The biological effects and thermal degradation of NPB-22, a synthetic cannabinoid

Synthetic cannabinoids (SCs) have been originally developed based on the chemical structure of tetrahydrocannabinol (THC) contained in cannabis to study the endocannabinoid system [1]. They are functionally similar to THC, bind to cannabinoid receptor type 1 (CB₁) and cannabinoid receptor type 2 (CB₂), and are expected to have cannabis-like effects. They were mixed with or sprayed on plant materials and sold as herbs; SCs were used to obtain cannabis-like effects around the mid-2000s.

The governments of various nations have regulated the amount of SCs detected in products; however, illicit manufacturers have synthesized new SCs by replacing a part of the chemical structure of SCs distributed previously in drug markets to circumvent the regulation. NPB-22 (quinolin-8-yl 1-pentyl-1H-indazole-3-carboxylate), an indazole analog of PB-22 (quinolin-8-yl 1-pentyl-1H-indole-3-carboxylate), was first found in Hungary in January 2014 [2] and in Ankara, Turkey, in the same year [3]. Moreover, it was detected in authentic blood samples obtained in cases of driving under the influence of drugs between January 2013 and November 2015 in Germany [4]. These new SCs have not been adequately researched on in terms of their biological effects, and NPB-22 has not been thoroughly examined. Accordingly, we evaluated the CB₁ and CB₂ activities of NPB-22 using [³⁵S]guanosine-5’-O-(3-thio)-triphosphate ([³⁵S]GTPγS) binding assays. Furthermore, because the abusers of SCs have created handmade cigarettes by rolling the plant material in cigarette paper and inhaled the smoke from ignited cigarettes to absorb SCs [5‐7], we have also evaluated the biological effects of these new SCs using the inhalation exposure test in which mice are exposed to smoke from burning plants sprayed with a solution containing SCs similar to cigarettes [8].

In this study, it was revealed that NPB-22 possessed a strong binding activity on the same level with the positive control, CP-55,940 (2-((1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)-5-(2-methyloctan-2-yl)phenol), and was expected to exhibit cannabis-like effects. However, the biological effects of NPB-22 were weaker than expected. Accordingly, the smoke rising from plants sprayed with NPB-22 burned in the inhalation exposure test was collected using midget impingers and was analyzed by liquid chromatography. The results of the analysis showed the degradation of NPB-22 by combustion. Conventional cigarettes are directly ignited, the temperature of the combustion point reaches 800–900 °C, and the thermal degradation of SCs has been reported [9, 10]. In this paper, we report the binding activity on cannabinoid receptors and the biological effects of NPB-22 and its degradation by combustion.

NPB-22 possessed CB₁ activity equivalent to that of CP-55,940, the positive control, and Adamantyl-THPINACA. However, the influence of NPB-22 was weaker than that of Adamantyl-THPINACA in the inhalation exposure test. The smoke from the burned NPB-22 was analyzed, and it was revealed that NPB-22 was degraded to 8-quinolinol and pentyl indazole 3-carboxylic acid by combustion. Therefore, it was presumed that the actual amount of NPB-22 absorbed into the body was decreased by combustion, and its biological effects on the inhalation exposure test were weaker than the estimation on the [35S]GTPγS binding assays.

We prepared the calibration curves of NPB-22, 8-quinolinol, and pentyl indazole 3-carboxylic acid to calculate rate of thermal degradation of NPB-22. The correlation coefficients of the calibration curves of NPB-22, 8-quinolinol, and pentyl indazole 3-carboxylic acid were 0.99960, 0.99996, and 0.99997, respectively, in the range of 0.5–10 μg/mL; therefore, the calibration curves indicated satisfactory linearity. The molarity of NPB-22, 8-quinolinol, and pentyl indazole 3-carboxylic acid in the solutions of the midget impingers was calculated using the calibration curves. In the first midget impinger, the molarity of NPB-22, 8-quinolinol, and pentyl indazole 3-carboxylic acid was approximately 0.03, 0.05, and 0.08 mM, respectively, and in the second midget impinger, the molarity of the compounds under study was approximately 0.02, 0, and 0.03 mM, respectively. Incidentally, 8-quinolinol was not detected in the second midget impinger. Therefore, approximately 0.05, 0.05, and 0.11 μmol of NPB-22, 8-quinolinol, and pentyl indazole 3-carboxylic acid were recovered from the smoke of burned NPB-22. When NPB-22 degrades to 8-quinolinol and pentyl indazole 3-carboxylic acid, it is presumed that the thermal degradation compounds are generated in equal amounts. However, the amount of 8-quinolinol in the midget impingers was lower than that of pentyl indazole 3-carboxylic acid. Because it was presumed that 8-quinolinol was lost through the smoke pathway, the rate of thermal degradation of NPB-22 was estimated using the molarity of NPB-22 and pentyl indazole 3-carboxylic acid. As a result, it was estimated that 68% of NPB-22 was degraded by combustion. In addition, the degradation products of NPB-22, 8-quinolinol, and pentyl indazole 3-carboxylic acid did not possess CB₁, and CB₂ agonist activity. Therefore, absorbing NPB-22 like a cigarette was expected to affect CB₁, and CB₂ less than the results of in vitro assays, and the converted EC₅₀ of the burned NPB-22 was found to be 1.56 × 10⁻⁸ and 8.47 × 10⁻⁸ mol/L for CB₁ and CB₂ respectively.

When the converted EC₅₀ of the burned NPB-22 on CB₁ was compared with the EC₅₀ of Adamantyl-THPINACA and CUMYL-4CN-B7AICA, the order of them was Adamantyl-THPINACA > burned NPB-22 > CUMYL-4CN-B7AICA. CUMYL-4CN-B7AICA was also degraded by combustion; however, the amount of degraded CUMYL-4CN-B7AICA was of minimal quantity, and it was estimated that the thermal degradation of CUMYL-4CN-B7AICA did not affect the order of the converted EC₅₀ on CB₁. Grooming and verticalness were suppressed more strongly in this converted order on the inhalation exposure test. In addition, aggressiveness, sound response, and pain response were suppressed or promoted by Adamantyl-THPINACA temporarily; however, NPB-22 and CUMYL-4CN-B7AICA had no significant effects on these items. Adamantyl-THPINACA strongly lowered the body temperature of the mice under study. NPB-22 and CUMYL-4CN-B7AICA lowered it temporarily; however, this behavior was approximately equivalent to that of the negative control. The order of these items can also be explained by the converted order. Piloerection was observed only in mice that inhaled NPB-22. According to the reported results of a previous inhalation exposure test, Adamantyl-THPINACA and CUMYL-4CN-B7AICA, including other SCs (5F-MDMB-PICA, 5F-EMB-PINACA, AMB-CHMICA, 5F-MDMB-PINACA, THJ, and the herb products containing AB-CHMINACA and 5F-AMB) did not lead to piloerection [8, 13]. In addition, it has been reported that 8-quinolinol and pentyl indazole 3-carboxylic acid did not lead to piloerection; therefore, piloerection was specific to only NPB-22.

Drug abusers absorb SCs by inhaling the smoke from ignited cigarettes that are made by rolling the plant material in cigarette paper. It is reported that the ignition point of cigarettes reaches 950 °C, and some SCs undergo thermal degradation at the high temperature of cigarettes [9]. For example, the cyclopropyl ring in XLR-11 ([1-(5-fluoropentyl)-1H-indole-3-yl](2,2,3,3-tetramethylcyclopropan-1-yl)methanone) and UR-144 ((1-pentyl-1H-indole-3-yl)(2,2,3,3-tetramethylcyclopropan-1-yl)methanone) opens by thermal degradation; moreover, the thermal degradation compounds have been reported to possess higher CB₁ activity than the parent compounds [10, 14]. In contrast to their SCs, the target compound of this study, NPB-22, was subjected to hydrolysis in its ester bond by thermal degradation, and the thermal degradation compounds did not possess CB₁/CB₂ activities.

Electronic cigarettes (e-cigarettes) are used recently by SC abusers [15, 16]. E-cigarettes heat some solutions to generate steam containing compounds (e.g., nicotine, and perfume). In fact, the solution that contains SCs has been reported to be sold in drug markets [17]. The range of heating temperature of e-cigarettes is 30–250 °C [18]. The degradation of NPB-22 was not observed at 250 °C with gas chromatography–mass spectrometry; therefore, when NPB-22 is absorbed using e-cigarettes whose heating temperature is 30–250 °C, it is not expected to undergo thermal degradation, and abusers could absorb more NPB-22 than smoking conventional cigarettes. Therefore, it is suggested that some SCs induce stronger biological effects when being absorbed using e-cigarettes even if they undergo thermal degradation using conventional cigarettes.

NPB-22 had the same functional activity at cannabinoid receptors as Adamantyl-THPINACA. Meanwhile, the inhalation exposure tests showed that some biological effects of NPB-22 were weaker than those of Adamantyl-THPINACA. When analyzing the solution in the midget impingers recovering the smoke from the burned NPB-22 during the inhalation exposure test, approximately 70% of NPB-22 was degraded to 8-quinolinol and pentyl indazole 3-carboxylic acid by combustion. The results showed that the biological effects of NPB-22 were weakened by combustion. However, it has been recently reported that drug abusers absorb SCs using e-cigarettes. Because e-cigarettes heat solutions containing SCs at lower temperatures than conventional cigarettes, the compounds are expected not to undergo thermal degradation. Therefore, even if some new psychoactive substances are degraded thermally by conventional cigarettes and their biological activity is decreased, they could cause health hazards when using new devices for absorbing, such as e-cigarettes.