Cannabinoid CB1/CB2 receptors; hCB1-R ( pKi = 7.93 ); hCB2-R ( pKi = 9.06 )

AZD1940 exhibits complete agonism at both receptors in all three species after binding with high affinity to the CB1 and CB2 receptors in humans, rats, and mice[1][2].

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AZD1940 binds with high affinity to human, rat and mouse CB1 and CB2 receptors and displays full agonism at both receptors in all three species [1].AZD1940 was radiolabeled with carbon-11 in the benzimidazole moiety. The radioactive precursor, lithium [11C]pivalate was obtained via 11C-carboxylation of tert-butyl lithium. The target compound, [11C]AZD1940, was in turn obtained by the microwave assisted reaction between lithium [11C]pivalate and the o-phenylene diamine analog of AZD1940 (N-(3-amino-4-((4,4-difluorocyclohexyl)methylamino)phenyl)ethanesulfonamide) in neat phosphorous oxychloride [2].

AZD1940 exhibits a strong analgesic effect in a variety of inflammatory and neuropathic pain models when administered orally to rats[1][2]. It has been shown that rats and primates exhibit low brain uptake of AZD1940 at analgesic doses[1].

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The overall radiochemical yield of final formulated radiochemically pure (>99%) [(11)C]AZD1940 was 0.4% (uncorrected for decay) and the specific radioactivity was 13GBq/μmol at time of administration (58min after end of bombardment). After intravenous injection to cynomolgus monkey, the maximum concentration of radioactivity detected in the brain region of interest was 0.7% of the total injected radioactivity. The regional distribution of radioactivity within brain was homogenous.[2]

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Conclusions: AZD1940 was radiolabelled with carbon-11 and its brain exposure, assessed using PET, was relatively low in comparison to peripheral organ exposure. Brain PET imaging[2]
In PET measurements in cynomolgus monkey after intravenous injection of [11C]AZD1940 radioactivity in brain was low relative to that in surrounding extra-cerebral tissues (Fig. 1). At the time of maximum concentration (Tmax), 0.70% of the total radioactivity injected (0.59 SUV; not corrected for radioactivity in blood; Fig. 2A) was present in brain. Radioactivity was homogeneously distributed in brain with similar concentration in all of the regions investigated (Fig. 2B).

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The aim of the present study was to investigate the effects of AZD1940, a novel peripherally acting cannabinoid CB(1) /CB(2) receptor agonist, on capsaicin-induced pain and hyperalgesia, as well as on biomarkers of cannabinoid central nervous system (CNS) effects. The present study was a randomized, double-blind, placebo-controlled, four-sequence, two-period, cross-over study in 44 male healthy volunteers aged 20-45 years. The effects of two single oral doses of AZD1940 (400 and 800 μg) were compared with placebo. Pain intensity after intradermal capsaicin injections in the forearm was assessed on a continuous visual analogue scale (VAS; 0-100 mm). Primary and secondary hyperalgesia induced by application of capsaicin cream on the calf were assessed by measuring heat pain thresholds and the area of mechanical allodynia, respectively. The CNS effects were assessed at baseline and up to 24 h after dosing using a visual analogue mood scales (VAMS) for feeling 'stimulated', 'high', 'anxious', 'sedated' or 'down'. AZD1940 did not significantly attenuate ongoing pain or primary or secondary hyperalgesia compared with placebo. Mild CNS effects for AZD1940were observed on the VAMS for 'high' and 'sedated'. Dose-dependent mild-to-moderate CNS-related and gastrointestinal adverse events were reported following treatment with AZD1940. No evidence of analgesic efficacy was found for a peripherally acting CB(1)/CB(2) receptor agonist in the human capsaicin pain model. The emergence of mild dose-dependent CNS effects suggests that the dose range predicted from preclinical data had been attained [1].

Brain PET imaging [2]
Anaesthesia was induced and maintained by repeated intramuscular injections of a mixture of ketamine hydrochloride (3.75 mg/kg h Ketalar® and xylazine hydrochloride (1.5 mg/kg h) for the duration of the entire measurements. Body temperature was maintained by Bair Hugger Model 505 and monitored by a rectal thermometer. Heart and respiration rates were measured every 20 min throughout the experiment. The monkey was observed continuously during the PET experimental day.
A head fixation system was used to secure a fixed position of the monkey head during the PET measurement. Radioactivity in brain was measured with the Siemens ECAT Exact HR 47 system. The data was acquired in 3D-mode. The three-ring detector block architecture gives a 15 cm wide field of view. The transversal resolution in the reconstructed image is about 3.8 mm full width half maximum (FWHM) and the axial resolution is 3.125 mm. The attenuation correction of the data was obtained with the three rotating 68Ge line sources. Raw PET data were then reconstructed using the standard filtered back projection.
A sterile physiological phosphate buffer (pH = 7.4) solution containing 44 MBq of [11C]AZD1940 was injected as a bolus into a sural vein during 5 s with simultaneous start of PET-data acquisition. Radioactivity in brain was measured continuously for 93 min according to a pre-programmed series of 20 frames starting immediately after an intravenous injection of [11C]AZD1940.

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Study design [1]
A randomized, double-blind, placebo-controlled, four-sequence, two-period, crossover study was conducted to investigate the effects of single doses of an oral solution of AZD1940 (400 and 800 μg) compared with placebo on topical and intradermal (i.d.) capsaicin-evoked pain symptoms, as well as on CNS psychomotor and/or cognitive functions. Each subject was randomized to one of four treatment sequences (Fig. 1). Dose selection was based on a preceding single ascending dose study in healthy volunteers in which a dose of 800 μg AZD1940 was found to be the maximal well-tolerated dose.30 The dose-limiting side-effects in that study were postural dizziness, hypotension and mild-to-severe psychiatric adverse events at higher doses.

Subjects visited the clinic on five occasions: an enrolment visit (Visit 1), followed by a training visit for the congitive testing and capsaicin-evoked pain models (Visit 2), two treatment visits (Visits 3 and 4) and a follow-up visit (Visit 5). At the enrolment visit, ≤ 28 days before randomization, each subject underwent a health examination, including a semistructured interview by a psychiatrist to judge whether it was acceptable to expose the subject to AZD1940. Subjects with positive tests for hepatitis B/C, human immunodeficiency virus or a positive urine drug screen were excluded from the study. Visit 2 occurred 7–14 days before randomization. The two treatment visits (Visits 3 and 4) were separated by 14 days and consisted of 2 days of residential stay. The follow-up visit was 10–14 days after the last treatment day.

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Pain models [1]
Two different pain challenges were used: (i) topical capsaicin cream (0.075% AXSAIN); and (ii) i.d. capsaicin (in 20% cyclodextrin; 0.3 μg; injection volume 10 μL). Intradermal capsaicin was administered in the volar forearm, whereas the topical capsaicin cream was applied on the medial side of the calf for 90 min, covering a 5.5 × 3 cm2 area. At the training visit, one injection of capsaicin was given and capsaicin was also applied topically to familiarize the subjects with the study procedures. The times for measurement of pain end-points were based on pharmacokinetic and CNS pharmacodynamic data from the preceding single ascending dose study, because the maximum effect of AZD1940 was expected to be obtained some time after tmax (occurring ~ 2 h after dosing in the fasting condition).

At each treatment visit (Visits 3 and 4), three i.d. injections of capsaicin were given in different areas in the volar forearm. The injection sides (left/right) were changed between Visits 2 and 3. The first injection was given before (baseline) and the second and third injections 3 h and 4 h 45 min after administration of the study drug. The intensity of pain after capsaicin injections was measured using a continuous electronic visual analogue scale (eVAS), deriving the variables eVASmax (= maximal) and eVASAUC (= area under the curve) over a period of 5 min.

At 3 h 15 min after administration of the study drug, capsaicin was applied topically on the medial side of the calf for 90 min. Primary hyperalgesia was assessed by measuring the heat pain threshold (HPT) at the centre of the area where capsaicin was applied using a Peltier thermode at baseline and immediately after removal of capsaicin from the skin, at 4 h 45 min after administration of the study drug. The mean of five consecutive measurements was calculated. The area of secondary hyperalgesia (i.e. brush allodynia) was measured every 20 min (4 h 5 min, 4 h 25 min, 4 h 45 min after study drug administration) by stroking the skin with a standardized brush for moving tactile stimulation at approximately 1 cm/s. The borders of the allodynia area were delineated by stimulating clockwise from the periphery along eight radial axes. Subjects were asked to indicate when the sensation of moving touch changed into a painful or unpleasant perception (i.e. allodynia). The distance (cm) of each point from the centre of the application site was recorded.

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Pharmacokinetic analysis [1]
Pharmacokinetic (PK) samples were collected before dosing and 20 and 40 min and 1, 1.5, 2, 2.5, 3, 4, 6, 13, 24 and 48 h after administration of study drug to follow concentrations of AZD1940 over time during each treatment visit. The PK parameters (Cmax, maximum plasma concentration; tmax, time after dose of maximum plasma concentration; AUC, area under curve (plasma concentration × time); and t½, half-life) of AZD1940 were derived using a non-compartmental model in WinNonlin.

Pharmacokinetics [1]
The mean Cmax of AZD1940 in plasma following oral dosing of 400 and 800 μg was 5.3 and 10.6 nmol/L, respectively. Corresponding tmax estimates were 2.7 and 2.3 h, respectively. The half-life for the 400 and 800 μg doses of AZD1940 was 39.6 and 35.0 h, respectively. Owing to the long half-life in relation to the sampling time, t½ and other PK parameters were uncertain.

Safety and tolerability [1]
There were no SAEs in the study. In all, 20 of 22 subjects reported AE when treated with 800 μg AZD1940compared with 10 of 22 when these subjects received placebo. After 400 μg AZD1940, 13 of 20 subjects reported AE compared with 10 of 20 when they received placebo treatment. Central nervous system AE (mainly dizziness, somnolence and headache) were most frequent and increased with the higher dose of AZD1940 (Table 1). Gastrointestinal AE (mainly dry mouth and nausea) also increased with the higher dose of AZD1940 (Table 1). The intensity of the AE was also higher with the higher dose of AZD1940; for example, AE of moderate intensity were reported by six subjects on 400 μg AZD1940 (three while on placebo) and by eight subjects on 800 μg AZD1940 (two while on placebo). One subject treated with 800 μg AZD1940 had one AE of severe intensity, a vasovagal syncope that was provoked by standing blood pressure measurement.

[1]. Evaluation of the analgesic efficacy and psychoactive effects of AZD1940, a novel peripherally acting cannabinoid agonist, in human capsaicin-induced pain and hyperalgesia. Clin Exp Pharmacol Physiol. 2013 Mar;40(3):212-8.

[2]. Radiolabeling of the cannabinoid receptor agonist AZD1940 with carbon-11 and PET microdosing in non-human primate. Nucl Med Biol. 2013 Apr;40(3):410-4.

A method for the radiolabeling of AZD1940 with carbon-11 in a metabolically stable position was developed. The method development comprised preparation of two new radiolabeled precursors, namely lithium [11C]pivalate and [11C]ethanesulfonyl chloride. These agents, as well as the method for radiolabeling the benzimidazolyl moiety with carbon-11, may find wider utility in PET radiochemistry. PET imaging with [11C]AZD1940 confirmed a relatively low CNS exposure of AZD1940 after administration of an intravenous microdose in cynomolgus monkey.[2]

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The CB1/CB2 receptor agonist AZD1940 did not reduce capsaicin-induced pain or primary or secondary hyperalgesia. As expected, mild subjective CNS-related effects were observed on VAMS and the AE profile showed mild-to-moderate dose-dependent CNS effects.
Pain and hyperalgesia after capsaicin administration have been used extensively as a model for the initial evaluation of analgesic drugs for acute pain. Studies of cannabinoid effects on acute pain, including capsaicin models, have shown inconsistent results. Most studies of orally administered cannabinoids have shown negative effects on capsaicin-induced pain. However, attenuation of pain and/or hyperalgesia have been observed in placebo-controlled studies of a topically administered cannabinoid16 and of smoked cannabis. The discrepancy in efficacy between different cannabinoid agonists may be due to a number of factors, such as PK effects, mode of administration, differences in the distribution of central and peripheral sites of action, tolerability issues limiting the dose that can be given, differences in CB1/CB2 receptor selectivity and the intrinsic efficacy of the molecules. The Cmax of AZD1940 occurred, as expected, 2–3 h after drug administration and the plasma concentrations declined very slowly due to the drug's long half-life. Thus, the pain models and the timing of measurements in the present study were considered to be appropriate. For the area of mechanical allodynia, differences favouring both placebo versus 400 μg AZD1940 and 800 μg AZD1940 versus placebo at single points in time were found. In general, the variability of the areas of allodynia was high within and between individuals. Therefore, these apparently conflicting observations are likely due to random variation.

Patients receiving AZD1940 felt more ‘sedated’ and ‘high’ than on placebo, with maximal effects on VAMS of < 30 mm occurring at the time of the maximal plasma concentration of the drug. These effect sizes can be compared with those observed with the centrally acting cannabinoid nabilone, for which the maximal effect sizes were up to approximately 60 mm on VAMS ‘sedated’, 20–40 mm on VAMS ‘anxious’ and 20–25 mm on VAMS ‘down’. Nabilone also had dose-dependent effects on a number of other subjective and cognitive measures that were more pronounced than those that have been observed with AZD1940 (AstraZeneca R&D, unpubl. data, 2007).

Adverse events classified as nervous system related (headache, dizziness, somnolence) were reported on AZD1940, with an increase in frequency and intensity with the higher dose. Similar but more pronounced AE, including severe CNS-related AE, were reported at clinically recommended doses of nabilone. Both doses of AZD1940 induced orthostatic effects, with a reduction in mean BP on standing compared with placebo and a corresponding increase in mean pulse rate. It is conceivable that some of the AE termed as ‘dizziness’ could be related to haemodynamic effects.

The characterization of AZD1940 has shown high-affinity binding and full agonism at human, rat and mouse CB1 and CB2 receptors. Preclinical studies have shown that the analgesic effects of AZD1940 are reversed by local administration of a CB1 receptor antagonist. Thus, the preclinical data suggest that the analgesic effects of AZD1940 are mediated by peripheral CB1 receptors. AZD1940 has low brain uptake and a low propensity of adverse CNS effects at analgesic doses in the rat, consistent with a window for analgesic effects before adverse CNS effects are observed. Because dose-dependent mild CNS effects were observed in the present study, it is likely that the appropriate dose range for testing the efficacy of AZD1940 was covered.

Thus, there were no obvious methodological explanations to the negative efficacy outcome of the present study. Recent negative data on AZD1940 in postoperative dental pain, which can be considered as a model of acute inflammatory pain, are also in agreement with the findings of the present study. According to meta-analyses of studies on different cannabinoid compounds (e.g. cannabis extracts, nabilone, △-9-tetrahydrocannabinol), the evidence for cannabinoid efficacy in acute pain is rather weak, with several examples of absent or weak analgesic effects in acute pain treatment or experimental pain in humans. Evidently, AZD1940, a synthetic peripherally acting CB1/CB2 receptor agonist, does not provide any better efficacy than centrally acting cannabinoids. Because the preclinical data on the importance of peripheral CB1 receptors seem strong, this highlights that the translation between preclinical and clinical data is poorly understood. There are several examples in the literature of failures in translation of positive animal efficacy data into humans (e.g. NK1 receptor antagonists, N-methyl-d-aspartate glycine site antagonists). However, it should be pointed out that most positive data on cannabinoid efficacy in human pain conditions come from multiple dose studies in multiple sclerosis and other types of neuropathic pain. The pathophysiological mechanisms behind chronic inflammatory, neuropathic and visceral pain are different from those mediating acute pain and may involve plasticity of peripheral and central neurons. Thus, it is conceivable that cannabinoids could affect certain pain mechanisms that are mainly of importance in chronic pain conditions. Therefore, multiple dose studies in, for example, chronic neuropathic and visceral pain conditions could be considered to explore the analgesic potential of peripherally acting cannabinoids. Other strategies of exploiting the endogenous cannabinoid systems for analgesia may also be considered; for example, instead of treatment with an exogenous receptor agonist, the levels of endogenous cannabinoids (‘endocannabinoids’) could be elevated through inhibition of a degrading enzyme. Endocannabinoids have rather complex effects on receptors; for example, palmitoylethanolamide (PEA), which has shown efficacy in human pain conditions, may act mainly through the peroxisome proliferator-activated receptor α and has only weak affinity for CB1/CB2 receptors. In addition, PEA has been identified as an agonist of two orphan G-protein-coupled receptors with cannabinoid-like pharmacology, namely the GPR55 and GPR119 receptors. Thus, the systems activated by endocannabinoids seem to be more complex than merely those acting via the CB1/CB2 receptors.

In conclusion, no evidence of analgesic efficacy was found for a peripherally acting CB1/CB2 receptor agonist in the human capsaicin pain model. However, it is as yet unknown to what extent peripheral CB1/CB2 receptor agonists may be useful in the treatment of human chronic pain conditions. [1]

C20H29F2N3O2S

413.52

413.195

C, 58.09; H, 7.07; F, 9.19; N, 10.16; O, 7.74; S, 7.75

881413-29-2

881413-29-2

11675994

White to off-white solid powder

6.074

1

6

6

28

635

0

O=S(CC)(NC1C=C2N=C(C(C)(C)C)N(C2=CC=1)CC1CCC(F)(F)CC1)=O

ZAGGGZCIFUQHOH-UHFFFAOYSA-N

InChI=1S/C20H29F2N3O2S/c1-5-28(26,27)24-15-6-7-17-16(12-15)23-18(19(2,3)4)25(17)13-14-8-10-20(21,22)11-9-14/h6-7,12,14,24H,5,8-11,13H2,1-4H3

N-[2-tert-butyl-1-[(4,4-difluorocyclohexyl)methyl]benzimidazol-5-yl]ethanesulfonamide

AZD-1940; AZD 1940; AZD1940; UNII-0J0035E9FT; ART-27.13; 881413-29-2; AZD1940; Ethanesulfonamide, N-(1-((4,4-difluorocyclohexyl)methyl)-2-(1,1-dimethylethyl)-1H-benzimidazol-5-yl)-; CHEMBL4550236; 0J0035E9FT; N-[2-tert-butyl-1-[(4,4-difluorocyclohexyl)methyl]benzimidazol-5-yl]ethanesulfonamide; Ethanesulfonamide, N-[1-[(4,4-difluorocyclohexyl)methyl]-2-(1,1-dimethylethyl)-1H-benzimidazol-5-yl]-; ART 27.13; ART27.13; ART-2713; ART 2713; ART2713

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~241.8 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)