ß-Caryophyllene Inhibits Monoacylglycerol Lipase Activity and Increases 2-Arachidonoyl Glycerol Levels In Vivo: A New Mechanism of Endocannabinoid-Mediated Analgesia?

The mechanisms of ß-caryophyllene (BCP)-induced analgesia are not well studied. Here, we tested the efficacy of BCP in an acute postsurgical pain model and evaluated its effect on the endocannabinoid system. Rats were treated with vehicle and 10, 25, 50, and 75 mg/kg BCP. Paw withdrawal responses to mechanical stimuli were evaluated using an electronic von Frey anesthesiometer. Endocannabinoids, including 2-arachidonoylglycerol (2-AG), were also evaluated in plasma and tissues using high-performance liquid chromatography-tandem mass spectrometry. Monoacylglycerol lipase (MAGL) activity was evaluated in vitro as well as ex vivo. We observed a dose-dependent and time-dependent alleviation of hyperalgesia in incised paws up to 85% of the baseline value at 30 minutes after administration of BCP. We also observed dose-dependent increases in the 2-AG levels of about threefold after administration of BCP as compared with vehicle controls. Incubations of spinal cord tissue homogenates from BCP-treated rats with isotope-labeled 2-arachidonoylglycerol-d8 revealed a reduced formation of the isotope-labeled MAGL product 2-AG-d8 as compared with vehicle controls, indicating MAGL enzyme inhibition. In vitro MAGL enzyme activity assessment using 2-AG as the substrate revealed an IC50 of 15.8 µM for MAGL inhibition using BCP. These data showed that BCP inhibits MAGL activity in vitro and in vivo, causing 2-AG levels to rise. Since the endocannabinoid 2-AG is a CB1 and CB2 receptor agonist, we propose that 2-AG-mediated cannabinoid receptor activation contributes to BCP's mechanism of analgesia. SIGNIFICANCE STATEMENT: ß-Caryophyllene (BCP) consumption is relatively safe and is approved by the Food and Drug Administration as a flavoring agent, which can be used in cosmetic and food additives. BCP is a potent anti-inflammatory agent that showed substantial antihyperalgesic properties in this study of acute pain suggesting that BCP might be an alternative to opioids. This study shows an additive mechanism (monoacylglycerol lipase inhibition) by which BCP might indirectly alter CB1 and CB2 receptor activity and exhibit its pharmacological properties.

Short-Term Cannabidiol with Δ-9-Tetrahydrocannabinol in Parkinson's Disease: A Randomized Trial.

BACKGROUND: Cannabis use is frequent in Parkinson's disease (PD), despite inadequate evidence of benefits and risks. OBJECTIVE: The aim is to study short-term efficacy and tolerability of relatively high cannabidiol (CBD)/low Δ-9-tetrahydrocannabinol (THC) to provide preliminary data for a longer trial. METHODS: Persons with PD with ≥20 on motor Movement Disorder Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS) who had negative cannabis testing took cannabis extract (National Institute of Drug Abuse) oral sesame oil solution for 2 weeks, increasing to final dose of 2.5 mg/kg/day. Primary outcome was change in motor MDS-UPDRS from baseline to final dose. RESULTS: Participants were randomized to CBD/THC (n = 31) or placebo (n = 30). Mean final dose (CBD/THC group) was 191.8 ± 48.9 mg CBD and 6.4 ± 1.6 mg THC daily. Motor MDS-UPDRS was reduced by 4.57 (95% CI, -8.11 to -1.03; P = 0.013) in CBD/THC group, and 2.77 (-4.92 to -0.61; P = 0.014) in placebo; the difference between groups was non-significant: -1.80 (-5.88 to 2.27; P = 0.379). Several assessments had a strong placebo response. Sleep, cognition, and activities of daily living showed a treatment effect, favoring placebo. Overall adverse events were mild and reported more in CBD/THC than placebo group. On 2.5 mg/kg/day CBD plasma level was 54.0 ± 33.8 ng/mL; THC 1.06 ± 0.91 ng/mL. CONCLUSIONS: The brief duration and strong placebo response limits interpretation of effects, but there was no benefit, perhaps worsened cognition and sleep, and there was many mild adverse events. Longer duration high quality trials that monitor cannabinoid concentrations are essential and would require improved availability of research cannabinoid products in the United States. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Cognitive Safety Data from a Randomized, Double-Blind, Parallel-Group, Placebo-Controlled Phase IIb Study of the Effects of a Cannabidiol and Δ9-Tetrahydrocannabinol Drug on Parkinson's Disease-Related Motor Symptoms.

BACKGROUND: Cannabis is increasingly available worldwide but its impact on cognition in Parkinson's disease (PD) is unknown. OBJECTIVE: Present cognitive safety data from study of an oral high-dose cannabidiol (CBD; 100 mg) and low-dose Δ9-tetrahydocannabinol (THC; 3.3 mg) drug in PD. METHODS: Randomized, double-blind, parallel-group, placebo-controlled study of a CBD/THC drug administered for 16.3 (SD: 4.2) days, with dosage escalating to twice per day. Neuropsychological tests were administered at baseline and 1-1½ hours after final dose; scores were analyzed with longitudinal regression models (alpha = 0.05). Cognitive adverse events were collected. RESULTS: When adjusted for age and education, the CBD/THC group (n = 29) performed worse than the placebo group (n = 29) on Animal Verbal Fluency. Adverse cognitive events were reported at least twice as often by the CBD/THC than the placebo group. CONCLUSION: Data suggest this CBD/THC drug has a small detrimental effect on cognition following acute/short-term use in PD. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Sex differences in endocannabinoid tone in a pilot study of cannabis use disorder and acute cannabis abstinence.

Cannabis use disorder (CUD) presents differently in men and women, particularly in symptoms of cannabis withdrawal. Novel pharmacotherapeutic interventions for CUD, such as those that target the endocannabinoid (eCB) system, must be developed in a manner consistent with these sex differences. The present pilot study sought to prospectively assess sex differences in cannabis withdrawal in a small sample of adults with moderate-to-severe CUD and to determine if withdrawal was associated with peripheral eCB and eCB congener tone. Men and women (n = 5/sex) completed 2 weeks of study participation separated by 1 month; in the latter week, participants abstained from cannabis use. Each week, participants attended in-person laboratory visits during which blood was drawn repeatedly to assess plasma eCB and eCB congener tone. Participants also completed multiple daily ambulatory assessments to assess cannabis use and withdrawal symptoms. As anticipated, women reported a greater increase in withdrawal symptoms during the abstinent week [Δ = 9.4 (SE = 1.1); p < 0.001] than men [Δ = 1.2 (SE = 1.2); p = 0.35]. Sex differences in levels of the eCB N-arachidonoylethanolamide (AEA), as well as the eCB congeners stearoylethanolamide (SEA) and linoleylethanolamide (LEA), were evident during abstinence at the morning time point only (p's < 0.05). LEA was associated with withdrawal symptom expression in both sexes [ß = 0.16 (SE = 0.09)] and palmitoylethanolamide (PEA) [ß = 0.22 (SE = 0.13)] and 2-arachidonoylglycerol (2-AG) [ß = 0.32 (SE = 0.15)] were associated with withdrawal symptoms in women only. Pharmacotherapeutic development for CUD should consider evident sex differences in eCB and eCB congener tone during abstinence and their associations with cannabis withdrawal, as eCB-based interventions may produce differential effects by sex.

Metabolic reprogramming in a slowly developing orthologous model of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and affects 1 in 1,000 individuals. There is accumulating evidence suggesting that there are shared cellular mechanisms responsible for cystogenesis in human and murine PKD and that reprogramming of metabolism is a key disease feature. In this study, we used a targeted metabolomics approach in an orthologous mouse model of PKD (Pkd1RC/RC) to investigate the metabolic modifications a cystic kidney undergoes during disease progression. Using the Kyoto Encyclopedia of Genes and Genomes pathway database, we identified several biologically relevant metabolic pathways that were altered early in this disease (in 3-mo-old Pkd1RC/RC mice), the most highly represented being arginine biosynthesis and metabolism and tryptophan and phenylalanine metabolism. During the next 6 mo of disease progression, multiple uremic solutes accumulated in the kidney of cystic mice, including several established markers of oxidative stress and endothelial dysfunction (allantoin, asymmetric dimethylarginine, homocysteine, malondialdehyde, methionine sulfoxide, and S-adenosylhomocysteine). Levels of kynurenines and polyamines were also augmented in kidneys of Pkd1RC/RC versus wild-type mice, as were the levels of bacteria-produced indoles, whose increase within PKD kidneys suggests microbial dysbiosis. In summary, we confirmed previously published and identified novel metabolic markers and pathways of PKD progression that may prove helpful for diagnosis and monitoring of cystic kidney disease in patients. Furthermore, they provide targets for novel therapeutic approaches that deserve further study and hint toward currently understudied pathomechanisms.NEW & NOTEWORTHY This report delineates the evolution of metabolic changes occurring during autosomal dominant polycystic kidney disease (ADPKD) progression. Using an orthologous model, we performed kidney metabolomics and confirmed dysregulation of metabolic pathways previously found altered in nonorthologous or rapidly-progressive PKD models. Importantly, we identified novel alterations, including augmentation of kynurenines, polyamines, and indoles, suggesting increased inflammation and microbial dysbiosis that provide insights into PKD pathomechanisms and may prove helpful for diagnosing, monitoring, and treating ADPKD.

N-Oleoyl dopamine induces IL-10 via central nervous system TRPV1 and improves endotoxemia and sepsis outcomes.

BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1) participates in thermosensation and inflammatory pain, but its immunomodulatory mechanisms remain enigmatic. N-Oleoyl dopamine (OLDA), an endovanilloid and endocannabinoid, is a TRPV1 agonist that is produced in the central nervous system and the peripheral nervous system. We studied the anti-inflammatory effects and TRPV1-dependent mechanisms of OLDA in models of inflammation and sepsis. METHODS: Mice were challenged intratracheally or intravenously with LPS, or intratracheally with S. aureus to induce pneumonia and sepsis, and then were treated intravenously with OLDA. Endpoints included plasma cytokines, leukocyte activation marker expression, mouse sepsis scores, lung histopathology, and bacterial counts. The role of TRPV1 in the effects of OLDA was determined using Trpv1-/- mice, and mice with TRPV1 knockdown pan-neuronally, in peripheral nervous system neurons, or in myeloid cells. Circulating monocytes/macrophages were depleted using clodronate to determine their role in the anti-inflammatory effects of OLDA in endotoxemic mice. Levels of exogenous OLDA, and of endovanilloids and endocannabinoids, at baseline and in endotoxemic mice, were determined by LC-MS/MS. RESULTS: OLDA administration caused an early anti-inflammatory response in endotoxemic and septic mice with high serum levels of IL-10 and decreased levels of pro-inflammatory cytokines. OLDA also reduced lung injury and improved mouse sepsis scores. Blood and lung bacterial counts were comparable between OLDA- and carrier-treated mice with S. aureus pneumonia. OLDA's effects were reversed in mice with pan-neuronal TRPV1 knockdown, but not with TRPV1 knockdown in peripheral nervous system neurons or myeloid cells. Depletion of monocytes/macrophages reversed the IL-10 upregulation by OLDA in endotoxemic mice. Brain and blood levels of endovanilloids and endocannabinoids were increased in endotoxemic mice. CONCLUSIONS: OLDA has strong anti-inflammatory actions in mice with endotoxemia or S. aureus pneumonia. Prior studies focused on the role of peripheral nervous system TRPV1 in modulating inflammation and pneumonia. Our results suggest that TRPV1-expressing central nervous system neurons also regulate inflammatory responses to endotoxemia and infection. Our study reveals a neuro-immune reflex that during acute inflammation is engaged proximally by OLDA acting on neuronal TRPV1, and through a multicellular network that requires circulating monocytes/macrophages, leads to the systemic production of IL-10.

Endocannabinoid System in Polycystic Kidney Disease.

INTRODUCTION: Autosomal dominant polycystic kidney disease (ADPKD) is a commonly inherited disorder characterized by renal cyst formation. A major pathological feature of ADPKD is the development of interstitial inflammation. The endocannabinoid (EC) system is present in the kidney and has recently emerged as an important player in inflammation and the pathogenesis of progressive kidney disease. METHODS: Data on ECs were collected using a validated mass spectrometry assay from a well-characterized cohort of 102 ADPKD patients (at baseline and after 2- and 4 years on standard vs. rigorous blood-pressure control) and compared to 100 healthy subjects. RESULTS: Compared to healthy individuals, we found higher interleukins-6 and -1b as well as reduced plasma levels of anandamide (AEA), 2-arachidonoyl-glycerol (2-AG), and their congeners in ADPKD patients. Baseline AEA concentration negatively associated with the progression of ADPKD as expressed by the yearly percent change in height-corrected total kidney volume and positively with the yearly change in renal function (measured as estimated glomerular filtration rate, ΔeGFR). AEA analog palmitoylethanolamide (PEA) is also associated positively with the yearly change in eGFR. DISCUSSION AND CONCLUSION: The results of the present study suggest that ADPKD patients present with lower levels of ECs and that reestablishing the normality of the renal EC system via augmentation of AEA, PEA, and 2-AG levels, either through the increase of their synthesis or through a reduction of their degradation, could be beneficial and may present a promising therapeutic target in said patients.

Pharmacokinetics of cannabichromene in a medical cannabis product also containing cannabidiol and Δ9-tetrahydrocannabinol: a pilot study.

PURPOSE: Cannabichromene (CBC) is a phytocannabinoid commonly found in cannabis, yet its acute post-dose pharmacokinetics (PK) have not been examined in humans. This is a secondary data analysis from a trial investigating Spectrum Yellow oil, an oral cannabis product used for medical purposes that contained 20 mg cannabidiol (CBD), 0.9 mg Δ9-tetrahydrocannabinol (THC), and 1.1 mg CBC, per 1 mL of oil. METHODS: Participants (N = 43) were randomized to one of 5 groups: 120 mg CBD, 5.4 mg THC, and 6.6 mg CBC daily; 240 mg CBD, 10.8 mg THC, and 13.2 mg CBC daily; 360 mg CBD, 16.2 mg THC, and 19.8 mg CBC daily; 480 mg CBD, 21.6 mg THC, and 26.4 mg CBC daily; or placebo. Study medication was administered every 12 h for 7 days. Plasma CBC concentrations were analyzed by a validated two-dimensional high-performance liquid chromatography-tandem mass spectrometry assay. RESULTS: After a single dose and after the final dose, the Cmax of CBC increased by 1.3-1.8-fold for each twofold increase in dose; the tmax range was 1.6-4.3 h. Based on the ratio of administered CBD, THC, and CBC to the plasma concentration, the dose of CBD was 18 times higher than the dose of CBC, yet the AUC0-t of CBD was only 6.6-9.8-fold higher than the AUC0-t of CBC; the dose of THC was similar to the dose of CBC, yet THC was quantifiable in fewer plasma samples than was CBC. CONCLUSIONS: CBC may have preferential absorption over CBD and THC when administered together. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry #ACTRN12619001450101, registered 18 October 2019.

Effects of cannabidiol in cannabis flower: Implications for harm reduction.

Using a federally compatible, naturalistic at-home administration procedure, the present study examined the acute effects of three cannabis flower chemovars with different tetrahydrocannabinol (THC) to cannabidiol (CBD) ratios, in order to test whether chemovars with a higher CBD content produce different effects. Participants were randomly assigned to ad libitum administration of one of three chemovars (THC-dominant: 24% THC, 1% CBD; THC+CBD: 9% THC, 10% CBD; CBD-dominant: 1% THC, 23% CBD); 159 regular cannabis users (male = 94, female = 65) were assessed in a mobile pharmacology lab before, immediately after, and 1 h after ad libitum administration of their assigned chemovar. Plasma cannabinoids as well as positive (e.g., high, elation) and negative (e.g., paranoia and anxiety) subjective effects were assessed at each time points. Participants who used the CBD-dominant and THC + CBD chemovars had significantly less THC and more CBD in plasma samples compared to participants who used the THC-dominant chemovar. Further, the THC + CBD chemovar was associated with similar levels of positive subjective effects, but significantly less paranoia and anxiety, as compared to the THC-dominant chemovar. This is one of the first studies to examine the differential effects of various THC to CBD ratios using chemovars that are widely available in state-regulated markets. Individuals using a THC + CBD chemovar had significantly lower plasma THC concentrations and reported less paranoia and anxiety while also reporting similar positive mood effects as compared to individuals using THC only, which is intriguing from a harm reduction perspective. Further research is needed to clarify the harm reduction potential of CBD in cannabis products.

Using Population Pharmacokinetic Modeling to Estimate Exposure to Δ9-Tetrahydrocannabinol in an Observational Study of Cannabis Smokers in Colorado.

BACKGROUND: Self-report questionnaires, weighing products consumed, and Δ9-tetrahydrocannabinol (THC) biomarkers are established techniques for estimating cannabis exposure. Population pharmacokinetic modeling of plasma THC and metabolite concentrations by incorporating self-reported and weighed products as covariates could improve estimates of THC exposure in regular cannabis users. METHODS: In this naturalistic study, blood samples were obtained from 36 regular smokers of cannabis for analysis of THC and its 2 metabolites at 4 time points: recruitment and during an experimental mobile laboratory assessment that included 3 time points: before, immediately after, and 1 hour after ad libitum legal market flower use. These data were analyzed using an established model of population pharmacokinetics developed from laboratory-controlled cannabis administration data. Elimination and metabolite production clearances were estimated for each subject as well as their daily THC doses and the dose consumed during the ad libitum event. RESULTS: A statistically significant correlation existed between the daily THC dose estimated by self-report questionnaire and population pharmacokinetic modeling (correlation coefficient = 0.79, P < 0.05) between the weighed cannabis smoked ad libitum and that estimated by population pharmacokinetic modeling (correlation coefficient = 0.71, P < 0.05). CONCLUSION: Inclusion of self-reported questionnaire data of THC consumption improved pharmacokinetic model-derived estimates based on measured THC and metabolite concentrations. In addition, the pharmacokinetic-derived dose estimates for the ad libitum smoking event underestimated the THC consumption compared with the weighed amount smoked. Thus, the subjects in this study, who smoked ad libitum and used cannabis products with high concentrations of THC, were less efficient (lower bioavailability) compared with computer-paced smokers of low potency, NIDA cannabis in a laboratory setting.

Analysis of 14 endocannabinoids and endocannabinoid congeners in human plasma using column switching high-performance atmospheric pressure chemical ionization liquid chromatography-mass spectrometry.

The endocannabinoid system (ECS) is a complex cell-signaling system. To address the growing need of analytics capturing endocannabinoid levels to investigate the ECS, we developed and validated an assay for the quantitative analysis of 14 endocannabinoids and congeners. A simple extraction using protein precipitation with acetonitrile followed by online-trapping high-performance liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) was used to monitor the levels of 14 endocannabinoids in plasma. The assay was validated and intra-run and inter-run accuracies and imprecisions as well as matrix effects, recoveries, and sample stabilities were determined. As a proof of concept, a subset of study samples after naturalistic administration of Cannabis flower and concentrate was analyzed. With the exception of N-oleoyl dopamine and oleamide, all endocannabinoids fulfilled the predefined acceptance criteria. Reproducible recoveries and no significant matrix effects were observed. Sample stability was an issue. Analysis of the proof-of-concept study samples revealed a significantly (p = 0.006) higher concentration of docosatetraenoyl ethanolamide in concentrate users (300 ± 13 pg/mL) compared to flower users (252 ± 11 pg/mL). A robust, sensitive high-throughput assay for the quantitation of 14 endocannabinoids and congeners was successfully validated. Our study showed that it is mandatory to (A) appropriately stabilize samples and (B) separate and separately quantify 1-AG and 2-AG; otherwise, study results are unreliable. The analysis of study samples from Cannabis flower users versus Cannabis concentrate users revealed higher levels of docosatetraenoyl ethanolamide and anandamide (n.s.) in high THC concentrate users in accordance with the existing literature, supporting the validity of the assay measurements. Graphical abstract.

Population pharmacokinetic modeling of plasma Δ9-tetrahydrocannabinol and an active and inactive metabolite following controlled smoked cannabis administration.

AIMS: Population pharmacokinetic models of Δ9-tetrahydrocannabinol (THC) have been developed for THC plasma and blood concentration data. Often, only the metabolites of THC are measurable when blood samples are obtained. Therefore, we performed a population pharmacokinetic analysis of THC, 11-OH-THC and THCCOOH plasma concentration data from a Phase I clinical trial of THC smoking. METHODS: Frequently obtained plasma THC, 11-OH-THC and THCCOOH concentration data were obtained over 168 h from 6 subjects who smoked low (15.8 mg) and high dose (33.8 mg) THC cigarettes on 2 occasions. Bayesian estimates of the THC pharmacokinetic model from each individual for each dose were fixed prior to the sequential pharmacokinetic analysis of the metabolites. RESULTS: A 3-compartment model of THC was developed that has a steady-state volume of distribution (VdSS ) of 3401 ± 788 L and a clearance of 0.72 ± 0.10 L/min. 11-OH-THC was characterized by 50 ± 6% of the THC being directly cleared to a 3-compartment model with a VdSS of 415.2 ± 4.3 L and clearance of 0.78 ± 0.05 L/min. The THCCOOH model shared the central compartment of the 11-OH-THC model with a VdSS of 29.1 ± 0.05 L and a clearance of 0.12 ± 0.02 L/min. First order kinetics were observed for THC and THCCOOH between the low and high doses, but a nonlinear pattern was observed for 11-OH-THC. CONCLUSION: We describe the pharmacokinetics of THC, 11-OH-THC and THCCOOH including inter- and intraindividual variability of the parameter estimates of the model.

Alterations in Oscillatory Behavior of Central Medial Thalamic Neurons Demonstrate a Key Role of CaV3.1 Isoform of T-Channels During Isoflurane-Induced Anesthesia.

Although the central medial nucleus (CeM) of the thalamus is an essential part of the arousal system for sleep and anesthesia initiation, the precise mechanisms that regulate its activity are not well studied. We examined the role of CaV3.1 isoform of T-type calcium channels (T-channels) in the excitability and rhythmic activity of CeM neurons during isoflurane (ISO)-induced anesthesia by using mouse genetics and selective pharmacology. Patch-clamp recordings taken from acute brain slices revealed that CaV3.1 channels in CeM are inhibited by prototypical volatile anesthetic ISO (250 and 500 µM) and selective T-channels blocker 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2). Both TTA-P2 and ISO attenuated tonic and burst firing modes, and hyperpolarized CeM neurons from wild type (WT) mice. These effects were greatly diminished or abolished in CaV3.1 null mice. Our ensuing in vivo local field potential (LFP) recordings from CeM indicated that the ability of TTA-P2 and anesthetic concentrations of ISO to promote δ oscillation was substantially weakened in CaV3.1 null mice. Furthermore, escalating ISO concentrations induced stronger burst-suppression LFP pattern in mutant than in WT mice. Our results demonstrate for the first time the importance of CaV3.1 channels in thalamocortical oscillations from the non-specific thalamic nuclei that underlie clinically important effects of ISO.

Temsirolimus metabolic pathways revisited.

Temsirolimus, a derivative of sirolimus, exhibits potent antitumor properties. It was the goal of this study to identify yet unknown temsirolimus metabolites generated after incubation with human liver microsomes. Previously, 23-hydroxy-, 24-hydroxy, 12-hydroxy, hydroxy-piperidine and 27-O-desmethyl temsirolimus had been described.Metabolite structures were identified using high-resolution mass spectrometry, MS/iontrap (MSn) and comparison of fragmentation patterns of the metabolites with those of temsirolimus and other known sirolimus derivatives. Moreover, enzyme kinetic parameters of temsirolimus metabolite formation as well as the contribution of individual recombinant cytochrome P450 (CYP) enzymes to temsirolimus metabolism were investigated.Human liver microsomes mainly hydroxylated and/or demethylated temsirolimus. The structures of the following metabolites were identified: O-demethylated metabolites: 39-O-desmethyl, 16-O-desmethyl and 27-O-desmethyl temsirolimus; hydroxylated metabolites: hydroxy piperidine temsirolimus, 11-hydroxy, 12-hydroxy, 14-hydroxy, 23-hydroxy, 24-hydroxy, 25-hydroxy, 45/46-hydroxy and 49-hydroxy temsirolimus; demethylated-hydroxylated metabolites: 16-O-desmethyl, 24-hydroxy; 16-O-desmethyl, 23-hydroxy and 16-O-desmethyl 46-hydroxy temsirolimus; didemethylated metabolite: 27,39-O-didesmethyl temsirolimus; and dihydroxylated metabolite: 12,24-dihydroxy temsirolimus. It was confirmed that CYP3A4 represents the predominant enzyme responsible for temsirolimus metabolism. Moreover, CYP3A5 as well as CYP2C8 also showed significant activities especially resulting in the formation of 27-O-desmethyl, 25-hydroxy and hydroxy-piperidine temsirolimus.It is concluded that temsirolimus is metabolized to more than 20 metabolites, not counting metabolism via the sirolimus pathway. Eighteen of these metabolites could be structurally identified using ion trap MSn and high-resolution mass spectrometry. Moreover, the present study showed that, in addition to CYP3A4, metabolism via CYP3A5 and CYP2C8 also represent significant metabolic pathways.

Cyclophilin D knockout protects the mouse kidney against cyclosporin A-induced oxidative stress.

Mitochondrial dysfunction and oxidative stress have been implicated in cyclosporin A (CsA)-induced nephrotoxicity. CsA interacts with cyclophilin D (CypD), an essential component of the mitochondrial permeability transition pore and regulator of cell death processes. Controversial reports have suggested that CypD deletion may or may not protect cells against oxidative stress-induced cell death. In the present study, we treated wild-type (WT) mice and mice lacking CypD [peptidylprolyl isomerase F knockout (Ppif-/-) mice] with CsA to test the role and contribution of CypD to the widely described CsA-induced renal toxicity and oxidative stress. Our results showed an increase in the levels of several known uremic toxins as well as the oxidative stress markers PGF2α and 8-isoprostane in CsA-treated WT animals but not in Ppif-/- animals. Similarly, a decline in S-adenosylmethionine and the resulting methylation potential indicative of DNA hypomethylation were observed only in CsA-treated WT mice. This confirms previous reports of the protective effects of CypD deletion on the mouse kidney mediated through a stronger resistance of these animals to oxidative stress and DNA methylation damage. However, a negative effect of CsA on the glycolysis and overall energy metabolism in Ppif-/- mice also indicated that additional, CypD-parallel pathways are involved in the toxic effects of CsA on the kidney. In summary, CsA-mediated induction of oxidative stress is associated with CypD, with CypD deletion providing a protective effect, whereas the reduction of energy production observed upon CsA exposure did not depend on the animals' CypD status.

Assessment of tacrolimus intrapatient variability in stable adherent transplant recipients: Establishing baseline values.

The purpose of this study was to determine the intrapatient (within the same patient) variability of tacrolimus in adherent patients. Daily tacrolimus trough levels were obtained at home using dried blood spot technology in kidney and liver transplant recipients. Patients were randomized to receive 3 formulations of tacrolimus, each for two 1-week periods. Adherence was monitored by patient diary, pill counts, and use of the Medication Event Monitoring System (MEMS). Variability was quantified as the coefficient of variation (CV). Comparison of CV between groups was by independent t test or one-way ANOVA as appropriate. The population was found to be adherent with a rate of 99.9% with a mean interval between the evening and morning dose of tacrolimus of 11.86 hours. The median CV for the entire population was 15.2% (range 4.8%-110%). There were no differences in CV by allograft type or tacrolimus formulation. The multivariate analysis did not identify any demographic characteristics associated with a CV > 30%. In a highly adherent population, tacrolimus did not display high intrapatient variability. Given the association between IPV and poor allograft outcomes, future studies are needed to quantitate the influence of adherence and establish target IPV goals.

Depressed prostaglandins and leukotrienes in veterans with Gulf War illness.

Background: There is need to understand biological markers and mechanisms in Gulf War illness (GWI). Goal: To examine whether and how eicosanoids - prostaglandins and leukotrienes - are altered in veterans with GWI. Methods: Seventy participants including 37 GWI and 33 healthy controls, shared exposure information, and had plasma eicosanoids assessed - prostaglandin F2 alpha (pgf2α), prostaglandin D2 (pgd2), leukotriene B4 (lb4) among others. Values were compared for GWI versus controls. Eicosanoid intercorrelations were compared in cases vs. controls. For the most significantly altered eicosanoid in GWI, exposure and symptom relations were assessed. Results: Prostaglandins and leukotrienes were depressed in GWI, strongest for pgf2α, then lb4. Eicosanoid intercorrelations differed in GWI vs. controls. Fuel-solvent, pesticide, radioactive chemicals and metal exposures related negatively to pgf2α; as, in GWI, did chemical attack and vaccines. Multivariate predictors included fuels-solvents and radioactive chemicals (negative); tetanus vaccine and herbicides (positive). Fuels-solvents and radioactive chemicals predicted lower pgf2α in cases, controls, and all participants controlled for case status. Lower pgf2α related to GWI "Kansas criteria" domains of pain, respiratory, and (borderline significantly) skin symptoms. Conclusion: Multiple eicosanoids are depressed in GWI, particularly pgf2α and lb4. Prior fuel-solvent exposures, radioactive chemicals, and (in GWI cases) vaccines were linked to lower pgf2α.

Bone marrow-derived cPLA2α contributes to renal fibrosis progression.

The group IVA calcium-dependent cytosolic phospholipase A2 (cPLA2α) enzyme directs a complex "eicosanoid storm" that accompanies the tissue response to injury. cPLA2α and its downstream eicosanoid mediators are also implicated in the pathogenesis of fibrosis in many organs, including the kidney. We aimed to determine the role of cPLA2α in bone marrow-derived cells in a murine model of renal fibrosis, unilateral ureteral obstruction (UUO). WT C57BL/6J mice were irradiated and engrafted with donor bone marrow from either WT mice [WT-bone marrow transplant (BMT)] or mice deficient in cPLA2α (KO-BMT). After full engraftment, mice underwent UUO and kidneys were collected 3, 7, and 14 days after injury. Using picrosirius red, collagen-3, and smooth muscle α actin staining, we determined that renal fibrosis was significantly attenuated in KO-BMT animals as compared with WT-BMT animals. Lipidomic analysis of homogenized kidneys demonstrated a time-dependent upregulation of pro-inflammatory eicosanoids after UUO; KO-BMT animals had lower levels of many of these eicosanoids. KO-BMT animals also had fewer infiltrating pro-inflammatory CD45+CD11b+Ly6Chi macrophages and reduced message levels of pro-inflammatory cytokines. Our results indicate that cPLA2α and/or its downstream mediators, produced by bone marrow-derived cells, play a major role in eicosanoid production after renal injury and in renal fibrinogenesis.

Ablation of Cyclophilin D Results in an Activation of FAK, Akt, and ERK Pathways in the Mouse Heart.

Cyclophilin D (CypD) is a mitochondrial chaperone that regulates the mitochondrial permeability transition pore. Metabolically, deletion of Ppif (the gene encoding CypD) in mice is associated with elevated levels of mitochondrial matrix Ca2+ that leads to increased glucose as relative to fatty acid oxidation. Here, we characterized the adaptive mechanisms involved in the regulation of glucose metabolism including the regulation of Akt and ERK kinases that we evaluated by Western blot analysis of Ppif-/- in comparison to wild type (WT) mouse hearts. CypD loss led to adaptive mechanisms in the heart resulting in an upregulation of focal adhesion kinase (phosphorylated at Tyr925) and increased phosphorylation of Akt at S473. The increased activity of this pathway (pAktS473 increased to 170% and 145% in Ppif-/- versus WT males and females, respectively) could be responsible for the observed metabolic switch towards glycolysis. Furthermore, the phosphorylation of ERK1/2 proteins was elevated following CypD ablation. In addition, we observed differences in protein expression and activity in male versus female hearts that were independent of CypD expression. This included an upregulation of pAktS473 (to 273% and 269% in Ppif-/- and WT females as compared to their corresponding males, respectively). Furthermore, decreased levels of endothelial nitric oxide synthase (eNOS) inhibitor asymmetric dimethylarginine were accompanied by an upregulation of eNOS in female mice. The higher extent of kinases phosphorylation may be responsible for the reported lowered tolerance of CypD animals to stress. Moreover, the higher nitric oxide production could be responsible for the cardioprotective properties observed only in female hearts. J. Cell. Biochem. 118: 2933-2940, 2017. © 2017 Wiley Periodicals, Inc.

Bioequivalence between innovator and generic tacrolimus in liver and kidney transplant recipients: A randomized, crossover clinical trial.

BACKGROUND: Although the generic drug approval process has a long-term successful track record, concerns remain for approval of narrow therapeutic index generic immunosuppressants, such as tacrolimus, in transplant recipients. Several professional transplant societies and publications have generated skepticism of the generic approval process. Three major areas of concern are that the pharmacokinetic properties of generic products and the innovator (that is, "brand") product in healthy volunteers may not reflect those in transplant recipients, bioequivalence between generic and innovator may not ensure bioequivalence between generics, and high-risk patients may have specific bioequivalence concerns. Such concerns have been fueled by anecdotal observations and retrospective and uncontrolled published studies, while well-designed, controlled prospective studies testing the validity of the regulatory bioequivalence testing approach for narrow therapeutic index immunosuppressants in transplant recipients have been lacking. Thus, the present study prospectively assesses bioequivalence between innovator tacrolimus and 2 generics in individuals with a kidney or liver transplant. METHODS AND FINDINGS: From December 2013 through October 2014, a prospective, replicate dosing, partially blinded, randomized, 3-treatment, 6-period crossover bioequivalence study was conducted at the University of Cincinnati in individuals with a kidney (n = 35) or liver transplant (n = 36). Abbreviated New Drug Applications (ANDA) data that included manufacturing and healthy individual pharmacokinetic data for all generics were evaluated to select the 2 most disparate generics from innovator, and these were named Generic Hi and Generic Lo. During the 8-week study period, pharmacokinetic studies assessed the bioequivalence of Generic Hi and Generic Lo with the Innovator tacrolimus and with each other. Bioequivalence of the major tacrolimus metabolite was also assessed. All products fell within the US Food and Drug Administration (FDA) average bioequivalence (ABE) acceptance criteria of a 90% confidence interval contained within the confidence limits of 80.00% and 125.00%. Within-subject variability was similar for the area under the curve (AUC) (range 12.11-15.81) and the concentration maximum (Cmax) (range 17.96-24.72) for all products. The within-subject variability was utilized to calculate the scaled average bioequivalence (SCABE) 90% confidence interval. The calculated SCABE 90% confidence interval was 84.65%-118.13% and 80.00%-125.00% for AUC and Cmax, respectively. The more stringent SCABE acceptance criteria were met for all product comparisons for AUC and Cmax in both individuals with a kidney transplant and those with a liver transplant. European Medicines Agency (EMA) acceptance criteria for narrow therapeutic index drugs were also met, with the only exception being in the case of Brand versus Generic Lo, in which the upper limits of the 90% confidence intervals were 111.30% (kidney) and 112.12% (liver). These were only slightly above the upper EMA acceptance criteria limit for an AUC of 111.11%. SCABE criteria were also met for the major tacrolimus metabolite 13-O-desmethyl tacrolimus for AUC, but it failed the EMA criterion. No acute rejections, no differences in renal function in all individuals, and no differences in liver function were observed in individuals with a liver transplant using the Tukey honest significant difference (HSD) test for multiple comparisons. Fifty-two percent and 65% of all individuals with a kidney or liver transplant, respectively, reported an adverse event. The Exact McNemar test for paired categorical data with adjustments for multiple comparisons was used to compare adverse event rates among the products. No statistically significant differences among any pairs of products were found for any adverse event code or for adverse events overall. Limitations of this study include that the observations were made under strictly controlled conditions that did not allow for the impact of nonadherence or feeding on the possible pharmacokinetic differences. Generic Hi and Lo were selected based upon bioequivalence data in healthy volunteers because no pharmacokinetic data in recipients were available for all products. The safety data should be interpreted in light of the small number of participants and the short observation periods. Lastly, only the 1 mg tacrolimus strength was utilized in this study. CONCLUSIONS: Using an innovative, controlled bioequivalence study design, we observed equivalence between tacrolimus innovator and 2 generic products as well as between 2 generic products in individuals after kidney or liver transplantation following current FDA bioequivalence metrics. These results support the position that bioequivalence for the narrow therapeutic index drug tacrolimus translates from healthy volunteers to individuals receiving a kidney or liver transplant and provides evidence that generic products that are bioequivalent with the innovator product are also bioequivalent to each other. TRIAL REGISTRATION: ClinicalTrials.gov NCT01889758.