Development and validation of genetic markers for sex and cannabinoid chemotype in Cannabis sativa L.

Hemp (Cannabis sativa L.) is an emerging dioecious crop grown primarily for grain, fiber, and cannabinoids. There is good evidence for medicinal benefits of the most abundant cannabinoid in hemp, cannabidiol (CBD). For CBD production, female plants producing CBD but not tetrahydrocannabinol (THC) are desired. We developed and validated high‐throughput PACE (PCR Allele Competitive Extension) assays for C. sativa plant sex and cannabinoid chemotype. The sex assay was validated across a wide range of germplasm and resolved male plants from female and monoecious plants. The cannabinoid chemotype assay revealed segregation in hemp populations, and resolved plants producing predominantly THC, predominantly CBD, and roughly equal amounts of THC and CBD. Cultivar populations that were thought to be stabilized for CBD production were found to be segregating phenotypically and genotypically. Many plants predominantly producing CBD accumulated more than the current US legal limit of 0.3% THC by dry weight. These assays and data provide potentially useful tools for breeding and early selection of hemp.     

Effects of infection of EGFP-expressing Escherichia coli on haemocytes in Ciona intestinalis

The effects of infection of EGFP-expressing Escherichia coli on the haemocytes of the ascidian Ciona intestinalis were investigated. The results showed that THC of the infected individuals changed significantly. Hyaline amoebocytes phagocytosed E. coli in 5 min and excreted lysosome particles that attached to the surface of the bacteria. Granular amoebocytes released lots of particles for humoral immunity while stem-cell-like haemocytes remained intact. With the increase in THC, the stem-cell-like haemocytes showed division and proliferation. A small portion of hyaline amoebocytes was at early apoptosis stage 1 h after infection and typical apoptosis bodies emerged in granular amoebocytes. A few of the infected haemocytes showed DNA damage using SCGE assay. Flow cytometry analysis revealed an obvious apoptosis peak in infected haemocytes. In conclusion, apoptosis was found to be an important immune response of ascidian haemocytes response to bacterial infection. To our best knowledge, this is the first report of the occurrence of apoptosis of haemocytes in ascidians.     

The psychoactive substance of cannabis Δ9-tetrahydrocannabinol (THC) negatively regulates CFTR in airway cells

Background

Marijuana consumption is on the rise in the US but the health benefits of cannabis smoking are controversial and the impact of cannabis components on lung homeostasis is not well-understood. Lung function requires a fine regulation of the ion channel CFTR, which is responsible for fluid homeostasis and mucocilliary clearance. The goal of this study was to assess the effect that exposure to Δ9-tetrahydrocannabinol (THC), the psychoactive substance present in marijuana, has on CFTR expression and function.

The Endocannabinoid System and Extinction Learning

The endocannabinoid system has emerged as a versatile neuromodulatory system, implicated in a plethora of physiological and pathophysiological processes. Cannabinoid receptor type 1 (CB1 receptor) and endocannabinoids are widely distributed in the brain. Their roles in learning and memory have been well documented, using rodents in various memory tests. Depending on the test, the endocannabinoid system is required in the acquisition and/or extinction of memory. In particular, the activation of CB1 receptor-mediated signaling is centrally involved in the facilitation of behavioral adaptation after the acquisition of aversive memories. As several human psychiatric disorders, such as phobia, generalized anxiety disorders, and posttraumatic stress disorder (PTSD) appear to involve aberrant memory processing and impaired adaptation to changed environmental conditions, the hope has been fuelled that the endocannabinoid system might be a valuable therapeutic target for the treatment of these disorders. This review summarizes the current data on the role of the endocannabinoid system in the modulation of extinction learning.     

Preformulation Studies of a Prodrug of Δ9-Tetrahydrocannabinol

Preformulation studies were performed on a hemiglutarate ester prodrug of Δ⁹-tetrahydrocannabinol (THC-HG), to facilitate the development of stable formulations by hot-melt methods. The various studies performed included solid-state thermal characterization, pKa, logP, aqueous and pH dependent solubility, pH stability and effect of moisture, temperature and oxygen on solid-state stability. A hot-melt method was utilized to fabricate THC-HG incorporated poly (ethylene oxide) (PEO) matrices and the bioadhesive properties, release profiles and post-processing stability of these matrices were assessed as a function of the polymer molecular weight. The prodrug exhibited a T _g close to 0°C, indicating its amorphous nature. Thermogravimetric analysis revealed a rapid weight loss after 170°C. The prodrug exhibited a seven-fold higher aqueous solubility as compared to the parent drug (THC). Also, the solubility of the compound increased with increasing pH, being maximum at pH 8. The prodrug exhibited a v-shaped pH-rate profile, with the degradation rate minimum between pH 3 and 4. The moisture uptake and drug degradation increased with an increase in relative humidity. Solid-state stability indicated that the prodrug was stable at −18°C but demonstrated higher degradation at 4°C, 25°C and 40°C (51.6%, 74.5% and 90.1%, respectively) at the end of 3-months. THC-HG was found to be sensitive to the presence of oxygen. The release of the active from the polymeric matrices decreased, while bioadhesion increased, with an increase in molecular weight of PEO.     

Synthesis and pharmacology of the isomeric methylheptyl-Δ-tetrahydrocannabinols

The synthesis of the 3-heptyl, and the eleven isomeric 3-methylheptyl-Δ⁸-tetrahydrocannabinols (3–7, R and S methyl epimers, and 8) has been carried out. The synthetic approach entailed the synthesis of substituted resorcinols, which were subjected to acid catalyzed condensation with trans-para-menthadienol to provide the Δ⁸-THC analogue. The 1′-, 2′- and 3′-methylheptyl analogues (3–5) are considerably more potent than Δ⁸-THC. The 4′-, 5′- and 6′-methylheptyl isomers (6–8) are approximately equal in potency to Δ⁸-THC.     

The inhibitory action of exo- and endocannabinoids on [³H]GABA release are mediated by both CB₁and CB₂receptors in the mouse hippocampus

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.     

Cannabinoid-hormone interactions in the regulation of motivational processes

There is a bi-directionality in hormone-cannabinoid interactions: cannabinoids affect prominent endocrine axes (such as the hypothalamic-pituitary-gonadal), and gonadal hormones modulate cannabinoid effects. This review will summarize recent research on these interactions, with a specific focus upon their implications for motivated behavior. Sexual behavior will serve as a “case study.” I will explore the hypothesis that ovarian hormones, in particular estradiol, may serve to release estrous behavior from endocannabinoid inhibition. Hormonal regulation of the endogenous cannabinoid system also affects processes that underlie drug abuse. This review will briefly discuss sex differences in behavioral responses to cannabinoids and explore potential mechanisms by which gonadal hormones alter cannabinoid reward. An examination of this research informs our perspective on how hormones and endocannabinoids may affect drug-seeking behavior as a whole and the development of addiction.     

Male-female differences in the effects of cannabinoids on sexual behavior and gonadal hormone function

The putative role of the endocannabinoid system and the effects of cannabis use in male and female sexual functioning are summarized. The influence of cannabis intake on sexual behavior and arousability appear to be dose-dependent in both men and women, although women are far more consistent in reporting facilitatory effects. Furthermore, evidence from nonhuman species indicate somewhat more beneficial than debilitating effects of cannabinoids on female sexual proceptivity and receptivity while suggesting predominantly detrimental effects on male sexual motivation and erectile functioning. Data from human and nonhuman species converge on the ephemeral nature of THC-induced testosterone decline. However, it is clear that cannabinoid-induced inhibition of male sexual behavior is independent of concurrent declines in testosterone levels. Investigations also reveal a suppression of gonadotropin release by cannabinoids across various species. Historical milestones and promising future directions in the area of cannabinoid and sexuality research are also outlined in this review.