COVID-19: Focusing on the Link between Inflammation, Vitamin D, MAPK Pathway and Oxidative Stress Genetics.

An uncontrolled inflammatory response during SARS-CoV-2 infection has been highlighted in several studies. This seems to be due to pro-inflammatory cytokines whose production could be regulated by vitamin D, ROS production or mitogen-activated protein kinase (MAPK). Several genetic studies are present in the literature concerning genetic influences on COVID-19 characteristics, but there are few data on oxidative stress, vitamin D, MAPK and inflammation-related factors, considering gender and age. Therefore, the aim of this study was to evaluate the role of single nucleotide polymorphisms in these pathways, clarifying their impact in affecting COVID-19-related clinical features. Genetic polymorphisms were evaluated through real-time PCR. We prospectively enrolled 160 individuals: 139 patients were positive for SARS-CoV-2 detection. We detected different genetic variants able to affect the symptoms and oxygenation. Furthermore, two sub-analyses were performed considering gender and age, showing a different impact of polymorphisms according to these characteristics. This is the first study highlighting a possible contribution of genetic variants of these pathways in affecting COVID-19 clinical features. This may be relevant in order to clarify the COVID-19 etiopathogenesis and to understand the possible genetic contribution for further SARS infections.

A Novel UHPLC-MS/MS Method for the Quantification of Seven Opioids in Different Human Tissues.

BACKGROUND: Opioids are considered the cornerstone of pain management: they show good efficacy as a first-line therapy for moderate to severe cancer pain. Since pharmacokinetic/pharmacodynamic information about the tissue-specific effect and toxicity of opioids is still scarce, their quantification in post-mortem autoptic specimens could give interesting insights. METHODS: We describe an ultra-high-performance liquid chromatography coupled with tandem mass spectrometry method for the simultaneous quantification of methadone, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone and fentanyl in several tissues: liver, brain, kidney, abdominal adipose tissue, lung and blood plasma. The presented method has been applied on 28 autoptic samples from different organs obtained from four deceased PLWH who used opioids for palliative care during terminal disease. RESULTS: Sample preparation was based on tissue weighing, disruption, sonication with drug extraction medium and a protein precipitation protocol. The extracts were then dried, reconstituted and injected onto the LX50 QSight 220 (Perkin Elmer, Milan, Italy) system. Separation was obtained by a 7 min gradient run at 40 °C with a Kinetex Biphenyl 2.6 µm, 2.1 × 100 mm. Concerning the analyzed samples, higher opioids concentrations were observed in tissues than in plasma. Particularly, O-MOR and O-COD showed higher concentrations in kidney and liver than other tissues (>15-20 times greater) and blood plasma (>100 times greater). CONCLUSIONS: Results in terms of linearity, accuracy, precision, recovery and matrix effect fitted the recommendations of FDA and EMA guidelines, and the sensitivity was high enough to allow successful application on human autoptic specimens from an ethically approved clinical study, confirming its eligibility for post-mortem pharmacological/toxicological studies.

COVID-19: A Possible Contribution of the MAPK Pathway.

BACKGROUND: COVID-19 is characterized by an uncontrolled inflammatory response with high pro-inflammatory cytokine production through the activation of intracellular pathways, such as mitogen-activated protein kinase (MAPK). Viruses are able to exploit the MAPK pathway to their advantage; this pathway relevance to severe COVID-19 is poorly described. The aim of this study was to quantify biomarkers involved in the MAPK pathway and to clarify its possible role in affecting some COVID-19-related clinical features. METHODS: H-RAS, C-RAF, MAPK1, MAPK2, and ERK were quantified through ELISA, and genetic polymorphisms were evaluated through real-time PCR. RESULTS: We prospectively recruited 201 individuals (158 positive and 43 negative for SARS-CoV-2): 35 were male, and their median age was 65 years. MAPK-related biomarker levels were increased in SARS-CoV-2-positive participants (n = 89) compared to negative ones (n = 29). Dyspnea was reported by 48%; this symptom was associated with PBMC C-RAF levels in positive participants (p = 0.022) and type of ventilation (p = 0.031). The highest degree of ventilation was used by 8% for invasive ventilation and 41% for continuous positive airway pressure (CPAP). CONCLUSIONS: This is the first study that showed a possible contribution of MAPK-related biomarkers in affecting COVID-19 clinical features, and this may be relevant for identifying COVID-19 positive participants at risk of serious complications.

A description of Cannabinoid levels in Cannabis oil by high-performance liquid chromatography-mass spectrometry in a reference laboratory of North-Italy.

BACKGROUND: Cannabis oils from FM2®, Bedica®, Bediol®, Bedrocan®, Bedrolite® and Pedanios 22/1® are largely used for medical purposes such as spasticity, chronic pain and appetite stimulating. Several studies showed cannabinoids action on CB1 and CB2 receptors reduces the hyperalgesic phase in inflammatory pain, leading to an improvement of conditions. The active compounds of these galenic preparations show a high variability making titration mandatory. For this reason, the exact oil composition knowledge is fundamental for personalizing therapy. This amis at adapting the correct dose to the patient, improving safety and efficacy of the galenic formulation, choosing the best preparation for each patient. PURPOSE: The aim of this study was to investigate oil preparations variability among different galenic laboratories in order to highlight the importance of titration activity. METHODS: Cannabis pharmacological active compounds titration has been performed in a large cohort of galenic laboratories in Italy. CBD, CBN, THC, THCA and CBDA quantification was carried out by a previous validated method in UHPLC-MS/MS. RESULTS: A number of 4318 samples of Cannabis oil from 83 pharmacies between January 2021 and February 2022 were evaluated. All galenic preparation specialities showed statistically significant differences among galenic laboratories (p-value < 0.001). THCA and CBDA concentrations were investigated as percentage of the extration yelds for total THC and CBD: these compounds had different values in the same specialities among distinct galenic laboratories. Moreover, seasonal variability in analytes concentrations was observed. CONCLUSION: This study described a wide range of oily samples from a large number of galenic laboratories, compared to published papers. In conclusion, knowledge of the exact oil composition is fundamental in the perspective of personalized therapy. Further studies aiming at the correlation between galenic composition and cannabinoids pharmacokinetics, clinical outcomes and toxic effects could be useful to improve our knowledge.

Pregnancy and COVID-19: The Possible Contribution of Vitamin D.

BACKGROUND: Vitamin D deficiency has been associated with the severity of COVID-19. The role of vitamin D in pregnant women with COVID-19 has been poorly investigated to date. The aim of this study was to evaluate the influence of vitamin D in affecting some clinical features in pregnancy between SARS-CoV-2 positive and negative patients. METHODS: Vitamin D pathway related polymorphisms and 25-hydroxyvitamin D levels were quantified in pregnant women followed from the first to the third trimester of pregnancy. Vitamin D deficiency was considered with values ≤ 30 ng/mL. RESULTS: In total, 160 women were enrolled: 23 resulted positive for at least one SARS-CoV-2 related test (molecular swab or antibody tests). Vitamin D-associated polymorphisms were able to affect vitamin D levels in SARS-CoV-2 negative and positive subjects: remarkably, all the VDR TaqICC genotype patients were negative for SARS-CoV-2. In a sub-population (118 patients), vitamin D levels correlated with pregnancy-related factors, such as alpha-fetoprotein levels. Third-trimester vitamin D levels were lower in preterm births compared to full-term pregnancy: this trend was highlighted for SARS-CoV-2 positive patients. CONCLUSIONS: This is the first study demonstrating a role of vitamin D in affecting the clinical characteristics of pregnant women during the COVID-19 era. Further studies in larger and different cohorts of patients are required to confirm these findings.

Long-Term Pharmacokinetics of Dalbavancin in ABSSSI and Osteoarticular Settings: A Real-Life Outpatient Context.

Dalbavancin is a lipoglycopeptide approved for treatment of Gram-positive infections of skin and skin-associated structures (ABSSSI). Currently, off-label use at high dosages for osteoarticular infections deserves attention. This work aimed to study the long-term plasma pharmacokinetics of dalbavancin in outpatients with ABSSSI or osteoarticular infections, treated either with one or two 1500 mg doses of dalbavancin. A liquid chromatography-tandem mass spectrometry method was used to measure total dalbavancin concentrations in plasma samples. The results were analyzed through a non-compartmental analysis (NCA). Breakpoint minimum inhibitory concentration (MIC) was used to calculate AUC/MIC and T > MIC parameters, adjusted by 93% protein binding. A total of 14 patients were enrolled, 11 with osteoarticular infection and 3 with ABSSSI. Long-term pharmacokinetics showed median T > MIC (0.125 mg/L) of 11.9 and 13.7 weeks for single and dual dose, respectively. Similarly, median AUC0-2w/MIC ratios of 20,590 and 31,366 were observed for single and dual dose regimens, respectively. No adverse events were observed, and treatment success was achieved in 12/14 patients. Failure was associated with the worst clinical conditions, bone infections, and single dose. The results of this study show that dalbavancin exposure exceeds previously suggested pharmacodynamic targets. Optimization of these targets is needed for the osteoarticular setting.

Seasonal Variation of Antiretroviral Drug Exposure during the Year: The Experience of 10 Years of Therapeutic Drug Monitoring.

Although studies show an annual trend for immunosuppressive drugs, particularly during different seasons, no data are available for antiretroviral drugs exposures in different periods of the year. For this reason, the aim of this study was to investigate an association between seasonality and antiretroviral drugs plasma concentrations. Antiretroviral drugs exposures were measured with liquid chromatography validated methods. A total of 4148 human samples were analysed. Lopinavir, etravirine and maraviroc levels showed seasonal fluctuation. In detail, maraviroc and etravirine concentrations decreased further in summer than in winter. In contrast, lopinavir concentrations had an opposite trend, increasing more in summer than in winter. The etravirine efficacy cut-off value of 300 ng/mL seems to be affected by seasonality: 77.1% and 22.9% of samples achieved this therapeutic target, respectively, in winter and summer, whereas 30% in winter and 70% in summer did not reach this value. Finally, age over 50 years and summer remained in the final multivariate regression model as predictors of the etravirine efficacy cut-off. This study highlights the seasonal variation in antiretroviral drugs plasma concentrations during the year, leading to a better understanding of inter-individual variability in drug exposures. Studies are required in order to confirm these data, clarifying which aspects may be involved.

Analysis of Cannabinoids Concentration in Cannabis Oil Galenic Preparations: Harmonization between Three Laboratories in Northern Italy.

Medical cannabis is increasingly being used in the treatment and support of several diseases and syndromes. The quantitative determination of active ingredients (delta-9 tetrahydrocannabinol, THC, and cannabidiol, CBD) in galenic oily preparations is prescribed by law for each produced batch. The aim of this work is to describe the organization of the titration activity centralized at three regional reference laboratories in Northern Italy. Pre-analytical, analytical, and post-analytical phases have been defined in order to guarantee high quality standards. A cross-validation between laboratories allowed for the definition of the procedures that guarantee the interchangeability between reference laboratories. The risk management protocol adopted can be useful for others who need to undertake this activity.