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

PURPOSE: NPB-22 (quinolin-8-yl 1-pentyl-1H-indazole-3-carboxylate), Adamantyl-THPINACA (N-(1-adamantantyl)-1-[(tetrahydro-2H-pyran-4-yl)methyl]-1H-indazole-3-carboxamide), and CUMYL-4CN-B7AICA (1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)-1H- pyrrolo[2,3-b]pyridine-3-carboxamide), synthetic cannabinoids were evaluated in terms of CB1 (cannabinoid receptor type 1) and CB2 (cannabinoid receptor type 2) activities, and their biological effects when inhaled similar to cigarettes were examined. METHODS: The half maximal effective concentration values of the aforementioned synthetic cannabinoids at the CB1 and CB2 were investigated using [35S]guanosine-5'-O-(3-thio)-triphosphate binding assays. In addition, their biological effects were evaluated using the inhalation exposure test with mice. The smoke generated was recovered by organic solvents in the midget impingers, and the thermal degradation compounds of the smoke components were identified and quantified using a liquid chromatography-photo diode array detector. RESULTS: NPB-22 and Adamantyl-THPINACA had equivalent CB1 activity in in vitro assays. Meanwhile, NPB-22 had a weaker biological effect on some items on the inhalation exposure test than Adamantyl-THPINACA. When analyzing organic solvents in the midget impingers, it was revealed that NPB-22 was degraded to 8-quinolinol and pentyl indazole 3-carboxylic acid by combustion. In addition, these degradation compounds did not have CB1 activity. CONCLUSION: It was estimated that the biological effects of NPB-22 on the inhalation exposure test weakened because it underwent thermal degradation by combustion, and the resultant degradation compounds did not have any CB1 activity in vitro.

Different effects of crizotinib treatment in two non-small cell lung cancer patients with SDC4::ROS1 fusion variants.

The possibility of stratifying patients according to differences in ROS proto-oncogene 1 (ROS1) fusion partners has been discussed. This study aimed to clarify the clinicopathological differences between two SDC4::ROS1 positive NSCLC cases who had different responses to crizotinib. Cytology and pathology samples from two NSCLC cases with SDC4::ROS1 who were diagnosed and treated with crizotinib at Nihon University Itabashi Hospital were obtained. Case 1 has been well-controlled with crizotinib for over 5 years, but case 2 was worse and overall survival was 19 months. Sequencing analysis of ROS1 fusion genes was performed by reverse-transcription-PCR and Sanger's sequencing methods. In addition, thyroid transcription factor (TTF)-1, ROS-1, Ki67, and phosphorylated extracellular signal-regulated kinase (pERK)1/2 expression were investigated using immunohistochemistry. Sequencing analysis showed SDC4 exon2::ROS1 exon 32 (exon33 deleted) in case 1, and coexistence of SDC4 exon2::ROS1 exon 34 and SDC4 exon2::ROS1 exon35 in case 2. The Ki67 index was not different, but ROS1 and pERK1/2 expression levels tended to be higher in the tumor cells of case 2 than in case 1. Therapeutic response to crizotinib and patients' prognosis in ROS1 rearranged NSCLC may be related to the activation of ROS1 signaling, depending on ROS1 and pERK1/2 overexpression status, even if the ROS1 fusion partner is the same.

SPARC is a decoy counterpart for c­Fos and is associated with osteoblastic differentiation of bone marrow stromal cells by inhibiting adipogenesis.

Secreted protein acidic and rich in cysteine (SPARC), also called basement­membrane protein 40 or osteonectin, is a matricellular protein that is abundant not only in bone tissue as a non­collagenous protein but is also ubiquitously expressed in non­calcified tissue. SPARC is located intracellularly and disruption of the Sparc gene has been reported to reduce bone formation and increase fat tissue; however, the mechanism by which SPARC inhibits adipogenesis remains unclear. The present study evaluated the intracellular function of SPARC in adipogenesis using the bone marrow stromal cell line ST2. When ST2 cells with low SPARC production were cloned, intrinsic activator protein­1 (AP­1) activity was markedly higher, mineralized nodule formation was significantly lower and lipid accumulation was significantly increased compared with in the parental ST2 cells. Forced expression of secreted SPARC with the signal peptide­coding sequences of wild­type Sparc or preprotrypsin in SPARC­low ST2 cells significantly reduced AP­1 transcription activity; however, these reductions were not observed in the absence of signal peptide sequences. Recombinant SPARC, produced using Brevibacillus brevis, specifically bound to c­Fos but not c­Jun and inhibited the binding of c­Fos/c­Jun to a TPA­response element sequence. These data suggested that SPARC was incorporated into the cells from the extracellular spaces and serves an intracellular role as a decoy counterpart for c­Fos, as well as being associated with osteoblastogenesis through the inhibition of adipogenesis. These findings may provide new insights into regenerative medicine.

MRI Contrasting Agent Based on Mn-MOF-74 Nanoparticles with Coordinatively Unsaturated Sites.

PURPOSE: The development of magnetic resonance imaging (MRI) contrasting agents (CAs) that are safer and have a higher relaxivity than Gd(III)-based agents is a significant research topic. Herein, we propose the use of a Mn-based metal organic framework (MOF), Mn-MOF-74, characterized by a safe paramagnetic center, a coordinatively unsaturated site (CUS) for aquation, and a long rotational correlation time, endowing high relaxivity. Furthermore, biocompatibility and delivery to the tumor are generally expected for MOFs that are obtainable in the nanometer size range. PROCEDURE: Drop-wise mixing of 2,5-dihydroxyterephthalic acid (DHTP) and Mn(II) acetate yielded Mn-MOF-74 with a diameter of < 150 nm, which was then modified with 1-fivefold higher amounts of poly(ethylene glycol) (M.W. = 5000) to afford MOFs stably dispersed in water for at least 24 h. RESULTS: The longitudinal and transverse relaxivity of the PEG-modified MOF was in the range of r1 = 8.08-13.5 and r2 = 32.7-46.8 mM-1 s-1, respectively (1.0 T, 23.7-23.9 °C), being larger than those of typical Gd(III)- and Mn(II)-based CAs of single-nuclear metal complexes. The in vivo imaging of a tumor-bearing mouse clearly showed that the tumor could be readily recognized due to signal enhancement (117%) in T1-weighted images, whereas other tissues showed small signal changes. CONCLUSIONS: These results suggest that PEG-Mn-MOF-74 can be passively delivered to tumors and can act as a high-relaxivity T1 agent.

Emergence of the super antenna effect in mixed crystals of ytterbium and lutetium complexes showing near-infrared luminescence.

The synthesis of luminescent molecular crystalline materials requires a good understanding of the luminescence properties of crystals in which many molecules are densely packed. Previously, we studied the near-infrared (NIR) luminescence of a trivalent ytterbium (Yb(iii)) complex with a Schiff base ligand, tris[2-(5-methylsalicylideneimino)ethyl]amine (H3L). Herein, we extended our study on the Yb complex (YbL) to enhance and understand its solid-state luminescence via mixed crystallization with the lutetium complex (LuL). We prepared (YbL) x (LuL)1-x mixed crystals (x = 0.01, 0.05, 0.1, 0.2, 0.3, 0.5, and 0.7) and studied their NIR luminescence properties. The NIR luminescence intensity per Yb(iii) ion for (YbL)0.01(LuL)0.99 was determined to be two orders of magnitude larger than that for YbL. The excitation spectral shape of (YbL)0.01(LuL)0.99 was different from the absorption spectral shape of YbL but similar to that of LuL. We attribute this observation to the emergence of an intermolecular energy-migration path. In the mixed crystals, LuL molecules acted as a light-harvesting super antenna for Yb(iii) luminescence. Decay measurements of the NIR luminescence for (YbL) x (LuL)1-x with x > 0.2 showed mono-exponential decay, while (YbL) x (LuL)1-x with x < 0.1 showed a grow-in component, which reflected the lifetime of the intermediate state for energy migration. The decay lifetime values tended to increase with decreasing x, suggesting that Yb(iii) isolation resulted in a reduction in concentration quenching. We propose that the luminescence enhancement in the highly Yb-diluted conditions was mainly caused by an increase in the super antenna effect.

Selective crystallization of dysprosium complex from neodymium/dysprosium mixture enabled by cooperation of coordination and crystallization.

Designing a molecular-level Ln3+ separation system remains a challenge for developing next-generation separation methodologies. Herein, we report crystallization-based Nd3+/Dy3+ separation using a tripodal Schiff base ligand. Highly selective crystallization of the Dy3+ complex was enabled by cooperation between the coordination and crystallization processes.

Short Radiative Lifetime and Non-Triplet Sensitization in Near-Infrared-Luminescent Yb(III) Complex with Tripodal Schiff Base.

We prepared Ln(III) (Ln=Eu, Gd, and Yb) complexes with a tripodal Schiff base, tris[2-(5-methylsalicylideneimino)ethyl]amine (H3 L) and studied their photophysical properties. Upon ligand excitation, YbL showed Yb(III)-centered luminescence in the near-infrared region. While the overall quantum yield (0.60(1)%) of YbL in acetonitrile was moderate among the reported values for Yb(III) complexes, its radiative lifetime (0.33(2) ms) was significantly shorter than those reported previously. We propose that the ligand-to-metal charge-transfer (LMCT) state mediated the sensitization in YbL. The emission and excitation spectra of EuL indicated the participation of the LMCT state in the sensitization. The radiative lifetime (0.84(7) ms) for EuL in the solid state was rather short compared to those of reported Eu(III) complexes. Our results show that the Yb(III) complex with the Schiff base ligand has two features: the short radiative lifetime and the non-triplet sensitization path.

Intracellular zinc-dependent TAS2R8 gene expression through CTCF activation.

Taste-2 receptors (TAS2Rs), which belong to the G-protein coupled receptor (GPCR) family, are receptors for bitter taste perception. The aim of this study was to investigate whether zinc deficiency affects the expression of TAS2R genes. The promoter activity of the TAS2R7, TAS2R8, and TAS2R42 genes were determined in Ca9-22 oral squamous cell carcinoma cells cultured in the presence or absence of zinc. Luciferase reporter assays showed that zinc deprivation inhibited TAS2R8 promoter activity, but not the promoter activity of the other two genes. Treatment of the cells with N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), an intracellular chelator of Zn2+, in the presence of 10% fetal bovine serum reduced TAS2R8 promoter activity. Truncation/deletion mutants of TAS2R8 promoter-luciferase constructs showed that the region from nucleotide -1152 to nucleotide -925 was critical for intracellular zinc dependency and contained a CCCTC-binding factor (CTCF) binding motif. A chromatin immunoprecipitation (ChiP) assay showed that CTCF bound specifically to this region, a binding abrogated by zinc deficiency, suggesting that CTCF plays a critical role in zinc-dependent bitter taste perception through TAS2R8.

Influence of cigarette smoking on biventricular systolic function independent of respiratory function: a cross-sectional study.

BACKGROUND: Cigarette smoking harms nearly every organ, including the heart and lungs. A comprehensive assessment of both cardiac and respiratory function is necessary for evaluating the direct effects of tobacco on the heart. However, few previous studies examining the effects of cigarette smoking on cardiac function included an assessment of lung function. This cross-sectional study investigated the influence of cigarette smoking on cardiac function, independent of respiratory function. METHODS: We retrospectively reviewed the medical records of 184 consecutive cases that underwent both spirometry and transthoracic echocardiography around the same time (within 1 month) in one hospital from April 2019 to March 2020. Participants were classified into three groups based on lifetime smoking exposure (pack-years): non-smoker (n = 49), low exposure (1-20 pack-years, n = 40), and high exposure (≥ 20 pack years, n = 95). Multiple linear regression analysis was used to assess the relationship among cigarette smoking, and cardiac and respiratory functions. The relationship between selected dependent variables and lifetime pack-years was assessed in two models with multiple linear regression analysis. Model 1 was adjusted for age and male sex; and Model 2 was adjusted for Model 1 plus forced expiratory volume percentage in 1 s and forced vital capacity percentage. RESULTS: Compared with the non-smokers, the participants with high smoking exposure had lower left ventricular (LV) systolic function and larger LV size. Multiple linear regression analysis revealed a negative association of cumulative lifetime pack-years with LV and right ventricular (RV) systolic functions, even after adjustment for age, sex, and spirometric parameters (forced expiratory volume percentage in 1 s and forced vital capacity percentage). Meanwhile, there was no significant association of smoking exposure with LV diastolic function (E/e' and E/A) and RV diastolic function (e't and e't/a't). CONCLUSIONS: Cumulative smoking exposure was associated with a negative effect on biventricular systolic function in patients with relatively preserved cardiac function, independent of respiratory function.

Cardiac events in Patients in their forties with Kawasaki disease and regression of coronary artery aneurysms.

Over a 50-year period from the first description of Kawasaki disease, we encountered three male patients with a history of Kawasaki disease, who had their first cardiac events in their forties. They were considered to have almost normal coronary arteries in the coronary angiograms when they were children and adolescents. They had no follow-up examinations after 20 years old. The 1st patient had an acute myocardial infarction, and the 2nd was a new appearance of coronary aneurysm and stenotic lesions with coronary artery calcification. The 3rd patient had unexpected sudden death. The interval from the onset of Kawasaki disease to the cardiac events ranged from 37 to 38 years. In the former two patients, coronary artery lesions could not be evaluated immediately after Kawasaki disease. Although the 3rd patient had bilateral medium-sized coronary artery aneurysms, his coronary aneurysms regressed 1 year after acute Kawasaki disease. The intimal thickening at a previous coronary aneurysm at the age of 19 was mild. The patients with regressed coronary aneurysms were asymptomatic for about 40 years after Kawasaki disease, prior to their cardiac events. Coronary artery calcification of the proximal portion of the major coronary arteries was a predictable marker in such patients. To prevent serious cardiac events in middle-aged adult patients, reevaluation of coronary artery lesions and restarting of anti-thrombotic therapy are needed. We must be aware that there are some differences in the clinical course and time of cardiac events between patients with giant aneurysms and those with medium aneurysms.

Adaptation to chronic acidic extracellular pH elicits a sustained increase in lung cancer cell invasion and metastasis.

Acidic extracellular pH (pHe) is an important microenvironment for cancer cells. This study assessed whether adaptation to acidic pHe enhances the metastatic phenotype of tumor cells. The low metastatic variant of Lewis lung carcinoma (LLCm1) cells were subjected to stepwise acidification, establishing acidic pHe-adapted (LLCm1A) cells growing exponentially at pH 6.2. These LLCm1A cells showed increased production of matrix metalloproteinases (MMPs), including MMP-2, -3, -9, and -13, and pulmonary metastasis following injection into mouse tail veins. Although LLCm1A cells exhibited a fibroblastic shape, keratin-5 expression was increased and α-smooth muscle actin expression was reduced. Despite serial passage of these cells at pH 7.4, high invasive activity through Matrigel® was sustained for at least 28 generations. Thus, adaptation to acidic pHe resulted in a more invasive phenotype, which was sustained during passage at pH 7.4, suggesting that an acidic microenvironment at the primary tumor site is important in the acquisition of a metastatic phenotype.

Photostable near-infrared-absorbing diradical-platinum(ii) complex solubilized by albumin toward a cancer photothermal therapy agent.

A hydrophobic diradical-platinum(ii) complex was solubilized in aqueous solutions by using bovine serum albumin and exhibited photothermal conversion under near-infrared (NIR) light irradiation. The complex was introduced into cancer cells and induced cell death upon absorption of NIR. These results imply that the complex can function as a photothermal therapeutic agent.

A new class of pentapeptide KISS1 receptor agonists with hypothalamic-pituitary-gonadal axis activation.

The kisspeptin (Kp, Kp-54, metastin)/KISS1R system plays crucial roles in regulating the secretion of gonadotropin-releasing hormone. Continuous administration of nonapeptide Kp analogs caused plasma testosterone depletion, whereas bolus administration caused strong plasma testosterone elevation in male rats. To develop a new class of small peptide drugs, we focused on stepwise N-terminal truncation of Kp analogs and discovered potent pentapeptide analogs. Benzoyl-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (16) exhibited high agonist activity for KISS1R and excellent metabolic stability in rat serum. A single injection of a 4-pyridyl analog (19) at the N-terminus of 16 into male Sprague Dawley rats caused a robust increase in plasma luteinizing hormone levels, but unlike continuous administration of nonapeptide Kp analogs, continuous administration of 19 maintained moderate testosterone levels in rats. These results indicated that small peptide drugs can be successfully developed for treating sex hormone deficiency.

Efficacy of a Moisturizer for Pruritus Accompanied With Asteatosis in Dialysis Patients: An Open-Label, Randomized, Exploratory Study.

BACKGROUND: In dialysis patients, skin disorders (dryness and itching) are frequently observed and treated with a moisturizer, in the absence of clear evidence of efficacy. STUDY DESIGN: An open-label, randomized, before/after, parallel-group, comparative/exploratory study. SETTING & PARTICIPANTS: 12 Japanese patients with chronic kidney failure undergoing maintenance hemodialysis who presented with dry skin and itching. INTERVENTION: Patients received a topical heparinoid moisturizer as the study drug for 2 weeks from the first day of the study treatment, followed by either a 2-week washout (group A: 6 participants) or further 2-week treatment (group B: 6 participants). OUTCOMES: The primary end point was change in water content in the stratum corneum in the hypochondrium. Secondary end points included change in visual analogue scale itching score and subjective evaluations of symptoms. To evaluate safety, adverse events were also investigated. MEASUREMENTS: Water content of the stratum corneum, dryness/itching improvement rating, itching visual analogue scale/duration of itching, photographic evaluation of skin symptoms, principal investigator's overall assessment of study drug, and adverse events. RESULTS: Mean water content of the stratum corneum in the combined groups significantly increased at week 2 (51.2 arbitrary units [AU] vs treatment start day, 31.6 AU; P<0.001), but significantly decreased at week 4 in group A, in which patients discontinued treatment with the study drug (39.4 AU; P = 0.005). Other efficacy end points, including the visual analogue scale itching score, were also improved by treatment with the study drug, but such improvement was not sustained after discontinuation of treatment. There were no adverse events related to the study treatment. LIMITATIONS: Only Japanese patients were included in the study, with a small sample size. CONCLUSIONS: Continuous application of the topical heparinoid moisturizer increased water content in the stratum corneum and lessened itching in dialysis patients. FUNDING: Maruho Co, Ltd. TRIAL REGISTRATION: Registered at the University Hospital Medical Information Network Clinical Trials Registry with study number UMIN000017016.

Effect of Asymptomatic Severe Aortic Stenosis on Outcomes of Individuals Aged 80 and Older.

OBJECTIVES: To examine the effect of asymptomatic severe aortic stenosis (AS) on mortality late in life. DESIGN: Retrospective cohort study. SETTING: Large medical center. PARTICIPANTS: Asymptomatic adults aged 80 and older (mean age 86 ± 4; N=1,060, n=569 women) with preserved left ventricular ejection fraction (LVEF; > 50%); 927 (87.5%) with no AS, 70 (6.6%) with nonsevere AS, and 63 (5.9%) with severe AS. MEASUREMENTS: Information was collected on demographic characteristics, comorbidities, and laboratory and echocardiographic data. Survival was assessed according to longest follow-up available. RESULTS: During a mean follow-up of 2.2 ± 2.3 years, there were 203 (19%) deaths, 51 of which were from cardiovascular causes. Four-year estimates of survival were 72% for no AS, 58% for nonsevere AS, and 23% for severe AS (p<.001). Univariable analysis showed that asymptomatic severe AS was significantly associated with greater risk of all-cause mortality (hazard ratio (HR)=3.06, 95% confidence interval (CI)=1.96-4.58, p<.001). After adjustment for age, sex, LVEF, hypertension, dyslipidemia, diabetes, atrial fibrillation, chronic kidney disease, and coronary artery disease, asymptomatic severe AS was an independent predictor of all-cause mortality (HR=3.16, 95% CI=1.97-4.88, p<.001). CONCLUSION: Asymptomatic severe AS has a major effect on prognosis even in very old adults.

Cancer metabolism: New insights into classic characteristics.

Initial studies of cancer metabolism in the early 1920s found that cancer cells were phenotypically characterized by aerobic glycolysis, in that these cells favor glucose uptake and lactate production, even in the presence of oxygen. This property, called the Warburg effect, is considered a hallmark of cancer. The mechanism by which these cells acquire aerobic glycolysis has been uncovered. Acidic extracellular fluid, secreted by cancer cells, induces a malignant phenotype, including invasion and metastasis. Cancer cells survival depends on a critical balance of redox status, which is regulated by amino acid metabolism. Glutamine is extremely important for oxidative phosphorylation and redox regulation. Cells highly dependent on glutamine and that cannot survive with glutamine are called glutamine-addicted cells. Metabolic reprogramming has been observed in cancer stem cells, which have the property of self-renewal and are resistant to chemotherapy and radiotherapy. These findings suggest that studies of cancer metabolism can reveal methods of preventing cancer recurrence and metastasis.

Prolyl isomerase Pin1 is required sperm production by promoting mitosis progression of spermatogonial stem cells.

A prolyl isomerase Pin1 deficient (Pin1-/-) male mice had severe testicular atrophy. We investigated the function of Pin1 in spermatogenesis by analyzing the Pin1-/- mice at reproductive age. Pin1-/- mice had lessαPLZF positive spermatogonia (undifferentiated spermatogonia) than wild type (WT). Nevertheless, the Pin1-/- testis contained approximately the same number of GFRα1 positive spermatogonia (SSCs in steady state) as the WT testis. Furthermore, degeneration of the spermatogenia appeared in seminiferous tubules of 10 months old Pin1-/- mouse testis, and abnormal shape GFRα1 positive spermatogonia were observed. In Pin1-/- spermatogonia, the ratio of the phospho-histone H3 positive cells (mitotic cells) in GFRα1-positive spermatogonia was higher than that of WT. These results suggest that Pin1 promotes the progression of the mitotic cell cycle of SSC in steady-state, which is required for the sperm production from SSCs.

TRPM5 mediates acidic extracellular pH signaling and TRPM5 inhibition reduces spontaneous metastasis in mouse B16-BL6 melanoma cells.

Extracellular acidity is a hallmark of solid tumors and is associated with metastasis in the tumor microenvironment. Acidic extracellular pH (pH e ) has been found to increase intracellular Ca2+ and matrix metalloproteinase-9 (MMP-9) expression by activating NF-κB in the mouse B16 melanoma model. The present study assessed whether TRPM5, an intracellular Ca2+-dependent monovalent cation channel, is associated with acidic pH e signaling and induction of MMP-9 expression in this mouse melanoma model. Treatment of B16 cells with Trpm5 siRNA reduced acidic pH e -induced MMP-9 expression. Enforced expression of Trpm5 increased the rate of acidic pH e -induced MMP-9 expression, as well as increasing experimental lung metastasis. This genetic manipulation did not alter the pH e critical for MMP-9 induction but simply amplified the percentage of inducible MMP-9 at each pH e . Treatment of tumor bearing mice with triphenylphosphine oxide (TPPO), an inhibitor of TRPM5, significantly reduced spontaneous lung metastasis. In silico analysis of clinical samples showed that high TRPM5 mRNA expression correlated with poor overall survival rate in patients with melanoma and gastric cancer but not in patients with cancers of the ovary, lung, breast, and rectum. These results showed that TRPM5 amplifies acidic pH e signaling and may be a promising target for preventing metastasis of some types of tumor.

Inhalation Exposure Method for Illegal Drugs.

We developed a new inhalation exposure method to evaluate effects of synthetic cannabimimetics that are being distributed as new, unregulated drugs in the Tokyo area. We selected the commercial product "SOUTOU" containing AB-CHMINACA and 5F-AMB as the test drug and dried marshmallow (Althaea officinalis) leaves as the negative control. A half cigarette packed with dried marshmallow leaves or SOUTOU was ignited, then mainstream smoke from each was delivered to five mice in an exposure box. After the cigarettes were fully consumed, neurobehavioral observations and a catalepsy test were performed at 15, 30 and 60 min after exposure. The effluent air from the exposure box was poured into impingers containing acetonitrile (first impinger) and dimethyl sulfoxide (second impinger). The resulting solutions were analyzed to assess decomposition of the synthetic cannabimimetics. Mice exposed to SOUTOU smoke showed many excitement behaviors and some suppressive behaviors at 15, 30 and 60 min. These clearly included cannabimimetic specific pharmacological actions. Negative control mice also showed some suppressive behaviors at 15 min but these were attenuated at later times, nearly disappearing at 60 min. In addition, the behavioral effects observed in controls were less pronounced than those in SOUTOU exposed mice. The inhalation exposure method developed in our study would be effective for determining cannabinoid specific pharmacological effects of illegal drugs, as well as for assessing the presence of active compound(s) by comparing the test substance with a negative control.