Mouse Brain Homogenates Research Articles

ODM-201, a new generation androgen receptor inhibitor for castration-resistant prostate cancer: Preclinical and phase I data.

65^ Background: Castration resistant prostate cancer (CRPC) is characterized by persistent, high level androgen receptor (AR) expression and resistance to conventional AR inhibitors. ODM-201 is a novel AR inhibitor with unique pharmacologic properties that has shown promising results in preclinical and clinical studies. Methods: AR binding affinity of ODM-201 to wild type AR was determined in cytosolic lysates obtained from ventral prostates of castrated rats using a competition binding assay. Additionally, effects of ODM-201 on the growth of castration resistant VCaP tumors was evaluated. Tumors were established by subcutaneous injection of VCaP prostate cancer cells into male nude mice. After initial tumor growth, mice were castrated. ODM-201 (50 mg/kg QD or BID orally) was initiated upon tumor regrowth. Since the risk of seizures has been reported for some second generation AR inhibitors, ODM-201 concentrations in mouse brain homogenates were studied after repeated oral administration to assess the risk. The clinical effects of ODM-201 (100, 200, 300, 500, 700, 900 mg BID) was examined in a Phase I/II dose-escalation trial in patients (N=24) with progressive metastatic CRPC. Results: ODM-201 binds to wild type AR with superior affinity compared to enzalutamide (Ki of 9 and 39 nM, respectively). In the VCaP CRPC model, ODM-201 significantly (p<0.001 compared to castrated control), and more efficiently than enzalutamide, inhibited tumor growth. After oral administration, tissue/plasma ratio of ODM-201 in mouse brain homogenates was negligible in all studied doses. In the Phase I/II clinical study, ODM-201 (100 to 700 mg results available) was well tolerated, and most commonly reported adverse events were asthenia, diarrhea and nausea. A PSA response (defined as ≥ 50% decrease) was obtained in 13/15 patients evaluable at 12 weeks with all patients achieving partial response or stable disease by RECIST/PCWG2 criteria. Conclusions: ODM-201 is a new generation AR inhibitor with superior preclinical efficacy compared to enzalutamide and bicalutamide, it does not enter the brain in preclinical studies, and it shows very promising activity and no significant toxicity in patients with CRPC. Clinical trial information: NCT01317641.

Neuroprotective effects of forsythiaside on learning and memory deficits in senescence-accelerated mouse prone (SAMP8) mice

Forsythiaside (3,4-dihydroxy-β-phenethyl-O-α-L-rhamnopyranosyl-(1→6)-4-O-caffeo yl-β-d-glucopyranoside, C29H36O15), which is isolated from air-dried fruits of Forsythia suspensa, has been shown to possess anti-oxidant, anti-bacterial and anti-inflammatory activities. The aim of this study is to investigate the neuroprotective effects of forsythiaside on learning and memory deficits in the senescence-accelerated mouse prone 8 (SAMP8, a model of age-dependent neurodegenerative disorders such as Alzheimer's disease). Forsythiaside (60, 120 and 240mg/kg) was orally administered to aged (8months old) SAMP8 mice for 45days followed by evaluating cognitive impairment (Morris water maze and step-through passive avoidance), inflammation (interleukin-1β (IL-1β) and interleukin-6 (IL-6) levels), oxidative stress (glutathione peroxidase (GSH-Px) and total superoxide dismutase (T-SOD) activities; malondialdehyde (MDA) and nitric oxide (NO) contents) and neurotransmitter such as norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT), glutamate (GLU) gamma-aminobutyric acid (GABA) and acetyl choline (ACh). In Morris water maze, forsythiaside had significantly reduced the latency time, the crossing numbers and time spent in target quadrant compared to aged SAMP8 mice. In passive avoidance test, a significant decline in number of errors while increase in latency was observed when compared with aged SAMP8 mice. Furthermore, a significant decrease in IL-1β, NO, MDA and NE levels, and an increase in T-SOD and GSH-Px activities and GLU and Ach levels were evident in the brain homogenates of forsythiaside-treated mice compared to aged SAMP8 mice. These findings demonstrated that forsythiaside may be a useful treatment against amnesia.

Protective Effect of Luteolin on an Oxidative-Stress Model Induced by Microinjection of Sodium Nitroprusside in Mice

Accumulating lines of evidence showed that luteolin, a polyphenolic compound, has potent neuroprotective effects. The purpose of this study was to examine whether luteolin can protect against sodium nitroprusside (SNP)-induced oxidative damage in mouse brain. Intrastriatal co-injection of luteolin (3 – 30 nmol) with SNP (10 nmol) dose-dependently protected against brain damage and motor dysfunction. Oral administrations of luteolin (600 – 1200 mg/kg) dose-dependently protected against brain damage and motor dysfunction induced by striatal injection of SNP. Furthermore, luteolin (30 – 100 μM) concentration dependently protected against Fe2+-induced lipid peroxidation in mouse brain homogenate. Luteolin (1 – 100 μg/ml) showed potent DPPH radical scavenging ability, when compared with ascorbic acid and glutathione. Finally, a ferrozine assay showed that luteolin (30 – 100 μg/ml) has Fe2+-chelating ability, but this was weaker than that of ethylenediaminetetraacetic acid. These results suggest that intrastriatal or oral administration of luteolin protected mice brain from SNP-induced oxidative damage by scavenging and chelating effects.

Simultaneous Measurement of Serotonin, Dopamine and Their Metabolites in Mouse Brain Extracts by High-Performance Liquid Chromatography with Mass Spectrometry Following Derivatization with Ethyl Chloroformate

In order to measure the levels of serotonin (5-hydroxyltryptamine, 5-HT), dopamine (DA), 3,4-hydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) simultaneously, an effective derivatization method followed by high-performance liquid chromatography (HPLC) coupled to electrochemical ionization mass spectrometry was used. The derivatization reaction of biological samples with ethyl chloroformate occurred rapidly at room temperature in aqueous conditions, and the resulting derivatives were isocratically separated with good selectivity using a C18 reversed-phase column within 30 min. The study results showed that the new derivatization procedure offers an excellent means of simultaneous determination of 5-HT, DA and their metabolites in mouse brain homogenates, which are important in a number of physiological and behavioral functions.

Low-molecular-mass metal complexes in the mouse brain

The presence of labile low-molecular-mass (LMM, defined as <10 kDa) metal complexes in cells and super-cellular structures such as the brain has been inferred from chelation studies, but direct evidence is lacking. To evaluate the presence of LMM metal complexes in the brain, supernatant fractions of fresh mouse brain homogenates were passed through a 10 kDa cutoff membrane and subjected to size-exclusion liquid chromatography under anaerobic refrigerated conditions. Fractions were monitored for Mn, Fe, Co, Cu, Zn, Mo, S and P using an on-line ICP-MS. At least 30 different LMM metal complexes were detected along with numerous P- and S- containing species. Reproducibility was assessed by performing the experiment 13 times, using different buffers, and by examining whether complexes changed with time. Eleven Co, 2 Cu, 5 Mn, 4 Mo, 3 Fe and 2 Zn complexes with molecular masses <4 kDa were detected. One LMM Mo complex comigrated with the molybdopterin cofactor. Most Cu and Zn complexes appeared to be protein-bound with masses ranging from 4-20 kDa. Co was the only metal for which the "free" or aqueous complex was reproducibly observed. Aqueous Co may be sufficiently stable in this environment due to its relatively slow water-exchange kinetics. Attempts were made to assign some of these complexes, but further efforts will be required to identify them unambiguously and to determine their functions. This is among the first studies to detect low-molecular-mass transition metal complexes in the mouse brain using LC-ICP-MS.

Subcellular Fractionation of Mouse Brain Homogenates

This subcellular fractionation protocol is used for separation of cellular organelles based on their density. We have designed and optimized the protocol for separation of subcellular compartments of brain homogenates with focus on the localization and trafficking of transmembrane proteins, but we have also successfully used this protocol for fractionation of other types of tissue. The protocol has two major steps 1) preparation of homogenate from dissected tissue and 2) separation of organelles by centrifugation of homogenates using a continuous sucrose gradient.

The active hunt for ribosomes

This study describes a way to establish the molecular profiles of neurons that respond to a particular stimulus. The authors found that, in mice, stimulus-induced neural activity often leads to a rise in the level of phosphorylated ribosomal protein S6 (pS6). Using specific anti-pS6 antibodies and mouse brain homogenates, they specifically immunoprecipitated ribosomes and their associated mRNAs from neurons in which S6 phosphorylation had been induced. This method might help to reveal the neural mechanisms that underlie various behaviours. For example, through pS6 capture, the authors showed that fasting in mice is linked to galanin neuron activity.

Molecular Profiling of Activated Neurons by Phosphorylated Ribosome Capture

The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, thereby enriching directly for the mRNAs expressed in discrete subpopulations of activated cells. We use this approach to identify neurons in the hypothalamus regulated by changes in salt balance or food availability. We show that galanin neurons are activated by fasting and that prodynorphin neurons restrain food intake during scheduled feeding. These studies identify elements of the neural circuit that controls food intake and illustrate how the activity-dependent capture of cell-type-specific transcripts can elucidate the functional organization of a complex tissue.

Calpain-mediated ataxin-3 cleavage in the molecular pathogenesis of spinocerebellar ataxia type 3 (SCA3)

Spinocerebellar ataxia type 3 (SCA3) is pathologically characterized by the formation of intranuclear aggregates which contain ataxin-3, the mutated protein in SCA3, in a specific subtype of neurons. It has been proposed that ataxin-3 is cleaved by proteolytic enzymes, in particular by calpains and caspases, eventually leading to the formation of aggregates. In our study, we examined the ability of calpains to cleave ataxin-3 in vitro and in vivo. We demonstrated in cell culture and mouse brain homogenates that cleavage of overexpressed ataxin-3 by calpains and in particular by calpain-2 occur and that polyglutamine expanded ataxin-3 is more sensitive to calpain degradation. Based on these results, we investigated the influence of calpains on the pathogenesis of SCA3 in vivo. For this purpose, we enhanced calpain activity in a SCA3 transgenic mouse model by knocking out the endogenous calpain inhibitor calpastatin. Double-mutant mice demonstrated an aggravated neurological phenotype with an increased number of nuclear aggregates and accelerated neurodegeneration in the cerebellum. This study confirms the critical importance of calcium-dependent calpain-type proteases in the pathogenesis of SCA3 and suggests that the manipulation of the ataxin-3 cleavage pathway and the regulation of intracellular calcium homeostasis may represent novel targets for therapeutic intervention in SCA3.

Structure-Based Design, Synthesis and Molecular Modeling Studies of Thiazolyl Urea Derivatives as Novel Anti-Parkinsonian Agents

Synthesis of 1-(substituted aryl)-3-(thiazol-2-yl)urea derivatives was undertaken as our efforts to discover novel antiparkinsonian agents with improved pharmacological profile in haloperidol-induced catalepsy and oxidative stress in mice. Furfuryl, 2- and/or 3-methoxy substituted phenyl derivatives emerged as potent agents. With exception of 2-chloro,5-trifluoromethyl substituted analog, halogen substituted derivatives exhibited moderate antiparkinsonian activity. The results of biochemical investigations from brain homogenate of mice outline the importance of neuroprotective/antioxidant therapy for Parkinson's disease (PD), supporting the notion that the oxidative stress may play a significant role in the pathophysiological mechanisms underlying PD. Molecular docking studies of these compounds with adenosine A(2A) receptor exhibited very good binding interactions and warrants further studies to confirm their binding with human A(2A) receptor for the design and development of potent antagonists. Parameters for Lipinski's rule of 5 were calculated computationally because pharmacokinetic and metabolic behaviors in the body often are linked to the physical properties of a compound. None of the synthesized compounds violated Lipinski's rule, making them suitable drug candidate for the treatment of PD.

Interaction of 2-mercaptobenzimidazole derivatives with sigma-1 receptors

The interaction of afobazole and its main metabolite M-11 {2-[2-(3-oxomorpholin-4-yl)ethylthio]-5-ethoxybenzimidazole hydrochloride} with sigma-1 (σ1) receptors was studied in vitro in radioligand binding experiments on human T-cell lymphoblast-like cell line. Afobazole exhibited ligand properties towards σ1 receptors with IC50 = 7.1 × 10–6 M. For M-11, the corresponding value was IC50 = 9.6×10–4 M. Experiments in vitro on CD-1 mouse brain homogenate established that afobazole interacted with σ1 receptors (IC50 = 1.37×10–5 M) whereas M-11 did not show affinity towards σ1 receptors (IC50 > 5×10–4 M). Radioligand analysis results on mongrel CD-1 mice confirmed the results obtained on human T-cell lymphoblast-like cell line and led to the conclusion that σ1 receptors did not contribute to the pharmacological effects of M-11.

Amelioration of melatonin on oxidative stress and genotoxic effects induced by cisplatin in vitro

In this study, we made an effort to evaluate the possible protective actions of melatonin on cisplatin-induced oxidative damage in mice brain homogenate and genotoxic effects in human lymphocytes under in vitro conditions. The tissue homogenate was divided into three parts. The first portion was kept as control treated with dimethyl sulphoxide (DMSO) (group 1) while the second and third portion were treated with 24 µg/g tissue cisplatin alone (group 2) and 24 µg/g tissue cisplatin in combination with 3 mM melatonin (group 3), respectively. We measured the oxidative stress biomarkers such as lipid peroxidation, 8-hydroxy 2′ deoxyguanosine (8-OHdG) and antioxidant parameters such as reduced glutathione, superoxide dismutase, glutathione peroxidase, and glutathione reductase in brain homogenate. Likewise peripheral venous blood was collected from healthy donors and human lymphocyte culture was done using karyotyping medium. Cultures were divided into three groups. Group 1 was the control i.e. lymphocytes treated with DMSO 5 µg/mL. In group 2, lymphocytes were treated with 2 µg/mL cisplatin and group 3 with a combination of 2 µg/mL cisplatin and 0.3 mM melatonin. Incubation of tissue homogenates with cisplatin elevated the malondialdehyde and 8-OHdG levels which were then reversed by melatonin. Reduction in antioxidant parameters with respect to corresponding controls were also restored by melatonin treatment. Furthermore, supplementation of melatonin was found to modulate the chromosome damage elicited by cisplatin which was determined using Giemsa (GTG) banding and karyotyping. These findings suggest that melatonin improves the cellular function and helps them to survive in the belligerent environment created by free radicals.

Ultrasensitive detection of scrapie prion protein derived from ARQ and AHQ homozygote sheep by interspecies in vitro amplification

Prions, infectious agents causing TSEs, are composed primarily of the pathogenic form (PrP(Sc)) of the PrP(C). The susceptibility of sheep to scrapie is determined by polymorphisms in the coding region of the PRNP, mainly at codons 136, 154, and 171. The efficiency of in vitro amplification of sheep PrP(Sc) seems to be linked also to the PrP genotype. PrP(Sc) derived from sheep with V(136)R(154)Q(171)-associated genotypes can be amplified efficiently by PMCA in the presence of additional polyanion such as poly A, but there are no reports that cite ultrasensitive detection of PrP(Sc) derived from sheep of other PrP genotypes. We report here that sheep PrP(Sc) derived from ARQ and AHQ homozygotes was amplified efficiently by serial PMCA using mouse brain homogenate as PrP(C) substrate. ARQ/ARQ PrP(Sc) was detected in infected brain homogenates diluted up to 10(-10) after five rounds of amplification, and AHQ/AHQ PrP(Sc) was detected in samples diluted up to 10(-8) after four rounds of amplification. On the other hand, amplification of PrP(Sc) from VRQ/ARQ sheep seemed to be less efficient under the experimental conditions used. The interspecies PMCA developed in this study may be useful in the detailed analysis of PrP(Sc) distribution in classical scrapie-infected ARQ and AHQ homozygote sheep.

Novel Indanone Derivatives as Potential Imaging Probes for β‐Amyloid Plaques in the Brain

Molecular imaging probes to detect senile plaques (SPs) might help the early diagnosis of Alzheimer's disease (AD). In this study, a novel series of indanone derivatives were synthesized and characterized. In in vitro binding studies, compound 2e exhibited a K(i) value of 16 nM with a human AD brain homogenate. Although they displayed relatively low affinities for 2i and 2j--with K(i) values of 99 and 237 nM, respectively--the SPs in AD brain sections were positively stained by 2j. A method for in situ micro-autoradiography of AD brain was developed in this study and showed clear labeling of SPs by [(125)I]2i and [(125)I]2j. Both [(125)I]2i and [(125)I]2j had suitable lipophilicities and displayed high initial uptake and rapid clearance from the mouse brains. Furthermore, [(125)I]2i and [(125)I]2j were more stable in human brain homogenates than in mouse brain homogenates. These data suggest that such indanone derivatives might represent potential amyloid imaging agents for the detection of SPs in AD.

A New Class of Highly Potent and Selective Endomorphin-1 Analogues Containing α-Methylene-β-aminopropanoic Acids (Map)

A new class of endomorphin-1 (EM-1) analogues were synthesized by introduction of novel unnatural α-methylene-β-amino acids (Map) at position 3 or/and position 4. Their binding and functional activity, metabolic stability, and antinociceptive activity were determined and compared. Most of these analogues showed high affinities for the μ-opioid receptor and an increased stability in mouse brain homogenates compared with EM-1. Examination of cAMP accumulation and ERK1/2 phosphorylation in HEK293 cells confirmed the agonist properties of these analogues. Among these new analogues, H-Tyr-Pro-Trp-(2-furyl)Map-NH(2) (analogue 12) exhibited the highest binding potency (K(i)(μ) = 0.221 nM) and efficacy (EC(50) = 0.0334 nM, E(max) = 97.14%). This analogue also displayed enhanced antinociceptive activity in vivo in comparison to EM-1. Molecular modeling approaches were then carried out to demonstrate the interaction pattern of these analogues with the opioid receptors. We found that, compared to EM-1, the incorporation of our synthesized Map at position 4 would bring the analogue to a closer binding mode with the μ-opioid receptor.

Striatal-enriched Protein-tyrosine Phosphatase (STEP) Regulates Pyk2 Kinase Activity

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr(1472). Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr(402). In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr(402). STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr(402) and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP.

Sialidase NEU4 Hydrolyzes Polysialic Acids of Neural Cell Adhesion Molecules and Negatively Regulates Neurite Formation by Hippocampal Neurons

Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.

Berberine ameliorates β-amyloid pathology, gliosis, and cognitive impairment in an Alzheimer's disease transgenic mouse model

The accumulation of β-amyloid (Aβ) peptide derived from abnormal processing of amyloid precursor protein (APP) is a common pathological hallmark of Alzheimer's disease (AD) brains. In this study, we evaluated the therapeutic effect of berberine (BBR) extracted from Coptis chinensis Franch, a Chinese medicinal herb, on the neuropathology and cognitive impairment in TgCRND8 mice, a well established transgenic mouse model of AD. Two-month-old TgCRND8 mice received a low (25 mg/kg per day) or a high dose of BBR (100 mg/kg per day) by oral gavage until 6 months old. BBR treatment significantly ameliorated learning deficits, long-term spatial memory retention, as well as plaque load compared with vehicle control treatment. In addition, enzyme-linked immunosorbent assay (ELISA) measurement showed that there was a profound reduction in levels of detergent-soluble and -insoluble β-amyloid in brain homogenates of BBR-treated mice. Glycogen synthase kinase (GSK)3, a major kinase involved in APP and tau phosphorylation, was significantly inhibited by BBR treatment. We also found that BBR significantly decreased the levels of C-terminal fragments of APP and the hyperphosphorylation of APP and tau via the Akt/glycogen synthase kinase 3 signaling pathway in N2a mouse neuroblastoma cells stably expressing human Swedish mutant APP695 (N2a-SwedAPP). Our results suggest that BBR provides neuroprotective effects in TgCRND8 mice through regulating APP processing and that further investigation of the BBR for therapeutic use in treating AD is warranted.

Proteomic identification of aldolase A as an autoantibody target in patients with atypical movement disorders

We tried to identify the target/s of autoantibodies to basal ganglia neurons found in a patient with hyperkinetic movement disorders (HMD) characterized by rapid, rhythmic involuntary movements or spasms in both face and neck. Patient and control sera were used in Western blot to probe mouse brain homogenates. Two-dimensional gel electrophoresis (2-DE) SDS-PAGE protein spots recognized by the patient's antibodies were excised and sequenced by mass spectrometry analysis, and the glycolytic enzyme aldolase A was identified as the antigen recognized by the patient's autoantibodies. To assess relevance and specificity of these antibodies to the identified targets as biomarkers of autoimmunity in movement disorders, autoantibody responses to the identified target were then measured by ELISA in various diseases of the central nervous system. Anti-aldolase A autoantibodies were associated mainly with HMD (7/17, 41%) and Parkinson's disease (4/30, 13%) patients, and undetectable in subjects with other inflammatory and non-inflammatory central nervous system diseases. We, thus, identified aldolase A as an autoantigen in a sub-group of patients with HMD, a clinically ill-defined syndrome. Anti-aldolase A antibodies may represent a useful biomarker of autoimmunity in HMD patients.

Monohydroxylated metabolites of the K2 synthetic cannabinoid JWH-073 retain intermediate to high cannabinoid 1 receptor (CB1R) affinity and exhibit neutral antagonist to partial agonist activity

K2 and several similar purported “incense products” spiked with synthetic cannabinoids are abused as cannabis substitutes. We hypothesized that metabolism of JWH-073, a prevalent cannabinoid found in K2, contributes to toxicity associated with K2 use. Competition receptor binding studies and G-protein activation assays, both performed by employing mouse brain homogenates, were used to determine the affinity and intrinsic activity, respectively, of potential monohydroxylated (M1, M3–M5) and monocarboxylated (M6) metabolites at cannabinoid 1 receptors (CB1Rs). Surprisingly, M1, M4 and M5 retain nanomolar affinity for CB1Rs, while M3 displays micromolar affinity and M6 does not bind to CB1Rs. JWH-073 displays equivalent efficacy to that of the CB1R full agonist CP-55,940, while M1, M3, and M5 act as CB1R partial agonists, and M4 shows little or no intrinsic activity. Further in vitro investigation by Schild analysis revealed that M4 acts as a competitive neutral CB1R antagonist (Kb∼40nM). In agreement with in vitro studies, M4 also demonstrates CB1R antagonism in vivo by blunting cannabinoid-induced hypothermia in mice. Interestingly, M4 does not block agonist-mediated responses of other measures in the cannabinoid tetrad (e.g., locomotor suppression, catalepsy or analgesia). Finally, also as predicted by in vitro results, M1 exhibits agonist activity in vivo by inducing significant hypothermia and suppression of locomotor activity in mice.In conclusion, the present study indicates that further work examining the physiological effects of synthetic cannabinoid metabolism is warranted. Such a complex mix of metabolically produced CB1R ligands may contribute to the adverse effect profile of JWH-073-containing products.