Artificial intelligence-enhanced electrocardiogram analysis for identifying cardiac autonomic neuropathy in patients with diabetes.

AIM: To develop and employ machine learning (ML) algorithms to analyse electrocardiograms (ECGs) for the diagnosis of cardiac autonomic neuropathy (CAN). MATERIALS AND METHODS: We used motif and discord extraction techniques, alongside long short-term memory networks, to analyse 12-lead, 10-s ECG tracings to detect CAN in patients with diabetes. The performance of these methods with the support vector machine classification model was evaluated using 10-fold cross validation with the following metrics: accuracy, precision, recall, F1 score, and area under the receiver-operating characteristic curve (AUC). RESULTS: Among 205 patients (mean age 54 ± 17 years, 54% female), 100 were diagnosed with CAN, including 38 with definite or severe CAN (dsCAN) and 62 with early CAN (eCAN). The best model performance for dsCAN classification was achieved using both motifs and discords, with an accuracy of 0.92, an F1 score of 0.92, a recall at 0.94, a precision of 0.91, and an excellent AUC of 0.93 (95% confidence interval [CI] 0.91-0.94). For the detection of any stage of CAN, the approach combining motifs and discords yielded the best results, with an accuracy of 0.65, F1 score of 0.68, a recall of 0.75, a precision of 0.68, and an AUC of 0.68 (95% CI 0.54-0.81). CONCLUSION: Our study highlights the potential of using ML techniques, particularly motifs and discords, to effectively detect dsCAN in patients with diabetes. This approach could be applied in large-scale screening of CAN, particularly to identify definite/severe CAN where cardiovascular risk factor modification may be initiated.

Pharmacokinetics of Oral Minor Cannabinoids in Blood and Brain.

Introduction: Minor cannabinoids are increasingly being consumed in oral formulations (i.e., edibles, tinctures) for medical and nonmedical purposes. This study examined the pharmacokinetics (PKs) of cannabinoids tetrahydrocannabivarin (THCV), cannabichromene (CBC), cannabinol (CBN), and delta-8-tetrahydrocannabinol (D8-THC) after the first and last oral dose during a 14-day administration period. Materials and Methods: Sprague-Dawley rats (N=6 animals/dose, 50% female) were given an assigned dose of one of four cannabinoids (THCV=3.2-100 mg/kg, CBC=3.2-100 mg/kg, CBN=1-100 mg/kg, or D8-THC=0.32-10 mg/kg) or vehicle (medium-chain triglyceride oil) through oral gavage once daily for 14 days. Blood was collected 45 min and 1.5, 3, and 24 h following the first dose (day 1) and the last dose (day 14) of repeated oral cannabinoid treatment for PK analysis. Outcomes of interest included time to maximum concentration (Tmax), maximum concentration (Cmax), and area under the concentration versus time curve (AUClast). Dose-normalized (DN) Cmax and DN AUClast were also calculated. Brain tissue was collected 24 h post-administration of the first (day 1) and the last (day 14) dose of each cannabinoid to determine concentrations in brain. Results: All cannabinoids tested were detectable in plasma after single and 14-day repeated dosing. DN Cmax and DN AUClast were highest for D8-THC, followed by CBC, CBN, and THCV. There was no sex difference observed in cannabinoid kinetics. Accumulation of D8-THC in plasma was observed after 14 days of administration. THCV levels in plasma were lower on day 14 compared to day 1, indicating potential adaptation of metabolic pathways and increased drug elimination. Cannabinoids were detected in brain tissue 24 h post-administration of the first and the last dose of 17-100 mg/kg THCV, 3.2-100 mg/kg CBC, 10-100 mg/kg CBN, and 10 mg/kg D8-THC. Conclusions: THCV, CBC, CBN, and D8-THC produced detectable levels in plasma and translocated to brain tissue after the first dose (day 1) and the last dose (day 14) of repeated oral dosing. Examination of PKs of these minor cannabinoids in blood and brain provides a critical step for informing target dose ranges and dosing schedules in future studies that evaluate the potential effects of these compounds.

Toxicological Evaluation and Pain Assessment of Four Minor Cannabinoids Following 14-Day Oral Administration in Rats.

Introduction: Despite growing consumer interest and market availability, the safety of minor cannabinoids, generally present in low concentrations in Cannabis sativa L., is not well understood. Materials and Methods: Cannabichromene (CBC; 3.2, 10, 17, 22, 32, or 100 mg/kg-bw/day), cannabinol (CBN; 1, 3.2, 10, 17, 32, or 100 mg/kg-bw/day), delta-8-tetrahydrocannabinol (D8-THC; 0.32, 1, 3.2, or 10 mg/kg-bw/day), tetrahydrocannabivarin (THCV; 3.2, 10, 17, 22, 32, or 100 mg/kg-bw/day), and vehicle (medium-chain triglyceride oil) preparations were administered via oral gavage once daily for 14 days to Sprague Dawley rats. Changes in behavior, body weight, food consumption, clinical pathology, organ weights, body temperature, and thermal pain sensitivity (tail flick assay) were assessed. Select organ tissues were collected at terminal necropsy and fixed for histopathological examination. Results: No treatment-related deaths were observed throughout the study, and cannabinoids were generally well tolerated. While some significant trends in body weight differences from controls (increases and decreases) were observed, these occurred independently of food consumption. Overall, differences in serum chemistry and hematology parameters between cannabinoid groups and their respective control groups were considered to occur due to biological variation among rats. No treatment-related gross abnormalities were observed in examined organs. Significant changes in absolute and relative organ weights occurred primarily in males and were generally of negligible magnitude. There were no biologically significant histopathological observations. While pain tolerance was significantly improved in animals treated with D8-THC (3.2 and 10 mg/kg-bw/day, day 14), results across minor cannabinoids were inconsistent and warrant further study. Conclusion: Minor cannabinoids were well tolerated across 14 days of daily oral administration at the doses assessed. Modest, dose-dependent trends in relative organ weights and serum chemistry parameters warrant exploration at higher oral doses. These data will assist in dose selection for future studies investigating the long-term safety and effects of CBC, CBN, D8-THC, and THCV.

Oral Cannabidiol Treatment in Two Postmenopausal Women with Osteopenia: A Case Series.

Introduction: Cannabidiol (CBD), a nonintoxicating cannabinoid, may be involved in bone remodeling, but human studies are limited. In this case series, we explored the effects of oral CBD administration on bone turnover. Materials and Methods: Two postmenopausal women with osteopenia (T-score=-1 to -2.5) were randomized to receive 100 or 300 mg CBD daily (oral, bis in die [twice per day]) for 12 weeks. Serum markers of bone resorption (carboxyl-terminal collagen crosslinks [CTx]) and bone formation (procollagen type 1 N-terminal propeptide [P1NP], bone-specific alkaline phosphatase [BSAP], and osteocalcin [OC]); safety measures; plasma concentrations of CBD and metabolites; sleep disturbance; symptoms of depression, anxiety, and stress; and quality of life, were assessed. Results: CBD was well tolerated, with no clinically significant change in vital signs, hematology, chemistry, or urinalysis, and no adverse events reported. Reductions (% change vs. baseline) in CTx (-8.5%, -28.1%), P1NP (-9.9%, -39.5%), BSAP (-12.7%, -74.8%), and OC (-16.0%, -6.7%) were observed after 12 weeks of oral administration of 100 or 300 mg CBD daily, respectively. The two participants self-reported consuming 95.3% and 98.8% of CBD doses, respectively. CBD and select metabolites were measurable in plasma after 4 and 12 weeks of CBD treatment. No notable changes in sleep disturbance, depression, anxiety, stress, or quality of life were observed. Conclusions: CBD was well tolerated after 12 weeks of twice-daily oral administration and was associated with reduction in measured markers of bone turnover. Compliance with CBD treatment was good. Large-scale randomized clinical trials into the bone protective effects of CBD in postmenopausal women are warranted.

Machine learning profiles of cardiovascular risk in patients with diabetes mellitus: the Silesia Diabetes-Heart Project.

AIMS: As cardiovascular disease (CVD) is a leading cause of death for patients with diabetes mellitus (DM), we aimed to find important factors that predict cardiovascular (CV) risk using a machine learning (ML) approach. METHODS AND RESULTS: We performed a single center, observational study in a cohort of 238 DM patients (mean age ± SD 52.15 ± 17.27 years, 54% female) as a part of the Silesia Diabetes-Heart Project. Having gathered patients' medical history, demographic data, laboratory test results, results from the Michigan Neuropathy Screening Instrument (assessing diabetic peripheral neuropathy) and Ewing's battery examination (determining the presence of cardiovascular autonomic neuropathy), we managed use a ML approach to predict the occurrence of overt CVD on the basis of five most discriminative predictors with the area under the receiver operating characteristic curve of 0.86 (95% CI 0.80-0.91). Those features included the presence of past or current foot ulceration, age, the treatment with beta-blocker (BB) and angiotensin converting enzyme inhibitor (ACEi). On the basis of the aforementioned parameters, unsupervised clustering identified different CV risk groups. The highest CV risk was determined for the eldest patients treated in large extent with ACEi but not BB and having current foot ulceration, and for slightly younger individuals treated extensively with both above-mentioned drugs, with relatively small percentage of diabetic ulceration. CONCLUSIONS: Using a ML approach in a prospective cohort of patients with DM, we identified important factors that predicted CV risk. If a patient was treated with ACEi or BB, is older and has/had a foot ulcer, this strongly predicts that he/she is at high risk of having overt CVD.

Sex Differences in the Pharmacokinetics of Cannabidiol and Metabolites Following Oral Administration of a Cannabidiol-Dominant Cannabis Oil in Healthy Adults.

Introduction: Oral cannabidiol (CBD) product use is increasingly growing among women; however, there is a lack of data on sex differences in the pharmacokinetics (PKs) of CBD and its primary metabolites, 7-hydroxy-CBD (7-OH-CBD) and 7-carboxy-CBD (7-COOH-CBD), after repeated doses. Materials and Methods: The present study is a secondary analysis of data from a randomized, double-blind, placebo-controlled multiple-dose trial of a commercially available, CBD-dominant oral cannabis product. Healthy participants (n=17 males and 15 females) were randomized to receive 120 to 480 mg of CBD daily for 7 days. Dosing groups were pooled for all analyses due to sample size limitations. Analyses compared plasma PK parameters by sex, day, and sex×day. Results: For raw PK parameters for CBD and metabolites, there were no statistically significant effects of sex×day or sex (all p-values >0.05). For metabolite-to-parent ratios (MPRs) of AUC0-t, there were significant effects of the sex×day interactions for 7-OH-CBD (F=6.89, p=0.016) and 7-COOH-CBD (F=5.96, p=0.021). For 7-OH-CBD, follow-up analyses showed significant simple effects of day within females (t=4.13, p<0.001), but not within males (t=0.34, p=0.73), such that 7-OH-CBD MPRs increased significantly from day 1 to 7 for females, but not for males. For 7-COOH-CBD, follow-up analyses revealed significant simple effects of day within females (t=8.24, p<0.001) and males (t=5.20, p<0.001), therefore 7-COOH-CBD MPRs increased significantly from day 1 to 7 in both sexes, but the increase was significantly greater among females than among males. Within dosing days, there were no statistically significant simple effects of sex on MPRs of 7-OH-CBD or 7-COOH-CBD. Conclusions: Females exhibited greater relative exposure to CBD metabolites in plasma over time, which may reflect sex differences in CBD metabolism or elimination. Further research assessing the safety implications of higher relative exposure to CBD metabolites over longer periods of time is warranted to mirror typical consumer use patterns.

Characterization of aerosols from hemp-derived pre-roll joints.

OBJECTIVE: Availability and consumer use of hemp products is rapidly increasing, but little work has been done to assess aerosol emissions of hemp pre-rolls. The objective of this research was to characterize the aerosol of pre-rolled joints from hemp material enriched for production of cannabigerol (CBG) that were smoked on a test system mimicking human use patterns. MATERIALS AND METHODS: Aerosol emissions were collected and analyzed using glass microfiber filters and charcoal cartridges. The aerosol was screened for nine phytocannabinoids and 19 terpenes. RESULTS: Three phytocannabinoids (CBG, cannabichromene (CBC), and delta-9-tetrahydrocannabinol (THC)) were detected and quantified at a mean (SD) concentration of 19.4 (4.7), 0.48 (0.01), and 0.40 (0.04) mg per pre-roll, respectively. Five terpenes ((-)-α-bisabolol, (-)-guaiol, ß-caryophyllene, nerolidol, and α-humulene) were detected and quantified at an average concentration of 352.7 (112.0), 194.3 (66.4), 106.0 (50.4), 28.3 (9.3), and 27.7 (11.2) µg per pre-roll, respectively. Particle size distribution testing via aerodynamic particle sizer and inertial impactor showed that average size of emitted aerosols was 0.77 (0.0) and 0.54 (0.1) µm, respectively. CONCLUSIONS: This study describes methodology for characterization of cannabinoid and terpene dose in emitted aerosols and aerosolization efficiency from hemp pre-rolls. It also presents these data for one of the marketed products.

Diabetic Peripheral Neuropathy is Associated With Diabetic Kidney Disease and Cardiovascular Disease: The Silesia Diabetes-Heart Project.

Microvascular complications of diabetes seem to be clustered and put patients at higher risk of developing cardiovascular disease (CVD). This was a questionnaire-based study designed to screen for the presence of diabetic peripheral neuropathy (DPN), defined as the score in the Michigan Neuropathy Screening Instrument (MNSI) above 2, and to evaluate its association with other complication of diabetes, including CVD. There were 184 patients included into the study. The prevalence of DPN in the study group was 37.5%. The regression model analysis revealed that the presence of DPN was significantly associated with the presence of diabetic kidney disease (DKD) (P = 0.0034;) and patient's age (P < 0.0001). Thirty-four patients (49.3%) with MNSI score >2 were diagnosed with CVD in comparison to 24 (20.1%) subjects with MNSI score ≤ 2 (P = 0.00006). In case of having one diabetes complication diagnosed, it is important to screen for others, including macrovascular ones.

Pharmacokinetics of Cannabidiol in Sprague-Dawley Rats After Oral and Pulmonary Administration.

Introduction: Cannabidiol (CBD) is primarily consumed through ingestion and inhalation. Little is known about how CBD pharmacokinetics differ between routes of administration, and duration of pulmonary exposure. Methods: Pharmacokinetics, brain distribution, and urinary elimination of CBD and its major metabolites (6-hydroxy-cannabidiol [6-OH-CBD], 7-hydroxy-cannabidiol [7-OH-CBD], 7-carboxy-cannabidiol [7-COOH-CBD], and CBD-glucuronide) were evaluated in adult Sprague-Dawley rats following a single oral CBD ingestion (10 mg/kg in medium chain triglyceride oil; 24 male animals), and 1 or 14 days of repeated inhalation (0.9-13.9 mg/kg in propylene glycol [41%/59% by weight]; 5 male and 5 female animals per dose). Blood and brain tissue were collected at a single time point from each animal. Collection times were staggered from 5 min to 24 h postoral gavage or first (blood only) and final inhalation. Urine was collected 24 h postoral gavage or final inhalation. Samples were analyzed through liquid chromatography-mass spectrometry (LC-MS/MS). Results: CBD was more rapidly absorbed following inhalation than ingestion (Tmax=5 min and 2 h, respectively). Inhalation resulted in a dose-responsive increase in CBD Cmax and AUClast. CBD Cmax was 24-fold higher following the highest pulmonary dose (13.9 mg/kg) versus an oral dose of comparable concentration (10 mg/kg). Cmax and AUClast (0-16 h) trended higher following repeated exposure. Elimination was notably faster with repeated CBD inhalation (t1/2=5.3 and 2.4 h on days 1 and 14, respectively). While metabolites were detectable in plasma, AUClast (0-2 h) was at least 10- (7-OH-CBD, 7-COOH-CBD) to 100- (6-OH-CBD) fold lower than the parent compound. Metabolite concentration trended higher following repeated inhalation (6.7 mg/kg CBD); AUClast (0-16 h) was ∼1.8-, ∼1.4-, and ∼2.4-fold higher following 14 days of exposure for 6-OH-CBD, 7-OH-CBD, and 7-COOH-CBD, respectively. CBD was detectable in brain homogenate tissue 24-h after 14-day inhalation (>3.5 mg/kg deposited dose) or a single oral administration. CBD metabolites were only measurable in brain tissue following the highest inhaled dose (13.9 mg/kg CBD). CBD, but not metabolites, was detectable in urine for all dose groups following 2 weeks of CBD inhalation. Neither CBD nor metabolites were present in urine after oral administration. Conclusion: CBD pharmacokinetics differ across oral and pulmonary routes of administration and acute or repeated dosing.

Serum Markers of Bone Turnover Following Controlled Administration of Two Medical Cannabis Products in Healthy Adults.

Introduction: Cannabidiol (CBD) has been shown to maintain bone integrity in pre-clinical models, but little is known about the effects of delta-9-tetrahydrocannabinol (THC) on bone turnover. In this study we explored the effects of two oral medical cannabis products on normal bone homeostasis through evaluation of markers of bone resorption (carboxyl-terminal collagen crosslinks, CTx) and bone formation (procollagen type 1 N-terminal propeptide, P1NP; alkaline phosphatase, ALP). Methods: This study is an analysis of secondary data from two Phase 1 double-blind, placebo-controlled trials of Spectrum Yellow (0.9 mg THC, 20 mg CBD/mL of oil) and Spectrum Red (2.5 mg THC, 0.3 mg CBD/softgel). Healthy participants (n=38 men, 45 women) were randomized to receive 5-20 mg THC (CBD levels varied as a function of administered product) or placebo daily (BID) for 7 days. Bone markers were assessed at baseline, upon completion of product administration (day 8), and after a 5-day washout (day 13). Results: All bone markers were significantly higher in men at baseline (p≤0.008). For CTx, there was a significant day×group interaction (F=3.23, p=0.04); CTx levels were significantly lower in participants treated with Spectrum Red (b=-164.28; 95% confidence interval [CI], -328 to -0.29; p=0.04) and marginally lower in participants treated with Spectrum Yellow (b=-157.31; 95% CI, -323 to 8.68; p=0.06) versus placebo on day 8. For P1NP and ALP, there were no significant differences between treatments across study days. Bone marker values outside the reference range (RR) were observed; CTx > RR (n=71) was predominantly (85.9%) observed in male participants, whereas P1NP > RR (n=100) was more evenly distributed between sexes (53.0% in men). These were not considered clinically significant and did not differ between treatment groups. Conclusions: These are the first interventional human data on the effect of cannabinoids on biomarkers of bone turnover. Short-term treatment with CBD- or THC-dominant medical cannabis products resulted in attenuation of a marker of bone resorption. Although the attenuation was not clinically significant, this finding may be indicative of protective properties of cannabinoids in bone. Further research over longer dosing durations in individuals exhibiting bone-specific conditions (e.g., osteoporosis) is warranted. ClinicalTrials.gov IDs: ACTRN12619001723178 and ACTRN12619001450101.

Randomized, placebo-controlled, 28-day safety and pharmacokinetics evaluation of repeated oral cannabidiol administration in healthy dogs.

OBJECTIVE: To determine the safety and pharmacokinetics of various doses of plant-derived cannabidiol (CBD) versus placebo following repeated oral administration. ANIMALS: 20 healthy adult Beagles. PROCEDURES: In a randomized, blinded, placebo-controlled trial, dogs were randomized to 5 groups balanced in body weight and sex (n = 4 dogs/group) and received a CBD (1, 2, 4, or 12 mg/kg; from cannabis extract) or placebo oil formulation PO once daily for 28 days. Outcome variables were assessed through daily health observations, veterinary examinations, CBC, and serum biochemical analysis. Blood samples were collected at various time points to estimate 24-hour pharmacokinetic profiles of CBD and selected metabolites (7-carboxy-CBD and 7-hydroxy-CBD). RESULTS: Repeated CBD administration was well tolerated by dogs, with no clinically important changes in measured safety outcomes. Veterinary examinations revealed no clinically important abnormal findings. Adverse events were mild in severity. Relative to placebo administration, CBD administration at 12 mg/kg/d resulted in more gastrointestinal adverse events (mainly hypersalivation) and significantly higher serum alkaline phosphatase activity. Total systemic exposure to CBD increased on a dose-dependent basis following both acute (first dose) and chronic (28 days) administration. Within each CBD dose group, repeated administration increased total systemic exposure to CBD 1.6- to 3.3-fold. The 24-hour trough plasma CBD concentrations were also dose dependent, with a steady state reached following 2 weeks of administration. CONCLUSIONS AND CLINICAL RELEVANCE: Repeated, daily oral administration of the CBD formulation led to dose-dependent increases in total systemic exposure to CBD and 24-hour trough plasma concentrations in healthy dogs. These findings could help guide dose selection.

Safety and tolerability of escalating cannabinoid doses in healthy cats.

OBJECTIVES: The aim of this study was to determine the safety and tolerability of escalating doses of orally delivered cannabis oils predominant in cannabidiol (CBD), tetrahydrocannabinol (THC), or both CBD and THC in healthy cats. METHODS: In this placebo-controlled, blinded study, 20 healthy adult cats were randomized to one of five treatment groups (n = 4 per group): two placebo groups (sunflower oil [SF] or medium-chain triglyceride oil [MCT]), or three plant-derived cannabinoid oil groups (CBD in MCT, THC in MCT or CBD/THC [1.5:1] in SF). Up to 11 escalating doses of each formulation were delivered orally via syringe to fasted subjects, with at least 3 days separating doses. Safety and tolerability were determined from clinical observations, complete blood counts (CBCs) and clinical chemistry. Plasma cannabinoids (CBD, THC) and metabolites (7-COOH-CBD, 11-OH-THC) were assessed. RESULTS: Titration to maximum doses of 30.5 mg/kg CBD (CBD oil), 41.5 mg/kg THC (THC oil) or 13.0:8.4 mg/kg CBD:THC (CBD/THC oil) was safely achieved in all subjects. All observed adverse events (AEs) were mild, transient and resolved without medical intervention. Gastrointestinal AEs were more common with formulations containing MCT. Constitutional (lethargy, hypothermia), neurologic (ataxia) and ocular (protrusion membrana nictitans) AEs were more common with oils containing THC (CBD/THC and THC oils). There were no clinically significant changes in CBC or clinical chemistry across treatment groups. Higher plasma levels of the cannabinoids and their metabolites following administration of the CBD/THC combination product are suggestive of a pharmacokinetic interaction. CONCLUSIONS AND RELEVANCE: This is the first feline study to explore the safety and tolerability of CBD and THC, alone and in combination, in a controlled research setting. These findings will inform veterinarians of the safety profile of cannabinoids, particularly when considering the potential therapeutic use of CBD in cats or recognizing clinical signs associated with accidental exposure to THC-containing products.

Preliminary Investigation of the Safety of Escalating Cannabinoid Doses in Healthy Dogs.

Objective: To determine the safety and tolerability of escalating doses of three cannabis oil formulations, containing predominantly CBD, THC, or CBD and THC (1.5:1) vs. placebo in dogs. Design: Randomized, placebo-controlled, blinded, parallel study. Animals: Twenty healthy Beagle dogs (10 males, 10 females). Methods: Dogs were randomly assigned to one of five treatment groups (n = 4 dogs per group balanced by sex): CBD-predominant oil, THC-predominant oil, CBD/THC-predominant oil (1.5:1), sunflower oil placebo, medium-chain triglyceride oil placebo. Up to 10 escalating doses of the oils were planned for administration via oral gavage, with at least 3 days separating doses. Clinical observations, physical examinations, complete blood counts, clinical chemistry, and plasma cannabinoids were used to assess safety, tolerability, and the occurrence of adverse events (AEs). AEs were rated as mild, moderate, or severe/medically significant. Results: Dose escalation of the CBD-predominant oil formulation was shown to be as safe as placebo and safer than dose escalation of oils containing THC (CBD/THC oil or THC oil). The placebo oils were delivered up to 10 escalating volumes, the CBD oil up to the tenth dose (640.5 mg; ~62 mg/kg), the THC oil up to the tenth dose (597.6 mg; ~49 mg/kg), and the CBD/THC oil up to the fifth dose (140.8/96.6 mg CBD/THC; ~12 mg/kg CBD + 8 mg/kg THC). AEs were reported in all dogs across the five groups and the majority (94.9%) were mild. Moderate AEs (4.4% of all AEs) and severe/medically significant AEs (0.8% of all AEs) manifested as constitutional (lethargy, hypothermia) or neurological (ataxia) symptoms and mainly occurred across the two groups receiving oils containing THC (CBD/THC oil or THC oil). Conclusions and clinical significance: Overall, dogs tolerated dose escalation of the CBD oil well, experiencing only mild AEs. The favorable safety profile of 10 escalating doses of a CBD oil containing 18.3-640.5 mg CBD per dose (~2-62 mg/kg) provides comparative evidence that, at our investigated doses, a CBD-predominant oil formulation was safer and more tolerated in dogs than oil formulations containing higher concentrations of THC.

Design, synthesis and evaluation of antiestrogen and histone deacetylase inhibitor molecular hybrids.

The combination of antiestrogens and histone deacetylase inhibitors (HDACi) has been found to be antiproliferative in breast cancer models. We designed and synthesized hybrid structures which combined structural features of the pure antiestrogen ICI-164,384 and HDACi's SAHA and entinostat in a single bifunctional molecule. The hybrids retained antiestrogenic and HDACi activity and, in the case of benzamide hybrids, were selective for Class I HDAC3 over Class II HDAC6. The hybrids possessed low micromolar to high nanomolar activity against both ER+ MCF-7 and ER- MDA-MB-231 breast cancer cell models.

Rapid changes in cardiac myofilament function following the acute activation of estrogen receptor-alpha.

Estrogens have well-recognized and complex cardiovascular effects, including altering myocardial contractility through changes in myofilament function. The presence of multiple estrogen receptor (ER) isoforms in the heart may explain some discrepant findings about the cardiac effects of estrogens. Most studies examining the impact of estrogens on the heart have focused on chronic changes in estrogen levels, and have not investigated rapid, non-genomic pathways. The first objective of this study was to determine how acute activation of ERα impacts cardiac myofilaments. Nongenomic myocardial estrogen signaling is associated with the activation of a variety of signaling pathways. p38 MAPK has been implicated in acute ER signaling in the heart, and is known to affect myofilament function. Thus, the second objective of this study was to determine if acute ERα activation mediates its myofilament effects through p38 MAPK recruitment. Hearts from female C57Bl/6 mice were perfused with the ERα agonist PPT and myofilaments isolated. Activation of ERα depressed actomyosin MgATPase activity and decreased myofilament calcium sensitivity. Inhibition of p38 MAPK attenuated the myofilament effects of ERα activation. ERα stimulation did not affect global myofilament protein phosphorylation, but troponin I phosphorylation at the putative PKA phosphorylation sites was decreased. Changes in myofilament activation did not translate into alterations in whole heart function. The present study provides evidence supporting rapid, non-genomic changes in cardiac myofilament function following acute ERα stimulation mediated by the p38 MAPK pathway.

Relaxin alters cardiac myofilament function through a PKC-dependent pathway.

The pregnancy hormone relaxin (RLX) is a powerful cardiostimulatory peptide. Despite its well-characterized effects on the heart, the intracellular mechanisms responsible for RLX's positive inotropic effects are unknown. Cardiac myofilaments are the central contractile elements of the heart, and changes in the phosphorylation status of myofilament proteins are known to mediate changes in function. The first objective of this study was to determine whether RLX stimulates myofilament activation and alters the phosphorylation of one or more myofilament proteins. RLX works through a variety of intracellular signaling cascades in different tissue types. Protein kinases A (PKA) and C (PKC) are two common molecules implicated in RLX signaling and are known to affect myofilament function. Thus the second objective of this study was to determine whether RLX mediates its myocardial effects through PKA or PKC activation. Murine myocardium was treated with recombinant H2-RLX, and cardiac myofilaments were isolated. RLX increased cardiac myofilament force development at physiological levels of intracellular Ca(2+) without altering myofilament ATP consumption. Myosin binding protein C, troponin T, and troponin I phosphorylation levels were increased with RLX treatment. Immunoblot analysis revealed an increase in myofilament-associated PKC-delta, decreases in PKC-alpha and -beta(II), but no effect on PKC-epsilon. Inhibition of PKC with chelerythrine chloride or PKC-delta with rottlerin prevented the RLX-dependent changes in myofilament function and protein phosphorylation. PKA antagonism with H-89 had no effect on the myofilament effects of RLX. This study is the first to show that RLX-dependent changes in myofilament-associated PKC alters myofilament activation in a manner consistent with its cardiostimulatory effects.