V116517) in Healthy Volunteers and Comparison with Preclinical Profile Lars Arendt-Nielsena,B,*, Steve Harrisc, Garth T

Research Paper

A randomized, double-blind, positive-controlled, 3-way cross-over human experimental pain study of a TRPV1 antagonist (V116517) in healthy volunteers and comparison with preclinical profile Lars Arendt-Nielsena,b,*, Steve Harrisc, Garth T. Whitesidec, Michele Hummelc, Terri Knappenbergerc, Sarah O’Keefec, Ram Kapilc, Don Kylec

Abstract This experimental, translational, experimental pain, single-center, randomized, double-blind, single-dose, 3-treatment, 3-period cross-over proof-of-concept volunteer trial studied the efficacy of a novel TRPV1 antagonist (V116517) on capsaicin- and UV-B- induced hyperalgesia. Heat and pressure pain thresholds, von Frey stimulus–response functions, and neurogenic inflammation were assessed together with safety. Each treatment period was 4 days. The 3 single oral treatments were 300 mg V116517, 400 mg celecoxib (a COX-2 inhibitor), and placebo. The heat pain detection and tolerance thresholds were increased significantly (P , 0.0001) by V116517. Heat pain detection and tolerance thresholds showed significantly less capsaicin hyperalgesia after V116517 (P 5 0.004 and P , 0.0001, respectively). Celecoxib reduced UV-B-provoked pressure pain sensitization (P 5 0.01). Laser Doppler flowmetry and erythema index after UV-B were significantly (P , 0.0001) reduced by celecoxib. Stimulus–response function in capsaicin-treated areas showed significant differences between both celecoxib and placebo and between V116517 and placebo. The body temperature showed no change, and no side effects were reported for any of the treatments. The TRPV1 antagonists and the COX-2 inhibitor showed different antihyperalgesic profiles indicating different clinical targets. In addition, the preclinical profile of V116517 in rat models of UV-B and capsaicin-induced hypersensitivity was compared with the human experimental data and overall demonstrated an alignment between 2 of the 3 end points tested. The TRPV1 antagonist showed a potent antihyperalgesic action without changing the body temperature but heat analgesia may be a potential safety issue. Keywords: Experimental pain, Sensitization, UV-B, Capsaicin, Pain thresholds, TRPV1 antagonist, Phase I, Proof-of-concept, Preclinical

1. Introduction failed in clinical trials.33,38,50 One clinical study showed analgesia after third molar tooth extraction.44 An unwanted side effect of The unmistakable pungency of capsaicin has led to substantial TRPV1 antagonists has been hyperthermia42 although new research into TRPV1 (transient receptor potential vanilloid sub- 32 family, member 1) function and an appreciation of this ion channel “modality-specific” molecules seem to show less of this effect. on pain signaling pathways. The TRPV1 channel is distributed in The discussion concerning clinical potency and safety of TRPV1 antagonists has been intense,31 and hence other TRPV channels the peripheral and central nervous system and shown in 24,29 preclinical studies to play a role in nociceptive pathways.34 Direct such TRPV3 have been targeted for pain relief. Because only blockade of this channel interrupts initiation of various sensory one clinical study has confirmed analgesic actions of TRPV1 receptor signals as it is activated by a range of mechanical, antagonists in humans, the use of human mechanistic quantita- thermal, or chemical stimuli.28 tive sensory testing may help profiling the TPRV1 antagonist in proof-of-concept studies to target the most optimal patient The TRPV1 expression on sensory neurons is known to be 2 increased in persistent inflammation and has been a target for population for later clinical trials. drug development20,47 although most TRPV1 antagonists have As TRPV1 receptors are involved in pain and sensitization associated with tissue injury and inflammation, human pain models should incorporate those phenomena in an efficacy test Sponsorships or competing interests that may be relevant to content are disclosed platform. Chizh et al.11 investigated the antihyperalgesic activity at the end of this article. a of a TRPV1 receptor antagonist (SB-705498). The compound SMI, Department of Health Science and Technology, Faculty of Medicine, Aalborg showed significant effects on heat-evoked pain, cutaneous University, Aalborg, Denmark, b C4Pain, Aalborg, Denmark, c Purdue Pharma, Stamford, CT, USA hyperalgesia, and neurogenic inflammation induced by capsaicin *Corresponding author. Address: SMI, Department of Health Science and or UV-B irradiation. Similarly, laser-evoked pain from UV-B- Technology, Faculty of Medicine, Aalborg University, Fredrik Bajersvej 7D3, DK- sensitized skin has shown to be modulated by a TRPV1 receptor 9220 Aalborg, Denmark. Tel.: 145 9940 8837; fax 145 98154008. E-mail address: antagonist (ABT-102).48 [email protected] (L. Arendt-Nielsen). These findings support the many preclinical studies of novel PAIN 157 (2016) 2057–2067 TRPV1 antagonists showing significant antihyperalgesia against © 2016 International Association for the Study of Pain thermal and mechanical end points in standard models of pain- http://dx.doi.org/10.1097/j.pain.0000000000000610 like behavior in rodents. Furthermore, this underlines the

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suggestion that translational experimental quantitative pain The production and packaging of the study medication were assessment tools, representing isolated pain mechanisms, may performed as follows: V116517 was produced by Purdue be useful for proof-of-concept studies4 although a direct Pharmaceuticals L.P. and GMP packed by HB- Medical ApS, correlation between rodent models and human experimental Hvidovre, Denmark. Placebo for V116517 was produced by pain has not been ascertained. A possible side effect of TRPV1 Purdue Pharmaceuticals L.P. and GMP packed by HB-Medical antagonists could be burn injuries by an elevated intraoral heat ApS, Hvidovre, Denmark. Celebrex was produced by Pfizer and pain threshold (HPT). Therefore, a human experimental pain GMP packed by HB-Medical ApS, Hvidovre, Denmark. Placebo study on the TRPV1 antagonist ABT-102 evaluated changes in for Celebrex was produced and GMP packed by HB-Medical intraoral HPTs and found an elevation of 4.4˚C.46 ApS, Hvidovre, Denmark. This healthy volunteer, proof-of-concept, randomized study aimed to investigate mechanistically the antihyperalgesic profile of 4. Experimental study drug a new-generation TRPV1 antagonist (V116517) in comparison with a COX-2 inhibitor (celecoxib) in healthy subjects and to compare V116517 was demonstrated to be a potent TRPV1 antagonist of the effects with the preclinical profile from analogous rodent both pH- and capsaicin-activated recombinant human TRPV1 53 models. Cutaneous sensitizations were provoked with capsaicin receptor in vitro (IC50 5 39.6 nM and 35.1 nM, respectively). In and UV-B, and thermal and/or mechanical stimuli were used for a specificity profile run against 65 total enzymes, cell surface pharmacodynamic assessments in humans and rats together with receptors, ion channels, and other proteins (NovaScreen Profile), recordings of neurogenic inflammation in humans. The hypothesis V116517 showed more than 50% inhibition against only the was that the different sensitization models, based on the adenosine transporter target (actual value 5 51.09%). However, mechanisms involved, responded differently to the 2 drugs. the IC50 for this transporter (8.68 mM) was more than 200-fold greater than the IC50 values for blockade of capsaicin- or pH-activated TRPV1 channels. Evaluation of the specificity toward TRPV1 vs other 2. Material and methods members of the TRPV family also showed that V116517 is not Healthy male subjects aged 18 to 45, inclusive, with no clinically a potent blocker of heat-induced activation of recombinant human significant medical history, who were deemed suitable to take part in TRPV3 channels, or 4alpha-PDD-induced activation of recombinant this clinical study by the investigator, were screened in consecutive human TRPV4 channels. The in vivo efficacy of V116517 has been order after signing the informed consent form when visiting the clinic. demonstrated in a series of preclinical studies with standard rodent The sample size was estimated to 32 based on previous TRPV1 and models of capsaicin- and Freund’s complete adjuvant (FCA)- COX-2 studies, and hence a total of 63 were screened and 37 induced inflammatory pain, FCA-induced arthritis pain, postsurgical volunteers were included as some drop-outs were to be expected. pain, and neuropathic pain. In these models, V116517 demonstrated Volunteers were recruited from the databases of C4Pain, a high potency and a broad-spectrum efficacy that was similar to, or Aalborg, Denmark, the study was conducted in the C4Pain clinic, better than, the standard comparators such as indomethacin, and all study files were kept at these premises. The study was celecoxib, morphine, and gabapentin, in the respective models. approved by the ethics committee, the Danish Medicine Agency (reg. no. 2011-0714-56) with the attributed EudraCT No. 2011- 5. Inclusion and exclusion criteria 002399-18, and registered at ClinicalTrials.gov (Identifier: NCT02695745). Because the study was registered as a phase I During the first visit, demographic information was obtained from trial, it was not made public in the EU Clinical Trials Register. The all patients. Vital signs (blood pressure, pulse rate, and respiratory full EudraCT registered and locked protocol can be downloaded rate, oral temperature, 12-lead ECG) were recorded and blood from: https://smiadm.hst.aau.dk/protocols/search.php. and urine samples collected for laboratory analysis (chemistry, hematology, and urine). The inclusion criteria were: Caucasian male aged 18 to 45, body 3. Clinical study design weight ranging from 50 to 100 kg, body mass index (BMI) ranging Experimenter and subjects were blinded to the treatment. from 18 to 32 (kg/m2), partner should use contraception in the Subjects were randomized to one of the 3 treatment sequences: study period, should be healthy and free of significant abnormal (1) V116517 5 V116517 (300 mg) oral suspension 1 placebo findings (as determined by medical history, physical examination, capsules (2 3 capsules) clinical laboratory values, vital signs, ECG), and should understand (2) Celecoxib 5 placebo oral suspension 1 celecoxib (Celebrex, and provide reliable outcomes of the experimental pain tests. 400 mg [2 3 200 mg capsules]) The exclusion criteria were: current or recent (within 5 years) (3) Placebo 5 placebo oral suspension 1 placebo capsules (2 3 history of drug or alcohol abuse, history or current conditions that capsules) might interfere with drug absorption, distribution, metabolism, or All doses were administered in the fasted state. Fasted doses excretion, history of allergic-type reactions to drugs, did not were preceded by an overnight fast (ie, at least 8 hours) from food develop erythema at the maximum dose of the UV-B irradiation, (not including water) and was followed by a 4-hour fast (not any abnormal clinical findings. including water). The study drug was administered in each period according to 6. Clinical assessments the study randomization schedule. The randomization was generated by an external provider. The subjects were confined Before each visit, volunteers were clinically evaluated (inclusion and to the clinic from 2 days before dosing to 24 hours after drug exclusion criteria, medical history, vital signs, 12-lead ECG, administration during each period. Subjects returned to the clinic chemistry, hematology, urine, and drug, alcohol, cotinine screens). for 48 and 72 hours of procedures during each of the treatment Vital signs and ECG were also assessed just before dosing and 3 periods. There was a washout period of at least 6 days between hours after dosing, and adverse events were monitored throughout treatment periods. Subjects came for end study procedures 7 to the stay at the clinic. At the end of the study visit or after 14 days after the last dose or upon discontinuing from the study. discontinuing from the study, a full screening was again performed.

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Oral temperature was measured during each treatment period area (contralateral leg, same position). On the day of dosing at -24, -23, -22, -21, -20, -18, -16, and -12 hours before dose and (approximately 24 hours after UV-B induction), predose measure- at 1, 2, 3, 4, 6, 8, 12, 24, 48, and 72 hours after study drug ments were performed at the irradiated site and control area and administration. Blood samples were collected at predose (within 30 then repeated 3.5 hours after dose. minutes before dosing) and at 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 48, and 72 hours after dose. For each pharmacokinetic blood sample, 4 8.2. Topical capsaicin mL of venous blood was drawn through an indwelling catheter and/or direct venipuncture into tubes containing K2 EDTA. Capsaicin cream (capsaicin 1%, approx. 0.5 mL; Aalborg Hospital Pharmacy, Aalborg, Denmark) was applied to a 3- 3 7. Pharmacokinetics 3-cm area on the volar aspect of one forearm (randomized to left or right). The cream was applied under occlusion for 30 Plasma concentrations of analyte metabolite (S)-V116517 were minutes and then gently wiped off. The capsaicin test was applied analyzed. For each subject, the following pharmacokinetic metrics on the day of dosing 2.5 hours after dose. were calculated, whenever possible, based on the plasma concen- Before capsaicin application, the skin temperature was trations of V116517 according to the model independent approach16: measured using an infrared thermometer (TH03F Thermometer). AUCt: area under the plasma concentration–time curve from hour The skin temperature was attempted within the range 32˚C 6 1˚C 0 to the last measurable plasma concentration calculated by for all subjects before capsaicin application. If a lower skin the linear trapezoidal method. temperature was observed, the area was covered by a blanket AUCinf: area under the plasma concentration–time curve until the requested skin temperature was reached and vice versa extrapolated to infinity, calculated using the formula: in case of a high skin temperature. The skin temperature was also assessed after capsaicin application. Pharmacodynamic measures (see below) were performed ¼ 1 Ct AUCinf AUCt l predose at the site where capsaicin was later applied and at z a corresponding control area (contralateral arm, same position)

where Ct is the last measurable plasma concentration and lZ is and then repeated 3.5 hours after dose (equal to 1 hour after the apparent terminal phase rate constant. capsaicin application). Cmax: maximum observed plasma concentration. T : time to maximum plasma concentration. max 9. Experimental pain t : apparent terminal phase half-life: t 5 (ln2)/lz. 1/2 1/2 assessment (pharmacodynamics) Plasma concentration values below the level of quantification were set to equal zero for the analysis. Actual sampling times To assess the pharmacodynamics, specific pain test platforms were used for the calculations. were established for assessment at UV-B, capsaicin and control test sites. The time points for the assessments were scheduled to match T of V116517. 8. Experimental pain models of sensitization max Two models were used for provoking cutaneous sensitization; the 9.1. Laser Doppler flowmetry UV-B model and the topical capsaicin model. Neurogenic inflammation was assessed by monitoring cutaneous blood flow using Laser Doppler imaging (LDI-2, Moor Instruments 8.1. UV-B model Ltd; Devon, United Kingdom). The device produced an output During the screening phase, the minimum erythema dose (MED) signal proportional to the blood cell perfusion (or flux). An area of for irradiation for each subject was using a calibrated UV source (B 7.5- 3 7.5-cm around the stimulation site (either capsaicin or spectrum, 290-320 nm; Saalmann Multitester SBB LT 400 TM; UV-B irradiation) was scanned at a distance of 30 cm. The mean Saalmann Medizintechnik, Herford, Germany). Five circular spots perfusion was calculated using relative flux (arbitrary units). with a diameter of 1.5 cm in the ventral side of the upper left leg were irradiated with increasing intensities of UV-B light to 9.2. Erythema index determine the MED value (the quantity of UV-B light to evoke redness and irritation). Approximately 24 hours later, erythema was Erythema was assessed using colorimetry (DSM II ColorMeter; assessed using colorimetry (DSM II ColorMeter, Cortex Technol- Cortex Technology) providing an erythema index of the skin as ogy, Hadsund, Denmark). Subjects who did not develop erythema another indicator for neurogenic inflammation. The probe of the at the maximum UV-B dose were excluded from the study. DSM II ColorMeter was placed on the skin surface within the area Freckles, burns, tattoos, and similar skin marks were not of stimulation (either capsaicin or UV-B irradiation). The erythema allowed at the site of skin irradiation. After the exposure for MED index was recorded as displayed on the instrument. determination, subjects should avoid sun bathing or sun exposure at the site of irradiation. Using soothing agents and 9.3. Secondary pin-prick hyperalgesic area lotions at the site of irradiation was not allowed during MED determination unless treatment was needed. The MED value was The area of secondary pin-prick hyperalgesia around the the basis for the UV-B induction in all treatment periods. UV-B capsaicin-sensitized areas was assessed by a 60-g von Frey hair irradiations (3 3 MED) were performed 24 hours before drug (Somedic, Horby, ¨ Sweden) by stimulating along 8 linear paths administration. A circular spot (5 cm diameter) on the upper leg arranged vertically, horizontally, and obliquely around the zone of (randomized to left or right) was exposed. the capsaicin application. Stimulations were initiated outside the Baseline pharmacodynamic measures (see below) were primary hyperalgesic area (area of capsaicin application) where no performed before UV-B induction approximately 24 hours before pain was experienced and continued centripetally in 5-mm steps at dose at the area to be irradiated and at a corresponding control 4-second intervals. The subjects were asked to report when the

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sensation changed to a “different sensation,” “unpleasant” or The procedure for pressure pain tolerance threshold (PPTolT) “burning pain” sensation. This was repeated for each vector and was identical to PPT measurements except that the subjects the marks adjoined. The defined area was transferred onto a clear were instructed to press the button when the highest acceptable transparency film (prelabeled with study number, subject screening pain level was reached. The PPTolT measurement was not number, time point), and the area in cm2 of secondary hyperalgesia performed at the UV-B irradiation test site to avoid too much was calculated using a digitizer. Subjects were not allowed to see irritation of the irradiated area. the area tested. The test was performed 3.5 hours after dose. 10. Statistics 9.4. Stimulus–response function to graded von Frey The null hypothesis was that there was no difference among the 3 hair stimulation treatments, against the alternative that they were different. Within the primary hyperalgesic area (UV-B and capsaicin), the Because the study was an experimental explorative trial, no stimulus–response function to graded von Frey hair stimulation specific primary and secondary outcome parameters were defined. was determined. Mechanical pain sensitivity was assessed using The analysis of variance (ANOVA) model was used to ascertain a set of 8 weighted pin-prick stimuli to obtain a stimulus–response whether there were statistically significant differences among the 3 function for pin-prick evoked pain. The custom-made weighted treatments. This was followed up by the Dunnet multiple comparison pin-prick stimuli were performed with a set of 8 pin-prick test to investigate whether there was a difference between placebo stimulators (Aalborg University, Denmark) exerting forces of 8, and V116517, placebo and celecoxib, and V116517 and celecoxib. 16, 32, 64, 128, 256, 512, and 800 mN applied to a flat contact The Dunnet test included an adjustment for multiplicity. The area with a diameter of 0.2 mm. The stimuli were applied at a rate pharmacodynamic response variable was the differences from of 2-second on and 2-second off. baseline before dosing and the measurement after dosing and the The weights were applied in randomized order and each statistical comparison of those differences between active and weight was applied 3 times with a 10-second interval. The placebo. To exclude the influence of systemic drug effects on subjects were asked to rate the mean pain intensity of the 3 pharmacodynamic measures, the ANOVA model used the assess- stimulations on a 0 to 10 electronic visual analog scale (VAS), ments from the control area as covariates. where “0” indicated “no pain” and “10” indicated “the most Possible covariates, such as age and BMI, were not included intense pain imaginable.” The middle of the scale represented the due to the ensured homogeneity of the healthy young males. The pin-prick pain threshold. The subjects were instructed to rate the results from the statistical analysis are reported as estimates, P intensity of the nonpainful sensations on the lower half of the scale values (P , 0.05 considered significant), 95% confidence intervals, and the pain intensity on the upper half of the scale. The pain if relevant, and the size of the differences between treatments. rating score was transformed into a numeric value. Statistical analyses were performed using SAS version 9.2.

9.5. Heat pain threshold and heat pain tolerance threshold 11. Animals and compounds A computer-controlled 3- 3 3-cm Peltier device (ATS, Pathway Male Sprague-Dawley rats (180-200 g on arrival; Harlan, IN) were System, Medoc, Israel) was positioned either on the volar aspect used for all preclinical studies. The rats (2 per cage) were maintained of the forearm (capsaicin test site or control site) or on the upper in an AAALAC accredited temperature- and humidity-controlled thigh (UV-B test site or UV-B control site). The baseline vivarium on a 12-hour light–dark cycle. All experiments were temperature of the probe was 32˚C. For the HPT measurements, conducted during the day. Food and water were available ad libitum the temperature was gradually increased at a rate of 1˚C/s until except when compounds were dosed orally, in which case the food the subject pushed the button when perceiving the first sensation was removed 12 hours before the start of the experiment. All studies of pain (pricking pain). The temperature then returned to baseline. were approved by the Purdue Pharma L.P. Institutional Animal Care The determination of heat pain tolerance thresholds (HPTolTs) and Use Committee in accordance with the NIH GuidefortheCare was performed identically except that the subjects were and Use of Laboratory Animals and the Ethical Guidelines of the instructed to push the button when they felt the maximum International Association for the Study of Pain. tolerable pain (burning pain). The cutoff temperature for both tests V116517 was synthesized by Purdue Pharma (Cranbury, NJ), was 55˚C to prevent skin injury. The HPTolT measurements were whereas naproxen was purchased from Sigma-Aldrich (St. Louis, not performed at the UV-B irradiation test site to avoid too much MO). All drugs were administered as an oral suspension in 0.5% provocation of the irradiated area. methylcellulose as free base equivalents in a volume of 10 mL/kg unless otherwise stated.

9.6. Pressure pain threshold and pressure pain tolerance threshold 12. Rat UV-B model A handheld pressure algometer (Somedic) was used to assess The analgesic efficacy of V116517 was investigated using the UV-B pressure pain threshold (PPT). The 1-cm2 diameter probe was irradiation model as adapted from previous reports.7,12,22 Briefly, the placed perpendicularly to the skin, and pressure was applied at baseline paw withdrawal thresholds and latency (PWT and PWL) a rate of 30 kPa/second. The subject was instructed to press were first assessed to a noxious mechanical stimulus using an a button when defining the pressure as pain. When the subject analgesymeter (model 7200, Ugo Basile, Gemonio, Italy). This pressed the button, the display would freeze the value. The device applies a linearly increasing mechanical force to the third and pressure was terminated immediately and the probe removed. fourth metatarsus of the left paw. The cutoff was set to 250 g, and Pressure pain threshold was measured 3 times with a minimum of the nociceptive withdrawal threshold was defined as the force at 10 seconds between measurements at the UV-B-irradiated site, which the rat withdrew its paw from the device. After baseline PWT capsaicin test site, and both control sites. The mean of 3 assessment, the rats were anaesthetized with 2% isoflurane in measurements was used in the statistical analysis. oxygen. Under nose cone anesthesia, the plantar surface of the left

hind paw was exposed to UV-B radiation (500 mJ/cm2; Daavlin, GraphPad Prism. Significant effects were analyzed further by Bryan, OH). The rats were allowed to recuperate in a recovery station subsequent least significant difference analysis. The level of after which time they were returned to their home cages. Forty-eight significance was set at P , 0.05. hours after UV-B exposure, predrug PWTs were measured, and the rats were dosed with oral (p.o.) vehicle, V116517 (3-30 mg/kg) or 30 mg/kg naproxen (positive control). The animals were subsequently 14. Results tested 1, 3, and 5 hours after dosing. Of the 37 volunteers included, 36 received V116517, 36 received In a separate group of rats, the paw withdrawal latency to celecoxib, and 35 received placebo. The mean (SD) age of the a radiant heat stimulus was determined by placing the rats in volunteers included was 22.2 (2.66) years, weight 80.4 (9.74) kg, a Plexiglas box on a glass surface maintained at 32˚C (IITC; and BMI 24.3 (2.4) kg/m2. Woodland Hills, CA). The plantar surface of the paw was then exposed to a beam of radiant heat. The latency to paw withdrawal was measured to the nearest 0.1 seconds with a maximal cutoff 15. Safety and pharmacokinetics of 20 seconds to avoid tissue damage. Baseline latencies were No serious adverse events were reported. Two subjects (1002 determined after which rats received an exposure to UV-B and 1054) discontinued because of adverse events. Subject radiation as described above. Forty-eight hours after UV-B 1002 had generalized rash (exanthema) possibly related to exposure, the rats received 3, 10, or 30 mg/kg V116517 p.o., 30 V116517. Subject 1054 stopped permanently because of Gilbert mg/kg naproxen (positive control), or vehicle. The animals were syndrome, which is a congenital genetic disorder unrelated to the retested to determine whether the compounds had alleviated study drug. Three other subjects turned out to have Gilbert plantar hypersensitivity 1, 3, and 5 hours after dosing. syndrome when they were retested. No changes in oral In a final group of rats, the tactile thresholds were assessed using temperature were observed for any of the drugs, and none of a series of calibrated von Frey monofilaments (Stoelting, Wood the clinical parameters were affected. Dale, IL). The assessment of tactile allodynia was measured as the The skin temperature for the different interventions and PWT, which produced a 50% likelihood of a response using the 49 treatments are listed in Table 1. The V116517 group showed up–down method as previously described. Baseline thresholds a slight but significant (P , 0.05) increase in skin temperature 3.5 were first assessed, and then the rats received an exposure of hours after treatment as compared with placebo. UV-B radiation as described above. Subsequent PWTs were The mean (6SD) analyte metabolite (S)-V116517 pharmaco- determined 24 and 48 hours after UV-B exposure. From previous kinetic values were analyzed from the 35 volunteers. The values experience with TRPV1 antagonists and the UV-B-induced tactile related to AUCt (hr ng/mL), AUCinf (hr ng/mL), Cmax (ng/mL), Tmax allodynia model in rats, it was determined that this end point is (hr), t1/2 (hr) were 70,721 (34,400), 72,543 (35,261), 5083 (2444), intractable. To maximize the chance of seeing a reversal of this pain 2.2 (1.0), and 13.75 (1.78), mean (SD), respectively. The analyte state, a local administration of V116517 was included in addition to (S)-V116517 was expected to be washed out after 68.75 hours. following up with a systemic administration at a higher dose than that was tested against the pressure and thermal end points. As such, V116517 was administered 1 hour before UV-B (intrader- 16. Pharmacodynamics mally 500 mg/50 mL) and 1 hour before testing (100 mg/kg p.o.). The testing was conducted 48 hours after UV-B exposure. 16.1. Neurogenic inflammation (laser Doppler flowmetry and erythema index) Naproxen was not included in this study because it has previously been found to be a nonsuitable positive control for this end point As expected, both UV-B and topical capsaicin strongly increased because of limited and inconsistent reversals (data not shown). the vasomotor responses (Tables 2 and 3). The laser Doppler flowmetry and the erythema index (redness of the inflammation) in the UV-B inflamed area were very significantly reduced by 13. Statistical analysis (rodent models) celecoxib as compared with placebo (P , 0.0001). No effect of Data are shown as mean 6 SEM. The statistical significance was V116517 was found on UV-B or capsaicin-induced neurogenic determined on untransformed data using a 2-way ANOVA with inflammation (Tables 2 and 3).

Table 1 Averaged (6SD) skin temperature assessed for the 3 different treatments. Skin temperature (˚C) Drug Before UV-B Baseline 2.5 h after drug 3.5 h after drug UV-B (leg) Placebo 33.2 (0.8) 36.4 (0.6) 36.9 (0.4) Celecoxib 33.2 (0.8) 36.3 (0.6) 36.6 (0.4) V116517 33.1 (0.9) 36.5 (0.7) 37.9 (0.6) Capsaicin (arm) Placebo 34.4 (0.6) 32.0 (0.5) 36.2 (0.6) Celecoxib 34.4 (1.0) 32.2 (0.8) 36.1 (0.9) V116517 34.5 (0.8) 32.0 (0.5) 36.8 (1.0)* For the UV-B model, the skin temperature at the leg was measured before UV-B irradiation, at baseline (24 hours after UV-B irradiation), at baseline just before drug administration, and 3.5 hours after drug administration. For the topical capsaicin model, the skin temperature at the arm was measured at baseline just before drug administration, 2.5 hours after drug administration and just before capsaicin application (capsaicin applied for 30 minutes 2.5 hours after drug administration), and 3.5 hours after drug administration (30 minutes after removal of the capsaicin cream). * Significant difference from placebo.

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Table 2 Averaged (6SD) parameters assessed for the 3 different treatments at baseline (before UV-B irradiation of the leg), after 24 hours when the UV-B inflammation was developed, and 3.5 hours after drug administration (24 hours after UV-B irradiation 1 3.5 hours) UV-B Test Drug Baseline After 24 h 3.5 h after drug QST HPT (˚C) Placebo 45.1 (2.2) 39.8 (1.4) 39.5 (1.2) Celecoxib 44.8 (2.6) 39.4 (1.3) 39.5 (1.2) V116517 44.7 (2.3) 39.4 (1.4) 45.0 (2.2) PPT (kPa) Placebo 511 (136) 308 (118) 306 (110) Celecoxib 541 (146) 283 (89) 321 (85)* V116517 502 (161) 290 (106) 324 (151) Neurogenic inflammation Erythema Placebo 5.8 (1.8) 19.0 (2.9) 19.7 (2.7) index (a.u.) Celecoxib 5.5 (1.9) 19.4 (4.3) 16.6 (3.9)* V116517 5.6 (1.8) 19.6 (3.5) 19.5 (3.6) Laser Placebo 116 (34) 1419 (476) 1529 (452) Doppler (a.u.) Celecoxib 110 (32) 1422 (444) 1039 (356)* V116517 114 (33) 1524 (453) 1538 (428) The neurogenic inflammation is measured in arbitrary units (a.u.). * Significant differences from placebo. HPT, heat pain threshold; PPT, pressure pain threshold; QST, quantitative sensory testing.

16.2. Intrinsic analgesic action (heat pain thresholds) 16.4.2. Stimulus–response function to graded von Frey hair stimulation The intrinsic analgesic action was evaluated as changes in HPTs and HPTolT assessed from the control points on the arm and leg Generally the stimulus response curve was a right shift of the before and after drug administration. There was a highly curve for the highest stimulus intensities for both drugs. For the significant effect of treatment on the thresholds (P , 0.0001). 256-mN stimulus, both V116517 (P 5 0.085) and celecoxib (P 5 The post hoc analysis showed that both HPT and HPTolT were 0.014) reduced the response, the same for 512-mN stimulus (P 5 significantly (P , 0.0001) increased by V116517 as compared 0.009 and P 5 0.05), and similar for 800-mN stimulus (P 5 0.04 with placebo and celecoxib (Fig. 1). The HPT increased by and P 5 0.02). approximately 6˚C and the HPTolT by approximately 2˚C by V116517. The reason why the SD is low for the HPTolT after 16.4.3. Heat pain threshold and heat pain tolerance threshold V116517 is that that the temperature limit for the thermal stimulation (55˚C) was reached for some volunteers. Significantly less heat pain hyperalgesia (HPT [P 5 0.004] and HPTolT [P , 0.0001]) were shown after V116517 as compared with placebo (Table 3). No HPT differences were found between 16.3. UV-B model celecoxib and placebo. 16.3.1. Stimulus–response function to graded von Frey hair stimulation 16.4.4. Pressure pain threshold and pressure pain tolerance None of the treatments modulated the responses to graded threshold mechanical von Frey hair stimulation. Neither the PPT nor the PPTolT was affected by V116517 (P 5 0.08 and P 5 0.838) or celecoxib (P 5 0.477 and P 5 0.970) as 16.3.2. Heat pain threshold compared with placebo. The developed hyperalgesia assessed by HPT was not different from placebo for any of the drugs. 17. Preclinical results Exposure of the hind paw to UV-B radiation resulted in the 16.3.3. Pressure pain threshold development of mechanical hyperalgesia as indicated by a decreased PWT to a noxious pressure stimulus (Fig. 2), thermal A significant antihyperalgesic effect of celecoxib as compared hyperalgesia as indicated by a decreased PWL to a noxious thermal with placebo was found for PPT (P 5 0.01) (Table 2). stimulus (Fig. 3), and tactile allodynia as indicated by decreased PWT to a nonnoxious tactile stimulus (Fig. 4). Oral administration of 16.4. Capsaicin model V116517 produced a significant effect on mechanical hyperalgesia (PWT)afterasingledoseandatasingletimepoint(ie,3hoursafter 16.4.1. Secondary pin-prick hyperalgesic area 10 mg/kg) (Fig. 2). Because this effect only occurred at a single time There were no significant changes in the size of the secondary point, and it was not dose-dependent, it may not constitute a true pin-prick hyperalgesic area for any of the treatments. reversal of the pain state. In contrast to mechanical hyperalgesia,

Table 3 mode of actions. Mechanistic drug profiling in human volunteers Averaged (6SD) parameters assessed for the 3 different provides the ability to determine the potency by which treatments at baseline (before topical capsaicin application a compound exerts its action on a specific target and translates on the arm and 3.5 hours after drug administration). this information into a meaningful clinical context. Capsaicin Test Drug Baseline 3.5 h after drug 19. The challenge of developing new analgesics QST Many new compounds are currently in clinical development and most HPT (˚C) Placebo 44.3 (2.9) 38.4 (2.7) of them are designed to target very specific receptors or selected 23,30 Celecoxib 44.2 (2.5) 38.5 (2.6) mechanisms. More recently, the interest in the TRP channel for pain management has faded slightly,30 but TRPV1, TRPV3, TRPA1, V116517 44.5 (2.9) 41.9 (4.0)* and TRPM8 antagonists are still of interest as novel analgesics.29 HPTolT (˚C) Placebo 49.8 (1.3) 44.3 (4.1) The focus on the TRPV1 receptor has been 2-fold. The TRPV1 Celecoxib 49.9 (1.3) 44.9 (4.4) agonist (capsaicin) has been developed for topical application in, V116517 49.8 (1.6) 50.3 (2.1)* eg, neuropathic pain conditions,41 and ultrapotent capsaicin 25 PPT (kPa) Placebo 457 (142) 429 (131) analogues are under development. TRPV1 antagonists have been developed to block nociceptive Celecoxib 432 (120) 429 (128) pathways throughout the nervous system, and several programs V116517 434 (140) 455 (197) have been terminated because of side effects (hyperthermia).29 PPTolT (kPa) Placebo 918 (252) 914 (260) This unwanted effect was not observed in the present study although this was seen for V116517 in preclinical studies.53 Celecoxib 871 (232) 939 (289) A COX-2 inhibitor was selected as a control compound V116517 867 (229) 904 (305) because it has previously shown to increase HPTs and HPTolTs 51 Neurogenic from UV-B-sensitized areas. Furthermore, COX-2 inhibitors are inflammation assumed to exert both peripheral anti-inflammatory effects and central antinociceptive effects,54 and a single dose in otherwise Erythema Placebo 9.4 (4.7) 14.4 (3.9) healthy subjects is considered safe. index (a.u.) Celecoxib 7.9 (3.5) 13.9 (3.6)

V116517 8.5 (3.6) 14.2 (3.5) 20. Mechanistic pain assessment in drug Laser Placebo 89 (32) 649 (265) development and preclinical relations Doppler (a. Celecoxib 88 (33) 655 (267) Human mechanistic pain assessment tools can provide an u.) understanding of transduction, transmission, modulation, and V116517 93 (28) 622 (274) perception under normal and pathophysiological conditions 27 The topical capsaicin was applied 2.5 hours after baseline recordings and left on the skin for 30 minutes. The and as such contribute to mechanism-based patient profiling neurogenic inflammation is measured in arbitrary units (a.u.). and drug profiling.3 In addition, experimental conditions applied * Significant differences to placebo. HPT, heat pain threshold; HPTolT, heat pain tolerance threshold; PPT, pressure pain threshold; PPTolT, to humans can be very closely replicated in animals to test the pressure pain tolerance threshold; QST, quantitative sensory testing. translation of the results as performed in this study. The approach was to use 2 different sensitization models in both human and animals and a platform of mechanistic quantitative sensory tests oral administration of V116517 produced a dose-dependent to probe and quantify the drug effects. 8,15 reduction in thermal hyperalgesia (F(4,225) 5 15.83, P , 0.0001) The UV-B model is well-established, working in animals and after administration (Fig. 3). At the 1- and 5-hour time points, humans6 with reliable and reproducible effects.40 The model statistically significant increases in PWL were seen after the 30 causes local release of cytokines,1 and the cutaneous sensitiza- mg/kg dose (Fig. 3), while at the 3-hour time point, both 10 and 30 tion develops over 24 hours21 with a demarked area of mg/kg produced statistically significant (P , 0.001) effects. The neurogenic inflammation6 as also seen in this study. The model maximum percent reversal, 83 6 11%, was achieved 3 hours after has been used extensively in drug-profiling studies for assessing the 30 mg/kg dose. Oral administration of naproxen (30 mg/kg) also anti-inflammatory and antihyperalgesic effects26 of traditional produced a statistically significant reversal of thermal hyperalgesia 3 NSAIDs52 and COX-2 inhibitors.51 and 5 hours after administration (Fig. 3). Administration of a high In this human study, the UV-B-induced heat hyperalgesia was dose of V116517 (100 mg/kg, p.o. subsequent to a local in- not affected by the COX-2 inhibitor celecoxib, whereas pressure tradermal administration of 500 mg) before induction did not hyperalgesia was modulated. Is not known whether there is significantly cause a preemptive effect by reducing the UV-B- a different mode of action between different COX-2 inhibitors, but induced tactile allodynia (Fig. 4). previous studies showed that rofecoxib reduced heat hyperalgesia, whereas this study found that celecoxib inhibited pressure hyperalgesia for the dose used. In the preclinical 18. Discussion studies, the nonselective COX inhibitor, naproxen, inhibited both This study showed how a receptor-selective TRPV1 antagonist thermal hyperalgesia and tactile allodynia. increased the HPTs and inhibited heat hyperalgesia in a capsaicin The UV-B model was also selected as preclinical data suggest sensitized area. The COX-2 inhibitor specifically modulated that TRPV1 receptors are essential for inflammatory-induced mechanical hyperalgesia in a UV-B-sensitized area. thermal hyperalgesia,13 and it has been used in a previous study Mechanistic quantitative sensory testing in combination with evaluating the effect of a TRPV1 antagonist.11 The data from this specific experimental pain sensitization models in both animals study showed similar drug effects on heat hyperalgesia as and humans showed the ability to separate between distinct assessed by heat pain and HPTolTs.

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Figure 1. Heat pain threshold and heat pain tolerance threshold measured from the control points on the arm and leg in healthy volunteers before and after dosing (celecoxib, placebo, V116517). There was a significant effect of dosing on the thresholds and the post hoc analysis showed highly significant (*P , 0.0001) increases (analgesia) after V116517. SD bars are given.

No effects were found on neurogenic inflammation as shown view, celecoxib was expected to reduce the UV-B-induced by Chizh et al.11 In this study, quantitative measures (laser neurogenic inflammation in this study. Doppler flowmetry and erythema index) were used, whereas Topical application of capsaicin was selected as the other Chizh et al.11 used the flare area, which is based on a subjective model to develop sensitization as it acts as an agonist on the evaluation. Recently, a pressure pain threshold technique for peripheral target of interest (TRPV1) and causes heat hyper- screening hyperalgesia in UV-B-sensitized areas has been algesia involving heat nociceptors where the TRPV1 receptors developed,35 and for the first time, this technique proved are expressed34 and hence should be the most optimal a sensitive model to profile the antihyperalgesic effect of translational model as it is also known to induce hyperalgesia celecoxib. From a mechanistic and anti-inflammatory point of and neurogenic inflammation in animals14,18 and humans9,36

Figure 2. Evaluation of V116517 (3-30 mg/kg, p.o.) in a UV-B-induced pain model in Sprague-Dawley rats. V-116517 (10 mg/kg, p.o.) and the positive control, naproxen (30 mg/kg, p.o.), produced a reduction of mechanical hyperalgesia (paw withdrawal threshold, Randall–Selitto method) at the 3-hour time point (n 5 10 per group; ***P , 0.001 and ****P , 0.0001 vs vehicle control).

Figure 3. Evaluation of V116517 (3–30 mg/kg, p.o.) in a UV-B-induced pain model in Sprague-Dawley rats. V-116517 (10 and 30 mg/kg, p.o.) and the positive control, naproxen (30 mg/kg, p.o.), produced a dose-dependent reduction of thermal hyperalgesia (paw withdrawal latency) (n 5 10 per group; *P , 0.05, **P , 0.01, ***P , 0.001, and ****P , 0.0001 vs vehicle control).

with association between sensory changes and the vascular 21. Translating preclinical data into humans responses.39 The capsaicin model may be sensitive to both A major obstacle for successful development of new analgesics is sexes17 and ethnicity,55 but as the study included only the unpredictable gap in translation from animals into humans.37,43 Caucasian males, these factors did not add to the variability It has been estimated that the lack of efficacy contributes to 51% of of the data. The duration of action for the TRPV1 antagonist 5 10 phase II trial failures and emphasized that translational predictive studied was short so the alternative heat-capsaicin model pain models efficiently may bridge the gap.45 This study extensively was not selected as this is more adequate for drug-profiling used the pain models and assessment modalities in both animals studies requiring a longer monitoring period. The topical and humans, and preclinical and clinical data are presented side by capsaicin model has previously been used for evaluating side and compared. One limitation in both animal and human 11 a TRPV1 antagonist (SB-705498) where no antiheat hyper- experimental pain trials is that efficacy measures are mainly based ‐ algesia was found but only a reduction of capsaicin evoked on evoked reactions, whereas the main problem for patients is the flare area. This was surprising because in vitro data showed ongoing or transient clinical pain. As such, the experimental that this specific compound was effective in blocking the mechanistic approach may not fully predict the clinical effect, but it 19 activation of TRPV1 by capsaicin and heat. provides evidence that a drug in the given concentration reaches As expected from a mechanistic thermosensitive TRP the target and exerts the expected action. Because clinical trials on receptor interaction point of view, this study found a significant healthy volunteers are much faster and cheaper than clinical trials effect of the TRPV1 antagonist on HPTs in the capsaicin- on patients, such studies provide useful information in the early sensitized area. Furthermore, significant antihyperalgesia to phase of development.4 the strongest pin-prick stimulus intensities was found for both The preclinical profiling of V116517 showed a dose-dependent active drugs. It has previously been shown that V116517 is block of capsaicin-induced irritation and of thermal hyperalgesia effective against capsaicin-induced pain in animals,53 in- after inflammation without affecting body temperature.53 In the dicating an association between the preclinical and the human study, it was confirmed for a single dose that the human findings in the present study. capsaicin-provoked thermal hyperalgesia and the pin-prick

Figure 4. Evaluation of V116517 (500 mg/rat intradermally followed by 100 mg/kg, p.o.) in a UV-B-induced pain model in Sprague-Dawley rats. Local and systemic V-116517 given before (preemptive) the induction of the sensitization did not produce a reduction of tactile allodynia (paw withdrawal threshold) (n 5 10 per group).

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Harris, Thompson M, Rami HK, Randall A, Davis JB. Characterization of SB- S. O’Keefe, R. Kapil, and D. Kyle are employees of Purdue 705498, a potent and selective vanilloid receptor-1 (VR1/TRPV1) antagonist that inhibits the capsaicin-, acid-, and heat-mediated Pharma. The study was sponsored by Purdue Pharma. The other activation of the receptor. J Pharmacol Exp Ther 2007;321:1183–92. authors have no conflicts of interest to declare. [20] Gunthorpe MJ, Szallasi A. Peripheral TRPV1 receptors as targets for drug development: new molecules and mechanisms. Curr Pharm Des 2008; 14:32–41. Acknowledgements [21] Gustorff B, Sycha T, Lieba-Samal D, Rolke R, Treede RD, Magerl W. The pattern and time course of somatosensory changes in the human UVB The staff at C4Pain is acknowledged for their dedication and help sunburn model reveal the presence of peripheral and central executing the trial. sensitization. PAIN 2013;154:586–97. [22] Harrison GI, Young AR, McMahon SB. 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