Cannabinoid Receptor Type II Ligands from Sandalwood Oil and Synthetic α-Santalol Derivatives.

Bioassay-guided fractionation of the essential oil of Santalum album led to the identification of α-santalol (1) and ß-santalol (2) as new chemotypes of cannabinoid receptor type II (CB2) ligands with Ki values of 10.49 and 8.19 µM, respectively. Nine structurally new α-santalol derivatives (4a-4h and 5) were synthesized to identify more selective and potent CB2 ligands. Compound 4e with a piperazine structural moiety demonstrated a Ki value of 0.99 µM against CB2 receptor and did not show binding activity against cannabinoid receptor type I (CB1) at 10 µM. Compounds 1, 2, and 4e increased intracellular calcium influx in SH-SY5Y human neuroblastoma cells that were attenuated by CB2 antagonism or inverse agonism, supporting the results that these compounds are CB2 agonists. Molecular docking showed that 1 and 4e had similar binding poses, exhibiting a unique interaction with Thr114 within the CB2 receptor, and that the piperazine structural moiety is required for the binding affinity of 4e. A 200 ns molecular dynamics simulation of CB2 complexed with 4e confirmed the stability of the complex. This structural insight lays a foundation to further design and synthesize more potent and selective α-santalol-based CB2 ligands for drug discovery.

Neuronal and Astrocyte Insulin-like Growth Factor-1 Signaling Differentially Modulates Ischemic Stroke Damage.

Ischemic stroke is a leading cause of death and disability, as therapeutic options for mitigating the long-term deficits precipitated by the event remain limited. Acute administration of the neuroendocrine modulator insulin-like growth factor-1 (IGF-1) attenuates ischemic stroke damage in preclinical models, and clinical studies suggest IGF-1 can reduce the risk of stroke and improve overall outcomes. The cellular mechanism by which IGF-1 exerts this protection is poorly defined, as all cells within the neurovascular unit express the IGF-1 receptor. We hypothesize that the functional regulation of both neurons and astrocytes by IGF-1 is critical in minimizing damage in ischemic stroke. To test this, we utilized inducible astrocyte-specific or neuron-specific transgenic mouse models to selectively reduce IGF-1R in the adult mouse brain prior to photothrombotic stroke. Acute changes in blood brain barrier permeability, microglial activation, systemic inflammation, and biochemical composition of the brain were assessed 3 hours following photothrombosis, and significant protection was observed in mice deficient in neuronal and astrocytic IGF-1R. When the extent of tissue damage and sensorimotor dysfunction was assessed for 3 days following stroke, only the neurological deficit score continued to show improvements, and the extent of improvement was enhanced with additional IGF-1 supplementation. Overall, results indicate that neuronal and astrocytic IGF-1 signaling influences stroke damage but IGF-1 signaling within these individual cell types is not required for minimizing tissue damage or behavioral outcome.

Age-related neuroendocrine, cognitive, and behavioral co-morbidities are promoted by HIV-1 Tat expression in male mice.

In the U.S. about half of the HIV-infected individuals are aged 50 and older. In men living with HIV, secondary hypogonadism is common and occurs earlier than in seronegative men, and its prevalence increases with age. While the mechanisms(s) are unknown, the HIV-1 trans-activator of transcription (Tat) protein disrupts neuroendocrine function in mice partly by dysregulating mitochondria and neurosteroidogenesis. We hypothesized that conditional Tat expression in middle-aged male transgenic mice [Tat(+)] would promote age-related comorbidities compared to age-matched controls [Tat(-)]. We expected Tat to alter steroid hormone milieu consistent with behavioral deficits. Middle-aged Tat(+) mice had lower circulating testosterone and progesterone than age-matched controls and greater circulating corticosterone and central allopregnanolone than other groups. Young Tat(+) mice had greater circulating progesterone and estradiol-to-testosterone ratios. Older age or Tat exposure increased anxiety-like behavior (open field; elevated plus-maze), increased cognitive errors (radial arm water maze), and reduced grip strength. Young Tat(+), or middle-aged Tat(-), males had higher mechanical nociceptive thresholds than age-matched counterparts. Steroid levels correlated with behaviors. Thus, Tat may contribute to HIV-accelerated aging.

Safety and Pharmacokinetics of Intranasally Administered Heparin.

PURPOSE: Intranasally administered unfractionated heparin (UFH) and other sulfated polysaccharides are potential prophylactics for COVID-19. The purpose of this research was to measure the safety and pharmacokinetics of clearance of intranasally administered UFH solution from the nasal cavity. METHODS: Double-blinded daily intranasal dosing in C57Bl6 mice with four doses (60 ng to 60 µg) of UFH was carried out for fourteen consecutive days, with both blood coagulation measurements and subject adverse event monitoring. The pharmacokinetics of fluorescent-labeled UFH clearance from the nasal cavity were measured in mice by in vivo imaging. Intranasal UFH at 2000 U/day solution with nasal spray device was tested for safety in a small number of healthy human subjects. RESULTS: UFH showed no evidence of toxicity in mice at any dose measured. No significant changes were observed in activated partial thromboplastin time (aPTT), platelet count, or frequency of minor irritant events over vehicle-only control. Human subjects showed no significant changes in aPTT time, international normalized ratio (INR), or platelet count over baseline measurements. No serious adverse events were observed. In vivo imaging in a mouse model showed a single phase clearance of UFH from the nasal cavity. After 12 h, 3.2% of the administered UFH remained in the nasal cavity, decaying to background levels by 48 h. CONCLUSIONS: UFH showed no toxic effects for extended daily intranasal dosing in mice as well as humans. The clearance kinetics of intranasal heparin solution from the nasal cavity indicates potentially protective levels for up to 12 h after dosing.

Safety and Pharmacokinetics of Intranasally Administered Heparin.

PURPOSE: Intranasally administered unfractionated heparin (UFH) and other sulfated polysaccharides are potential prophylactics for COVID-19. The purpose of this research was to measure the safety and pharmacokinetics of clearance of intranasally administered UFH solution from the nasal cavity. METHODS: Double-blinded daily intranasal dosing in C57Bl6 mice with four doses (60 ng to 60 µg) of UFH was carried out for fourteen consecutive days, with both blood coagulation measurements and subject adverse event monitoring. The pharmacokinetics of fluorescent-labeled UFH clearance from the nasal cavity were measured in mice by in vivo imaging. Intranasal UFH at 2000 U/day solution with nasal spray device was tested for safety in a small number of healthy human subjects. RESULTS: UFH showed no evidence of toxicity in mice at any dose measured. No significant changes were observed in activated partial thromboplastin time (aPTT), platelet count, or frequency of minor irritant events over vehicle-only control. Human subjects showed no significant changes in aPTT time, international normalized ratio (INR), or platelet count over baseline measurements. No serious adverse events were observed. In vivo imaging in a mouse model showed a single phase clearance of UFH from the nasal cavity. After 12 hours, 3.2% of the administered UFH remained in the nasal cavity, decaying to background levels by 48 hours. CONCLUSIONS: UFH showed no toxic effects for extended daily intranasal dosing in mice as well as humans. The clearance kinetics of intranasal heparin solution from the nasal cavity indicates potentially protective levels for up to 12 hours after dosing.

6,5-Fused Ring, C2-Salvinorin Ester, Dual Kappa and Mu Opioid Receptor Agonists as Analgesics Devoid of Anxiogenic Effects.

Current common analgesics are mediated through the mu or kappa opioid receptor agonism. Unfortunately, selective mu or kappa receptor agonists often cause harmful side effects. However, ligands exhibiting dual agonism to the opioid receptors, such as to mu and kappa, or to mu and delta, have been suggested to temper undesirable adverse effects while retaining analgesic activity. Herein we report an introduction of various 6,5-fused rings to C2 of the salvinorin scaffold via an ester linker. In vitro studies showed that many of these compounds have dual agonism on kappa and mu opioid receptors. In vivo studies on the lead dual kappa and mu opioid receptor agonist demonstrated supraspinal thermal analgesic activity while avoiding anxiogenic effects in male mice, thus providing further strong evidence in support of the therapeutic advantages of dual opioid receptor agonists over selective opioid receptor agonists.

Identification of an Orally Bioavailable, Brain-Penetrant Compound with Selectivity for the Cannabinoid Type 2 Receptor.

Modulation of the endocannabinoid system (ECS) is of great interest for its therapeutic relevance in several pathophysiological processes. The CB2 subtype is largely localized to immune effectors, including microglia within the central nervous system, where it promotes anti-inflammation. Recently, a rational drug design toward precise modulation of the CB2 active site revealed the novelty of Pyrrolo[2,1-c][1,4]benzodiazepines tricyclic chemotype with a high conformational similarity in comparison to the existing leads. These compounds are structurally unique, confirming their chemotype novelty. In our continuing search for new chemotypes as selective CB2 regulatory molecules, following SAR approaches, a total of 17 selected (S,E)-11-[2-(arylmethylene)hydrazono]-PBD analogs were synthesized and tested for their ability to bind to the CB1 and CB2 receptor orthosteric sites. A competitive [3H]CP-55,940 binding screen revealed five compounds that exhibited >60% displacement at 10 µM concentration. Further concentration-response analysis revealed two compounds, 4k and 4q, as potent and selective CB2 ligands with sub-micromolar activities (Ki = 146 nM and 137 nM, respectively). In order to support the potential efficacy and safety of the analogs, the oral and intravenous pharmacokinetic properties of compound 4k were sought. Compound 4k was orally bioavailable, reaching maximum brain concentrations of 602 ± 162 ng/g (p.o.) with an elimination half-life of 22.9 ± 3.73 h. Whether administered via the oral or intravenous route, the elimination half-lives ranged between 9.3 and 16.7 h in the liver and kidneys. These compounds represent novel chemotypes, which can be further optimized for improved affinity and selectivity toward the CB2 receptor.

Insulin-Like Growth Factor-1 Differentially Modulates Glutamate-Induced Toxicity and Stress in Cells of the Neurogliovascular Unit.

The age-related reduction in circulating levels of insulin-like growth factor-1 (IGF-1) is associated with increased risk of stroke and neurodegenerative diseases in advanced age. Numerous reports highlight behavioral and physiological deficits in blood-brain barrier function and neurovascular communication when IGF-1 levels are low. Administration of exogenous IGF-1 reduces the extent of tissue damage and sensorimotor deficits in animal models of ischemic stroke, highlighting the critical role of IGF-1 as a regulator of neurovascular health. The beneficial effects of IGF-1 in the nervous system are often attributed to direct actions on neurons; however, glial cells and the cerebrovasculature are also modulated by IGF-1, and systemic reductions in circulating IGF-1 likely influence the viability and function of the entire neuro-glio-vascular unit. We recently observed that reduced IGF-1 led to impaired glutamate handling in astrocytes. Considering glutamate excitotoxicity is one of the main drivers of neurodegeneration following ischemic stroke, the age-related loss of IGF-1 may also compromise neural function indirectly by altering the function of supporting glia and vasculature. In this study, we assess and compare the effects of IGF-1 signaling on glutamate-induced toxicity and reactive oxygen species (ROS)-produced oxidative stress in primary neuron, astrocyte, and brain microvascular endothelial cell cultures. Our findings verify that neurons are highly susceptible to excitotoxicity, in comparison to astrocytes or endothelial cells, and that a prolonged reduction in IGFR activation increases the extent of toxicity. Moreover, prolonged IGFR inhibition increased the susceptibility of astrocytes to glutamate-induced toxicity and lessened their ability to protect neurons from excitotoxicity. Thus, IGF-1 promotes neuronal survival by acting directly on neurons and indirectly on astrocytes. Despite increased resistance to excitotoxic death, both astrocytes and cerebrovascular endothelial cells exhibit acute increases in glutamate-induced ROS production and mitochondrial dysfunction when IGFR is inhibited at the time of glutamate stimulation. Together these data highlight that each cell type within the neuro-glio-vascular unit differentially responds to stress when IGF-1 signaling was impaired. Therefore, the reductions in circulating IGF-1 observed in advanced age are likely detrimental to the health and function of the entire neuro-glio-vascular unit.

Rotenoids and Other Specialized Metabolites from the Roots of Mirabilis multiflora: Opioid and Cannabinoid Receptor Radioligand Binding Affinities.

Mirabilis multiflora is an acclaimed hallucinogen consumed traditionally by the Hopi Indians to induce diagnostic visions. Its root extract afforded a new (3) and four known (2, 5, 6, and 7) 12a-hydroxyrotenoids, a known rotenoid (4), and two known secondary metabolites (1 and 8). The structures of the compounds were elucidated based on spectroscopic and spectrometric data analysis. Electronic circular dichroism data were used to define the (6aS,12aR) absolute configuration of the 12a-hydroxyrotenoids. Compounds 2-7 were screened for their radioligand binding affinities toward the opioid (δ, κ, and µ) and cannabinoid (CB1 and CB2) receptor subtypes. The 6-methoxy-substituted rotenoids 3, 4, and 7 showed the highest receptor binding affinity with moderate selectivity toward the δ-opioid receptor subtype, with negligible binding affinities for CB1 and CB2. Their binding affinities toward the δ-opioid receptor were 64.5% (4), 58.7% (7), and 55.3% (3) at 10 µM, respectively.

Preclinical and clinical evidence of IGF-1 as a prognostic marker and acute intervention with ischemic stroke.

Ischemic strokes are highly prevalent in the elderly population and are a leading cause of mortality and morbidity worldwide. The risk of ischemic stroke increases in advanced age, corresponding with a noted decrease in circulating insulin growth factor-1 (IGF-1). IGF-1 is a known neuroprotectant involved in embryonic development, neurogenesis, neurotransmission, cognition, and lifespan. Clinically, several studies have shown that reduced levels of IGF-1 correlate with increased mortality rate, poorer functional outcomes, and increased morbidities following an ischemic stroke. In animal models of ischemia, administering exogenous IGF-1 using various routes of administration (intranasal, intravenous, subcutaneous, or topical) at various time points prior to and following insult attenuates neurological damage and accompanying behavioral changes caused by ischemia. However, there are some contrasting findings in select clinical and preclinical studies. This review discusses the role of IGF-1 as a determinant factor of ischemic stroke outcomes, both within the clinical settings and preclinical animal models. Furthermore, the review provides insight on the role of IGF-1 in mechanisms and cellular processes that contribute to stroke damage.

First In Class (S,E)-11-[2-(Arylmethylene)Hydrazono]-PBD Analogs As Selective CB2 Modulators Targeting Neurodegenerative Disorders.

Newly designed pyrrolo[2,1-c][1,4]benzodiazepines tricyclic skeleton has shown potential clusters of cannabinoid receptors CB1/CB2 selective ligands. CB2 plays a critical role in microglial-derived neuroinflammation, where it modulates cell proliferation, migration, and differentiation into M1 or M2 phenotypes. Beginning with computer-based docking studies accounting the recently discovered X-ray crystal structure of CB2, we designed a series of PBD analogs as potential ligands of CB2 and tested their binding affinities. Interestingly, computational studies and theoretical binding affinities of several selected (S,E)-11-[2-(arylmethylene)hydrazono]-PBD analogs, have revealed the presence of potential selectivity in binding attraction towards CB1 and CB2. Reported here is the discovery of the first representatives of this series of selective binding to CB2. Preliminary data showed that this class of molecules display potential binding efficacy towards the cannabinoid receptors tested. Intriguingly, initial cannabinoid binding assay showed a selective binding affinity of 4g and 4h showed K i of 0.49 and 4.7 µM towards CB2 receptors while no binding was observed to CB1. The designed leads have shown remarkable stability pattern at the physiological pH magnifying their therapeutic values. We hypothesize that the PBD tricyclic structure offers the molecule an appropriate three-dimensional conformation to fit snugly within the active site of CB2 receptors, giving them superiority over the reported CB2 agonists/inverse agonists. Our findings suggested that the attachment of heterocyclic ring through the condensation of diazepine hydrazone and S- or N-heterocyclic aldehydes enhances the selectivity of CB2 over CB1.

IGF1R signaling regulates astrocyte-mediated neurovascular coupling in mice: implications for brain aging.

Aging is associated with a significant deficiency in circulating insulin-like growth factor-1 (IGF-1), which has an important role in the pathogenesis of age-related vascular cognitive impairment (VCI). Impairment of moment-to-moment adjustment of regional cerebral blood flow via neurovascular coupling (NVC) importantly contributes to VCI. Previous studies established a causal link between circulating IGF-1 deficiency and neurovascular dysfunction. Release of vasodilator mediators from activated astrocytes plays a key role in NVC. To determine the impact of impaired IGF-1 signaling on astrocytic function, astrocyte-mediated NVC responses were studied in a novel mouse model of astrocyte-specific knockout of IGF1R (GFAP-CreERT2/Igf1rf/f) and accelerated neurovascular aging. We found that mice with disrupted astrocytic IGF1R signaling exhibit impaired NVC responses, decreased stimulated release of the vasodilator gliotransmitter epoxy-eicosatrienoic acids (EETs), and upregulation of soluble epoxy hydrolase (sEH), which metabolizes and inactivates EETs. Collectively, our findings provide additional evidence that IGF-1 promotes astrocyte health and maintains normal NVC, protecting cognitive health.

Synthesis, biological evaluation, and NMR studies of 3-fluorinated derivatives of 3',4',5'-trihydroxyflavone and 3',4',5'-trimethoxyflavone.

Flavones are valuable scaffolds in medicinal chemistry, especially as they display activity as antioxidants and neuroprotective agents. The need to incorporate a fluorine atom on flavones has driven much of the recent synthetic work in this area. We now report a route for the production of 3-fluorinated derivatives of 3',4',5'-trihydroxyflavone and 3',4',5'-trimethoxyflavone. Biological evaluation of these agents, along with their non-fluorinated counterparts, demonstrate that antioxidant activity may be enhanced whereas neuroprotective activity is conserved. Also, the 3-fluoro-3',4',5'-trihydroxyflavone can act as an NMR probe to detect structural changes during its action as a radical scavenger.

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic, or prophylactic. As with other betacoronaviruses, attachment and entry of SARS-CoV-2 are mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin-converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third-generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in antiviral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH, and 6-O-desulfated enoxaparin with 50% inhibitory concentrations (IC50s) of 5.99 µg/liter, 1.08 mg/liter, 1.77 µg/liter, and 5.86 mg/liter, respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes.IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in antiviral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes.

HIV-1 Tat promotes age-related cognitive, anxiety-like, and antinociceptive impairments in female mice that are moderated by aging and endocrine status.

Hypogonadism is a common comorbidity associated with HIV-1 that is more prevalent among infected individuals over the age of 45. The underlying mechanisms are unknown, but both combined antiretroviral therapeutics and HIV-1 proteins, such as trans-activator of transcription protein (Tat), dysregulate steroid-synthetic mechanisms including lipid storage/synthesis and mitochondrial function. Thus, Tat expression may accelerate age-related comorbidities partly by impairing endocrine function. Few studies exist of Tat-mediated behavioral deficits in aged animals and effects of endocrine status have not been investigated. Accordingly, we tested whether conditional Tat expression in aged (~ 1.5 years old), female, Tat-transgenic [Tat(+)] mice increases anxiety-like behavior, impairs cognition, and augments mechanical allodynia, when compared to age-matched controls that do not express Tat protein [Tat(-)]. We further tested whether aged mice that maintained their endocrine status (pre-estropausal) were more resilient to Tat/age-related comorbidities than peri- or post-estropausal mice. Tat and endocrine aging status exerted separate and interacting effects that influenced anxiety-like and cognitive behaviors. Peri- and post-estropausal mice exhibited greater anxiety-like behavior in the elevated plus-maze and impaired learning in the radial arm water maze compared to pre-estropausal mice. Irrespective of estropause status, Tat(+) mice demonstrated impaired learning, reduced grip strength, and mechanical allodynia compared to Tat(-) mice. Tat exposure reduced circulating estradiol in post-estropausal mice and increased the estradiol-to-testosterone ratio in pre-estropausal mice. Changes in circulating estradiol, testosterone, and progesterone correlated with grip strength. Thus, endocrine status is an important factor in age-related anxiety, cognition, neuromuscular function, and allodynia that can be accelerated by HIV-1 Tat protein.

Age-dependent hormesis-like effects of the synthetic cannabinoid CP55940 in C57BL/6 mice.

Use of cannabis and cannabinoid-containing substances is increasing among geriatric patients, despite relatively sparse preclinical evidence in aged models. To better understand the effects of exogenous cannabinoids on aging male and female rodents, we compared the age- and dose-dependent physiological and behavioral effects of the synthetic cannabinoid CP55940 in young-adult and aged C57BL/6 mice. Locomotion, body temperature, thermal nociception, and fecal output were measured following CP55940 administration. Our findings indicate that CP55940 is more potent and efficacious in older mice, evidenced by exaggerated antinociception and locomotor inhibition when compared to younger adult mice. In addition, we report that low doses of CP55940 paradoxically stimulate locomotion in young-adult (4 m) mice; however, this hormesis-like response is not as evident in aged animals (21-24 m). These bidirectional effects appear to be mediated via the endocannabinoid CB1 and CB2 receptors.

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic or prophylactic. Like other betacoronaviruses, attachment and entry of SARS-CoV-2 is mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in anti-viral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH and 6-O-desulfated enoxaparin with an IC50 of 5.99 µg/L, 1.08 mg/L, 1.77 µg/L, and 5.86 mg/L respectively. The low serum bioavailability of intranasally administered UFH, along with data suggesting that the nasal epithelium is a portal for initial infection and transmission, suggest that intranasal administration of UFH may be an effective and safe prophylactic treatment.

Cannabis constituents reduce seizure behavior in chemically-induced and scn1a-mutant zebrafish.

Current antiepileptic drugs (AEDs) are undesirable for many reasons including the inability to reduce seizures in certain types of epilepsy, such as Dravet syndrome (DS) where in one-third of patients does not respond to current AEDs, and severe adverse effects that are frequently experienced by patients. Epidiolex, a cannabidiol (CBD)-based drug, was recently approved for treatment of DS. While Epidiolex shows great promise in reducing seizures in patients with DS, it is used in conjunction with other AEDs and can cause liver toxicity. To investigate whether other cannabis-derived compounds could also reduce seizures, the antiepileptic effects of CBD, Δ9-tetrahydrocannabinol (THC), cannabidivarin (CBDV), cannabinol (CBN), and linalool (LN) were compared in both a chemically-induced (pentylenetetrazole, PTZ) and a DS (scn1Lab-/-) seizure models. Zebrafish (Danio rerio) that were either wild-type (Tupfel longfin) or scn1Lab-/- (DS) were exposed to CBD, THC, CBDV, CBN, or LN for 24 h from 5 to 6 days postfertilization. Following exposure, total distance traveled was measured in a ViewPoint Zebrabox to determine if these compounds reduced seizure-like activity. Cannabidiol (0.6 and 1 µM) and THC (1 and 4 µM) significantly reduced PTZ-induced total distance moved. At the highest THC concentration, the significant reduction in PTZ-induced behavior was likely the result of sedation as opposed to antiseizure activity. In the DS model, CBD (0.6 µM), THC (1 µM), CBN (0.6 and 1 µM), and LN (4 µM) significantly reduced total distance traveled. Cannabinol was the most effective at reducing total distance relative to controls. In addition to CBD, other cannabis-derived compounds showed promise in reducing seizure-like activity in zebrafish. Specifically, four of the five compounds were effective in the DS model, whereas in the PTZ model, only CBD and THC were, suggesting a divergence in the mode of action among the cannabis constituents.

Developmental exposure to Δ9-tetrahydrocannabinol (THC) causes biphasic effects on longevity, inflammation, and reproduction in aged zebrafish (Danio rerio).

Increased availability of cannabis and cannabinoid-containing products necessitates the need for an understanding of how these substances influence aging. In this study, zebrafish (Danio rerio) were exposed to different concentrations of THC (0.08, 0.4, 2 µM) during embryonic-larval development and the effects on aging were measured 30 months later and in the offspring of the exposed fish (F1 generation). Exposure to 0.08 µM THC resulted in increased male survival at 30 months of age. As the concentration of THC increased, this protective effect was lost. Treatment with the lowest concentration of THC also significantly increased egg production, while higher concentrations resulted in impaired fecundity. Treatment with the lowest dose of THC significantly reduced wet weight, the incidence of kyphosis, and the expression of several senescence and inflammatory markers (p16ink4ab, tnfα, il-1ß, il-6, pparα and pparγ) in the liver, but not at higher doses indicating a biphasic or hormetic effect. Exposure to THC did not affect the age-related reductions in locomotor behavior. Within the F1 generation, many of these changes were not observed. However, the reduction in fecundity due to THC exposure was worse in the F1 generation because offspring whose parents received high dose of THC were completely unable to reproduce. Together, our results demonstrate that a developmental exposure to THC can cause significant effects on longevity and healthspan of zebrafish in a biphasic manner.

Shear Stress in Schlemm's Canal as a Sensor of Intraocular Pressure.

Elevated intraocular pressure (IOP) narrows Schlemm's canal (SC), theoretically increasing luminal shear stress. Using engineered adenoviruses containing a functional fragment of the shear-responsive endothelial nitric oxide synthase (eNOS) promoter, we tested effects of shear stress and elevated flow rate on reporter expression in vitro and ex vivo. Cultured human umbilical vein endothelial cells (HUVECs) and SC cells were transduced with adenovirus containing eNOS promoter driving secreted alkaline phosphatase (SEAP) or green fluorescent protein (GFP) and subjected to shear stress. In parallel, human anterior segments were perfused under controlled flow. After delivering adenoviruses to the SC lumen by retroperfusion, the flow rate in one anterior segment of pair was increased to double pressure. In response to high shear stress, HUVECs and SC cells expressed more SEAP and GFP than control. Similarly, human anterior segments perfused at higher flow rates released significantly more nitrites and SEAP into perfusion effluent, and SC cells expressed increased GFP near collector channel ostia compared to control. These data establish that engineered adenoviruses have the capacity to quantify and localize shear stress experienced by endothelial cells. This is the first in situ demonstration of shear-mediated SC mechanobiology as a key IOP-sensing mechanism necessary for IOP homeostasis.