LC-MS/MS quantification of Δ8-THC, Δ9-THC, THCV ISOMERS and their main metabolites in human plasma.

BACKGROUND: In recent years, potential therapeutic applications of several different cannabinoids, such as Δ9-tetrahydrocannabinol (Δ9-THC), its isomer Δ8-THC and Δ9-tetrahydrocannabivarin (Δ9-THCV), have been investigated. Nevertheless, to establish dose-effect relationship and to gain knowledge of their pharmacokinetics and metabolism, sensitive and specific analytical assays are needed to measure these compounds in patients. For this reason, we developed and validated an online extraction high-performance liquid chromatography- tandem mass spectrometry (LC/LC-MS/MS) method for the simultaneous quantification of 13 cannabinoids and metabolites including the Δ8 and Δ9 isomers of THC, THCV and those of their major metabolites in human plasma. METHODS: Plasma was fortified with cannabinoids at varying concentrations within the working range of the respective compound and 200 µL were extracted using a simple one-step protein precipitation procedure. The extracts were analyzed using online trapping LC/LC-atmospheric pressure chemical ionization (APCI)-MS/MS running in the positive multiple reaction monitoring (MRM) mode. RESULTS: The lower limit of quantification ranged from 0.5 to 2.5 ng/mL and the upper limit of quantification was 400 ng/mL for all analytes. Inter-day analytical accuracy and imprecision ranged from 82.9 to 109% and 4.3 to 20.3% (coefficient of variance), respectively. Of 534 plasma samples following controlled oral administration of Δ8-THCV, 236 were positive for Δ8-THCV (median; interquartile ranges: 3.5 ng/mL; 1.8 - 11.9 ng/mL), 383 for the major metabolite (-)-11-nor-9-carboxy-Δ8-tetrahydrocannabivarin (Δ8-THCV-COOH) (95.4 ng/mL; 20.7 - 328 ng/mL), 260 for (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabivarin (Δ9-THCV-COOH) (5.8 ng/mL; 2.5 - 16.1 ng/mL), 157 for (-)-11-hydroxy-Δ8-tetrahydrocannabivarin (11-OH-Δ8-THCV) (1.7 ng/mL; 1.0 - 3.7 ng/mL), 49 for Δ8-THC-COOH (1.7 ng/mL; 1.4 - 2.3 ng/mL) and 42 for Δ9-THCV (1.3 ng/mL; 0.8 - 1.6 ng/mL). CONCLUSIONS: We developed and validated the first LC/LC-MS/MS assay for the specific quantification of Δ8-THC, Δ9-THC and THCV isomers and their respective metabolites in human plasma. Δ8-THCV-COOH, 11-hydroxy-Δ8-THCV and Δ9-THCV-COOH were the major Δ8-THCV metabolites in human plasma after oral administration of 98.6% pure Δ8-THCV.

Using In Silico Molecular Docking to Explain Differences in Receptor Binding Behavior of HHC and THCV Isomers: Revealing New Binding Modes.

Even slight structural differences between phytocannabinoid isomers are usually enough to cause a change in their biological properties. In this study, we used in vitro CB1 agonism/antagonism assays to compare the receptor binding functionality of THCV (tetrahydrocannabivarin) and HHC (hexahydrocannabinol) isomers and applied molecular docking to provide an explanation for the difference in the activities. No CB1 agonism was observed for ∆9- and ∆8-THCV. Instead, both isomers antagonized CP 55940, with ∆9-THCV being approximately two times more potent than the ∆8 counterpart (IC50 = 52.4 nM and 119.6 nM for ∆9- and ∆8-THCV, respectively). Docking simulations found two binding poses for THCV isomers, one very similar to ∆9-THC and one newly discovered pose involving the occupation of side pocket 1 of the CB1 receptor by the alkyl chain of the ligand. We suggested the latter as a potential antagonist pose. In addition, our results established 9R-HHC and 9S-HHC among partial agonists of the CB1 receptor. The 9R-HHC (EC50 = 53.4 nM) isomer was a significantly more potent agonist than 9S (EC50 = 624.3 nM). ∆9-THC and 9R-HHC showed comparable binding poses inside the receptor pocket, whereas 9S-HHC adopted a new and different binding posture that can explain its weak agonist activity.

Chemistry, Crystal Structure, and In Vitro Receptor Binding of Δ10-THC Isomers.

Introduction: The psychoactive properties of Δ10-THC isomers (trans- and cis-Δ10-THC) are poorly understood. To shed more light on the biological effects of these compounds, we studied in vitro receptor binding of Δ10-THC isomers at cannabinoid CB1 and CB2 receptors. Materials and Methods: We first optimized and simplified catalytic synthesis of trans- and cis-Δ10-THC to allow their safe and cheap large-scale synthesis. In our synthesis, BuLi was replaced with KOtBu, and DMSO/anisole or NEt3/heptane solvent systems were used instead of HMPA/toluene. Single crystal X-ray analysis confirmed the structure of both isomers and the configuration of their chiral centers. Results: In the radioligand replacement assay, both isomers showed strong affinity toward the CB1 receptor, with IC50=29.1 nM for the trans isomer and IC50=294.2 nM for the cis counterpart. However, the IC50 values were significantly higher than that of Δ9-THC (2.1 nM), a naturally occurring psychoactive component of cannabis sativa, suggesting a lower affinity of Δ10-THCs toward this receptor. In function assays, in contrast to Δ9-THC, both isomers failed to show any agonist properties at concentrations up to 10 µM suggesting a lack of THC-like psychoactivity for trans- and cis-Δ10-THC. Conclusions: Our results established Δ10-THC isomers among antagonists of the CB1 receptor as both cis and trans isomers antagonized CP55,490 with IC50=460 nM for trans and IC50=1040 nM for cis. This functional property has not been previously observed for any other THC isomers.

Evaluation of the Modulatory Effects of Minor Cannabinoids and Terpenes on Delta-9-Tetrahydrocannabinol Discrimination in Rats.

Introduction: Cannabis contains a multitude of phytocannabinoids and terpenes in addition to its main psychoactive constituent, delta-9-tetrahydrocannabinol (D9-THC). It is believed that the combination of minor cannabinoids and terpenes with D9-THC may impact the subjective and physiological effects of D9-THC. In this study, select minor cannabinoids (cannabigerol [CBG], cannabidivarin [CBDV], cannabichromene [CBC], tetrahydrocannabivarin [THCV], cannabigerolic acid [CBGa], and cannabidiolic acid [CBDa]) and terpenes (beta-caryophyllene and linalool) were evaluated for their potential to decrease the interoceptive effects of D9-THC using drug discrimination methods. Materials and Methods: Male and female rats (n=16; 50% female) were trained to discriminate D9-THC from vehicle. Following training, D9-THC was administered 45 min pre-session, followed by administration of a minor cannabinoid or terpene (or vehicle) 15 min pre-session. CBG, CBDV, CBC, and THCV were administered at doses of 3-30 mg/kg; CBGa and CBDa were administered at doses of 10-100 mg/kg; beta-caryophyllene and linalool were administered at doses of 10-30 mg/kg. Percentage of D9-THC responding (%) was calculated to assess changes to D9-THCs interoceptive effects. Results: CBG, CBDV, CBC, THCV, CBGa, CBDa, beta-caryophyllene, and linalool had little effect on percent D9-THC responding in either sex. No compounds lowered percent D9-THC responding to 50% or below. THCV, CBC, CBDa, and beta-caryophyllene in combination with D9-THC decreased response rates compared with D9-THC alone. Conclusions: The minor cannabinoids and terpenes examined in the current study did not alter the discriminative stimulus effects of D9-THC. These results suggest that these compounds are unlikely to lower the psychoactive effects of D9-THC in human users.

Long and Short-term Metformin Consumption as a Potential Therapy to Prevent Complications of COVID-19.

Purpose: The aim of the study is to evaluate the effect of metformin in complication improvement of hospitalized patients with COVID-19. Methods: This was a randomized clinical trial that involved 189 patients with confirmed COVID-19 infection. Patients in the intervention group received metformin-500 mg twice daily. Patients who received metformin before admission were excluded from the control group. Patients who were discharged before taking at least 2000 mg of metformin were excluded from the study. Primary outcomes were vital signs, need for ICU admission, need for intubation, and mortality. Results: Data showed that patients with diabetes with previous metformin in their regimen had lower percentages of ICU admission and death in comparison with patients without diabetes (11.3% vs. 26.1% (P=0.014) and 4.9% vs. 23.9% (P≤0.001), respectively). Admission time characteristics were the same for both groups except for diabetes and hyperlipidemia, which were significantly different between the two groups. Observations of naproxen consumption on endpoints, duration of hospitalization, and the levels of spO2 did not show any significant differences between the intervention and the control group. The adjusted OR for intubation in the intervention group versus the control group was 0.21 [95% CI, 0.04-0.99 (P=0.047)]. Conclusion: In this trial, metformin consumption had no effect on mortality and ICU admission rates in non-diabetic patients. However, metformin improved COVID-19 complications in diabetic patients who had been receiving metformin prior to COVID-19 infection, and it significantly lowered the intubation rates.

A comprehensive review on methods for promotion of mechanical features and biodegradation rate in amniotic membrane scaffolds.

Amniotic membrane (AM) is a biological tissue that surrounds the fetus in the mother's womb. It has pluripotent cells, immune modulators, collagen, cytokines with anti-fibrotic and anti-inflammatory effect, matrix proteins, and growth factors. In spite of the biological characteristics, some results have been released in preventing the adhesion on traumatized surfaces. Application of the AM as a scaffold is limited due to its low biomechanical resistance and rapid biodegradation. Therefore, for using the AM during surgery, its modification by different methods such as cross-linking of the membrane collagen is necessary, because the cross-linking is an effective way to reduce the rate of biodegradation of the biological materials. In addition, their cross-linking is likely an efficient way to increase the tensile properties of the material, so that they can be easily handled or sutured. In this regard, various methods related to cross-linking of the AM subsuming the composite materials, physical cross-linking, and chemical cross-linking with the glutraldehyde, carbodiimide, genipin, aluminum sulfate, etc. are reviewed along with its advantages and disadvantages in the current work.

Comparison of losartan and amlodipine effects on the outcomes of patient with COVID-19 and primary hypertension: A randomised clinical trial.

BACKGROUND: Controversy exists regarding the drug selection in hypertension (HTN) management in patients with COVID-19. This study aimed to compare the effects of losartan and amlodipine in patients with primary HTN and COVID-19. METHODS: In this randomised clinical trial, hospitalised patients with COVID-19 and primary HTN were enrolled in the study. One arm received losartan, 25 mg, twice a day and the other arm received amlodipine, 5 mg per day for 2 weeks. The main outcomes were compare 30-day mortality rate and length of hospital stay. RESULTS: The mean age of patients treated with losartan (N = 41) and amlodipine (N = 39) was 67.3 ± 14.8 and 60.1 ± 17.3 years, respectively (P value = .068). The length of hospital stay in losartan and amlodipine groups was 4.57 ± 2.59 and 7.30 ± 8.70 days, respectively (P value = .085). Also, the length of ICU admission in losartan and amlodipine group was 7.13 ± 5.99 and 7.15 ± 9.95 days, respectively (P value = .994). The 30-day mortality was two and five patients in losartan and amlodipine groups, respectively (P value = .241). CONCLUSIONS: There was no priority in losartan or amlodipine administration in COVID-19 patients with primary HTN in decreasing mortality rate, hospital and ICU length stay. Further studies need to clarify the first-line anti-HTN medications in COVID-19.

New Insight for the Prognosis of CCHF: Clinical, Laboratory and Sonography Findings.

BACKGROUND: Crimean Congo Hemorrhagic Fever (CCHF) is an acute and fatal disease with various clinical and paraclinical characteristics. INTRODUCTION: In this article, we report data on confirmed CCHF cases from Iran and describe the association between studying factors and outcomes of the disease. METHODS: In the study design, we evaluated demographic characteristics, clinical, laboratory and sonographic findings of 160 CCHF confirmed cases during 2003 and 2012 in Zabol (A city in Sistan and Baluchestan province of Iran). The association between these factors and the fatal outcome were evaluated by regression analysis. RESULTS: The disease had a fatal outcome in 7 (4.4%) patients. Females had more severe symptoms and higher odds for death (odds ratio11.57, p=0.005). Leukocytosis (p<0.001), PT (p<0.001) and PTT (p=0.008) elongation, AST (p=0.010) and ALT (p>0.001) elevation were significantly associated with fatal outcome. CNS related symptoms (odds ratio 5.9, p=0.027) in clinical examination and ascites (odds ratio 38.4, p=0.012) and liquid in the pelvic cavity (odds ratio 24.2, p=0.004) were also identified as risk factors of death in this study. CONCLUSION: Our data suggest that in addition to clinical and laboratory findings practitioners consider sonography for CCHF prognosis.

A Comparison of Continuous Thoracic Epidural Analgesia with Bupivacaine Versus Bupivacaine and Dexmedetomidine for Pain Control in Patients with Multiple Rib Fractures.

BACKGROUND: The control of pain in traumatic patients with chest injury leading to rib fracture is one of the primary goals in traumatic patients. The efficacy of the thoracic epidural approach in comparison with other approaches for relieving post-thoracotomy pain is unknown. The goal of the present study was to compare thoracic epidural analgesia with bupivacaine alone and in combination with dexmedetomidine in patients with multiple rib fractures. METHODS: 64 traumatic patients with multiple rib fractures were selected and randomly assigned to two similar groups. For pain relief, a thoracic epidural catheter was inserted to infuse bupivacaine alone or the combination of bupivacaine and dexmedetomidine. Then, we recorded and analyzed pain scores and ABG changes. RESULTS: Based on the results, the two approaches could result in proper analgesia, but analgesia with the combination of bupivacaine and dexmedetomidine was significantly improved compared to bupivacaine alone (P < 0.05). In addition, ABG of patients significantly changed when the combination of bupivacaine and dexmedetomidine was used within 2 to 4 days (P < 0.05). CONCLUSIONS: The results of the present study showed that epidural infusion of a combination of bupivacaine and dexmedetomidine could provide better control of rib fracture pain in traumatic patients, and is a proper alternative for bupivacaine alone.

Double strain probiotic effect on Helicobacter pylori infection treatment: A double-blinded randomized controlled trial.

BACKGROUND: A decreased rate of successful helicobacter pylori (H.pylori) infection treatment has revealed serious demand for more effective regimens to eradicate infection. Therefore, probiotics have recently been considered to increase the rate of antibiotic regimens efficacy in H. pylori infections. In current randomized controlled trial, we evaluated the effect of double strain probiotic combination with standard triple therapy (STT), in the eradication rate of H. pylori infection. METHODS: In current randomized placebo-control study, all patients (176 subjects) underwent the STT for 10 days. However, the study group received triple therapy for the eradication of H. pylori with supplement of Lactobacillus probiotic for 4 weeks and placebo was administered to control group, as well. Adverse effects of the antibiotic regimen were recorded for all patients. Six weeks after the cessation of probiotic intake, all patients underwent H. Pylori with fecal antigen of test, followed by a recurrence evaluation six months later. RESULTS: There was no significant difference in demographic data and presenting symptoms between the study groups. The eradication rate of H. pylori infection was significantly higher in probiotic group (78.4%), compared to that of placebo group (64.8%) (P=0.033). In addition, adverse events were significantly less prevalent in patients that received probiotic (P=0.047). Nonetheless, there was no significant difference in terms of infection recurrence during a 6-month follow-up (P=0.07). CONCLUSION: Double strain probiotic in combination with STT increased the eradication rate of H. pylori infection, while the adverse events due to antibiotic therapy decreased.