was administered to evaluate whether the observed effects can be reversed.

The experiments were performed at the University Department of Anesthesiology and Pain Therapy, Inselspital Bern, Switzerland. Pharmacokinetic and pharmacogenomic investigations were performed at the Division of Clinical Pharmacology and Toxicology, University Hospital of Geneva.

Exclusion criteria were: current or past history of drug or alcohol abuse, intake of any psychotropic drug currently or in the last month, chronic alcohol intake, any concomitant illness, current or regular intake of any drugs that might affect pain or nociception.

Inclusion criteria were: european ethnicity (in order to minimize pharmacogenetic variations), male gender (in order to avoid possible variation due to hormonal changes during the menstrual cycle), age 18–55 years old, induction of static mechanical hyperalgesia by capsaicin as described below and status as non smoker or moderate smoker (≤10 cigarettes/day).

Volunteers were recruited by advertisement at the Inselspital and at the University of Bern by the first author (P.V.). Sixteen healthy volunteers were tested between December 2009 and July 2010 ( figure 1 ). They received 200 Swiss Francs for each session and a total of 800 Swiss Francs if they completed all the three experiments.

The subjects received the study medications in a randomized order. A computer-generated random list was prepared by the hospital pharmacy and was known only by the hospital pharmacy. The drugs were enclosed by the hospital pharmacy in the same type of capsule to assure blinding. The pharmacy delivered the blinded capsules to the investigators, numbered according to the defined randomization order. The first author (P.V.) assigned participants to the experimental sessions.

Flumazenil (Anexate®, Roche Pharma AG, Reinach, Switzerland) selectively antagonizes or attenuates the effects of benzodiazepines on GABA A receptors [14] . To minimize the risk of seizure, a dose of 0.2 mg iv was chosen.

Tolterodine (Detrusitol®, Pharmacia Gmbh, Pfizer Group, Berlin, Germany) is an anticholinergic compound [13] . Anticholinergic compounds usually cause some sedation and dry mouth; to our knowledge, they are devoid of analgesic effects. Because of its sedative properties, tolterodine has been chosen as an active placebo in order to keep double blinding.

The doses of clobazam and clonazepam were chosen based on the following considerations. Clonazepam is typically prescribed in chronic neuropathic pain at doses between 0.5 and 1 mg, and 2 mg a day is an average dose for this indication [11] . Typical anticonvulsant starting doses of clonazepam and clobazam are 0.5–2 mg and 10–20 mg, respectively [12] . A dose of 20 mg of clobazam has been shown to be less sedative than 1 mg of clonazepam and should be equipotent [8] . Therefore these doses have been chosen for the present study.

Clobazam (Urbanyl®, Sanofi-Aventis AG, Meyrin, Switzerland) is a benzodiazepine. To our knowledge, it has never been tested for analgesic or antihyperalgesic properties. Unlike 2 mg clonazepam, 20 mg clobazam did not affect significantly cognitive and psychomotor functions, possibly because of its unusual 1–5 chemical structure [9] . Clonazepam (Rivotril®, Roche Pharma AG, Reinach, Switzerland) is probably the benzodiazepine most widely used for the treatment of clinical pain, although the evidence behind this practice is weak [10] . It was therefore chosen as positive control.

Clobazam, clonazepam and tolterodine were given orally to the same volunteers in a randomized order on three different sessions, with a minimal washout interval between two consecutive sessions of two weeks. All subjects were tested in the morning and had been instructed to take a very light breakfast at home. The minimum interval between breakfast and drug intake was 3 h.

Tests

Endpoints. The primary endpoint was the area of static hyperalgesia induced by intradermal capsaicin injection. The additional measures were secondary endpoints.

General methodological aspects. Because of the very limited information on which experimental pain models would be sensitive to the effects of benzodiazepines, we applied a multimodal testing procedure that is expected to explore different aspects of nociceptive processes. The rationale is summarized in table 1. In particular, we applied models of hyperalgesia, tissue-specific pain sensitivity (cutaneous vs. muscular), sensitivity to short and ongoing painful stimulation, temporal summation (increase in pain perception during stimulation of constant intensity), stimulus-response function (dependency of pain rating from different stimulation intensities) and conditioned pain modulation (exploring endogenous pain modulation). PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Experimental pain tests employed. https://doi.org/10.1371/journal.pone.0043896.t001 Before starting each session, trainings of the pain tests were performed until the subjects were familiar with the testing procedures. All the tests were applied on the dominant side, except the cold pressor test and cuff algometry (see below for explanations). All subjects but one were right-handed. Each subject was tested on the same side in all three experimental sessions. Volunteers were not allowed to see the area tested and any read-outs from any instruments.

Intradermal capsaicin. Intradermal injection of capsaicin causes a brief stinging/burning pain at the injection site followed by development of secondary hyperalgesia, i.e. hyperalgesia detected at surrounding (not injected) skin [15], [16]. Secondary hyperalgesia after intradermal capsaicin is the result of sensitization of the central nervous system, which is one of the most relevant aspects of neuroplastic changes [17]. This model was sensitive to the action of GABAergic compounds in animal studies [18]. The capsaicin solution was prepared by the Hospital Pharmacy and underwent sterile filtration into sterile septum-sealed vials. The sterile solution (1 mg/ml) was placed into sterile syringes and used for intradermal injection at room temperature. The skin temperature was measured using a digital thermometer, and was 32°C+/−1 in all subjects. Subjects remained semi-supine during the experiment. Before capsaicin injection, the skin of the forearm was cleaned using an antiseptic wipe and allowed to air dry. The injection site was marked midway between the elbow and the wrist. Using a surgical skin marker, 4 lines were drawn through the injection site, so that they intersected the corners of a regular octagon (figure 2). These lines were marked every 0.5 cm from the center to the edges using a predefined grid. This resulted in the creation of triangles radiating from the intersection of the 4 lines outwards. We determined the area of hyperalgesia by adding the areas of each triangle in which hyperalgesia was recorded. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Capsaicin model. The dots represent the points where stimulation was applied. The circle in the center of the hectagon is the site of capsaicin injection. https://doi.org/10.1371/journal.pone.0043896.g002 Using a 1-ml tuberculin syringe fitted with a 27-gauge disposable needle, 100 µl capsaicin were injected epidermally into the skin, at the intersection of the 4 vectors previously drawn. A white skin elevation appeared during injection ensuring correct injection. Static mechanical hyperalgesia was judged to be present when the subjects reported that applying a 512 mN von Frey Hair, 5 and 30 minutes after capsaicin injection, elicited pain with an intensity of at least 4 using a numerical rating scale (NRS, whereby 0 = no pain and 10 = worst pain). Only these subjects were included in the study. In order to document pain induction, the subject recorded pain intensity at injection and 5 minutes post-injection using a 10 cm visual analogue pain scale (VAS), whereby 0 = no pain and 10 = worst pain imaginable. The area of secondary hyperalgesia was assessed using a calibrated 512 mN von Frey hair. The punctuated probe was moved along the 8 radial lines defined above, starting approximately 6 cm away from the site of injection, at each mark in steps of 0.5 cm. The weighted von Frey hair was placed gently on the skin and the load was applied for 2 seconds. The volunteers were asked to report when the pricking sensation changed to a pain sensation. At least 4 seconds elapsed between consecutive stimuli. This procedure was repeated for each vector. The number corresponding to the marker at which sensation changes as described above was noted and the individual numbers were used to calculate the area of hyperalgesia. The area of dynamic mechanical hyperalgesia was determined by gently stroking a hand-held cotton wool tip on a 1 cm strip of the skin, at a rate of approximately 1 cm/s. Subjects were asked to report when the sensation changed from a non-painful to a painful sensation. The borders of dynamic hyperalgesia were delineated similarly to the determination of static hyperalgesia. Additionally, mechanical pain sensitivity was assessed using a set of seven weighted pinprick stimuli to obtain a stimulus-response function. The test was applied 1 cm inside the outer border of the pinprick hyperalgesic area, using a set of 7 graded von Frey hair mechanical stimulators with fixed stimulus intensities (custom-made at Aalborg University, Denmark). The flat contact area of the stimulators has a 0.2 mm diameter, and the 7 stimulators exert forces of 8, 16, 32, 64, 128, 256 and 512 mN. The order of stimulation was defined randomly by computer. The stimulators were applied at a rate of 2 s on/2 s off. Subjects were asked to give a pain rating for each stimulus on a 10 cm VAS. Two assessments for each stimulation were made and the mean of these 2 measurements was used for the data analysis.

Pressure stimulation. Pain detection and tolerance thresholds were measured with an electronic pressure algometer (Somedic, Hörby, Sweden) applied at the center of the pulp of the 2nd toe. The probe had a surface area of 1 cm2. The pressure was increased from 0 at a rate of 30 kPa/s to a maximum pressure of 1200 kPa. Pain detection threshold was defined as the point at which the pressure sensation turned to pain. Pain tolerance threshold was defined as the point at which the subject felt the pain as intolerable. If the threshold was not reached at 1200 kPa, this value was considered as threshold. Three assessments were made and the mean of these 3 measurements were used for the data analysis.

Conditioned pain modulation (CPM). This method explores the endogenous modulation of nociceptive input. Under normal conditions, pain after application of a “test” nociceptive stimulus is attenuated by the application of an additional conditioning noxious stimulus to a remote body region, reflecting diffuse endogenous inhibition [19], [20]. In the present study, pressure pain detection threshold and cold pressure test (see below) were used as test and conditioning stimuli, respectively. An increase in pressure pain detection threshold immediately after cold pressure test was an indication of CPM.

Cold pressor test. The subjects placed their hand into a container filled with ice water. In order to maximize heterotopic stimulation, the hand contralateral to the side of pressure stimulation was used. The water was regularly mixed to maintain the temperature near to 0°C. The temperature of the water near the hand was monitored by a thermometer with a digital display (±0.1°C). The subjects were asked to keep the hand in the water until they felt an intolerable sensation of pain and were forced to remove the hand from the container, with a maximum time of 2 min.

Pressure stimulation after cold pressor test. Pressure pain detection threshold was measured again at the same time as the subject was withdrawing the hand from the water (one single measurement). CPM was measured as the difference in pressure pain detection threshold between measurements after and before the cold pressure test.

Cutaneous single electrical stimulation. Electrical stimulation was performed through electrodes placed distal to the lateral malleolus. A 25 ms, train-of-five, 1 ms, square-wave impulse (perceived as one stimulus), was delivered by a computer-controlled constant current stimulator (Digitimer DS7A, Neurospec, Letchworth Garden City, UK). The current intensity was increased from 1 mA in steps of 0.5 mA until a pain sensation was evoked. Three assessments were made and the mean of these 3 measurements was used for the data analysis.

Cutaneous repeated (5 stimuli) electrical stimulation. The stimulus burst used for single stimulus was repeated 5 times at 2 Hz, at constant intensity. The current intensity of the 5 stimuli was increased from 1 mA in steps of 0.5 mA until the subjects felt pain during the last 2–3 of the 5 stimuli (indicating temporal summation) [21]. Three assessments were made and the mean of these 3 measurements was used for the data analysis.

Cutaneous repeated (20 stimuli) electrical stimulation. The stimulus burst used for single stimulus was repeated for a train of 20 pulses at 2 Hz, at an intensity corresponding to the temporal summation threshold. During this 10 s stimulation, pain intensity was continuously rated by the subject with an electronic VAS, and the area under the curve (AUC) was computed. Additionally, the maximal VAS during stimulation was recorded.

Intramuscular electrical stimulation (1, 5 and 20 stimuli). A needle was placed in the tibialis anterior muscle, 14 cm distal from the caudal end of the patella and 20 mm in depth. The same single and repeated stimulation patterns and the same procedure described for cutaneous stimulation were used.

Cuff algometry. A tourniquet cuff was applied to the middle of the leg, at the level of the heads of the gastrocnemius and soleus muscle [22]. The cuff was applied at the side contralateral to the side of electrical stimulation in order not to interfere with the positioning of the intramuscular electrodes. During stimulation, the volunteer rated the pain intensity on an electronic VAS scale. The cuff was inflated with compressed air until VAS 6 was reached [22]. The maximum allowed inflating pressure was 200 kPa. The pressure was maintained for 10 min or until the subjects rated the pain as intolerable. The area under the curve VAS-time was computed. For those subjects who felt intolerable pain before 10 min, the time when the cuff was deflated was recorded and VAS 10 was extrapolated until 10 min. Additionally, the maximal VAS during inflation was recorded.

Psychomotor performance. The psychomotor performance was assessed by the digit symbol substitution test (DSST), a subscale of the Wechsler Adult Intelligence Scale. The DSST evaluates the ability to concentrate and modifications in processing information [23]. It is a two-minute paper-and-pencil test. The subject was asked to replace digits with corresponding symbols according to a code given on the same sheet of paper. The score consists in the total number and the correct number of symbols drawn.